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This is to certify that the
dissertation entitled

Heritable Somatic Instability in Hybrids Derived

from Protoplast Fusion of Petunia parodii (W.C.S.)
and Petunia inflata (Fries)
presented by

 

Linda Sue Davidson Schnabelrauch

yo" ..

has been accepted towards fulfillment
of the requirements for

Ph.D. . Horticulture
degree In

 

 

4.441

Major professor

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HERITABLE SOMATIC INSTABILITY IN HYBRIDS DERIVED

FROM PROTOPLAST FUSION OF PETUNIA PARODII (W.C.S.)

 

AND PETUNIA INFLATA (FRIES)

 

BY

Linda Sue Davidson Schnabelrauch

A DISSERTATION

submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1982

ABSTRACT
HERITABLE SOMATIC INSTABILITY IN HYBRIDS DERIVED FROM

PROTOPLAST FUSION OF PETUNIA PARODII (W.C.S.) AND

 

PETUNIA INFLATA (FRIES)

BY

Linda Sue Davidson Schnabelrauch

Somatic hybrid plants were regenerated following calcium-high
pH fusion of the unidirectional, sexually incompatible cross of

Petunia parodii wild-type leaf mesophyll protOplasts with

 

protoplasts from a cytoplasmic determined chlorophyll deficient
mutant of P, inflata grown in liquid suspension culture. Genic
complementation to chlorophyll synthesis and growth in selective
medium was used to visually select cell hybrids from parental cells
which, in the case of the P, inflata parent, remained colorless in
the selection medium, or exhibited a predetermined growth
inhibition, as in the case of the P. parodii parent; Hybrid calli
transferred to shoot regeneration medium were rooted and confirmed
as hybrids based on floral and leaf morphology, dominant
anthocyanin expression in the corolla limbs, chromosome number,
segregation for parental characters, and peroxidase and malic
dehydrogenase isozyme patterns. The frequency of somatic hybrid
plant production as a function of selection efficiency ranged from
3.5 to 5.3 hybrids per 106 protoplasts used, comparable to previous
reports, but parental reversion frequency approached 54%.

The phenotypic variation spectrum of regenerated hybrid plants
included subtle to moderate corolla variegation, corolla splitting,

chimeric leaf variegation, and various developmental anomalies such

LINDA SUE DAVIDSON SCHNABELRAUCH

as double styles, petaloid structures on the corolla limb and
ear-shaped projections at the corolla lobe junctions.

To quantitatively assess the level of anthocyanin instability,
counts were made of the number of variegated flecks and sectors on
flower limbs of somatic hybrid plants. Aceto-prOpiocarmine
squashes of pollen mother cells were also studied at various stages
of the division cycle to determine the number and behavior of
chromosomes in the hybrids.

Examination of selfed, sib-mated and testcrossed progeny
revealed that the expression of anthocyanin instability was
conditioned by two alleles segregating independently.

Morphological abnormalities in flowers of somatic hybrid plants
were attributed either to a type of hybrid dysgenic phenomenon or
nucleo-cyt0plasmic incompatibility following the loss of parental

chromosomes in a hybrid cytoplasm.

ACKNOWLEDGEMENTS

This dissertation is dedicated to the many workers in the area
of transposable genetic elements for providing the intellectual
stimulus by example, and to my husband, ROBERT L. SCHNABELRAUCH, my
father, HERBERT R. DAVIDSON, and my mother, HELEN L. DAVIDSON, for
influence and sacrifices that aided the completion of this degree.

Facilities and support of the Michigan State University
Department of Horticulture are gratefully acknowledged.

My thanks to K.C. SINK, major professor, L.C. EWART, J.
HANCOCK, J.W. HANOVER, H.H. MURAKISHI, and J.W. SAUNDERS for
guidance committee participation.

It is a pleasure to acknowledge the expert assistance of F.
KLOC-BAUCHAN, R.J. GRIESBACH and F.J. ZAPATA. Helpful discusssions

with BENJAMIN and FRANCIS BURR were appreciated.

ii

TABLE OF CONTENTS

LIST OF TABLES- —-

 

LIST OF FIGURES-

 

INTRODUCTION —

 

 

LITERATURE REVIEW;

 

Introduction

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
v
vii
l
4
4
Early observations on multinucleate cells 6
Plant protoplast fusion-- 7
Spontaneous fusion 8
Induced fusion—— 8
Models of cell fusion 9
Protoplast agglutination 10
Changes in membrane carbohydrates during fusion----11
Changes in membrane lipids during fusion 12
Fusion of cells at different stages of the cell
cycle 13
Premature chromosome condensation 14
Efficiency of plant protoplast fusion and hybrid cell
recovery: Selection systems 15
Hormone prototrophy and resistance to high hormone
concentration 16
Temperature sensitivity 16
Sensitivity to unusual metabolites 17
Complementation between recessive genes 17
Drug resistance and differential growth 19
Gene regulation patterns in hybrid cells 19
Progeny analysis of somatic hybrids—- 20
Variability in tissue culture 21
Phenotypic changes in regenerated plants 22
Organelle variation 23
Chromosome patterns in hybrid cells 24
Chromatin changes-- 25
Gene amplification 27
Somatic crossing—over and sister chromatid
exchange- 28

iii

TABLE OF CONTENTS, (cont'd.). Page

 

Transposable genetic elements 28

Relationship between transposable elements and

 

 

 

 

 

 

 

genetic instability—= 32
Maize genetic instability-— 33
,Drosophila genetic instabilityn 36
Nicotiana genetic instability 37
Petunia genetic instability 39
Origins of controlling elements 41

SECTION I

SOMATIC HYBRIDIZATION BETWEEN PETUNIA PARODII (W.C.S.)
AND PETUNIA INFLATA (FRIES)

 

 

 

 

 

 

 

 

Abstract 43

Introduction 44

Materials and Methods- 45

Results—— 52

Discussion 83

Literature Citedr- 88
SECTION II

HERITABLE SOMATIC INSTABILITY IN HYBRIDS DERIVED
FROM PROTOPLAST FUSION OF PETUNIA PARODII (W.C.S.)
AND PETUNIA INFLATA (FIRES)

 

 

 

 

 

 

 

 

 

Summary— 91
Introductionr- 92
Materials and Methods== 93
Results 96
Discussion== 123
Literature Cited-- 147
SUMMARY AND CONCLUSIONS 153
LIST OF REFERENCES-— 157

iv

LIST OF TABLES

Table ‘ Page

1.

1.

SECTION I

Yields and viabilities of Petunia parodii (wt) leaf
mesophyll protoplasts isolated from 51 day old plants
using 0, 0.5 and 1% Cellulase "Onozuka" R10, O-O.52
Driselase and 0-0.SZ Macerase for two hours 54

 

Effect of 24 hour dark incubation on division of
Petunia parodii (wt) leaf mesophyll protoplasts from
56 day old plants 55

 

P2 segregations for chlorophyll deficient seedlings

in Petunia parodii lines P-1043 and PD IR-40,

.3. inflata line INF IR-6l and g. parviflora line

P-1048 =67

 

F3 segregation ratios for green and white seedlings
in Petunia parodii line P-1043 and P, parviflora
line P-lO48 =69

 

Protoplast fusion of Petunia parodii (wt-m)CD
.P. inflata (ca-susp) using calcium-high pH _ 74

 

 

Morphological characteristics of somatic hybrid

 

 

 

 

plants 77

Corolla morphology of Petunia parodii®§. inflata

somatic hybrids 73

Mean quantitative measurements of corolla

characteristics in somatic hybrid plants 80
SECTION II

Morphological characteristics of Petunia parodii®
P: inflata somatic hybrids 98

 

 

Frequency profile of somatic instability in corolla
limbs of somatic hybrids expressed as mean sites
per flowers (based on 20 flowers/plant) 102

 

LIST OF TABLES, (cont'd.). Page

3.

4.

SF2 and TC segregations of progeny from somatic
hybrids 106

 

SF2 segregations for variegated corollas among
progeny from self— and cross-pollinations of somatic
hybrids 110

 

Testcross segregations for variegated corollas of
the cross 77-22-4-2 (Petunia parodii 43) x 15-6-------111

 

Reproductive characteristics of chloripetalous flowers
of somatic hybrid 15-2-- 114

 

Quantitative measurements of floral traits in
modified corolla phenotypes sectoring from somatic
hybrid 15-2 115

 

Association between split blossom characteristics
and type of corolla anthocyanin variegation in
somatic hybrid 15-2 - 117

 

vi

Figure

l.

2.

3.

4.

5.

LIST OF FIGURES
Page
SECTION I

The effect of Meicelase-P (O-SZ) with 0.152
Macerase, 92 mannitol, 40 ug/ml ampicillin,
10 ug/ml tetracycline and 10 ug/ml gentamicin,
in CPW salts at pH 5.8 on protoplast yield of
Petunia parodii leaf mesophyll protoplasts,

 

 

5 repliCations/treatment 53

Protoplast yield from liquid suspension and

callus cultures of Petunia inflata cytoplasmic
determined chlorphyll deficient mutant cell line

using O-SZ Cellulase "Onozuka" R10 with 1%

Macerase, lZ Driselase and 8% mannitol in CPW

salts at pH 5.8, 5 replications/treatment 57

 

Protoplast viability of Petunia inflata cytoplasmic
determined chlorophyll deficient mutant cell line

using O-SZ Cellulase "Onozuka" R10 with 12

Macerase, lZ Driselase, and 8X mannitol in CPW

salts at pH 5.8 . 58

 

Relationship between growth phase of Petunia

inflata cells in suspension culture and protoplast

yield. (a) Growth phase of cultured cells as

reflected by compacted cell volume and cell number,

(b) Protoplasts isolated at various times during

the development of cell suspension cultures.

Incubation mixture contained 2% Cellulase

"Onozuka" R10, 12 Macerase, lZ Driselase and 82
mannitol in CPU salts at pH 5.8 60

 

Protoplasts yields from in vitro shoot cultures

of Pp-nab——A, Pp-caH, Pi-naH, and
Pi-caO—O, using O-BZ Extractase with 2.52

Driselase and 42 mannitol in CPW salts at

pH 5.8 63

 

 

vii

LIST OF FIGURES, (cont'd.).

Figure Page

6.

7.

8.

l.

Viability of protOplasts isolated from shoot

cultures of Pp—naH, Pp—caO——0, Pi-naH,

and Pi-caD-——n, using 0-3Z Extractase with 2.5%
Driselase and 4% mannitol in CPW salts at pH 5.8

as determined by Evan's Blue staining 64

 

Yield and viability of Petunia inflata cytoplasmic
determined chlorophyll deficient leaf mesophyll
protoplasts isolated using O-SZ Driselase with

1% Extractase, 0.52 Macerase, 0.5% potassium

dextran sulfate and 42 mannitol in CPU salts at

pH 5.8 65

 

(a): Freshly isolated leaf mesophyll protOplasts
of_§gtunia parodii and protoplasts from liquid
suspension cultures of_§. inflata in M/S Pl 9M.
ca. 480x. (b): Heterokaryon produced after
calciumehigh pH fusion treatment. ca. ll45X.
(c): Cell colony after 12 days in M/S Pl 9M.

ca. 530x. (d): Callus with shoots produced on
M/S medium with 2 mg/l zeatin. ca. 2.4x.

(e): Adventitious root formation induced on M/S
medium with no exogenous growth regulators.

ca. 1.3x. 72

 

SECTION II

(a): Chimeric plastid distribution found on

somatic hybrids. (b): Anthocyanin variegation

patterns (left to right): stable, acyanic sectored,
minute acyanic flecks. (c): Anthocyanin variegation
patterns: p/m cyanic sectors. (d): Half-white,
half-magenta flower from somatic hybrid ll-l.

(e): Acyanic petaloid structure (arrow).

(f): Split blossom classes (left to right) narrow
split blossom, wide split blossom, deeply lobed
blossom 100

 

Viii

LIST OF FIGURES, (cont'd.).

Figure

2.

3.

4.

Page

(a): Floral types found on somatic hybrid 15-2

(top, left to right): stable, p/m cyanic sectored,
tobacco-like flower, (bottom) half-p/m sectored,
half-tobacco-like flowers, (b): Dilute floral

types found on albino branches of somatic hybrid

15-2 (left to right): stable, dilute anthocynanin
pigmentation, dilute anthocyanin pigmentation in
combination with tobacco-like flower form.

(c): Twinspot (circle with arrows) from corolla

of somatic hybrid 21-1. ca. 15X. 104

 

Regression of mean frequency profiles of progeny
on parental (a) acyanic sites (Table 3) and
(b) cyanic sites (Table 2) in somatic hybrids-------108

Possible genetic control of anthocyanin
biosynthesis in Petuniaghybrida (Kho et a1., 1977,
1978; Farcy and Cornu, 1979) 125

 

ix

Guidance Committee:

This dissertation is organized in journal style in accordance
with departmental and university requirements. Two sections were
prepared following the journal style formats of the Journal_gf

Heredity and Theoretical and Applied Genetics.

 

INTRODUCTION

Classical genetics is based on the segregation and analysis of
traits of individuals that arise from sexual crosses. Although it
is still the central theme of genetics, more recent technical
advances at the molecular, chromosomal and cellular levels have
established new areas of genetic analysis. Among these, somatic
cell genetics has advanced rapidly due to the impact of rapidly
expanding cell fusion technologies which make it possible to
generate cells and plants with altered genomes. It is now possible
to generate mutant cells harboring directed genetic alteration, to
create hybrid cells containing genetic information from two
different species, to construct cybrids where the nucleus and
cytoplasm of one species is combined with the cytOplasm from
another, and to transform cells with exogenously supplied DNA
sequences that become incorporated into the genome of recipient
cells. Such genetic alterations in somatic cells have underlined
the complexity of gene regulation systems in higher eucaryotes.

The fusion of somatic cells from two different species imposes
an added constraint on genetic control systems in that it requires
the coordinate functioning of both nuclei and cytoplasms. The
fusion of P. parodii®£. inflata protoplasts was undertaken to
explore this possibility. This study was designed to elicidate the
most efficient combination of nuclear and cytoplasmic determined

chlorophyll deficient mutants for fusion with wild-type somatic

cells which would lead to maximal heterokaryon production by
complementation in Petunia, However, regenerated progeny from this
somatic hybridization displayed perturbation in the genetic system
controlling anthocyanin biosynthesis that resulted in variegated
corollas. Subsequent investigations of the instability revealed
its inheritance into the SF2 generation, following segregation
ratios for two genes.

Repression of structural genes has been shown in some cases to
be caused by the insertion of small pieces of DNA at or near the
gene locus resulting in inactivation and mosaic or variegated
phenotypes. Insertions or deletions in gene families controlling
the expression of such genes also result in repressed gene product
synthesis. For example, the transposition of certain dispersed
repetitive gene families in Drosophila has been directly related to
Drosophila tissue culture systems. The mobility of such elements
may rely on the conditions supplied during culture, since the
mutants cannot survive normal conditions.

The P; parodii @3. inflata somatic hybrids also produced
other unexpected abnOrmalities. Nucleo-cytoplasmic incompatibility
developed as chromosome elimination progressed, evidenced by corolla
splitting in some of the hybrid plants. Such nucleo-cytOplasmic
incompatibility may have caused the instability in the anthocyanin
alleles. The onset of chromosomal-cytoplasm incompatibilies has
been correlated in DrOSOphila with the mobility of certain DNA
sequences resulting in hybrid dysgenesis. Traits often expressed
as a function of the dysgenic syndrome include a lack of
reciprocity in crosses between certain strains, the onset of

extensive chromosomal rearrangements, and the initiation of

unstable alleles.

The fusion process itself or other unidentified environmental
factors may subject genetic control systems to stress, suggesting
that certain control systems may not be effectively buffered and
protected against severe pressures such as those imposed by in.

vitro manipulations.

LITERATURE REVIEW

Introduction

Techniques which induce the fusion of somatic cells of plants
and animals to produce new cell hybrid combinations are being
refined rapidly. Applications to agriculture and medicine have
resulted in important contributions to our understanding of cell
biology and, in the case of plants, the creation of useable genetic
diversity (31,36,46,93,127,167,l70). Heterokaryons developing into
hybrid cells may result from synchronous division of both nuclei or
by direct nuclear fusion. Thus, analysis of gene regulation in
eucaryotic cells has been facilitated by the creation of cell
heterokaryons, their isolation and subsequent regeneration to
somatic hybrid plants. By fusing parental cells with unique
genetic markers, hybrids can be examined for expression of parental
characteristics and they can be used to study mechanisms governing
cell growth and differentiation during develOpment or tumor
formation. The fusion of terminally differentiated cells with
embryonic or nondifferentiated cultured cells can also be used to
elucidate whether cell specialization during maturation is due to
altered patterns of gene expression by repressor activation or due
to changes in the physical structure of DNA.

In addition, the fusion of plant cells enables studies of
segregation and expression of parental cytoplasmic organelles
co-functioning in one cell, heretofore impossible in sexually
produced hybrids. Once the cell wall has been enzymatically
degraded, the plant protoplast is amenable to genetic manipulations

which are rapidly becoming more sophisticated with the advent of

molecular and recombinant DNA techniques. Recent reviews
summarizing trends in protoplast technology have stressed its
potential for crop improvement (l5,3l,48,87,181).

However, only the Solanaceae and Cruciferae have provided
species in which protoplast isolation and regeneration to whole
plants, or fusion followed by plant regeneration has been
accomplished (9,57). Recent developments in in zi££g_culture of
crop species such as wheat, corn, pearl millet, and the legumes
(73,171,172) have renewed the optimism that major crap species will
benefit from the current cellular and genetic manipulation
techniques being used to successfully alter "model" species such as
petunia and tobacco.

Klesse and Duvick (91) related ten genetic needs of prime
concern to plant breeders which included incorporating resistance
to pests and stress into otherwise desirable genotypes while
simultaneously avoiding problems associated with polygenic traits.
What emerged was the need for manipulation of genetically complex
traits, which in turn demands further basic knowledge in the areas
of plant biochemistry, physiology and regulation of development.

In zitrg_manipulations in crops may lead to new approaches to these

problems by using potential advantages of the new techniques (55).

Many of the early findings of animal cell fusions were
reaffirmed by plant protoplast fusion. The animal workers found
through interspecies cell hybrids such as human®mouse and rat®
mouse that both parental genomes were expressed in the hybrid, and

furthermore, in long-lived interspecies hybrid cell lines, the

6

chromsomes of one species tended to be preferentially eliminated
(76,164). These findings have been replicated in plants and they
particular relevance in agricultural research as a means of
introgressing partial genomes.

The selective loss of chromosomes when one fuses whole cells has
lead to another promising approach to genetic modification which
involves the incorporation of only a selected part of one cellular
genome into another. Once a hybrid cell is made, the transfer of
favorable genes can be produced by gene recombination as a
consequence of allosyndetic meiotic pairing, translocations
involving nonhomologous segments of chromatin, or substitution of
alien chromosomes for another of the cultivated species (167).

EarLyobservationslgn_multinuc1eate cells:

 

 

The first observation of plant cell fusion was by Kuster (99)
on onion root and Elodea leaf cells. Upon plasmolysis and
deplasmolysis with calcium nitrate, he observed fusion of
subprotoplasts. In 1910, he fused freely isolated protoplasts by
the same process. After plasmolysis in calcium nitrate,
protoplasts were deplasmolyzed and upon expansion were seen to
fuse.

Mechanically isolated onion protoplasts were again used in
1929 by Lory (102). Fusion was observed after protoplasts were
held together by glass needles. By 1937, Michel (119) showed that
certain environmental pretreatments of plants influenced the
success of fusion and he also introduced the use of visual color
differences to follow the fusion process; radish and beet root
protoplasts containing native antho- and betacyanins were fused

with colorless onion and horseradish protoplasts, respectively.

7

In the 1960's, enzymatic degradation of plant cell walls made
possible the isolation of large numbers of protoplasts from many
tissue sources and offered a reliable method for isolating viable
protoplasts for use in plant cell biology studies and genetic
investigations.'

Plant protoplast fusion:

Power et al. (134) observed that subprotoplast fusion was
enhanced by sodium nitrate in cereal protoplasts. The first report
of successful regeneration of hybrid plants produced by sodium
nitrate fusion was by Carlson (18) between Nicotiana Llauca®fi.
langsdorffii. This combination was later reconfirmed by Smith et
al. (158) using polyethylene glycol (PEG) induced fusion. Several
other reports of observed fusion using sodium nitrate were made in
the 1970's (131,135).

In 1973, Keller and Melchers (94) described a procedure for
fusion based on reports with chicken erythrocytes which involved
the use of high concentrations of calcium buffered at a high pH
(10.5) and high temperatures (37°C). Subsequently, the high
pH-calcium fusion method was used by Melchers and Labib (115) to
create somatic hybrids between two light-sensitive mutants in
tobacco and by Schieder (145) to fuse two auxotrophic mutants of
Sphgerocarpus donnellii.

In 1974, Kao and Michayluk (84), and Wallin et al. (174)
reported the use of PEG to induce fusion in plant protOplasts that
did not simultaneously reduce protoplast viability. The use of PEG
coupled with calciumrhigh pH is currently used by many workers
(71,117).

Since the pioneering work with Nicotiana somatic hybrids,

8
fusion involving intra- and intergeneric crosses has resulted in
the regeneration of hybrids, but success has been restricted
primarily to the Solanaceae (57,170).

Spontaneous cell fusion:

 

Cells often fuse in organisms during ontogeny. Membranes fuse
in intracellular states, particuarly in cells involved in
endocytosis, bulk transport or in secretion. At fertilization,
male and female cells fuse to form the zygote. Fusion in higher
organisms appears to be ubiquitous and suggests that all tissues
retain a genetic capacity for effecting cell fusion (164). In
plants, vesicles of the Golgi apparatus coalesce to form the cell
plate during cytokinesis, secretory granules build up from fusion
of smaller vesicles and fuse to the plasma membrane. In fungi,
mononucleate haploid cells combine to produce binucleate cells
which can either remain as dikaryons or proceed into plasmogamy,
ultimately followed by nuclear fusion, meiosis and sporulation. In
animals, fusion of myoblasts to form myotubes, Osteoclasts and
giant cells creates polykaryons formed during inflamation. From
these examples, it becomes clear that membrane structure allows for
natural fusion and intracellular coalescence.

Induced fusion:

 

A fusogen was required to effect interspecies fusion with
regularity. The principle agents used to induce fusion in plant
cells include (1) sodium nitrate, (2) high calcium ions in solution
at high pH and exposed to high temperatures, (3) PEG, (4) a
combination of (2) and (3), and (5) various lipid derivatives.

Sodium nitrate was the first successful agent used to induce

fusion in plant cells that allowed retention of sufficient cell

9

viability so that regeneration of somatic hybrid plants was
possible (18,154). The second method employed calcium ions at a
concentration of 40-50mM which were buffered at pH 10.5 and kept at
elevated temperatures to induce fusion in meSOphyll protOplasts of
tobacco (88). Toister and Loyter (168) had earlier indicated
successful fusion upon exposure of chicken erythrocytes to 37°C at
a high pH, then cooling the erythrocytes in 40mM calcium chloride
and gradually rewarming them. Success using this method to produce
plant somatic hybrids has been amply demonstrated
(115,138,139,l45).

The use of PEG to induce fusion of plant cells is well
documented (27,40,43,84). This method has been highly successful
in the production of somatic hybrid plants between two chlorophyl

deficient mutants of_N. tabacum (115),_N. glauca and N. langsdorfii

 

(158), Petunia hybrida and P. parodii (135), and albino P. hybrida
and wild-type P. parodii (25).

A variety of other agents have also been used to induce fusion
between plant cells. Lysolecithin, a surface active lipoidal
substance, is a product of lecithin and has been reported to induce
fusion in hen erythrocytes (129). Polyvinyl alcohol, inorganic
salts, sea water, antibody reactions, and dextran sulfate (48,123)
have all been used as fusogens with some success, but none has so
far resulted in somatic hybrid plants.

Models.g§_gg11 fusion:

The plasmalemma is composed of 60-80% protein and 20-40%

lipid. The traditional model visualized the membrane as a

bimolecular leaflet of lipid molecules with the hydrophilic ends

oriented outward, inserted between two layers of protein molecules

10

extended in a B-configuration (93). The rigid bilateral symmetry of
such a model, however, does not allow for a tenable hypothesis
explaining fusion events, and it completely neglects the
carbohydrate component of the membrane.

In 1970, Lucy formulated the fluid mosaic model of membrane
structure (103) which provides greater membrane plasticity by
proposing the existence of two alternative forms of the
lipoproteins. These forms are the bimolecular leaflet form and the
micelle form. The lipids exist in the bilayer in a very fluid
state with membrane proteins embedded in them. This model allows
extensive movement of membrane proteins under certain stimuli as
required to explain fusion phenomenon.

A model proposed for membrane fusion (153) suggests that
fusogens and heat perturb the bilayer structure of the plasmalemma,
causing increased fluidity in localized lipid areas. Protein and
glycoprotein particles then become aggregated within the perturbed
membrane, leaving areas of the membrane free of protein. Calcium
may also aggregate these membrane proteins. The proteinefree areas
on closely apposed or agglutinated membranes are thought to be the
sites of fusion as the lipids interact and intermix. Electron
micrographs of fusing plant cells have shown agglutination to occur
over large area of the plasma membrane (177), but point to
localized membrane adhesions as the sites of initial fusion.

Protoplast agglutination:

 

PEG is thought to cause aggregation due to its slight negative
charge in solution, which in combination with calcium, may form
molecules providing electrostatic links between cells (84). The

divalent calcium ion may form bridges between PEG molecules and the

ll

negatively charged carbohydrates on the cell surface. In addition,
PEG plasmolyzes the protoplasts as well as the vacuoles of
differentiated cells.

Toister and Loyter (169) reported that agglutination by
calcium in chicken erythrocytes was probably mediated by calcium
ions quenching the negative surface charges caused by divalent
membrane ions.

Other lectins such as concanavaline A have been reported to
mediate adhesion in auxotrophic mutants of tobacco (70). Multiple
binding sites enable lectins to react with cells having suitable
membrane carbohydrates, thereby cross-linking the carbohydrates
located on adjacent cells (52). Other methods of agglutination
have involved polylysine, low speed centrifugation and
micromanipulation (66).

Changes in membrane carbohydrates during fusion:

Carbohydrates on membranes exist as neutral sugars such as
galactose, mannose or fucose, or as amino sugars or sialic acid.
’Such sugars are polymerized into free polysaccharides or are
covalentaly linked to lipids or proteins. Several lines of
evidence suggest that carbohydrates play a major role in cell
fusion. First, carbohydrates carry a characteristic negative
charge which is borne naturally by cells at physiological pHs, and
second, they play a part in the control of ion permeability. In
addition, carbohydrates have been shown to be the determinant
moieties for cell antigenic specificity and are often the receptor
sites for viruses, bacteria, and aggregating agents (164). Hence,
the carbohydrate moiety on the cell membrane plays a major role in

fusion mechanisms.

12

Calcium may react with the negatively charged cell surface
carbohydrates acting as a counter ion. Since carbohydrates seem to
play a negative role in fusion, after aggregation, the carbohydrate
containing coat must be removed or eliminated so underlying
hydrophobic layers of the two membranes can become contiguous.
Whithers and Cocking (177) provided some evidence for the presence
of glycoprotein in the outer surface of isolated plant protoplasts.

Changes in_membrane lipids during fusion:

 

The change from the bimolecular leaflet structure to a planar
array of micelles must occur if fusion is to be effected.. High
calcium concentration may cause fusion by influencing micelle
formation in lipid membranes (1,164,179). Divalent cations have a
- strong compacting effect on micelles, created by bridging
phosphoryl groups of adjacent lipid molecules, forming smaller
units of membrane which may intercalate easier. The high
calciumrhigh pH procedure may produce divalent bridges replacing
the monovalent cations such as sodium, potassium and other protons
as counter-ions on the anionic sites of the phospholipids, thereby
causing contraction of the lipid structures by water displacement.
and resulting in a greater likelihood of membrane fusion (21,143).
Membrane coalesence appears to be separate from fusion and the
merging of the cytoplasms requires microfilament activity,
involving not only morphological considerations but cytoplasmic
movement to open nascent cytoplasmic bridges into major connections
and creation of a single unit. Environmental conditions favoring
the functioning of microfilaments thus encourages colaescence

and fusion; high calciumehigh pH promotes membrane coalescence.

13
Fusion g§.g§11§.§£ different cell cycle stages:

When differentiated and cultured cells are used as fusion
partners, all combinations of G1, S G2 and mitotic cells can be
made. As a rule, homo- and heterokaryons show synchronous DNA
synthesis after fusion is completed (162,163). The trigger for DNA
synthesis appears to be dependent on the concentration of inducing
factors present in the cytoplasms of S phase cells. Synchronization
results from stimulation effects of S phase cytoplasms on Gl
nuclei (164).

Mechanisms for synchronization of mitosis are not fully
understood, but several generalizations can be made. Late G2 and
mitotic cells appear to have cytoplasmic factors that induce G1 or
S phase nuclei to enter mitosis more quickly and S phase cells
contain factors that prevent or delay entry of G2 nuclei into
mitosis (164). It appears that mitosis itself may be prolonged in
some instances so lagging nuclei catch up with the advanced nuclei
during extended metaphases.

Crucial events that follow cell fusion and lead to heterokaryon
formation are nuclear fusion, cytoplasmic mixing and stable
expression and replication of the combined nuclear and cytoplasmic
genomes. Often the formation of mononucleate hybrid cells results
in abnormal spindle organization and chromosome movements, so the
first division of heterokaryon and daughter cells fail. In
addition, multipolar divisions mey result. Interphase nuclei have
been observed to fuse in heterokaryons (28,29,40,82). Nuclear
fusion may also occur during the first mitotic divisions after
protoplast fusion in which division is synchronized. Overlapping
spindles can lead to the inclusion of both sets of chromosomes

within the new nuclear membrane. The fact that division has been

14

observed in heterokaryons arising from the cell fusion of
protoplasts from a wide variety of plants indicates that there may
be an absence of initial nuclear-cytoplasmic incompatibility
(28,40,72,82,86).

Coordination of nuclear events is frequent in heterokaryons,
but not always found. Some asynchrony and even negative synchrony
has been reported (10). It appears that nuclei can fuse to form
synkaryons and thus hybrid cell lines, or in the heterokaryons
nuclei can divide independently resulting in chimeral tissue (15).

In the intergeneric cross of soybean and corn, 50% of the
first divisions and 302 of the second divisions were found to be
synchronous (83) whereas division in Xigia and soybean hybrid cells
were not synchronous (86). Premitotic nuclear fusion as well as
mitosis has been detected cytologically in intergeneric plant
hybrids also (28,40,83).

Premature chromosome condensation:

The fusion of mitotic cells with cells in interphase often
results in precocious attempts by nuclei of the latter to enter
mitosis. The chromatin of interphase nuclei condense into small
chromosome-like structures unattached to a spindle and thus are
subject to loss in subsequent divisions. These forms have been
linked in HeLa cells and in plant cells to the stage of the cell
cycle at which interphase nuclei cells are fused with mitotic cells
(47,161,164). Cations that bind to DNA phosphate groups and
influence normal DNA-protein interaction have been linked to the

promotion or inhibition of mitotic chromosome condensation (164).

15
Efficiency g§_plant_p;ptoplast fusion and hybrid cell recovery:
Selection systems:

Hybrid cells do not always divide faster than parental lines
and must therefore either be enriched through selection schemes or
mechanically separated as single cells. Nonselective methods have
been used successfully in isolating plant hybrid cells between N.

glauca and soybean (86) and Arabidgpsis thaliana and Brassica

 

campestris (67,69,84). Visually identified heterokaryons are

 

followed individually at low plating densities in an enriched agar
medium or by the feeder layer technique (66,119). The advantage of
this system is that any two species can be combined and given
appropriate nutritional and environmental conditions to induce
division and eventually regeneration. However, the efficient
recovery of somatic hybrid cells requires that all potential fusion
products be visually or otherwise identified and induced to grow.
The low percentage of fused cells of the requisite combinations
the infrequent formation of synkaryons, and difficulties in
inducing division in hybrid cell lines all account for frequent low
production of hybrid cells (1 in 1 x 106) (15).

Identification of fusion products by selection techniques
can be done based on cytological differences in parental cell
morphology such as pigmentation, type of vacuolization of the
cytoplasms, or by the size and Structure of the nucleus (15,42,95).
Most selection methods rely on several of the above morphological
differences to check the functioning of the system.

Selection systems can be strict, resulting in the elimination
of both parental lines, or semi-selective, in which cells of one
parent are eliminated while heterokaryons and cells of the other

parent both divide. At the cellular level, these selection methods

16
can be used to study nucleo—cytoplasmic relationships. An added
selection process can be made at the whole plant level.

The following selection strategies have been devised for
working with plant cells which have high levels of biological
competence in terms of mutant production:

Hormone prototrophy and resistance 59 high hormone concentration:

Carlson, Smith and Dearing (18) and Smith (158) used
hereditary hormone prototrophy in combining N. glauca and N.

langsdorffii by selecting dividing hybrid cells in a medium.with no

 

auxin or cytokinin. Carlson reported the formation of stable

amphiploids similar to sexually produced plants, while Smith, upon

analysis of 23 somatic hybrids, found higher ploidy levels.
Differences in hormonal factor requirements were used for

selecting hybrid cells from Petunia hybr-ida® Parthenocissus

 

tricusgdata (134) and P. hybrida®§. parodii (136) combinations.
As a result of complementation, the hybrid between P. hybrida and
‘P. parodii displayed tolerance to high levels of hormones and made
possible the preferential growth of hybrid callus tissue which was
later regenerated into plants.

Temperature sensitivity:

Selection of interspecific hybrids may use the temperature
sensitivity of one parent coupled with differential tolerance to
extreme temperatures in the other. Dudits (39) used high
temperature for selection of hybrids in cell cultures of soybean
and rice.. At 37°C, rice was able to divide whereas soybean did
not. When plated in a medium that supported only soybean division
coupled with a shift toward higher temperatures, the heterokaryons
divided while the parental cells did not. The recent isolation of

temperature sensitive mutants in tobacco (107) has renewed interest

17

in the possible application of temperature sensitivity to selection
of somatic hybrid cells.

Sensitivity tg_unusual metabolites:

 

 

Conavanine is an unusual amino acid that has shown species
specificity in terms of resistance and may be applicable to a
selection system (154). Along these lines, propanyl resistance in
rice was used by Dudits and Nemet (41) to prepare a semi-selective
medium for fusion with wheat protoplasts. Propanyl inhibits the
enzyme arylacylamidase. The tolerant rice parental cells and
heterokaryons were shown to divide in the medium. Herbicides and
phytotoxins have been suggested for selection as well
(49,74,154,l70,) with hybrid formation dependent on the activation
of enzyme detoxification systems.

Complementation between recessive genes:

'The search for biochemical auxotrophs in higher plants has not
yet resulted in as much mutant material as the procaryotic systems.
However, several auxotrophic lines have been used to some extent in
plant hybridization studies.

Kao (89) has shown that several glycine auxotrophs can be
classified into four complementation groups. Schieder (145) used
auxotrOphic mutants of Sphaerocarpgs donnellii and after fusion and
plating on a simple mineral medium was able to select hybrids which
had complemented with respect to nicotinic acid and glucose
requirements. He also used complementation in pantothenate
deficient Datura lines to select hybrid cells (89).

Maliga et al. (105) used nitrate reductase deficient lines of

_N. tabacum, cix and nia, to complement the wild-type and showed on

 

the basis of chlorate resistance that the hybrids were the result

18

of complementation.

A selection process developed for human and animal cells by
Wright (178) allowed selective growth of fusion products by
complementation between artifically induced metabolic blocks
created by diethylysocarbonate and iodoacetate. Nehls (125)
applied this technique successfully to plant cells in the fusion of
.SQIQngm.nigrum and Petunia h brida, but only limited growth of the
heterokaryons occurred.

Chlorophyll deficient mutants have been most frequently
employed in selection schemes as a way of visually identifying
hybrid cells (25,115,146,l47). The first demonstration of albino
complementation was in maize using a green chloroplast containing
cells fused to a white chlorophyll deficient mutant cell line (63).
In the heterokaryons, greening was observed in the white
chlorOphyll deficient plastids.

Melchers and Labib (115) used complementation between two
chlorophyll deficient mutants in tobacco to select hybrid cells. In
both lines, the mutation involved sensitivity to high light
intensity, but was determined by non-allelic recessive genes, §_and
v. As a result of complementation and selection under high light
intensity, green pigmented colonies could be visually distinguished
from the yellowhgreen parental cells. The regenerated hybrids were
determined to be identical to the sexual hybrids.

Samsum tobacco and the sulfur mutant SQSQ, a homozygous
chlorophyll deficient tobacco line, were used by Gleba (64) to
isolate hybrid cells showing the restoration of photosynthetic
ability as a result of nuclear and cytoplasmic complementation. In

contrast, Dudits (45) using an albino carrot line fused with X-ray

l9

inactivated parsley, did not find complementation as evidenced by
restoration of chlorophyll synthesis in the hybrids, but rather a
rapid sorting out of each chloroplast type as well as a
preferential loss of parsley chromosomes from the synkaryons
resulted. However, cytological markers from the parsley parental
line made it clear that stable parasexual integration of part of
the parsley genome into a chromosome of the carrot line had indeed
occurred.

Drug resistance and differential growth:

Cocking et al. (25) and Power et al. (136) used suppression of
growth and actinomycin D sensitivity to produce hybrids between
Petunia hybrida and P, parodii. Complementation between the albino
P, hybrida line coupled with wild-type P, parodii leaf mesophyll
cells allowed visual identification of hybrid callus. A strict
selection scheme was designed with actinomycin D affecting the
growth of one parental line, 3. h brida, while the selection medium
employed did not allow,§. parodii to grow beyond the small colony
stage. Differential growth coupled with complementation to green
allowed Power at al. (138) to identify and remove cells at the
callus stage in the somatic hybrid between P. inflata and P.
parodii.

Gene regulation patterns 1g hybrid cells:

Data on the regulation of gene activity in somatic hybrid
cells are relatively scarce. Observations on the fate of
physiological and biochemical markers in hybrid cells and
heterokaryons have shown that in order to function in the same
nucleus and elicit normal morphogenesis, the genomes from two

different sources must be intercompatible with respect to

20

regulation of gene expression (15). Several markers have been used
to show that many heterokaryons express information coded by both
parental nuclei. These include male sterility or fertility
(6,79,180), the inheritance of peptides from the large subunit of
Fraction 1 protein coded by the chloroplast genome (24,96,116), and
the isozymes alcohol dehydrogenase or aspartate aminotransferase
(49,175). Many other types of heterokaryons reveal that prOperties
characteristic of one or the other parental cell lines are
eliminated or suppressed in the heterokaryotic state.

Progeny analysis g§_somatic hybrids:

Several progeny analyses have been performed on the

interspecific and intergeneric somatic hybrid plants and cybrids
following recovery and regeneration after protoplast fusion
(3,64,105,138,139,148,157). Generally, somatic hybrids have been
evaluated on the basis of chromosome number, chromosome size
differences, isozyme patterns, intermediate morphological
phenotype, peptide distribution of ribulose-l,5-bisphosphate
carboxylase-oxygenase, and restriction enzyme digests of
mitochondrial and chlorOplast DNA (78,96,98,99,
124,148,151). However, the breeding behavior of somatic hybrids in
subsequent generations has been reported in only a few instances
(137,149).

Schieder (149) found after four generations of selfing somatic

hybrids produced from the fusions of Datura innoxia®D. discolor

 

and Q. innoxia®D. stramonium, that chlorphyll deficient types
occurred regularly. He observed little variability among selfed
progeny with the exception of one plant which lacked the violet

striped throat contributed by D, discolor. That exceptional plant

21

was aneuploid.

Power at al. (137) compared flower color segregation in sexual
and somatic hybrids of Petunia hybrida x P, parodii. They observed
no cytological abnormalities in meiosis of hybrids produced by
protoplast fusion, yet minor segregational variations were detected
between individuals from both somatic and sexual hybrids. Overall,
both types of hybrids segregated for flower color in a similar
manner.

Variability ig_tissue culture:

.In_!itrg techniques have not had the impact on conventional
breeding that was anticipated at the beginning of the last decade.
Protoplast regeneration and successful regeneration following
somatic hybridization and the demonstration of universal plant
totipotency have been recalcitrant in most major crop species.
However, in the face of these seemingly insurmountable problems, it
has become increasingly evident that the in_yi£rg_culture condition
itself may be generating varying degrees of genetic variability
(4,100,126,154,l67). The genetic mechanisms behind such variation
has remained, to this point, speculative, but several different
mechanisms, chromosomal and genie, appear to be functioning at the
nondifferentiated state in cell cultures.

Although the chromosome complements often remain stable in
meristem tissue culture propagation systems, variability in plants
regenerated from callus has been directly or indirectly attributed
to changes in ploidy, gross chromatin structural changes, or other
chromosomal abnormalities such as formation of rings, multivalents,
chromosomal chains, and multipolar divisions (154). Yet wide

combinations for fusion have resulted in at least a few divisions,

22

indicating no somatic incompatibility exists early in the ontogeny
of fused cells (65,86).

Phenotypic changes in regenerated plants:

 

 

Culture induced variation after 32 yitrg propagation has been
exploited for use in crop species such as sugar cane, potato,
tobacco, rice, oats, maize, barley, Brassica species and others
(100). Different expressions of physiological and morphological
changes have been reported including habituation (114), changes in
biochemical sensitivity (23), alterations in growth habit (77), and
alterations and modifications of cellular constituents such as
pigmentation changes (159). Morphological changes have included
leaf and flower variegation, changes in quantitative characters
such as floral and leaf dimensions, and pollen fertility (105). In
addition to morphological traits, there have been instances of
enzyme and other biochemical alterations. Carrot cultures have
been isolated that show high levels of 5-methyltryptophan (176).
Partial resistance to methionine-sulfoximine has been reported in
toabcco after mutagenesis (18), and streptomycin resistance lines
in tobacco have been generated in culture using selective
techniques (113). Some carrot somatic hybrids have shown
cycloheximide resistance (101).

Somatic hybrids from the cross Nicotiana knightiana

 

(wild-type) ®N. tabacum (albino) showed variable but significant
numbers of plants with leaf plastid and flower pigment variegation
which was attributed to numerical instability and subsequent
somatic segregation of the chromosomes (105). These hybrid plants
displayed heterogeneity in overall morphology, while reports of the

hybrid combinations have indicated stable production of hybrid

23

plants with intermediate parental characteristics
(69,136,138,l39,l48).
Organelle variation:

Plastid segregation in somatic hybrids has been shown to
' follow the rapid and random sorting out of both parental types
(3,6,24,68,96,116,ll7). In interspecific hybrids, plastid
segregation may be the consequence of chromosomal-cytOplasmic
interactions (151). Selection systems used for the recovery of
heterokaryons could inadvertently be applying pressure to one or
the other parental types.

In addition, the differentiated state of wild-type plastids
with the mutant proplastids of the mutant protoplasts of the fusion
partner may be causing uniparental plastid predilection
. (25,lOS,llS,l38). However, in intrageneric fusions, chloroplast
DNA (chNA) has been shown to sort out randomly and completely to
one of the parental types, usually wild-type, even when the above
constraints of nucleo-cytoplasmic incompatibility, selection
pressures, and state of differentiation of isolated protOplasts
were intentionally eliminated (151).

A recent report of changes in the peptide patterns of the large
subunit of Fraction-l protein in plants regenerated from anthers of
somatic hybrids has shown that both parental types of the large
subunit peptides were translated in somatic hybrids (78). The
plastid genome of N. rustica appeared to be preserved in the
hybrids even though the initial predominant expression was from the
N, tabacum genome. In addition, restriction enzyme analysis of
chNA revealed a high frequency of chNA alteration in male

sterile, but not male fertile N. tabacum cytoplasms which suggests

24

recombination of chNA may have occurred (98).

Mitochondrial DNA (mtDNA) has also shown variation in tissue
cultured plants (6,59,124). Using restriction analysis of mtDNA in
progeny of selected, phytotoxin resistant male fertile plants,
Gengenbach and Connolly (59) found three lines with unique
restriction patterns, distinct from each other and from N cytoplasm
maize. Nagy, Torok and Maliga (124) found extensive rearrangement

of mtDNA in N. tabacum®§. lmightiana somatic hybrid plants.

 

Chromosome patterns 2f hybrid cells:

 

 

Early observations by Sorieul and Euphrussi (50) of
interspecific hybrid animal cells showed chromosome numbers which
approximate or were slightly less than the expected number if a l +
1 fusion had occurred. Only rarely did the mean values for the
cell population exactly equal the sum of the parental karyotypes
(50).

In plant somatic hybrids, conflicting reports of karyotype
stability (18,43,45,105,136,144,148,157) have lead to a
reassessment of potentials for complete genome retention and
expression in hybrid cells. Regenerates with aneuploid chromosome
numbers or higher ploidy numbers than expected from the fusion of
one nucleus of each parent has been reported in every case but two
(18,138). In the cross Daucus carota®_D. capillifolium (43),
departures from the expected chromosome numbers were attributed to
abnormal chromosome number in the cell cultures used for fusion,
the loss of chromosomes during development of the hybrids, or the
involvement of more than two protoplasts in the formation of a

fusion product.

25

In human®mouse hybrids, the preferential elimination of
chromosomes of one species with the retention of chromosomes of the
other species occurs frequently. Little is known about the
mechanism of chromosome loss and the factors which determine the
direction and rate of loss (164). However, the direction and rate
that the reduced chromosome number comes to predominate in the
hybrid can be controlled to some extent by culture conditions, the
species involved in the fusion, and also the state of
differentiation of the parental cells used in the fusion (89).
Chromosome loss is presumably a consequence of unequal distribution
of chromosomes during mitosis and cell division, and the growth
advantage that some of the resulting variants have over the
original hybrid clone (35,89).

Cell fusion has been a valuable technique for mapping human
chromosomes. With the identification of individual chromosomes by
standard chromosome morphology based on ratios of arm lengths,
location of secondary constrictions or banding with quinacrine, it
has,been possible to study the loss or retention of particular
enzymes in cultured cells induced to lose all but one specific
chromosome. By relating the expression of a human gene product to
the presence of specific human chromosomes, one can identify the
chromosome containing the gene for that particular product
(89,164). Such mapping methods have not been employed in plant
somatic cell hybrids.

Chromatin-changes:

Evidence points in some cases to culture induced variability

being generated by chromosome breakage and subsequent ligation

(100,132). Since the culture environment may be permissive in that

26

it may allow such events to survive, it may also provide an
environment which stimulates recombination in interspecific hybrid
cells. If this introgression could be controlled and directed, it
would have an immediate beneficial impact on the production of
desirable genotypes. Orton (126) found that tissue culture of
Hordeum caused polyploidy, aneuploidy and chromosomal
rearrangements as evidenced by an examination of pollen mother
cells and isozymes from leaf tissue of plants regenerated from
callus. Plantlets derived from cultures of the sexual cross of H,
vulgare x_H. jubatum had enhanced multivalent formation.

Chromosomal behavioral irregularities common to heterokaryons
included chromosomal "stickiness", bridges, fragments, rings,
branched chromosomes along with di- and triconstrictional
chromosomes observed in one parental genome (N. glauca) but not the
other (soybean) (46,86,105).

After 4 to 7 months in culture, Arabidobrassica hybrid cell
lines contained chromosomes of both species (65). However, a
significant percentage of cells with di- and multiconstrictional
chromosomes were observed. Isozyme studies using esterase, lactate
dehydrogenase and peroxidase stains supported the claim that
chromosomes of both parents were present.

Often phenotypically altered plants are not grossly aneuploid,
but show a normal karyotype. High yielding lines regenerated from
potato protoclones had normal karyotypes (152) and sorghum clones
displaying drastic phenotypic alterations also had normal
karyotypes (56). In somatic hybrids between_2. carota and

Aegopodiumgpodaggaria, karyotyping indicated that only Daucus

 

chromosomes were present, yet phenotypically, the plants displayed

27

the CAPO characteristics that could only be associated with a
hybrid genome (45). The rearrangement of small chromatin segments
have been substantiated by the observance of chromosomal
irregularities such as bridges, accompanying acentric fragments,
ring chromosomes and micronuclei. Such breaks may result in gene
position changes which influence exon splicing and thus
transcriptional efficiency. When structural genes are brought in
contact or at least in close proximity with heterochromatin as a
result of rearrangements, altered gene expression results (110).
When rearranged and normal chromatids occur in structural
heterozygotes, the wild-type gene function occurs in only part of
the cells, creating mosaic or variegated phenotypes with areas of
wild-type and mutant cells occurring in sectors of tissue. This
phenomenon appears to enforce the view that genes do not act
independently, and demonstrates that precise linear continuity of
genes is important in determining the pattern of gene action, or
coordinate gene function even with the excess of intervening and
repetitive sequences characteristic of eucaryotic DNA.

A rearrangement may also delete or inactivate a dominant
allele allowing the recessive allele to be expressed leading to a
condition known as culture-induced hemizygosity (22). Such
deletions of specific loci appear to have occurred in tobacco
clones (4). -
figggugmplification:

In eucaryotes, it has been documented that structural genes
may be amplified during differentiation or in response to
environmental pressures (150,173). Stepwise selection has been

used to increase and ultimately amplify the gene dihydrofolate

28

reductase in mouse cell lines by using the inhibitor methotrexate
(150). Also, it has been possible to select
N-(phosphoacetyl)-L-asparate resistant cell lines in hamster with
concomitant amplification of the CAD genes (172).

In plants, evidence for gene amplification is indirect, but
Gengenbach and Green (58) obtained resistance to Helminthosporium
maydis race T toxin by stepwise selection methods. Similarly,
Nabors et al. (122) achieved salinity tolerance in tobacco using a
successively higher salt concentration for selection. ££;§lEE
evidence for gene amplification has not yet been demonstrated in
plants.

Somatic crossing-over and sister chromatid exchange:

Somatic crossing-over in heterozygous tissue results in twin
spotting or sectors of dominant and recessive homozygous tissue
adjacent to each other. It has been reported in many plant species
such as tobacco, Antirrhinum majus, Tradescantia, Gossypium, and
soybean (100). In addition, twin spots have been reported in
homozygous cotton lines (5). One case of twin spotting was found
in tissue cultured-Sggg tobacco heterozygotes after irradiation
treatments (20). Tissue culture allowed the expression of somatic
crossing-over events induced by outside sources. Direct effects of
the tissue culture process on the frequency of somatic
crossing-over events have not been demonstrated.

Sister chromatid exchange leads to unequal genetic exchanges
through duplications and deficiencies. The induction of sister
chromatid exchange by tissue culture has not been shown.
Transposable ggnetic elements:

Over the past decade, develOpments in molecular genetics have

lead to the realization that chromosomal Spatial arrangements are

29

not static but constantly undergo significant sequence alteration.
The biological consequences of such genetic alterations can be

extensive.

First reports of agents that appeared capable of inducing
alteration in chromosome structure and gene expression were in
maize by McClintock (109). The drastic alteration in chromsome
organization such as deletions, inversions, and duplications
occurred at sites which mapped classically as distinct genetic
units but which were also capable of transposition to new sites
(111). Studies on transposable genetic elements have shown that
the fluid rearrangements of DNA may provide adaptive advantages
under some environmental conditions (16).

The function of mobile elements in the genome has been the
subject of considerable speculation. McClintock (109) and Calos
and Miller (16) proposed that translocatable elements might be
involved in control of developmental pathways during cell
specialization. Recent research has provided examples
substantiating this view, with the finding that mature immunoglobin
kappa light chain genes are formed during development from
separately coded V, J, and C domains (16). Kappa light chain gene
formation involves a directed rearrangement of sequences that
requires the excision of intervening DNA, which is partly
responsible for the diversity of immunoglobin molecules. Other
processes which generate diversity, such as antigenic variation by
parasites, may also involve DNA rearrangements (l6). Yeast mating

type conversion is another example of a directed rearrangement of

30

DNA sequences to perform a specific function (16).

The phenomenon of discontinuous coding sequences may also be
related to transposable elements. The intervening sequences are
generally terminated in short, imperfect direct repeats which may
have been derived from insertion sequences (16,90).

The presence of transposable elements in the eucaryotic genome
may allow adaptation of a cell to a new environment. Transposition
allows both movement of specific coding regions and duplication,
thereby increasing gene dosage and providing for genetic variation
without lethality (16,90). Movement of a sequence to a new
location can bring genes into a region where they would come under
the control of genes active in that region, or alter the expression
of genes near which it has inserted. Such events over time would
explain the gradual alteration in DNA sequence arrangement observed
in many species (54). Directed rearrangements could result in a
faster programmed evolution than is possible with single base
changes in structural genes.

Natural selection favors individuals and populations that have
acquired traits conducive to survival and reproduction. The
generation of biological variation which gives rise to new and
potentially advantageous combinations of genetic traits, is of
prime importance in this endeavor. Transposable genetic elements
possess the ability to induce chromosome reorganization, and thus
may make significant contributions to changes in gene expression
and genetic diversification.

Flavell (54) proposed a model for the structure and evolution
of higher plant genomes in which patterns are created and evolve by

transposition of short pieces of DNA 100 to 1000 base pairs in

31

length into different DNA sequences, creating three organizational
patterns: repetitive DNA, tandem arrays of repeating units, and
repeated DNA interspersed with short segments resulting in complex
permutations. Such movement is postulated to be relatively common,
and under stressful conditions, transposable elements may provide
the needed genetic plasticity required for survival.

Most sequences in cereal chromosomes have been created by
recombination of unrelated sequences into new linear combinations.
During evolution and divergence of the species, rounds of sequence
amplification and deletion may have occurred, creating major
sequence differences between chromosomes and related sequences
(54).

Controlling elements are transposed by recombinational
mechanisms which are not dependent on extensive genetic homology
between DNA sequences. General genetic recombination results in
genetic exchange of alleles, while controlling elements initiate
major structural permutations in DNA (12). Such alterations affect
adjacent genes by increasing or decreasing the activity of the
gene. Thus, the elucidation of information concerning controlling
element function may lead to further understanding of the
expression of plant genes.

Evidence for transposable genetic elements exists not only in
bacteria and higher plants, but similar genetic behavior has been

observed in other eucaryotic organisms including Drosophila

 

melanogaster (11) and Saccharomyces cerevisae (17); The sequences

 

Tyl in yeast and copia in DrOSOphila seem to be active in
transposition. In contrast to bacterial systems, however, there is

a lack of integration specificity. Although lack of specificity

32

may preclude the involvement of transposable elements in
development, they may, like procaryotic transposable elements,
modulate gene activity given specified environmental conditions.
Relationship between transposable elements and genetic instability:

The fate of genes and chromosomes following interspecific gene
transfer has been given detailed study in some species (60). Genes
from one species can often be introduced into another without
evident changes in their action and with little effect on their
stability as in hexaploid Triticum, where alien gene transfer is a
productive method for introducing new genetic variability into the
species.

On the other hand, genetic instability can often be induced
through interspecific crosses. In dicots, one of the earliest
known cases of genetic instability following hybridization was

described by Harland (76) in Gossypium purpurascens. A gene

 

controlling the appearance of petal spots was found to be unstable
upon backcrossing to Q, hirsutum. Also in this genus, an unstable

leaf shape allele in Q, arborensis became stable in hybrids with g,

 

anomalium. Such information suggests relative stability of a gene
may be under control of the residual genotypes.

Mosaic regions in plant tissues have been reported in a range
of species and have been related to a variety of genetic causes.
Sastry (144) hypothesized that naturally occurring cases of
instability in plants were the result of residual alien genetic
material left from evolution. Over time, residual DNA fragments
may have become permanently fixed in specific locations on the
chromosome, resulting in little or no transposition, or the new DNA

fragment may have been recruited for different functions, such as

33

gene regulation during deve10pment resulting in predictability of
controlling element behavior.

Maize genetic instability-

 

Mangelsdorf (108) pointed out in maize teosinte hybrids that
gene instability had its origins in the interaction of two alien
genomes or parts of genomes. While studying the inheritance of
pigment distribution in maize plants that had undergone cycles of
chromosome breakage, gene suppression or activation was observed at
certain times (110). The patterns of variegation were heritable
and were shown to move inactivating genes on other chromosomes.
Recently, further support for the biological switch hypothesis was
provided with the finding that mutator activity in maize arose late
in ontogeny and thus was developmentally rather than
chronologically triggered (165).

Chromosomal rearrangements are considered an essential
property for classification of mobile genetic elements as
controlling elements (16). McClintock's genetic analysis suggest
that three or more genes are often affected simultaneously,
indicating that the structure of the chromosome can be rearranged
at the site of the controlling element (111). Controlling
elements are often organized as systems consisting of a regulatory
and receptor elements. To detect the receptor, it must be present
at or near a gene with an identifiable gene product. Its presence
results in the suppression of gene activity. However, when the
receptor is in the same genome as the regulatory element, gene
expression is restored in some cells resulting in variegation of
gene expression. The regulator is thought to affect the receptor

through some trans-acting signal (53,126).

34

Two systems of controlling elements exist in maize: the
autonomous and nonautonomous mutable systems (125). In autonomous
mutable systems, the receptor and regulatory elements are both
located at the controlled gene. In the progeny of plants with
autonomous controlling element systems, the element appears to have _
been transposed to a new locus, resulting in a change in linkage
relationships.

In the nonautonomous systems, the receptor and regulatory
elements are located at distinct positions in the genome and
excision and subsequent reactivation of the receptor is dependent
on the presence of the regulator.

The Agzys system in maize is one example of a nonautonomous,
two element system. The receptor 22 can be identified after
insertion and subsequent suppression or alteration of gene action
at a locus. The location of D3 is stable in the absence of a
signal from the regulator Ag: When Ag_is introduced into the
genome by crosses to specific tester lines, 23 may transpose from
the locus leaving a stably altered or completely functional allele.

Excision is found in maize and Drosophila to be under genetic

 

control and may be influenced to a certain extent by the background
genome (110,126).

The chromosomal location of 2s may be determined when it is not
located at a defined locus by its ability to cause chromosome
breaks. In response to Ag, 2s can induce breaks at the point of
its insertion, resulting in acentric fragments carrying genes
between gs and the end of the chromosome. A dicentric chromosome
carrying the genes between the centromere and 2g is formed in the

daughter cells that receive the broken chromatid. This initiates

35

the breakage-fusion-bridge cycle. The dicentric chromsome breaks
in ensuing anaphases, resulting in random losses of marker alleles.
The position of 2;, therefore, can be determined relative to other
markers (14).

Besides transposition, there are-other known consequences of
events initiated by 2s that appear only when AS is present in the
nucleus. These include dicentric chromatid formation,
deficiencies, duplications of segments, inversions, ring
chromosomes and reciprocal translocations between chromosomes
(110).

Bacterial transposons and maize controlling elements have some
similarities, but are generally less complicated that those found
in maize. Both types are limited in number, indicating that the
transposition mechanism is the property of specific elements, using
specific sequence recognition sites for enzymes (38). Likewise,
both types exert gig effects on genes in their vicinity. If they
are integrated into a gene, the gene is completely inactivated. If
the site of integration lies adjacent to the gene, then increased
or decreased gene activity results (16,90,126). These effects are
heritable, however, and in maize, controlling elements occur in
pairs rather than singly.

Other complexities enter into the activity of maize
controlling elements which, unlike bacterial transposons, appear to
program and direct development. McClintock described controlling
elements that programmed the time and type of gene action during
development and which had the capacity to integrate the activity of

two or more genes (112).

36
Drosophila genetic instaibligy -

In Drosophila, DNA of some moderately repeated gene families

 

are known to be mobile. Transposition of the 412, 297 and copia
disperse repeated gene families have been reported in both fly
populations and in tissue cultured cell lines (132). Mbbile

sequences were more abundant in the genomes of Drosophila tissue

 

cultured cell lines than embryo DNA as detected by DNA—DNA

hybridizations (160). Potter demonstrated that these elements were

 

amplified and transposed in the Drosgphila genome during the course
of cell proliferation in culture (132). .Furthermore, the
additional elements which occurred in the tissue culture cells
transposed to different locations in the genome rather than being
amplified locally. The insertion frequency of the elements 412,
221, and ggpi§_appeared to be independently modulated in the
cultured vs. embryo genomes, however, each of the three elements
displayed differential increases in the number and location of
insertion sites as evidenced by $3 gigg hybridization using 3H-
labeled cDNA.. The tissue culture environment was judged more
permissive for such transpositions, since cells would tolerate more
mutation than the embryos which must develop into a viable
organism. All nonlethal transposition events were presumed to
remain in the cell population (160).

Studies on the transposition of elements in Drosophila cell

 

culture suggests many potential insertion sites exist and that the
only function of these elements may be to promote genetic
variability. Their gene products may only be necessary for the
maintenance and mobility of the elements themselves, rather than
for other cellular processes (160) as in the plant systems.

The moderately repeated gene families 412, 297 and COpia have

37

DNA sequence homology and thus may be evolutionarily conserved.
These DNA sequences are similar to procaryotic transposable
elements in that they contain AT-rich inverted repeats on the ends
(26,133). All bacterial tranposons studied to date have inverted
repeated sequences on the end.

Nicotiana ggnetic instability -

Genetic instability frequently arises following interspecific
hybridization and is of particularly common occurrence in hybrids
of the genus Nicotiana. Therein, a variety of phenotypic
expressions of hybrid instability are found including variegation
in the pigment of flowers or leaves, variation in morphology and
plant habit during development, and variability in growth among
hybrid plants (156). Several examples of instability in
interspecific Nicotiana hybrids were cited by Kostoff (94) where in
all of the hybrid combinations, one parent species was a member of
the Alatae section of Nicotiana..

Unstable genes have been found in Nicotiana with prOperties
resembling the controlling element systems described in maize
(142,157). High frequencies of the unstable gene 1 were produced
from the cross N. langsdorffii xIN. sanderae. The allele was not
observed in the parental lines which were assumed to be homozygous
for a dominant V conditioning uniformly colored non-variegating
flowers. Progeny with distinct patterns of anthocyanin distribution
in floral tissues were found. This somatic instability was fixed
into a genotype close to the N. sanderae parent by a series of
backcrosses followed by selection for the instability. Crosses
among the major distinct phenotypes revealed simple inheritance of

three distinct genotypes conditioning four phenotypic classes. Two

38

separate alleles at the y locus were required to account for the
different breeding behaviors of the variegated classes. The class
Var-l phenotypes occurred over a wide range of time during plant or
flower development and therefore, sectors involving only a few
cells, or major portions of the flower were found. Var-3
phenotypes were similar to Var-l but showed a difference in the
timing of the sectoring events. The genetic constitution of Var-3
was postulated to include a unique and independently segregating
regulator component in addition to the y_locus called flecktimer
(§1£_§). Flt (3) modified the timing of somatic sectoring events
and could clearly be distinguished by linkage with a stable marker
gene, E, a gene for corolla tube pigmentation, that was independent
of 1,

Both alleles of y_were unstable and somatic mutations occurred
in both directions, so a chromosomal deletion was apparently not
involved. Differences in frequency and developmetal timing of
these reversible mutations were shown to have both heritable and
environmental components (140). Higher temperatures decreased
somatic speckling but increased sectoring. In addition, the
phylotactic position of each flower borne on the plant affected the
instability of the y locus (141).

Nicotiana also displays floral variegation in N, tabacum x N,
otthora crosses which can be attributed to the frequent deletion
of the carmine allele of N, otophora located in the distal part of
a translocated chromsome segment (l3,60,61,62). Cytological
investigation revealed abnormal chromosome behavior with dicentric
chromsomes, fragments and anaphase bridges. Backcrossing the

hybrid to N, tabacum induced breakage of one large heterochromatic

39

block of N, thphgra, In plants marked 9992, corollas were
variegated due to deletions in the carmine allele (Cg) from N,
otophora, uncovering the recessive coral (92) condition.

Another Nicotiana cross between N, tabacum x N,
plumbaginifolia displayed flecked flowers (2,120). All
combinations of the parental lines showed somatic variegation of
dominant characters which were carried on the pgg_genome. It was
established that the variegation was due directly to the loss of
chromosomes.

Petunia genetic instability -

 

The occurrence of unstable anthocyanin alleles in Petunia
species were reported as early as 1935 when Malinowski described an
unstable race of P, violaceae which displayed variation in the
amount and distribution of pigmentation in the flowers (106).
Demerec (37) also listed the genus Petunia in his complilation of
unstable genes, describing gene in P, violaceae which affected both
color and size of the flowers at the same time.

Cornu (33) isolated five unstable systems related to
anthocyanin production in Petunig_hybrida flowers isolated after
mutagenic treatment (alleles Anl, An2, An3, An6, and Pgfl). In
addition, Bianchi found anthocyanin instability conditioned by
recessive dwarfing gene (7) and Potrykus reported an unstable
plastid mutation in P, hybrida (130).

Farcy and Cornu (57) identified a series of unstable 522:2.
alleles in P, hybrida characterized by the production of a large
number of sectors with colors intermediate between the dominant and
recessive phenotypes. They hypothesized that the unstable

mutations were the result of the integration of an insertion

40

segment into the 53;; locus at different sequence points, resulting
in varying amounts of decreased gene expression. The occasional
excision of this insertion segment was thought to give rise to new
gggzg stable alleles, depending on the precision of the excision
process.

Bianchi et al. (8) found upon selfing of a red inbred P.
hybrida line several red-spotted white flowers among the progeny.
This instability was attributed to the A21 locus. They postulated
that-Ag; consisted of both a structural gene responsible for
production of the enzyme UDP glucose:cyanidin
3-O-glucosyltransferase and a regulatory element built up from
intermediate repetitive DNA. The regulator element consisted of
two components: the "mutator", which activates of the structural
gene, and the "expressor" which modifies the rate of activation.
Mutations were considered to represent large or small deletions
within one or both of the components. Mutations in the mutator
were thought to increase or decrease spot density while mutations
in the expressor would result in changes in color intensity of the
spots. Examination of Anl_instabilites by Mulder (121) revealed
that the red-spotted white flower was trisomic, but the chromosome
present in triplicate was not the chromosome with the NgP_allele.
He concluded there must be an independently segregating factor
which influences the frequency of mutation to the dominant Ngl_
locus.

Cornu (32) and Maizonnier and Cornu (104) described another
unstable allele in P, hybrida, the NPl_allele. The gene NPl_causes
3' and 5' hydroxylation of the B-ring and the formation of

petunidin and paeonin glucosides. After gamma radiation, striped

41

mutants were frequently found in the progeny. Such striped
phenotypes were also found in the progeny of a haploid plant used
as the male parent. Mutants had intense zones of alternating light
and darkly pigmented areas on the lobes, correlated with the
presence of petunidin and paeonin, respectively. Karyotypic
analysis revealed an arm of chromosome I had been translocated to
the long arm of chromosome II in striped mutants. The breakage
point of the arm of chromosome I was concluded to be at the NP}.
locus.

Origig§_gf_gontrolling elements:

One question which has not be adequately answered concerning
controlling elements is their origin. The elements studied most
extensively have been the P§:Ag_and §2m_systems in maize. These
elements were discovered in maize lines after the introduction of
genetic instabilities. Such instabilities have been shown to
result from either the initiation of the breakage-fusion-bridge
cycle (109) or after exposure to atomic radiation (128).

There are three possible ways controlling elements could
originate. They could be formed gg.§gyg, by the introduction of
viral genes, or by the unmasking of an element present elsewhere in
the genome. While the first two possibilities have received
limited support (38), new occurrences of controlling elements as a
result of chromosomal damage supports the hypothesis that such
elements may have previously been located in or near
heterochromatin where they could not respond to regulatory signals.
Each new mutable allele arose from a functional allele and thus
represented an unstable inhibition of a previously functional gene.

The inhibition of gene activity is attributed to the insertion of

42

the controlling element or a derivative of it in or adjacent to the
gene.

It seems likely that highly mutable systems of higher plants
arise when a foreign piece of DNA, at least foreign to that part of
the genome, is inserted into a chromosome segment concerned with
regulating accessibility for transcription of a nearby gene. Such
insertions into a series of repeated elements could disrupt a
critical cooperative protein-DNA interaction to such an extent that
the adjacent chromosome segment never gets unfolded for
transcription (16). McClintock repeatedly suggested cell
differentiation involved controlled deletions or transpositions of

specific segments of DNA (112).

SECTION I
SOMATIC HYBRIDIZATION BETWEEN PETUNIA PARODII (W.C.S.)

AND PETUNIA INFLATA (FRIES)

SOMATIC HYBRIDIZATION BETWEEN PETUNIA PARODII (W.C.S.)

AND PETUNIA INFLATA (FRIES)

ABSTRACT:

Somatic hybrid plants were regenerated following calciumshigh
pH fusion of the unidirectional, sexually incompatible cross of
Petunia parodii wild-type leaf mesophyll protoplasts with
protoplasts from a cytoplasmic determined chlorophyll deficient
mutant of.P. inflata grown in liquid suspension culture. Genic
complementation to chlorophyll synthesis and growth in selective
medium was used to visually select cell hybrids from parental cells
which, in the case of the P. inflata parent, remained colorless in
the selection medium, or exhibited a predetermined growth
inhibition, as in the case of the P. parodii parent. Hybrid calli
transfered to shoot regeneration medium were rooted and confirmed
as hybrids based on floral and leaf morphology, dominant
anthocyanin expression in corolla limbs, chromOsome number,
segregation for parental characters, and peroxidase and malic
dehydrogenase isozyme patterns. The frequency of somatic hybrid
plant production as a function of selection efficiency ranged from

3.5 to 5.3 hybrids per 106

protoplasts used, comparable to previous
reports, but parental reversion frequency approached 54%. Somatic
hybrids displayed variable phenotypes, the most prominent being
distinct variegation in corollas and split corollas. These
characteristics were attributed to a type of hybrid dysgenic

phenomenon or nucleo-cytoplasmic incompatibility following the loss

of parental chromosomes in a hybrid cytoplasm.

43

44

INTRODUCTION

ProtOplast fusion is an Pg yiggg technique that can result in
the cellular integration and expression of both nuclear and
cytoplasmic DNA from two species in one cell. Development of such
cells into hybrid plants results in unique opportunities for the
investigation of gene regulation as well as providing for possible
practical outcomes following introgression of alien genes or gene
segments into desirable genotypic backgrounds.

The selection systems developed for identification of
heterokaryons in Petunia have exploited naturally occuring
differential drug sensitivities of cultured protoplasts (18),
differential growth response in standard media (18, 20), and the
use of spontaneous or induced chlorophyll deficient mutants (7,
22). One of the most successful combinations used the limited
growth response of one wild-type parental line (_. parodii) with a
chlorOphyll deficient mutant cell line (P. hybrida or.P. inflata).

The purpose of this investigation was to (i) elucidate the
conditions for optimal protoplast yield and viability in P.

arodii, wild-type leaf mesophyll (wt-m) and P. inflata,
cytOplasmic determined (ca-susp) chlorophyll deficient mutant cell
line to serve as a standard laboratory procedure for use in future
Solanaceous interspecific and intergeneric somatic hybridizations,
(ii) the determination of the inheritance patterns of chlorophyll
deficiency traits in five petunia mutant lines to provide the
framework for ongoing research, (iii) the application of a
previously described protocol for inducing fusion by the use of
high calcium-high pH technique selected as the preferred method in
Petunia to serve as a standard laboratory procedure, (iv) analysis

of fusion in terms of efficacy of somatic hybrid plant recovery as

45

a function of selection efficiency of the system used, and (v) the
assessment of the unexpected phenotypic variation spectrum
displayed in the somatic hybrid plants.

MATERIALS AND METHODS

Source of leaf mesophyll protoplasts of P.4parodii (wt):

Seeds of P, parodii were germinated biweekly on V.S.P.
soilless planting medium (Bay Houston Towing Co.). They were
maintained at 25i2°C, and received a photon fluence flux rate of 80 u
Em'zs'l (400-700 nm) (GE96T12CW) on a 16 hr photoperiod. After
transplanting to plastic cell packs, vegetative growth was
continued under greenhouse conditions of 25:2°C minimum night
temperature and fluctuating day temperatures under a photon fluence
flux rate of 680 uEszs'l (400-700 nm) (GE96T12CW) 10 hrs daily.
Plants were fertilized at each watering with lSOppm 20N-8.6P-l6.6K
aqueous solution adjusted to pH 6.5 with phosphoric acid. Standard
cultural procedures for insect and disease control were followed.
Leaf harvesting, sterilization using a 102 solution of commercial
5.252 NaOCl, and abaxial epidermis removal were performed as
described previously (11).

Isolation of leaf mesophyll protoplasts of P. parodii (wt):

Protoplasts of P, parodii (wt-m) were isolated as previously
described (11) but with the following enzyme modifications. Enzyme
mixtures tested for optimal release of viable leaf mesophyll
protoplasts under the environmental and cultural conditions at E.
Lansing, MI, involved commercial preparations of Cellulase
"Onozuka" RlO (Kinki Yakult Mfg. Co.), Driselase (Kyowa Hakko,
Kogyo Co.), Meicelase-P (Meiji Seika Kaisha LTD.), Macerase

(Calbiochem), and Pectinol AC (Rohm and Hass Co.).

46
Source of suspension cultures of the cytoplasmic determingg

chlorophyll deficient mutant of P. inflata:
In vitro shoot cultures -

In vitro shoot cultures of mutant P, inflata were obtained

 

from E.C. Cocking, University of Nottingham, on M/S basal medium
supplemented with 0.001 mg/L folic acid, 0.03 mg/L
6-furfuralaminopurine (kinetin) (K), 0.00875 mg/L indoleacetic acid
(IAA), 32 sucrose, and 0.8% agar at pH 5.6 (BGS medium) (21) and
were transferred to a modified Linsmaier and Skoog medium (14) with
0.3 mg/L K and 3 mg/L IAA, labeled P/C medium, where more vigorous
shoot growth occurred. Cultures were kept at 25i2°C under 28-34

23'1 (400-700 nm) (GE96T12CW) on a 16 hr photoperiod and

um-
subcultured monthly.
Establishment of P. inflata (ca) suspension cultures -

Leaves were removed from ;g_yiggg_shoot cultures and scored
prior to placing them onto Uchimiya and Murashige (U/M) (26) medium
solidified with 0.8% agar to induce callus. After approximately 2
months, 2 cm3 callus pieces were transfered to liquid U/M medium,
40 mls in 125 ml Erlenmyer flasks. The cell suspensions were
maintained at 28°C on a rotary shaker (9O cycles/min) and
subcultured weekly. To transfer the cells to fresh medium, large
cell clumps were removed by pouring the suspension through a 35 um
sieve. The resulting effluent was centrifuged at 80 x g_for 5
minutes in 16 ml sterile, screwbcapped test tubes. The old medium
was removed and 2 mls of compacted cells were resuspended in 38 mls
of fresh U/M medium.

Isolation of protoplasts from P. inflata (ca-susp):

The protoplast isolation procedures previously reported for P,

inflata (ca-susp) (21) were modified for more efficient and

47

reliable yields in our laboratory. Suspension cultures 5-9 days
old were transferred to 16 ml sterile, screwbcapped test tubes and
were centrifuged for 5 minutes at 80 x.g. The liquid U/M medium
was removed and the pellet was resuspended in approximately 5 of
the 25 ml enzyme solution, poured into 9 cm plastic petri dishes,
sealed with Parafilm‘gl and incubated at 50 rpm on a rotary shaker
at 28°C for 16-18 hrs. The enzyme solution consisted of 2.02
Driselase, lZ Macerase or Pectinol AC, and 82 mannitol in CPW salts
at pH 5.8. Digested cells were transfered using sterile Pasteur
pipettes to 16 m1 sterile, screwbcapped test tubes and centrifuged
at 80 x_g for 5-7 minutes, after which the enzyme solution was
removed and the pellet resuspended in 10 mls of CPW 8S and
centrifuged at 100 x_g for 10 minutes. Protoplasts floated to the
top of the test tube after this procedure and were collected,
counted and diluted with the appropriate medium.

Viability of isolated protoplasts was established by
fluorescein diacetate membrane exclusion, Evan's Blue membrane
exclusion, and by observing their behavior upon extended culture_;g
21—13131-

In vitro shoot cultures of Petunia chlorophyll deficient mutants:

Shoot cultures of four Petunia chlorophyll deficient mutants
were established in P/C medium. These were: P, parodii nuclear
and cytoplasmic determined chlorophyll deficient mutants (Pp-na and
Pp-ca), and P, inflata nuclear and cytoplasmic determined
chlorophyll deficient mutants (Pi-na and Pi-ca). The derivation
of Pi-ca was described previously (22). Pp-na was induced through

gamma radiation of P. parodii wild-type seeds. Pp-ca was

48

regenerated in vitro from white tissue of a periclinal chimera on
M/S Z selected after EMS treatment of seed. Pi-na was obtained by 60Co
irradiation of P. inflata wild-type seeds. Inheritance patterns

of chlorophyll deficiency in Pi-na, Pp-na and in an albino mutant

of.P.yp§rviflora were determined following F1 and F2 segregating

 

populations produced by standard emasculation, pollination, seed
collection procedures and storage methods employed for Petunia
(21).

Seeds of Pp-na, Pi-na, and P. parviflora were surface

 

sterilized using a 5% solution of 5.252 NaOCl for 25 minutes
followed by four sterile distilled water rinses. Seeds were sown
on BGS medium in 60 x 15mm plastic petri dishes sealed with
Parafilmcgh The dishes were kept at 2512°C under a photon fluence
flux rate of 28-34 uEm'zs‘l (400-700 nm) (GE96T12CW) on a 16 hr
photoperiod. Germination occurred after 5-7 days and white
seedlings were selected, numbered, transfered to P/C medium at
similar environmental conditions, and subcultured monthly by shoot
cuttings.

Protoplast fusion:

 

Calciumrhigh pH fusion treatment (12) of P, parodii (wt-m)
with P, inflata (ca-susp) protoplasts was modified from the
procedure used by Power et al. (21) for the fusion of these two
Petunia species. The experimental design conformed to that
outlined by Power (19, 20), except that initial numbers of parental
cells were as used in the protoplast fusion of P, hybrida®P.
parodii (7), i.e., 4 mls of each species at a density of 2.0 x 105
[ml in M/S P1 9M.

Protoplasts were pelleted in all tubes except the viability

49

controls by centrifugation at 35 x g_for 5 minutes. The fusion
solution was added as required and the tubes placed in a water bath
at 30°C. After 15 minutes, tubes were removed and centrifuged for
5 minutes at 35 x.g. Following two washes using Ca++/CPW 13M
solution (21), fusion treated protoplasts were resuspended in 16
mls of M/S Pl 9M, and viability and cross feeding controls were
prepared. The protoplasts were plated in liquid medium at a final
density of 5 x 104/ml in 5 cm plastic petri dishes, sealed with
ParafilmGD and cultured at 2512°C under a continuous photon fluence
flux rate of 18-22 uEm-z’s‘l (400-700 nm) (GE96T12CW).

Aliquots of M/S P1 6M and M/S Pl 3M (0.5 ml) were added after
44 days and 65 days, respectively. Transfer to M/S basal medium
supplemented with 2 mg/L zeatin (M/S 22) induced shoot regeneration
in putative hybrid calli.

Regeneration in the tetraploid sexual hybrid between P. parodii x
P. inflata {17-17):

Seeds from the sexual hybrid between P. parodii (wt) and P.
inflata (wt) at the tetraploid level were surfaced sterilized and
plated on BGS medium under the environmental conditions described
for chlorophyll deficient mutant seed germination. Plantlets were
subsequently transfered to M/S basal medium with no added growth
regulators where leaf expansion, internode elongation and rooting
occurred at 25i2°C under a photon fluence flux of 28-34 pEm729_1
(400-7OO nm) (GE96T12CW). Leaves were removed, scored, and callus
initiated on U/M solid medium as described for callus induction for
suspension cultures. Callus of the tetraploid sexual hybrid was
screened for shoot regeneration on M/S basal medium supplemented

with one of three cytokinins, K, 2, or 6-benzy1aminopurine (BAP),

50

each combined separately with four auxins, IAA, naphthalene acetic
acid (NAA), 2,4-dichlorophenoxyacetic acid (2-4-D), or
p-chlorophenoxyacetic acid (pCPA). Four concentrations of
cytokinin and auxin were tested in all possible combinations at
levels of O, 0.1, 1.0, and 2.0 mg/L. Media for shoot regeneration
experiments were prepared in 2.5 oz. bottles with metal screw lids.
One cm3 callus pieces were embedded on the various media and placed
at the environmental conditions of 25r2°C under a 16 hr photon

23-1 (400-700. nm) (GE96T12CW).

fluence flux rate of 28-34 uEm-
Cultures were evaluated after four weeks for the presence of
shoots. The resulting regeneration medium chosen was to be used to
induce regeneration in somatic hybrid calli.

Eyglggtion of somatic hybrids:

Chromosome determinatigg§_-

Chromosome counts of somatic hybrid plants were made by
observing meiosis in pollen mother cells. Immature flower buds,
approximately 0.2 cm in length, were fixed in Carnoy's solution,
followed by the removal of the anthers, and subsequent staining
with aceto-propiocarmine (20).

Pollgn viabiligy -

Pollen viabilities of somatic hybrid plants were determined by
counting the dark, fully round grains mounted in a solution of
iodine-potassium iodide. Flowers were collected one day before
anthesis and placed in vials of water until anther dehisence.
Counts of 5-10 random microscopic fields (lOOx) were made with a
minimum of 500 pollen grains per hybrid counted. Differences in

pollen color segregation and self-incompatibility were also noted.

51
Morphological traits -

Total floral length, corolla tube length, limb diameter, style
length and anther filament attachment length were measured in
selected somatic hybrid plants. In addition, variant phenotypes
were noted including changes in reproductive organs and in the
distribution of anthocyanin in corolla limbs. Leaf characteristics
were evaluated as to shape, pigmentation, presence of trichomes,

and ruffled or undulating leaf margins.

52

RESULTS
._§Qlation of leaf mesophyllyprotoplasts of P. pargdii (wt):

In order to refine previously established isolation techniques
for P. parodii (wt) leaf mesophyll protoplasts for the conditions
present at E. Lansing, MI, several enzyme combinations were
explored for the production of high yields of viable leaf mesophyll
protoplasts. Meicelase-P (1.52) was found to release maximum
yields of leaf mesophyll protoplasts at 25i2°C after 16 hours,
compatible with fusion experiment schedules (Fig. 1). Such
protoplasts proved viable in M/S basal medium supplemented with 0.5
mg/L NAA, 1.0 mg/L BAP and 92 mannitol, labeled M/S PD 9M, and
capable of regeneration into plants which were eventually grown to
maturity in the greenhouse.

Sixteen hour isolation procedures were not always practical
for some protoplasts studies, thus experiments to determine a rapid
2 hour isolation procedure were designed. Cellulase R10 was found
to produce adequate yields of P, parodii (wt) leaf mesophyll
protOplasts, but inhibitory effects expressed as delayed or
supressed cell division occurred (Table l). Eliminating Driselase
or Macerase did not increase division. High yields of viable leaf
mesophyll protoplasts were isolated using 0.52 Driselase and 0.52
Macerase after two hours.

Plating of protoplasts isolated by this enzyme combination in
either M/S Pl 9M or M/S PD 9M followed by 24 hours in the dark
before transfering to 25i2°C under 18-22 uEm-zs-1 (400—700 nm)
hastened the onset of division in both media (Table 2). Immediate
placement under a photon fluence flux of 18-22 11Em"23-1 {400-700 nm)

at 25i2°C resulted in delayed division, but after 4 days, small

. 6
leaf tissue 0: l0_)
'0

.0
(fl
1

Pplt. yield / g

 

Figure l.

53

I I 1 l A L l I

no 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Meicelase- P (7.)

The effect of Meicelase-P (0-52) with 0.152
Macerase, 92 mannitol, 40 ug/ml ampicillin,

10 ug/ml tetracycline and 10 ug/ml genta-
micin, 92 mannitol in CPW salts, pH 5.8, on
protoplast yield of Petggia parodii leaf
mesophyll protoplasts, 5 replications/treatment.

54

Table l. Yields and viabilities of Petunia pgrodii (wt)
leaf mesophyll protoplasts isolated from 51
day old plants using 0, 0.5 and 1.02 Cellulase
"Onozuka" R10, O-O.52 Driselase and O-O.52
Macerase for two hours.

 

Enzyme concn (2)* ' Growth
response

Cellulase/Driselase/Macerase Yield (4 days)

 

 

0.0 0.0 0.5 3.1 x 106 large

colonies
0.5 0.5 0.5 3.6 x 106 membrane
damage,
no growth
1.0 0.5 0.5 3.9 x 106 limited
division
no colony
formation

1.0 0.0 0.5 3.1 x 10 protoplasts
enlarged,
no division

1.0 0.5 0.0 2.8 x 10 limited
division

 

* Enzyme solution prepared with CPW salts, 92 mannitol,
40 ug/ml ampicillin, 10 ug/ml tetracycline, 10 ug/ml -
gentamicin and 0.52 potassium dextran sulfate, pH 5.8.

55

Table 2. Effect of 24 hour dark incubation on division
of Petunia pgrodii (wt) leaf mesophyll
protoplasts from 56 day old plants.

 

 

 

 

Observations
Dark Light
Plating
Media 24 hrs 96 hrs 24 hrs 96 hrs
M/S P1 9M lst div. colonies limited colonies
lst div.
M/S PD 9M lst div. large no div. limited

colonies

colony
formation

 

56

colonies had formed.

Psolation ofyprotoplasts from P. inflata (ca-susp) cells in liquid

 

suspension culture:

Concentration of enzymes - Established methods for the isolation

 

of P, inflata (ca-susp) did not yield sufficient quantities of
protoplasts on a routine basis to serve as a reliable source of
protoplasts for fusion experiments. Thus, preliminary enzyme
combination tests were carried out which indicated that a mixture
of 22 Cellulase R10, 12 Driselase, and 12 Macerase adequately
released protoplasts from P, inflata (ca-susp).

Experiments to further refine the enzyme concentrations
included testing Cellulase "Onozuka" R10 in concentrations from O
to 102 with Driselase and Macerase both at 12 in CPW salts with 82
mannitol. One ml of 16 day old compacted cell suspension with a
mean number of 5.05 x 105 aggregates/ml was resuspended in 10 mls
of enzyme. Two percent Cellulase "Ononzuka" R10 was chosen as
optimal, releasing adequate yields of viable protoplasts using the
least amount of Cellulase (Fig. 2). A lower Cellulase
concentration resulted in cellular debris with inadequate cell wall
degradation, while higher concentrations did not provide further
increases in yields. Furthermore, viability was impaired at the
higher Cellulase R10 concentrations (Fig. 3). Thus, 22 Cellulase
"Onozuka" R10 was adopted as the standard cellulytic enzyme
solution for protoplast release from liquid suspension cultured
cells.

a e o ult red cells - The isolation of protoplasts from
P, inflata (ca-susp) was influenced by the growth phase of the cell

culture at the time of isolation (Fig. 2). Initial 4 week-old

57

 

 

 

1 5" r5
:0 ' I
8 4- -4g
.2 :
o
m o
5% ' ,1
:3 -
m 3 -3'%
.3. ° ;
mo . I
z E
I 21 . 2'0
E ° :
2 ' :
a) _
"' . C1.
>. I Fla
3: l
‘3.
CL_ 0
l 1 1 l L 1 I l

 

 

05 Lo 1.5 so 25 3.0 3.5 4.0 4.5 5.0
Cellulose "Onozuka" RIO concn.(%)

Figure 2. Protoplast yield from liquid suspension and callus
cultures of Petunia inflata cytoplasmic determined
chlorophyll deficient mutant cell line using 0-52
Cellulase "Onozuka” R10 with 12 Macerase, 12 Driselase,
and 82 mannitol in CPW salts, pH 5.8, 5 replications/
treatment.

 

lOO ‘

Viability (96)

801

 

(D
(3

13 .

20"

 

58

I L 1 1 L 1 1 l l _J

O 05 LG l.5 2.0 2.5 .O 3.5 4.0 4.5 5.0

nge3.

Cellulose "Onozuka" RIO concnm

Protoplast viability of Petunia inflata cytoplasmic
determined chlorophyll deficient mutant cell line using
0-52 Cellulase "Onozuka" R10 with 12 Macerase, 12
Driselase, and 82 mannitol in CPW salts at pH 5.8.

59

suspensions from five 125 ml flasks were pooled to provide 5
replicates per day for sampling over a 13 day growth period. Cell
suspensions were sampled at 2 day intervals and their compacted
cell volume, cell concentration, yield and viability were
determined. The protoplasts were isolated using the enzyme
solution of 22 Cellulase "Ononzuka" R10, 12 Driselase, 12 Macerase
R10, 82 mannitol in CPW salts at pH 5.8.

The growth dynamics of the cells in liquid suspension culture
showed a linear increase over time (Fig. 4) as expressed by
compacted cell volume and number of aggregates/ml. Compacted cell
volume steadily increased over time in culture, while the number of
aggregates/ml increased until the 3rd day, remained approximately
constant until the 11th day, then decreased slightly until a sharp
increase occurred at the 13th day. Protoplast yields were not
dependent on the compacted cell volume or number of aggregates/ml,
as average yields of 25.8 x 106 protoplasts/ml were obtained by the
7th day when mean compacted cell volume had shown minimal change
(2.95 to 2.35 ml) and number of aggregates/ml likewise showed only
a slight increase (1.46 x 105 to 1.65 x 105/ml).

Multiple regression analysis of the effects of culture time
and compacted cell volume on the yield of.P. inflatg (ca-susp)
protoplasts indicated a significant (P=O.l) regression
relationship. The coefficient of multiple determination was 0.84.
Isolation of protoplasts from in vitro shoot cultures of P. parodii
and P. inflata nuclear and cytoplasmic determined chlorOphyll
deficient mutants -

An alternative method for protoplast isolation using EEUXEEEE

shoot cultures was investigated as a possible source of high

Figure 4.

60

Relationship between growth phase of Petunia inflata
cells in suspension culture and protoplast yield.
(a) Growth phase of cultured cells as reflected by
compacted cell volume and cell number, (b) Proto-
plasts isolated at various times during the
development of cell suspension cultures.

Incubation mixture contained 22 Cellulase "Onozuka"
R10, 12 Macerase, 12 Driselase, and 82 mannitol in
CPW salts at pH 5.8.

“All unfilnnmo lml‘

_.__.A___‘

61

I
663 22.8 \m__mo .oz

5

2
.

b

I

loo. 5 _E\o_m_> gen.

0
2

5 0 5

_ . .

 

 

 

l3.

 

 

2.5 9:29 :8 omaoanou

mu
8

m m m

are guano;

Time in culture (days)

Figure 4

62

yielding viable protoplasts of the mutant ngggig lines.

A combination of Extractase and Driselase was tested for
isolation efficiency. Extractase was prepared in a concentration
range of O to 32, while Driselase was held constant at 2.52 in CPW
salts with 42 mannitol at pH 5.8. Pp-na, Pp-ca, Pi-na and Pi-ca
all showed various response optimums (Fig. 5). Both cytoplasmic
mutants (Pp-ca and Pi-ca) showed a response to Extractase, but
neither nuclear mutant appeared to be affected by any of the
Extractase concentrations used.

Along with low yields of protoplasts isolated from leaf tissue
of.;g_y;§§g shoot cultures, protoplast viability was adversely
effected (Fig. 6). Most concentrations of Extractase gave less
than 302 unstained protoplasts using Evans Blue. Since Pi-ca
showed some positive response for protoplast release using this
method, the Driselase concentration was varied from O to 52 while
Extractase was held constant at 1.02. In addition, 0.52 Macerase
and 0.52 potassium dextran sulfate were included in the CPW salts
with 42 mannitol at pH 5.8. Optimal protoplast release was at 3.02
Driselase (Fig. 7). However, viability was severely affected (02)
at this concentration. It was concluded that $2 yiggg shoot
cultures did not warrant further investigation since high
protoplast yields were derived from suspension cultures.

Isolation of protoplasts from P. inflata cytoplasmic determined
chlorophyll deficient cells as callus cultures -

Five month old callus, subcultured at 3 week intervals,
produced maximal protoplast yields at 12 Cellulase "Onozuka" R10
with Driselase and Macerase held constant at 12 in CPW salts with

82 mannitol at pH 5.8 (Fig. 2). The maximal yields of 5.4 x 104/ml

Pplt. yield/0.59 leaf tissue mos)

63

 

 

 

3 -r

2 ..

I q
q .
A&' 1 1 ‘ 1
O 0.5 [.0 L5 2.0 2.5 3.0

Extractase concn. (7.)

Figure 5. Protoplast yields from in vitro shoot cultures
of Pp-nab—d, Pp-caO—O, Pi-naD—O and
Pi-ca O——O, using 0-32 Extractase with 2.52
Driselase and 42 mannitol in CPW salts at pH 5.8.

Viability (7.)

i?

64

ICC-1

80“

601

 

,‘ . ‘ «1“?

o 0.5 l.O. l.5 2o 25 3.0
Extractase concn. (‘2)

Figure 6. Viability of protoplasts isolated from shoot
cultures of Pp-naH, Pp-caO--O, Pi-naH, and
Pi-ca D———D , using 0-32 Extractase with 2.52
Driselase and 42 mannitol in CPW salts, pH 5.8,
as determined by Evan's Blue staining.

Pplt. yield /O.59 leaf tissue (xlo5)o—o

65

m
.4

rIOO

a:
1

i“

#50

Viability H

N.
I

 

 

 

 

   

L 1

0 0.5 l.0 LE) 2. 2.5 3.0 35 4.0 4.5 5.0

l L

 

Driselase concn. (’2)

Figure 7. Yield and viability of Petunia inflata cytoplasmic
determined chlorophyll deficient leaf meSOphyll
protoplasts isolated using 0-52 Driselase with 12
Extractase, 0.52 Macerase, 0.52 potassium dextran
sulfate and 42 mannitol in CPW salts, pH 5.8.

66

did not compare with those from suspension culture cells which were
in excess of 2.5 x 107/m1, probably reflecting increased secondary
wall formation in the callus cultures or the inaccessibility of
cell wall degrading enzymes to the interior cells in the callus
clumps. The second alternative received support from experiments
with suspension cultured cells, where suspensions had to be
routinely sieved to eliminate such clumps and thus promote fine
suspensions of 2-5 cells. Such suspension cultures were considered
the method of choice for maximal protoplast yields.
Inheritance-of chlorophyll deficiency in mutants of Petunia:
Segregation in the P. parodii chlorophyll deficient mutant
line PD IR 40 obtained by 60Co radiation fit the hypothesis of a
monogenically inherited recessive trait as tested using chi-square
analysis (Table 3). Petunia parodii line P-1043 produced by gamma
radiation produced green to white progeny at ratios more closely
approximating 4:1 than the 3:1 ratio expected for the segregation
of a single recessive gene. Since germination~ig_yiggg_did not
increase the appearance of white seedlings, lethality of the
recessive whites when germinated on filter paper as done in the F2
probably was not a factor in the appearance of this altered
ratio. The F2 seed germinated Pg yitrg gave the same 4:1 ratio as
F33 produced by selfing F2 heterozygotes and germinating the seed
on filter paper. Based on differences in the two P. parodii lines,
P-1043 and PD IR 40, it is assumed that these two lines result from
distinct nuclear mutations and that the chlorophyll deficiency
traits expressed are conditioned by two nonallelic genetic systems
ian, parodii. This hypothesis is supported by i2;yitrg culture

behavior of the two lines. .PD IR 40 appeared "leaky", often

67

 

 

 

 

 

Table 3. F2 segregations for chlorophyll deficient
seedlings in Petunia parodii lines P-lO43 and
PD IR-40,_P. inflata line INF IR-6l and
.P.gp§rviflora line P-1048.
Segregation
Petunia Total 'Chi-
line progeny green white square P
P-1043 344 233 111 (3:1) 0.05<P<0.01
9.30 sign.
PD IR-4O 753 563 190 (3:1) 0.9<P<O.95
0.01 n.s.
INF IR-6l 278 220 58 (3:1) 0.2<P<O.l
2.32 n.s.
P-1048 30 280 21 (15:1) 0.5<P<0.7
0.1 n.s.

 

68
producing a greenish phenotype, whereas P-1043 remained a stable
yellow-white as shoot cultures.
_Petnnia inflata line INF IR 61 fit the ratio for monogenic
recessive inheritance, although the probability level testing for
goodness of fit was lower than for_P. parodii line PD IR 40. INF IR

61 mutants also became green upon extended_ig vitro culture.

Petunia parviflora line P-lO48 did not approximate a 3:1

 

monogenic ratio, but gave a significant fit to a 15:1 segregation
ratio, indicating two interacting genes conditioning the recessive
phenotype. Dominant alleles at both loci produce the same
phenotype (green) resulting in recessive epistasis and thus, the
expected classical ratio for the segregation of two genes, 9:3:3:l,
is modified to 15:1. The F3 progeny segregation of selfed F2
heterozygotes also fit the 15:1 hypothesis, although there was a
slight deficiency in the recessive white class. Such deficiencies
may be the result of reduced vigor accompanying the recessive
phenotype so that germination on filter paper, as done for the F3
generation, resulted in fewer recessive individuals. The
chi-square for the deviation of the white class from the expected
number given a 15:1 ratio reflected this decrease, and was
determined to be contributing the most variation to the fit of the
15:1 ratio (Table 4).
Fusion of P. parodii (wt-m) protOplasts with P. inflata (ca-susp):
The production of somatic hybrids in this research was is
based on complementation for both sustained division and
chlorophyll synthesis in the somatic hybrid cells, restricted
growth of the P. parodii, and identification of P. inflata by

colorless cells. Thus, fusion between P. parodii (wt-m) and P.

69

Table 4. F3 segregation ratios for green and white
seedlings in Petunia parodii line P-1043 and
P, pgrviflora line P-lO48.

 

 

 

Prageny segregation

 

 

Petunia Total Chi-

line progeny green white Square P

P-1043 6,506 4,744 1,312 35.75 P<0.00l
sign.

P-lO48 1,531 1,472 59 14.59 P<0.00l

Sign.

 

70

inflata (ca-susp) was expected to yield dark green, actively
growing callus in liquid M/S P1 9M. Petunia inflata will grow in
M/S Pl 9M, but forms distinctively white callus (21) while P,
parodii (wt-m) will only grow to a predetermined size of about 50
cells before senescence ensues.

A total of 155 green calli were observed and recovered after 35
fusion experiments yielding a success rate of 312. In the first
fusion experiment, pale green colonies were also observed in the
post fusion cross-feeding indicator plates and in P, parodii
viability plates. Cross-feeding possibly occurred by secretion of
growth factors from the lysis products of the parental cells,
resulting in pseudo-prototrophy in the initial stages of the
experiment. Nonetheless, later experiments indicated that P,
parodii grows as a loose, pale green callus which could eventually
be screened out visually.

Selected calli were transfered from the 5 cm petri plates to
regeneration medium (Fig. 8) consisting of M/S 22 in 2.5 oz round
bottles, as determined by media screening for shoot regeneration
from callus cultures of the tetraploid sexual hybrid, 77-17. Of
192 auxin and cytokinin combinations tested, 2 mg/l Z without auxin
was the only medium which supported shoot regeneration in the
tetraploid callus and thus was used for regeneration in all
subsequent fusion experiments.

14.42 of the dark green tentative hybrid calli isolated by the
screening procedure regenerated into plants, and after flowering,
5.72 proved to be somatic hybrids. A reversion rate expressed as
parental plant regeneration was 54.82, resulting in a

complementation rate of 45.22 (Table 5).

PLEASE NOTE:
PAGE 71 IS MISSING IN

NUMBERING ONLY. FILMED
AS RECEIVED.

UNIVERSITY MICROFILMS INTERNATIONAL'

Figure 8.

72

(a) Freshly isolated leaf mesophyll protoplasts of
Petunia parodii and protoplasts from liquid

 

suspension cultured cells of.P. inflata in M/S Pl 9M.
ca. 480x. (b) Heterokaryon produced after calcium-
high pH fusion treatment. ca. 1145X. (c) Cell colony
after 12 days in M/S Pl 9M. ca. 530x. (d) Callus
with shoots produced on M/S medium with 2.0 mg/L
zeatin. ca. 2.4x. (e) Adventitious root formation
induced on M/S medium with no exogenous growth
regulators. ca. 1.3x.

73

 

Figure 8

74

Protoplast fusion of Petunia parodii (wt-m)
®_P. inflata (ca-susp) using calcium/high pH.

Table 5.

 

 

Fusion experiment

 

 

Fusion statistics P80-1 P80-2
Protoplast source:
(age - days)
.13- 2951.13. 28 44
_P. inflata 7 9
Protoplast yields: 5 6
_P. parodii 8.1 x 106 1.3 x 106
.P. inflata 15 x 10 2.7 x 10
Number of protoplasts
(x 106 :12)
1.5 1.4
Number of calli recovered
after selection 155 8
Frequency of selected
calli production
(per 106 protoplasts) 103.3 5.7
Number of calli
producing shoots (2) 62.7 75.0
Frequency of regenerating
calli production
(per 106 protoplasts) 24.6 4.3
Number of flowering
somatic hybrids (2) 13.6 62.5
Reverson rate (parental
regeneration) (2) 10.2 0
Frequency of somatic
hybrid production
(per 10 protoplasts) 5.34 3.57
Selection efficiency (2) 14.28 3.75

 

75

The frequency of selected calli produced per 106 protoplasts
of both parents proved to be variable between fusion experiments
(Table 3). The frequency of regenerating calli production per 106
protoplasts was also variable, 24.62 and 4.282. The first
experiment had an increased parental reverSion rate, 10.172
compared to 02. Yet the frequency of somatic hybrid production per
106 protoplasts was similar between experiments, 5.342 and 3.572.
Selection efficiency differed widely however with 14.282 for
experiment P80-l and 3.252 for experiment P80—2, which may be
attributable to the higher rates of parental reversion in
experiment P80-1. Selection efficiency is based on the number of
flowering somatic hybrids actually recovered as compared to the
theoretical maximum given the frequency of fusion. Based on a
fusion frequency of .00492 for experiment P80-1, for every 106
protoplasts subjected to fusion, 2.45 x 104 heterokaryons would be
produced. At an average plating efficiency of 252 for each of the
parental species in M/S Pl 9M, 1.53 x 103 viable heterokaryons
would result. Given a shoot regeneration rate of 372, 56
heterokaryons would be the highest maximum possible based on
experiment P80-l, resulting in a selection efficiency of 14.282.
Somatic hybrid plant variability spectrum:

Plants regenerated from selected green calli following high
calcium-high pH fusion treatment displayed various degrees of
aberrant phenotypes including variation in general plant
morphology, reproductive characteristics and corolla limb
pigmentation.

Leaf characteristics: P, inflata leaves are ovate with

obtuse apices and acute bases, while P, parodii leaves are ovate,

76

with obtuse apices, but have attenuate leaf bases. The regenerated
hybrids displayed primarily ovate shapes (1002), but varied in the
apex from rounded (3.82), obtuse (84.62) to acute (11.52), while
the leaf bases were rounded (26.92), acute (8.52) or attenuate
(34.62) (Table 6). Leaf margins showed differences in hybrids
compared to the parental types. While 76.92 of the hybrids had
entire leaf margins, undulate (19.22) and revolate (11.52) types
were also expressed. In addition, 26.92 showed a characteristic
chimeric leaf pattern consisting of a light green mid-rib area
outlined with a band of ivory cells, juxtaposed to a dark green
leaf margin. Hybrids also displayed variable degrees of surface
trichomes. Both 2, 9339011 and P, inflata were classified as
hirtellous, while hybrids were classified hirsululous (42.32) or
hirtellous (53.82). A branch of hybrid 15-2 sorted out with
subglabrous leaves.

Corolla morphology: Corollas of P, inflata are symmetrical,

 

magenta, with round lobes and a short corolla tubes. Petunia
(parodii corollas are slightly asymmertic, white, with pointed
lobes, and have a long corolla tube. Petunia parodii also has
purple venation in the outer throat area.

Corollas of somatic hybrids displayed the dominant magenta
coloration and varied from slight to pronounced asymmetry. Hybrids
also segregated for round and pointed corolla lobes (Table 7). In
addition, the majority of the somatic hybrids produced phenotypes
with anthocyanin variegation in the corolla limbs (71.42). Five
distinct variegation classes were observed: (1) uniform magenta,
(2) acyanic flecks, (3) acyanic sectors, (4) pale and dark magenta

cyanic flecks, and (5) pale and dark magenta highly sectored areas

77

 

 

 

 

Table 6. Morphological characteristics of somatic hybrid
plants.
Phenotype*
Plant Fusion Chrom. Leaf Corolla Pollen Compat. Po.
No. Plate No. No. Color Via.
14-3 2 36 0-L,G M9Cl.3 Ivory SC 71.7
15-1 2 28 O-L,G M+C1.l Blue SC 64.6
15-2 2 26-28 O-L,C M-Cl.2, Blue SC 44.4
& C1.4
15-3 2 27 0-L,C M-C1.1 Ivory SI sterile
15-4 2 - O-L,G M‘Clol - SC 65. 3
15-5 2 28 0-L,G M-C1.1 Ivory SC 66.3
15-7 2 26-28 O-L,G M-Cl.4 Ivory SC 45.2
20-1 3 - 0,C M-C1.3 Blue SI -
& C1.4
21-1 3 26 O-L,G M-Cl.2 Ivory SC 42.4
28-1 6 20 0-L,G M-C1.3 Blue SI 31.8
& Cl.4
& C1.4
* O = ovate SI = Self incompatible
G = green SC = Self compatible
L = lanceolate C1.1 = Solid
M = magenta 01.2 = acyanic flecks
W = white Cl.3 = acyanic sectors
C1.4 = p/m cyanic sectors

78

 

 

 

 

 

Table 7. Corolla morphology of Petunia pirodii ®
_P. inflata somatic hybrids.
Throat*
Plant Lobe Variegation
No. Morphology Symmetry Pigmentation Pattern
5-4 R S H ‘acyanic flecks
14-3 P AS L acyanic sectors
15-1 P S D p/m cyanic sectors
15-2 P AS D acyanic flecks,
p/m cyanic sectors
15-3 P AS D stable
15-4 P S D stable
15-5 P S D p/m cyanic sectors
15-6 R S D p/m cyanic sectors
15-7 R S D p/m cyanic sectors
20-1 P AS D acyanic sectors,
p/m cyanic sectors
21-1 R S L acyanic flecks
21-5 R S H acyanic flecks
28-1 R S D acyanic sectors
38-5 P AS L acyanic sectors,
p/m cyanic sectors
* P = pointed D = dark
R = round L = light
8 = symmertric H = halo

AS = asymmertric

79

usually comprising half or whole corolla lobes. The appearance of
twin spots were noted on the corolla limbs of 21-1 and 21-5. A
further floral modification seen was the production of split
blossom characteristics in some of the hybrids. The degree of
splitting was found to follow a linear progression from finely
divided to restored phenotypes in one hybrid.

Segregation for self-incompatibility alleles contributed by_P.
inflata approximated a 3:1 ratio (chi-square=0.3809, 0.7<P<0.5) in
somatic hybrid plants. Other reproductive anamolies were observed
on certain plants including the appearance of double and fasciated
styles, double ovaries, stigmoid anthers, staminoid pistils,
petaloid structures on the corolla limbs, increased numbers of
anthers and corolla lobes, ruffled corolla margins, and altered
corolla morphology.

Floristic data taken on somatic hybrids, white flowered.P.
.pargdii parental revertant types, the tetraploid sexual hybrid
77-17, and tetraploid P. parodii indicated parental revertants were
significantly different (P=0.01) in total corolla length and tube
length from magenta somatic hybrids. These plants were not
significantly different (P=0.01) from tetraploid‘P. parodii
(77-22-4-2) in total length, tube length, limb diameter, style
length and length of anther filament attachment (Table 8). Thus
based on these quantitative floristic data, white flowering plants
recovered from the fusion process (43-1, 43-7, 48-2, 3-1) appear to
be 4p_P, parodii, resulting from the growth of homokaryons produced
either by the fusion process or after spontaneous doubling in
culture.

Magenta flowered somatic hybrids had significantly shorter

80

 

 

 

Table 8. Mean quantitative measurements of corolla
characteristics in somatic hybrid plants.
Corolla (cm) (is.e.)
Plant Total ' Tube Limb Style Length Filament
No. Length Length Dia. Length Fil.Att. Att.(2)
(0.34) (0.11) (0.47) (0.06) (0.17)
(0.17) (0.20) (0.41) (0.29) (0.29)
110.3 6.44 4.50 4066 3.44 1.96 4306
(0.32) (0.22) (0.56) (0.08) (0.10)
15-1 5.49 3.40 4.80 1.29 2.47 37.8
(0.08) (0.09) (0.32) (0.10) (0.08)
15-2 5.35 3.40 4.80 2.37 1.33 39.1
(0.20) (0.08) (0.31) (0.24) (0.08)
(0.43) (0.30) (0.58) (0.31) (0.12)
15-4 5.84 3.62 5003 2.71 1.36 3707
(0.41) (0.21) (0.66) (0.23) (0.12)
15-5 4.43 2.82 3.66 2.02 1.07 , 38.1
(0.28) (0.12) (0.43) (0.10) (0.07)
15-6 5.87 5.59 5.35 2.55 1.39 38.7
(0.22) (0.12) (0.27) (0.25) (0.07)
15-7 4.38 2.85 3.85 2.01 1.09 38.4
(0.12) (0.15) (0.27) (0.04) (0.07)
21-1 4.73 2.95 4.22 1.90 0.97 32.8
(0.29) (0.14) (0.470 (0.32) (0.11)
21-5 4.88 2.71 4.81 2.04 1.10 40.6
(0.26) (0.13) (0.42) (0.07) (0.09)
28-1 4.37 2.63 4.46 1.55 0.91 34.5
(0.11) (0.08) (0.20) (0.16) (0.06)
(0.32) (0.26) (0.380 (0.24) (0.28)
38-5 4090 3.39 3067 2042 1056 4509
(0.42) (0.21) (0.53) (0.33) (0.22)

Table 8, (cont'd.).

81

 

43-1

43-7

48-2

3-1

77-17

Pp4p_

HSD

10.38
(0.85)

9.00
(0.53)

7.24
(0.20)

6.89
(0.35)

6.18
(0.33)

10.44
(0.23)

0.42

7.78
(0.67)

6.58
(0.380

5.44
(0.18)

5.15
(0.37)

3.92
(0.28)

7.98
(0.30)

1.16

5.22
(0.55)

4.92
(0.39)

3.88
(0.33)

3.93
(0.480

4.77
(0.45)

5.10
(0.25)

0.78

7.07
(0.31)

5.71
(0.34)

4.48
(0.12)

3.97
(0.23)

2.83
(0.18)

7.11
(0.19)

2.31

4.61
(0.40)

3.65

(0.31)

2.88
(0.13)

2.69
(0.31)

1.38
(0.19)

4.59
(0.20)

0.34

59.3

55.5

53.1

52.2

35.2

57.2

 

82

total corolla lengths, and in some plants shorter tube length,
notably 15-3, 15-5, 21-5, and 28-1. The length of anther filament
attachment was also shorter in some hybrids (28-1, 21-1, 21-5).
Chgomosome number:

Chromosome number ranged from 20 in plant 28-1 to 36 in 14-3
(Table 6). Five somatic hybrids had the expected tetraploid
chromosome number of 4pe28 (5-4, 15-1, 15-5, 15-6 and 21-5).
Variegated phenotype was found in hybrids with an aneuploid
chromsome number (14-3, 15-3, 15-2, 15-7, 38-5, 28-1, 21-1 and
20-1), but three plants with flecked phenotypes had 28 chromsomes
(5-4, 15-5, and 21-5).

Pollen color and viability:

 

Pollen color in Petunia is conditioned by two genes. Pg2pg
produces green (PEPE); or yellow (papa) pollen, and A24 introduces
anthocyanin into the pigmentation, resulting in blue (An4An4) or
white (an4an4) pollen (15). Hybrids showed segregation for these
two genes ranging from blue to white to yellow (Table 6). Pollen
viability ranged from complete sterility due to anther degeneration
(15-3), and from a low of 16.52 in 15-2 (phenotype
ruffled-sectored) to a high of 71.72 in 14-3.

Isozyme analysis:

Plants were verified as somatic hybrids by examination of
isozyme heterogenity for the enzymes peroxidase, malic
dehydrogenase, and glutamate oxaloacetate transaminase (F.
Kloc-Bauchan, personal communication). Induction of hybrid
peroxidase bands were noted for hybrids 11-1, 14-3, 15-4, 15-6,
21-1, 21-5 and 5-4 and were correlated to variegated corolla

phenotypes.

83

DISCUSSION

The successful isolation of prot0plasts has been described
previously for.£..nazgdii (wt-m) and.P..inflata (ca-susp) using
various cell and tissue sources (11, 19). The aim of this research
was to explore systematically the significance of conditions
prescribed by previous investigators for application in our
laboratory. The procedural adjustments determined in this study
provided reliable production of viable protoplasts of both species.

Petunia_p§rodii required lower Meicelase-P concentrations for
efficient isolations, presumably due to differences in greenhouse
growing conditions. A total change in the procedure of P. inflata
(ca-susp) isolation from suspension culture was required.
According to this investigation, tissue in the early growth phase
are the most suitable for protoplast isolation with respect to the
cell number increases. Throughout the late period of compacted
cell increase, as well as during the later phase of increasing cell
number, the yield of protoplasts was lower than during the early
period of cell division. The early logarithmic phase of increase
in cell number was observed to be characterized by a predominant
number of small, actively growing, and presumably thin-walled cells
which probably contributed to the higher yields of prot0plasts.
During subsequent periods, the cultures appeared to be composed of
larger and more highly vaculolated cells, with perhaps thicker
secondary walls.

The source and kind of enzymes used in the isolation of
prot0plasts is critical to survival and subsequent regeneration.
Protoplasts of P. parodii showed marked inhibition when isolated by

any concentration of Cellulase "Onozuka" R10 and delayed onset of

84

first division when cultured directly in the light. Petunia
inflata (ca-susp) was more tolerant of the variety of enzymes and
concentrations used for isolation. The optimal enzyme
concentrations of 22 Cellulase, 12 Driselase, 12 Macerase with 82
mannitol chosen for routine use did not impair division or the
regeneration capacity of the mutant cell lines, as evidenced by the
regeneration of white, presumably.P. inflata (ca), plants in
viability control plates from the fusion experiments.

Protoplasts of several Petunia species including P. parodii
and P. inflata vary with respect to the growth regulators required
for growth_1p_y;tgg culture (19). Such differences have been
exploited for selection systems in conjunction with complementation
t0 chlorophyll synthesis in cell hybrids (7, 20). Yet, even in
sexually compatible systems, the majority of the heterokaryons
produced as a result of protoplast fusion do not result in the
formation of hybrid cells capable of surviving the selection system
(7). While selection efficiency in this investigation was
comparable to previous reports, the increase noted (72 compared to
14.82) may be influenced by the high rate of P. parodii parental
reversion rates.

Another conspicuous difference in the plants regenerated here
was the high rate of anthocyanin instability expressed the in
corolla limbs of hybrids. Previous reports of anthocyanin
instability in_P. hybrida have linked unstable alleles to small
insertions and deletions in the regulatory component of the
anthocyanin biosynthetic control system (3, 9). High parental
reversion rates, induction of anthocyanin instability and the

morphological variability in the somatic hybrids of this study may

85

be attributable to either the subtle differences arising from
changes in the isolation conditions, most notably the change in
enzyme, or the fusion process itself.

A further possible source of the observed variability may be
the onset of hybrid dysgenic phenomenon, possibly being expressed
at the sexual level in the form of prezygotic unilateral
incompatibility. Somatic hybridzation has been shown to overcome
complete and partial sexual incompatibilites (5, 26, 28).
Previously existing crossing barriers may have been bypassed in
this investigation, causing induction of unstable alleles for
anthocyanin production and chromosomal-cytoplasmic
incompatibilities expressed as corolla splitting in conjunction
with parental chromosome loss in a hybrid cytoplasm.

Several authors have attributed phenotypic variability to (a)
mutagenic pr0perties of the culture conditions or (b) to the
exposure of covert elements pre-existing in the plant genome which
become activated or perpetuated in the permissive environment of a
tissue culture system (1). The first hypothesis has credence based
on the sometimes high levels of growth regulators used in culture
compared to effective endogenous levels_;p.y;y9. Supra-Optimal
levels of growth regulators have been shown to result in genetic
irregularities in relation to transcriptional regulation and DNA
amplification (2, 6, 23). The second hypothesis of mobile elements
within the genome being enhanced by tissue culture (17) and
involved with the nucleo-cytOplasmic incompatibility reactions (8)
has also been shown. The combination of the above two
possibilities may also exist.

The fusion process per_§g may provide conditions conducive to

86

DNA strand dissociation given the alkaline conditions of fusion.
The high temperature conditions used may produce heat-induced
strand clips, and the possiblity of mis-match upon ligation and
reannealing of the dissociated strands exists. The alkaline
conditions in conjunction with high temperature may also be acting
synergistically to create dissociation related sequence errors.
These may be the processes by which the chromosomal and phenotypic
irregularities seen in the somatic hybrids were initiated.

Selection efficiency in this investigation ranged from 3.75 to
14.282 for two fusion experiments successful in regenerating hybrid
plants. The higher selection efficiences in this experiment
compared to P. hybrida®P. parodii reported earlier (7, 18, 20)
probably reflect high parental reversion rates. Successful fusions
were produced at the rate of 31.42. In experiments giving rise to
somatic hybrids herein, the frequency of somatic hybrid plant'
production was 5.34 per 106 protoplasts. This is less than half
the rate reported earlier for this cross of 12.04 hybrids per 106
protoplasts (22).

Somatic hybrid plants showed intra- and interclonal
segregation for parental markers. Self-incompatibility alleles
segregated interclonally. Colonies orginating from the same fusion
plate, 14-3 and lS-series, segregated for pollen color alleles,
displaying ivory and blue pollen. Chimeric leaves and self
incompatibility in two plants of this group (15-3 and 15-2) may be
related to chromosome loss. 38-5 and 28-1 had reduced chromosome
numbers and segregated for white and blue pollen, respectively.

The possiblity of using somaclonal variation induced through

tissue culture systems or from interspecific somatic hybridization

87

has been discussed recently (13). Subtle variability such as
altered flowering times, pollen fertilities, etc., occurring after
passage of standard lines through tissue culture may be of value
when quantitative characters of agronomic importance such as yield
and certain forms of disease resistance are screened, providing an
extra hedge against germplasm vulnerability that is rapidly
occurring with the use of restricted numbers of genotypes. Such
subtle aspects for cr0p improvement may prove valuable in achieving
the goal of germplasm diversification.

The possibility of using protoplast fusion, not only to create
novel hybrids, but to open new areas of research in gene regulation
of higher organisms necessitates the need for identification of the
elements that induce gene instability. Development of procedures
to control and direct the extent of such disruptions would greatly

enhance efforts to engineer desired plant genotypes.

88

LITERATURE CITED

1.

5.

6.

7.

8.

9.

10.

ll.

12.

Barbier, M., Dulieu, H.L. Effets genetiques observes sue
des plantes de Tabac regenerees a parter de cotyledons
par culture_in vitgo. _Ann. Amelior. P1antes_§9:

321-344. 1980.

Bayliss, M.W. Chromosomal variation in plant tissues

in culture. _In: Inter. Rev. Cytol. Perspectives in Plant
Cell and Tissue Culture. I.K. Vasil, ed. pp.113-144.
Academic Press, New York. 1980.

Bianchi, F., et al. Regulation of gene action in Petunia
hybrida: unstable alleles of a gene for flower color.
Theor. Appl. Genet. 52:157-167. 1978.

Burr, B., Burr, F. Transposable elements and genetic
instabilities in cr0p plants. Stadler Symp. lg, 1981.

 

Butenko, R.G. Cultivation of isolated protoplasts and
hybridization of somatic plant cells. Inter. Rev.
Cytol. P23323-373. 1979.

Chang, S.E., Littlefield, J.W. Elevated dihydrofolate
reductase mRNA levels in methotrexate-resistant BHK
cells. Cell 13391-396. 1976.

Cocking, E.C. et al. Selection procedures for the
production of inter-species somatic hybrids of Petunia
hybrida and Petunia pgrodii. II. Albino complementation
selection. Plant S91, Lett. $957-12. 1977.

 

Engels, W.R. Hybrid dysgenesis in Drosophila and the
stochastic loss hypothesis. Cold Spring Harbor Pymp,
Quan_. Biol. 45:561-565. 1981.

 

Farcy, B., Cornu, A. Isolation and characterization of
anthocyanin variants originating from the unstable system
of an2-l in Petunia hybrida (Hort). Theor. App . Genet.
.§§:271-278. 1979.

Frearson, E.M., Power, J.B., Cocking, E.C. The isolation,
culture and regeneration of Petunia leaf protoplasts.
Dev. Biol. PP:130-137. 1973.

Hayward, C., Power, J.B. Plant production from leaf
protoplasts of Petunia parodii. Plant Sci. Lett.
.4:407-410. 1975.

 

Keller, W.A., Melchers, G. The effect of high pH and
calcium on tobacco leaf protoplast fusion. P, Naturforsch.
280:737-741. 1973.

 

13.

14.

15.

16.

l7.

18.

19.

20.

21.

22.

23.

24.

25.

26.

89

Larkin, P.J., Scrowcroft, W.R. Somaclonal variation - a
novel source of variability from cell cultures for plant
improvement. Theor. Appl. Genet._§Q:l97-214.

Linsmaier, E.M., Skoog, F. Organic growth factor
requirements of tobacco tissue cultures. Physiol. Plant.
P§:100-127. 1965. -

Maizonnier, D., Moessner, A.G. Localization of the
linkage groups on the seven chromosome of the Petunia
hybrida genome. Genetica §l:l43-148. 1979.

Murashige, T., Skoog, F. A revised medium for rapid
growth and bioassays with toabcco tissue cultures.
Physiol. Plant. 123473-497. 1962.

 

Potter, S.S. et a1. Transposition of elements of the
412, c0pia and 297 dispersed repeated gene families in
Drosophila. Cell £2,415-427. 1979.

 

 

Power, J.B. et al. The somatic hybridization of Petunia
hybrida and Petunia parodii. Nature 263:500-502. 1976.

 

 

Power, J.B. et al. The isolation, culture and
regeneration of leaf protoplasts in the genus Petunia.
Plant Sci. Lett. 1351-55. 1976.

Power, J.B. et a1. Selection procedures for the production
of inter-species somatic hybrids of Petunia hybrida and
Petunia parodii. I. Nutrient media and drug sensitivity
complementation selection. Plant Sci. Lett. 3941-6. 1977.

 

 

 

Power, J.B. et a1. Somatic and sexual hybrids of Petunia
hybrida and Petunia pgrodii. A comparision of flower
color segregation. .g, Hered. 923373-376. 1978.

Power, J.B. et a1. Somatic hybrids between unilaterial
cross-incompatible Petunia species. Theor. Appl. Genet.
2:97.99. 1979.

 

Skirvin, R.M. Natural and induced variation in tissue

Sink, K.C., Power, J.B., Natarella, N.J. The interspecific
hybrid Petunia parodii x.P. inflata and its relevance

to somatic hybridization in the genus Petunia. Theor.
.Anpl. Genet. 53:205-208. 1978.

Sink, K.C. Vegetative and floral characteristics of
Petunia species hybrids as related to the origin of
Petunia hybrida. Can..£. Genet. Qytol. P§:557-564. 1981.

Thomas, E., King, R.J., Potrykus, I. Improvement of crop
plants via single cells_;n vitro - an assessment.
.2. Pflanzenzuchtg._§P:1-30. 1979.

90

27. Uchimiya, H., Murashige, T. Influence of the nutrient
medium on the recovery of dividing cells from tobacco
protoplasts. Plant Physiol. 51:424-429. 1976.

28. Vasil, I.K. The progress, problems and prospects of
plant protoplast research. _AQ. Agron. P§:ll9-128. 1976.

SECTION II
HERITABLE SOMATIC INSTABILITY IN HYBRIDS DERIVED FROM
PROTOPLAST FUSION OF PETUNIA PARODII (W.C.S.)

AND PETUNIA INFLATA (FRIES)

HERITABLE SOMATIC INSTABILITY IN HYBRIDS DERIVED FROM
PROTOPLAST FUSION OF PETUNIA PARODII (W.C.S.) AND

PETUNIA INFLATA (FRIES)

SUMMARY: Plants were regenerated following interspecific
hybridization via protoplast fusion of leaf mesophyll protoplasts
of Petunia pgrodii, (W.C.S.) with suspension culture protoplasts of
a cytoplasmic determined chlorophyll deficient mutant of Petunia
inflata, (Fries) using calcium-high pH conditions. The phenotypic
variation spectrum of regenerated hybrid plants included subtle to
moderate corolla variegation, corolla splitting, chimeric leaf
variegation, and various developmental anomalies such as double
styles, petaloid structures on the corolla limb and ear-shaped
projections at the corolla lobe junctions.

To quantitatively assess the level of instability, counts were
made of the number of variegated spots and flecks on flower limbs
of somatic hybrid plants. Aceto-propiocarmine squashes of pollen
mother cells were also studied at various stages of the division
cycle to determine the number and behavior of chromosomes in the
hybrids.

Examination of selfed, sib-mated, and testcrossed progeny
revealed that the expression of anthocyanin instability was
conditioned by two unstable alleles segregating independently.
Morphological abnormalities in flowers of somatic hybrid plants
were attributed to either hybrid dysgenic phenomenon or nucleo-
cytoplasmic incompatibility following the loss of parental

chromosomes in a hybrid cytoplasm.

91

92

Key words: protoplast fusion, unstable genes, hybrid dysgenesis

 

INTRODUCTION

The literature dealing with tissue culture and, in some
instances, somatic cell hybridization is replete with documentation
of phenotypic variability in regenerated plants. The limitations
of such phenotypic observations lie in the lack of genetic analyses
necessary to distinguish between epigenetic and genetic bases of
change. A thorough understanding of underlying genetic mechanisms
that control and direct genetic modifications induced by cell
culture will required.

Several ;n_y1t§2_techniques have been successfully employed in
recent years to induce heritable changes in higher organisms.
Fusion of plant cells and their subsequent regeneration into hybrid
plants is one such technique that has demonstrated totipotency of
hybrid cells and coordination of nucleo-cytOplasmic functions. Yet
instances of preferential chromosome elimination and cytoplasmic
segregation of parental organelles in somatic hybrids have
accenuated the need for greater understanding of eucaryotic gene
regulation and nuclear spatial organization.

Variability generated by 3p;31£rg_manipulations has often been
dismissed as technical artifact with the palingenetic outcome
judged as the only desirable result. Reports of tissue culture
variability have been attributed to in 33239 conditions which may
provide a permissive environment for release of genetic elements
already present in the cell (Barbier, 1980). It has also been
hypothesized that culture conditions pe;_§g_generate variability

(Orton, 1981). In either case, the genetic mechanisms still remain

93

to be elucidated.

A high frequency of floral variegation in somatic hybrids
regenerated following fusion of protoplasts of g, parodii wild-type
mesophyll cells (wt-m) with a cytoplasmic determined chlorophyll
deficient mutant of g, inflata in suspension culture (ca-susp) has
provided the opportunity for genetic analysis of the variant
phenotypes. The objectives of this study were (i) to describe and
document hybrid phenotypic variation, (ii) to quantify flower
morphological and pigmentation instability, (iii) to determine
whether the instability was a heritable or epigenetic response,
(iv) to follow the transmission of the unstable alleles and assess
possible linkage patterns with other segregating genes, and (v) to
investigate chromosome behavior in pollen mother cells of selected
somatic hybrid plants.

Assessment of the instability of the interspecific somatic
hybrids has implications in the study of genetic transformation and
organelle transfer and has relevance in the study of
nucleo-cytoplasmic interrelationships. Current investigations in
the area of transposable genetic elements and their influence on
gene regulation have suggested that transposable elements may be
related to the unstable alleles found in higher organisms (Engels,
1981, Burr and Burr, 1981).

MATERIALS AND METHODS

Protoplast isolation, fusion, culture and plant regeneration:
Protoplasts from bulked inbred seedlings of g, parodii (wt-m)
(ZgélA) and g, inflata (ca-susp) (Zn§14) were isolated using
procedures described previously (Schnabelrauch et al., 1982).

Fusion of freshly isolated protoplasts was induced using the

94

calcium-high pH method of Keller and Melchers (1974). Selection
based on complementation between chlorOphyll deficient cells with
wild-type cells allowed visual separation of the green hybrid
callus from the colorless P, inflata parental callus, while pale
green 3. parodii colonies did not grow beyond the 50 cell stage in
a modified Murashige and Skoog medium (M/S P1 9M) (Power et al.,
1979). Selection efficiency based on this technique was 14.28%.
After an initial culture period of 5 weeks in M/S P1 9M, 0.5
ml of M/S P1 6M was added, and 0.5 ml of M/S Pl 3M added one week
later. Hybrid plant regeneration occurred after 3 weeks upon
transfer of selected green calli to M/S basal medium supplemented
with 2 mg/L zeatin (M/S 22) when cultures were kept at 2512°C under
a photon fluence flux rate of 15-20 Lam-23.l (400-700 nm)
(GE 96T12CW) on a 16 hr photoperiod. Cuttings from regenerating
colonies were excised and placed on a medium consisting of M/S
salts and vitamins with no exogenously supplied growth regulators.
These were placed in the same environmental conditions as for
plantlet regeneration from callus. After 3 weeks, plants with
roots were transferred to V.S.P. soilless planting medium (Bay
Houston Towing Co.) in 6/8 cell packs (Geo. J. Ball Co.) and
covered with clear polyethylene for humidification for 1 week.
Such plants were illuminated with 28-34 uEmrzs-l (400-700 nm)
(GE96T12CW) on a 16 hr photoperiod at 25: 2°C. Plants were then
transferred to the greenhouse and transplanted to 10 cm clay pots
using V.S.P. and were given 150 ppm 20N-8.6P-16.6K at each
watering. Plants were exposed for two week period to a photon
fluence flux of 550 uEm-gs-l (400-700 nm) (GE96T12CW) for 16 hrs.

After that period, they were returned to the natural photoperiodic

95

conditions present at East Lansing, MI, during the months of
February and March. A minimum night temperature of 22i2°C was
maintained; day temperature fluctuated with daily conditions.
Flowering occurred after approximately 1 month in the greenhouse.
Examination of flower color resulted in the tentative
identification of 14 somatic hybrid plants. These plants were
phenotypically evaluated and used for further genetic analysis.

Phenotypic variability spectrum: Overall plant variability was

 

visually assessed and categorized. Quantitative measures of floral
organs were made of the variant morphological phenotypes. A visual
assessment of developmental anomalies was noted for each somatic
hybrid.

Assessment of anthocyanin instability:

Quantification - To determine quantitatively the number of

 

unstable sites per flower, counts were made of the number of
acyanic and pale and dark magenta (p/m) cyanic flecks and sectors
present on the corolla limbs of 20 flowers from each somatic hybrid
plant.

Inheritance patterns - The somatic hybridization produced the

 

SF1 and the subsequent generations created by self, sib-mate, and
testcross pollinations constituted the progenies analyzed in this
study. Several crosses were made to follow segregation patterns of
the unstable anthocyanin phenotypes: (1) self pollinations of self
compatible somatic hybrid plants (15-1, 15-4, 15-6, 21-5), (2) a
testcross of 15-6 to 77-22-4-2 (An 2, parodii) as the female
parent, and (3) sib-mating 15-2 x 21-5. Crosses were carried out
by standard emasculation, pollination, seed collection and storage

methods for petunia (Sink, 1981).

96

Germinated seedlings were transplanted to V.S.P. soilless
planting medium in 12 cm x 8 cm x 5 cm deep plastic trays covered
with clear polyethylene and kept at 25:2°C under a photon fluence
flux of 64 11Emm23-1 (400-700 nm) (GE96T12CW) for 16 hrs. After full
expansion of the cotyledons, plants were transplanted to 6/8
plastic cell packs using V.S.P. and transferred to the greenhouse
where they were kept under temperature and light conditions already
described. Segregation for flower color, variegation pattern,
pollen color, corolla limb morphology, corolla tube pigmentation,
leaf morphology and leaf pigmentation were scored for each SF2 and
TC plant.

Cytggenetic studies: Meiosis in pollen mother cells was studied by
placing immature flower buds, approximately 0.2 cm in length, in
Carnoy's solution, followed by the removal of the anthers and
subsequent staining in aceto—propiocarmine (Power et al., 1978).
Chromosome number, meiotic behavior, chromosome associations,
frequency of meiotic anaphase bridges and frequency of micronuclei
were noted for selected somatic hybrid plants.

RESULTS

Phenotypic variation spectrum of somatic hybrid plants:

 

The somatic hybrid plants investigated in this study were from
fusion experiment P80-l. Each plant was evaluated in terms of leaf
pigmentation, shape, apex, base and margin characteristics, type of
trichomes present, corolla morphology and pigmentation, and, when
present, corolla variegation pattern.

Leaf characteristics - 0f the 14 plants regenerated from

fusion experiment P80-l, 9 were stable green while the remaining 5

97

displayed a distinct chimeric plastid distribution consisting of a
pale green lamina area around the midvein, surrounded by a narrow
band of off-white juxtaposed to dark green leaf margins (Fig.

la). In most cases, such chimeric plants produced variegated
flowers (15-2, 20-1, 21-5, 38-5). However, one plant (15-3) with
stable corolla pigmentation was observed with the chimeric leaf
pattern. The chimeric condition was not correlated to specific
types of variegation, as acyanic flecking and p/m cyanic sectoring
were both found associated with chimeric leaves (Table 1).

Leaf shape in somatic hybrid plants was generally ovate with
an obtuse leaf apex and round leaf base. Leaf margins were entire
and the surface of the leaf classified as hirtellous (Table 1).
These characteristics corresponded to those observed in the
tetraploid hybrid produced sexually between P, parodii and P.
inflata (77-17). Observed variations consisted of gradations in
apex and base angles of laminar expansion from the midvein,
reflexed and revolate leaf margins, and differences in degree of
leaf surface trichomes (Table 1).

Floral morphology - Somatic hybrid plants had flowers similar
in length, shape, and color to those of 77-17. Variation was
observed in morphology of corolla lobes and limb margins. However,
the most conspicuous difference between 77-17 and the somatic
hybrid plants was the presence of anthocyanin variegation in the
corollas of the latter.

Variegated floralgphenotypes - The anthocyanin variegation
observed in somatic hybrid plants involved a differential

distribution of pigmentation in the epidermal cells of the corolla

98

Table l. Morphological characteristics of Petunia
parodii®§. inflata somatic hybrids.

 

 

 

 

 

Leaf Characteristics* Variegation

Plant Limb
No. Apex Base Margins Trichomes margins Limb Leaf
5-4 Rd Rd Rv Ht ' En 2 G
14-3 Ac At Un Ht Rf 3 G
lS-l Ob Rd En Ht En 4 G
15-2 Ob Ac Un,Rv Ht En,Rv 2,4 C
15-3 Ac Rd Un Hs En,Rf 1 C
15-4 Ob Rd En Ht En 4 G
15-5 Ob Rd En Ht En 4 G
lS-6 Ob Rd En Hs En 4 G
15-7 Ob Rd En Ht En 4 G
20-1 Ob At Rv Ht . Rf 3,4 C
21-l Ob Ac En Ht En,Rf 2 G
21-5 Ob At En Ht En 2 C
28-1 Ob Rd En,Rv Ht En 4 G
77-1 Ob Ac Un Ht En 1 G
77-2 Ob At En Ht En l G
* Rd = round Rv = revolate l = stable

Ac = acute Un = undulate 2 = acyanic flecks

0b = obtuse Rf = ruffled 3 = acyanic sectors

At = attenuate Ht = hirtellous 4 = p/m cyanic sectors

En = entire Hs = hirsululous

G = green C = chimeric

99

limb. In contrast, the corolla limb of non-variegated flowers was
always uniformly magenta in color. Five classes of variegation
were distinguished: (1) minute acyanic flecks consisting of
scattered small white cells on a background of pigmented cells, (2)
acyanic sectors involving clearly defined sectors of solid white
tissue against a background of pigmented tissue, (3) p/m cyanic
flecks consisting of scattered cells of pale or dark magenta
pigmentation on a background of uniformly medium pigment intensity,
(4) p/m cyanic sectors consisting of fine alterations of light and
dark pigmentation encompassing entire corolla lobes, and (5)
various combinations of the previous 4 classes (Fig. 1b & c).

Mbst flowers on a given plant exhibited a consistent
variegation class but exceptions did occur. The acyanic
variegation occurred over a wide range of time during plant or
flower development, involving only a few petal cells or half a
flower, a whole flower, or an entire branch. Chimeral plants,
bearing more than one major variegation category were found (15-2),
indicating segregation or initiation of anthocyanin instability
could also occur upon extended growth and after the initial fusion
process.

Quantification of anthocyanin instability: Unstable sites on the
corolla limb varied in number and pigmentation (Table 2).
Anthocyanin instability was found in 25-100% of the flowers sampled
from each plant. Seventy-three percent.of the hybrids exhibited
corolla variegation. 14-3 and 38-5 had a predominance of acyanic
sectors, while 21-1 and 21-5 had minute acyanic flecks. In
addition, the latter two somatic hybrids were the only plants to

show twin spots in addition to corolla variegation. Twin spots

Figure 1.

100

(a): Chimeric plastid distribution found on somatic
hybrids. (b): Anthocyanin variegation patterns
(left to right): stable, acyanic sectored, minute
acyanic flecks. (c): Anthocyanin varigation
patterns: p/m cyanic sectors. (d): Half white,

half magenta flower from somatic hybrid ll-l.

(e) Acyanic petaloid structure (arrow). (f) Split
blossom classes (left to right): narrow split
blossom, wide split blossom, deeply lobed blossom.

101

 

Figure l

102

 

 

 

 

 

 

Table 2. Frequency profile of somatic instability in
corolla limbs of somatic hybrids expressed as
mean sites per flower (based on 20
flowers/plant).
Mean sites/flower
Unstable Acyanic Cyanic
Plant Phenotype Twin
No. (Z) Flecks Sectors Flecks Sectors Spots
5-4 89.0 0.55 0.11 2.44
14-3 100.0 11.35 0.35 4.80
15-1 56.3 0.26 0.16 1.42 12.6
15-2
(Nic.)* 95.0 0.70 4.25
(1) 87.5 62.5
(2) 100.0 100.0
(3) 89.0 0.05 0.15 44.4
(4) 85.0 0.25 0.50 36.0
(5) 35.0 0.05 17.0
15-3 45.0 0.15 0.07 2.0
15-5 25.0 0.05 1.70 1.0
15-6 35.7 0.04 0.21 22.8
15-7 31.3 0.13 0.25 6.3
21-1 100.0 8.30 0.11 7.56 0.15
21—5 100.0 3.15 0.03 11.00 2.6 0.05
38-5 100.0 3.63 0.12 3.37 25.0
i 73.4 9.54 1.04 2.15 20.5 0.01
(s) (29.4) (30.9) (2.68) (3.12) (28.3) (0.23)
* Nic. tobacco-like flowers (1) narrow split blossom

(2)

wide split blossom (3) = deeply lobed blossom
(4) =

"restored" blossom (5) "normal" blossom

103

averaging 0.15 sites/flower were observed on 21-1 (Fig. 2c).

Single magenta and white flecks on 21-1 occurred at frequencies of
0.37 and 8.30 sites/flower, respectively, while white sectors
occurred at a rate of 0.11 sites/flower. The frequency of twin
spots was less on 21-5 (mean 0.05 sites/flower), but reduced twin
spots were coupled with a concomitant increase in cyanic (p) flecks
(mean 0.82 sites/flower).

Plants generally had either acyanic flecks/sectors or p/m
cyanic sectors, but combinations of the two classes were also
observed. 15-2 initially showed p/m cyanic sectors, but after
extended growth in the greenhouse, branches with flowers having
both p/m cyanic sectors and newly appearing acyanic flecks
occurred. Another instance of delayed instability was seen in 5-4
which displayed acyanic flecks and sectors only after a progression
from very pale to dark magenta pigmentation had occurred uniformly
on the corolla limb surface.

Genetic behavior of anthocyanin instability: The SF2, sib-mated,
and testcrossed generations generally displayed reduced frequencies
of instabiliy compared to the respective parental means (Table 3).
The mean frequency of acyanic sites in the progeny linearly
regressed on the frequency of parental mean acyanic sites was
significant (P=0.05) (Fig. 3a), further substantiating the
hypothesis that the instability was determined by hereditary
factors. However, there was not a significant relationship between
the cyanic instability of the parents and progeny (P=0.05) (Fig.
3b); the sigmoidal curve of the plotted data points suggests a
polynomial equation would provide a better fit.

Of particular interest is the observation that 15-4 and 15-6,

Figure 2.

104

(a): Floral types found on somatic hybrid 15-2
(top, left to right): stable, p/m cyanic sectored,
tobacco-like flower, (bottom) half-p/m cyanic
sectored, half-tobacco-like flowers. (b): Dilute
floral types found on albino branches of somatic
hybrid 15-2 (left to right): stable, diluted
anthocyanin pigmentation, dilute anthocyanin
pigmentation in combination with tobacco-like
flower form. (c): Twin spot (circle with arrows)
from somatic hybrid 21-1. ca. 15X.

105

 

Figure 2

106

Table 3. SF2 and TC segregations of progeny from somatic

 

 

 

 

hybrids.
Mean sites/flower
Hybrid No. Acyanic Cyanic
- Plant
No. Flecks Sectors Flecks Sectors
15-1 ® 50 . 0
15-2 ®
-1 80.0
-2 0.0
-3 0.0
15-4 ®
-1 0.0
-2 ' 100.0
-3 0.0
-4 1.5
15-6 ®
-1 0.4 0.1 68.0
-2 20.0
-3 0.37 0.0 0.50 62.5
-4 0.75
-5 0.11 0.11
-6 1.14 0.28 0.57 40.0
-7 100.0
-8 11.1
15-2 x

21-1 1.00

Table 3, (cont'd.)

107

 

77-22-4-2

X

15-6

1.14
1.50
0.66
1.0

0.5

1.0

3.0

0.3 0.4 0.1
1.0 0.66

0.5

1.0

 

108

 

 

Mean progeny sites

    

LO 1 ®

0.8- '

0.6-

Y " OJZX :- .04
1.
4o.
1.
20 .

 

 

L
5 l0 I5 20 25 so 35
Mean parental sites

Figure 3. Regression of mean frequency profiles of progeny
on parental (a) acyanic sites (Table 3) and (b)
cyanic sites (Table 2) in somatic hybrids.

109

both with relatively stable corolla pigmentation, produced, after
self pollination, variegated progeny in the SF2. When testcrossed
to 4n P, parodii, 15-6 yielded both acyanic sectored and solid
flowers among the progeny. Progeny produced from the sib-mating of
15-2 ("normal" phenotype) x 21-5 (minute acyanic flecks) displayed
four variegation phenotypes: solid, acyanic sites (including
minute flecks and sectors) and cyanic sites (including pink flecks
and p/m cyanic sectored lobes) (Table 4). The segregation ratios
for corolla variegation are consistent with the hypothesis that the
occurrence of acyanic and cyanic variegation sites may be the
result of two unstable loci segregating independently in a 9:3:3:l
ratio (15-6 X and 15-2 x 21-5). Progeny from selfed pollination of
15-4 deviated from the two allele ratio and had a greater
proportion of solid flower phenotypes.

Testcross segregations using tetraploid P, parodii as the
female parent crossed to 15-6 gave rise to only light magenta and
dark magenta phenotypes (Table 5). Shades of light and dark
magenta can be explained by the dosage sensitivity of the
pigmentation allele described in petunia (Farcy and Cornu, 1979),
but there was a conspicuous lack of recessive white phenotypes in
the testcross progeny. The duplex and simplex genotypes (e.g. 5535.
and Aggg) would be medium and light magenta, respectively, due to
the dosage of the dominant A allele in the progeny. Testcross
progeny deviated significantly (P=0.05) from a l:l:1:l ratio and in
addition, showed a clear increase in the prOportion of variegated
as opposed to solid individuals (Table 5). The consistent excess
of variegated individuals above the expected may indicate a

preferential synapsis of chromosomes bearing acyanic instability

110

Table 4. SF2 segregations for variegated corollas among
progeny from self- and cross-pollinations of
somatic hybrids.

Progeny Segregations

Phenotype Total Solid Acyanic Cyanic Comb. Chi-

 

Cross of parent Progeny sites sites Square
15-4® stable 58 51 5 2 0 13:3
3.27

.l<P<.05

15-6® stable 55 39 6 8 29:3:3:1
5.02

.2<P<.1

15;i_§ stable x 16 7 5 3 l 9:3:3:l
acyanic 1.77

flecks .7<P<.5

 

111

Table 5. Testcross segregations for variegated corollas of
the cross 77-22-4-2 (Petunia parodii 49) x
15-6 0

 

 

Progeny Segregation

 

 

 

Variegated
Corolla Total Solid:
Color Solid Acyanic Cyanic Comb. Progeny Var.
Light 7 13 0 1 21 1:3
mag.

Magenta 1 1 1 2 5

 

112

alleles or segregation of trisomics in the allopolyploids carrying
the variegation alleles.

Corolla splitting - Somatic hybrid plant 15-2 upon further

 

growth in the greenhouse sectored out branches which displayed
flowers with chloripetalous characteristics, similar to the split
blossom morphology described in Nicotiana (Clayton, 1955). In
classifying these flower types, four main categories of corolla,
two principal stamen conditions, and three pistil conditions could
be distinguished (Fig. 1f). These categories are, following the
example in Nicotiana:

(1) narrow split blossom: corolla tubular only at base, and
dissected distally to form five extremely narrow petals.

(2) wide split blossom: corolla tubular for about half its

 

length, and dissected distally to form five petals.

(3) deeply lobed blossom: nearly normal corolla tube, with

 

one split proceeding across the limb to corolla tube and five petal
lobes more deeply dissected and ruffled than "normal".

(4) restored blossom: sympetalous with normal tubular corolla

 

and five petal lobes, limb slightly more ruffled than normal.
The stamen and pistil conditions include:

(81) terminal anther sac tissue degenerate, some

 

proliferation of subterminal tissue.

(32) terminal anther sac normally developed.

 

(P1) two separate ovaries and styles, generally shorter in

 

total length than "normal", with the stigmatic surface somewhat
cupped and elliptical in shape.

(P2) one large ovary with a fasciated double style

 

culminating in a bipartate stigmatic surface.

113

(P3) one ovary and one style in "normal" dimensions.

Flowers of corolla class 1 were significantly correlated
(P=0.05) with $1 anthers and P1 pistils (Table 6). Ninety percent
of class 2 corollas were associated with P1 pistils and $1 stamens.
A single instance of a flower with triple ovaries and styles was
encountered in this corolla class. Ten percent of class 2 corollas
were associated with P3 pistils and $1 anthers.

Ninety percent of class 3 corollas were associated with P3 and
pistils and $2 stamens. Restored ruffled blossom (class 4)
contained 202 PZ-Sl pistils and stamens. P1 pistils occurred at a
rate of 152 in this class. The remaining 65% involved P3 and 82
conditions. ‘

The "normal" corolla condition was significantly (P=0.05)
correlated with normal P3 pistils and gave 102 $1 stamens (Table
6).

Quantitative measures of five floral organ dimensions in
flower samples from the various chloripetalous corolla classes
showed significant (P=0.01) mean differences, supporting the
hypothesis of a successive developmental sequences of the split
blossom phenotypes to the "normal" phenotype (Table 7). Of the
floral organs analyzed, total corolla length and total style length
showed significant (P=0.01) linearity when regressed on the corolla
classes (r=96.12 and 99.11, respectively). Only the filament
attachment length showed low corolla class correlation. No
significant differences were found in this category between corolla
classes 1 and 2.

Modified corolla, stamen and pistil conditions were observed

together with the pattern of flower variegation characterized by

114

Table 6. Reproductive characteristics of chloripetalous
flowers of somatic hybrid 15-2.

 

Reproductive conditions (Z)

 

 

 

Pistils Anthers

Flower Pollen
phenotype P1 P2 P3 $1, $2 Viability
Narrow split

blossom 100.0 100.0 57.3
Wide split

blossom 90.0 10.0 100.0 70.1
Deeply lobed

blossom 10.0 90.0 100.0 84.2
Restored

ruffled

blossom 15.0 20.0 65.0 100.0 15.7
"Normal"

blossom 100.0 10.0 90.0 56.7
77-17 4n

sexual hybrid 100.0 100.0 90.6

 

115

Table 7. Quantitative measurements of floral traits in
modified corolla phenotypes sectoring from
somatic hybrid 15-2.

 

Mean organ dimensions (cm) (ise)

 

Total Tubular Total Fil. Filament
Flower corolla base Limb style att. attach.
phenotype length length dia. length length (Z)

 

Narrow

split

blossom 4.44 2.74 3.75 1.46 1.36 @ 49.6
(0.38) (0.25) (0.65) (0.18) (0.05)

Wide

split

blossom 5.09 2.93 4.40 1.71 1.35 @ 46.1
(0.23) (0.18) (0.49) (0.29) (0.11)

Deeply
lobed
blossom 5.24 3.05 4.76 2.12 1.25 41.1
(0.24) (0.13) (0.55) (0.27) (0.09)
"Normal"
blossom 5.47 3.16 4.98 2.52 1.32 40.2
(0.26) (0.10) (0.38) (0.10) (0.06)
77-17
4n sexual ~
hybrid 6.15 3.92 4.72 2.87 1.40 35.7
(0.33) (0.28) (0.45) (0.12) (0.19)
HSD (.01) 0.59 0.32 0.79 0.44 0.16

 

@ = means are not significantly different (P=0.01) when
compared by Tukey's method.

116

15-2. Distinct variegation patterns for the five corolla classes
were seen (Table 8). Narrow and wide split blossoms were
characterized by p/m cyanic sectoring in all five lobes.
Occasional white flecks were seen on corollas of these classes.
Deeply lobed split blossoms usually had.reduced lobe involvement
with p/m cyanic sectoring (402) but concomitant increases in the
occurrence of acyanic and cyanic flecking. The progression from
"normal" ruffled to "normal" unruffled blossom showed decreases in
both the percentage of unstable floral phenotypes and the presence
of acyanic flecks and p/m cyanic sectoring. In addition, acyanic
ear-shaped petal tissue projections were observed at the lobe
junctions of flowers in corolla class 4.

Developmental anomalies:

Along with corolla variegation and splitting, other
morphological abnormalies were apparent upon examination of somatic
hybrids. Various abnormalies associated with reproductive
structures were noted. The occurrence of stigmoid anthers was
found in 14-3 and in progeny of the cross of 15-2 x 21-5. A single
instance of anther sac formation on a stigmatic surface was noted
in 15-3. Flowers of 38-5 had anther sac formation of the corolla
lobe junctions on the limb surface, with adnate filaments extending
up the corolla tube. As mentioned previously, bipartate stigmas
(15-2), fasciated styles (15-2 and 38-5) and double stigmas, styles
and ovaries (15-2) were observed, and often associated with corolla
splitting. The occurrence of six anthers and six corolla lobes
together with anther fasciation were found in one flower from the
plant 15-6. Anthers were also seen with feather-like tissue

projections midway up the filament (20-1, 15-3).

117

Table 8. Association between split blossom characteris-
tics and type of corolla anthocyanin variegation
in somatic hybrid 15-2.

 

Floral variegation

 

p/m cyanic sectoring

 

 

 

. Ear-
Unstable (Z lobes involved) Flecking No. shaped
Floral phenotype project.
Class (Z) 2 3 4 5 cyanic acyanic (Z)
Narrow
split
blossom 100 100 0.0
Wide
split
blossom 100 100 0.0
Deeply
lobed
blossom 90 10 40 1.5 0.50 0.0
Restored
ruffled
blossom 85 15 30 1.67 0.67 10.0
"Normal"

blossom 35 15 10 1.00 . 0.0

 

118

Corolla modifications other than splitting were frequently
observed in somatic hybrids. Acyanic petaloid structures were
found along the midveins of plants 38-5, and 28-1 (Fig. 1e).
Suture-like vein incisions were common to corolla lobes of 38-5 and
28-1. Ruffled margined, asymmetric corollas were frequently
associated with p/m cyanic sectoring events and, various
combinations of ruffled margins, split corollas, acyanic sectors
and petaloid structures were found in some plants (38-5, 20-1).

One common corolla limb anomaly was the occurrence of acyanic
ear-shaped projections at the junction of corolla lobes (15-2,
21-1, 21-5, 15-3, 15-5, 5-4, 15-6, and TC progeny). Such
projections were frequently found at all 5 lobe junctions, but
cases of projections at only 1 or 2 lobes were also seen.
Individual somatic hybrid plant observations:

5:4} This plant flowered initially as a partial somatic
hybrid: white with blushes of magenta at the limb edges. As the
plant developed in the greenhouse, the flowers became darker
magenta until the intensity of the magenta coloration matched that
of other somatic hybrid plants. Once the intense coloration had
been reached, acyanic flecks and occasional acyanic sectors
appeared. Attempts to study the inheritance of the progressive
intensity increase by selfing 5-4 using both bud and cross
pollination were unsuccessful.

11:13 This plant was very weak under greenhouse culture
conditions but initial flowers proved to be a result of instability
early in floral primordia development, with several half white-half
magenta flowers developing (Fig. 1d). Other flowers from 11-1 had

large acyanic sectors and branches with very light magenta flowers

119

were observed.

‘1423: This plant proved consistently to be fertile, yet
seedlings were weak and difficult to raise to maturity.
Segregation of green to white seedlings at 2:1 ratios were
observed. Acyanic sectors in 14-3 flowers were large indicating an
anthocyanin instability occurred early in corolla primordia

development.

15-series: Plants of this series were from shoots regenerated
from the same hybrid callus and were phenotypically similar with
the exception of 15-2. All gave magenta flowers with p/m cyanic
sectoring. Different levels of stability were seen, with 15-5,
15-6 and 15-7 being the most stable in terms of uniform corolla
limb pigmentation. 15-2 and 15-3 also had a regular chimeric leaf
pigmentation pattern. 15-2 proved to be an extremely interesting
plant, sectoring into a variety of different phenotypes. A
progression of corolla splitting was seen on this plant as well as
all 5 classes of anthocyanin variegation. 15-2 sectored into
flowers that phenotypically resemble Nicotiana flowers (Fig. 2a).
These flowers were produced on glabrous green shoots. Flowers
equally p/m cyanic sectored and Nicotiana-like were frequently
observed during the course of branch development. Albino branches
also were found and such branches produced flowers with extremely
cupped corollas and a diluted anthocyanin pigmentation extending
from the lobe junctions into the center of the corolla (Fig. 2b).
Albino shoots isolated from 14-3 and placed in in viggg.culture on
shoot regeneration medium regenerated into green plants with a
stable floral phenotype (R.J. Griesbach, unpublished results),

indicating possible epigenetic basis for the sorting out of albino

120

branches.

21-1, 21-5, 38-5, 28-1: Flowers on these plants were ruffled
and often had acyanic petaloid structures developing from the
midvein of the limb. In addition, fasciated thick styles were
found. Adnated anther sacs with attenuating filaments were
frequently found at the lobe junctions in 38-5. The corolla of
38-5 was significantly (P=0.0l) reduced in diameter. 28-1
frequently had one acyanic midvein on the corolla limb that served
as a point for acyanic petaloid formation.

Cytology:
Meiosis in sexual hybrid 77-17:

In the sexually produced tetraploid hybrid, 77-17, meiosis
occurred regularly. Fourteen bivalent rings formed at metaphase I.
At anaphase I, 14-14 disjunction of chromosomes was followed by
cytoplasmic cleavage to give two daughter cells. An orientation of
14 dyads at the metaphase II equatorial plate was followed by
14-14 disjunction and finally four daughter cells at the quartet
stage.

Meiosis in somatic hybrids:

 

Cytological observations of microsporocytes revealed a number
of meiotic aberrations in somatic hybrid plants. While the
possibility of a correlation between meiotic aberrations and
variegated phenotypes may not be ruled out, the data do not support
loss of chromosomes as the only cause of variegation. In most
somatic hybrids, chromosomes were paired as ring bivalents. As the
rings separated at anaphase I and II, chromatin bridges were seen
extending from the termini of some chromosomes. Bridges were seen

in hybrids displaying flecking, and occurred in 40Z of the cells in

121

21-1 displaying minute acyanic flecks. Seventy-seven percent of
the cells in 14-3 with acyanic sectors and 39Z of the cells in p/m
cyanic sectored phenotypes of 15-2 had anaphase II bridges.
"Stable" somatic hybrids had bridge formation at a rate of 26Z in
anaphase II cells.

Chromosome associations at anaphase I and II varied
considerably. Common associations seen included 14-14, 13-15,
10-18, 12-16 and (6-5)-(8-4-5) in plants with 28 chromosomes. In
somatic hybrid 14-3 with 36 chromosomes, even more variability was
observed. Lagging chromosomes resulted in aneuploid quartets in
some instances, which was subsequently reflected in reduced pollen
viability. Micronuclei were observed in tetrads with 3 to 8
nuclei.

Narrow and wide split blossom phenotypes had chromosome
numbers of 24. Pollen mother cells from these floral types
progressed through meiosis to the formation of tetrads. Pollen
viabilities ranged from 57.3Z for narrow split blossom flowers to
70.12 for the wide split blossom flowers. Deeply lobed blossoms
had a chromosome number of 28, but chains of 3 or 4 chromosomes
were seen. Pollen viabilities for these flowers were 84.2Z. The
restored ruffled blossom phenotypes had 26-28 chromsomes, but
pollen viability was severely reduced (15.7Z). "Normal" flowers
had 26-28 chromsomes and their pollen viabilities averaged 56.72.
The tobacco-like flowers had a chromosomes number of 26 with pollen
viabilities of 52.5Z: dilutely pigmented flowers from albino shoots
of somatic hybrid 15-2 had chromosome numbers of 24 and pollen
viabilities of 63Z. The 49 sexual hybrid, 77-17, had 28

chromosomes and 90.62 pollen viability.

122
Segregation of other morphological traits:

A number of other traits were followed in the self, sib-mate
and testcross progeny in an attempt to detect co-segregation with
the anthocyanin instability. Segregation ratios for pollen color
(39), corolla limb margin and lobe morphology (03 and_PE,
respectively), corolla tube pigmentation (_D_, L, or H), leaf
pigmentation pattern (Eh), leaf margin morphology (3:) and
ear-shaped projections at corolla lobe junctions (E2) were noted
for solid phenotypes, acyanic and cyanic variegated phenotypes, and
acyanic/cyanic combination phenotypes.

Pollen color in self (15-4 and 15-6) and sib-mate progeny
approximated at 12:3:1 ratio (chi-square=l.72, 0.2<P<0.1),
indicating dominant epistasis between two pollen color loci. In
Petunia_hybrida, the two loci are Pg and A24, Undulated corolla
margins followed a 3:1 ratio in self and testcross progeny
(chi-square=3.36, 0.2<P<0.l, and 0.63, 0.5<P<O.3, respectively).
Corolla lobe morphology segregated at 15:1 ratibs in self progeny
(chi-square=3.39, 0.2<P<0.1), at 9:7 ratios in sib-mate progeny
(0.1<P<0.05) and 3:1 in testcross progeny (chi-square=1.84,
0.2<P<0.1). Corolla center pigmentation segregated at 15:1 ratios
in self progeny (chi-square=.42, 0.7<P<0.5), 1:1 in testcross
progeny (chi-square=0.38, 0.7<P<0.5) and 9:7 in sib mate progeny
(0.1<P<0.05). Chimeric plastid patterns in the leaves segregated
at 15:1 ratios in 15-6 self and sib-mate progeny (chi-square=3.04,
0.3<P<0.2), but showed no segregation in 15-4 self and testcross
progeny. Ruffled leaf margins segregated at 3:1 ratios in self
progeny (chi-square=0.31, 0.9<P<0.8), and at 15:1 in sib-mate

progeny. No segregation for ruffled leaf margin was seen in

123

testcross progeny. Ear-shaped projections similarly showed no
segregation in 15-4 self and testcross progeny, but segregated at
15:1 ratios in 15-6 self and sib-mate progeny (chi-square=2.78, 0.3<
P<0.2).

Only the ruffled leaf characteristic appears to be associated
with the varigation patterns seen in somatic hybrids. Further
observations are necessary to quantify this association.

DISCUSSION

Petunia parodii and P. inflata possess different alleles at a

 

large number of loci that determine both quantitative and

qualitative characteristics (Sink, 1975, 1981). Petunia parodii

 

carries the recessive white allele in a nearly homozygous genotype
while_§. inflata carries the dominant flower color allele in a

homozygous condition. Petunia parodii is further characterized by

 

a long corolla tube, pointed flower lobes, slightly asymmetic
corolla limb, and a tall slender growth habit. In the floral bud
1-2 days prior to anthesis, a light rose color is evident (Power et
al., 1978). This coloration fades upon opening and the mature
flower of_P. parodii remains white with purple veining in the
throat region. Petunia_inf1ata has a reduced limb diameter
compared to P. arodii, round lobes, a symmetric corolla limb and
short corolla tube length. The two species can also be separated
by the longer petioles, pedicels and stem pigmentation present in
.2. parodii. Several biochemical characters also distinguish these
two species (Natarella and Sink, 1974, 1975).

Anthocyanin biosynthesis in Petunia is conditioned by a series

of 9 separate alleles at various loci (Paris, 1956). While P.

parodii and_§. inflata have not been used in studies on the

124

regulation of anthocyanin biosynthesis per 33, white flowering
mutants of.P. hybrida and magenta colored P. violaceae can be
considered paradigms for the regulation of pigmentation in the

genus Petunia. Petunia parodii and P. inflata share biochemical

 

 

and genetic homology with P. hybrida, and thus may be considered as
part of the progenitor species of the cultivated petunia (Sink,
1981).

In Petunia, the synthesis of anthocyanins, which consist of
the aglycon, anthocyanidin, linked to one or more sugar or acid
groups, is governed by the complementary genes-gal, gag, and An;
(Kho et al., 1977, Kho, Kamsteeg and VanBrederode, 1978). Thus,
anthocyanin biosynthesis is strongly depressed when these three
genes are homozygous recessive and a completely white flower is
produced (Kho et al., 1977, 1978). Homozygous recessive anl or_an§

result in completely white flowers while homozygous recessive 322
creates only a partial block of anthocyanin synthesis and results
in faintly colored flowers (Kho et al., 1978, de Vlaming, 1981).

Genes A91 and_§§2 are located on chromosome VI and are
strongly linked with each other and with the gene RE which is
responsible for glycosylation and subsequent delphinidin and
cyanidin 3-rutinoside formation leading to magenta coloration (Kho
et al., 1977). A possible scheme for genetic control of
anthocyanin biosynthesis of_P. hybrida is given in Fig. 4 (Kho et
al., 1977, 1978, Farcy and Cornu, 1979).

Dominant_Anl and_Ang are required for the coversion of
dihydroquercitin, a precusor of anthocyanins and flavanols, into

cyanidin 3-glucoside. This reaction is thought to proceed in two

steps: (1) conversion of dihydroflavononal into anthocyanin by

125

Figure 4. Possible genetic control of anthocyanin biosynthesis
in Petunia h brida (Kho et al., 1977, 1978: Farcy
and Cornu, 1979 .

126

02303.96. EDEEEou
01.0.. o

IHOOUO.

I.

«53250. n 55:26

 

I

5.336825%

5398:0962?

010

:

«532:0- .v 5896:6835“.

 

 

. Io .
s .0 s
:o

v 9505.

2532.3 .. 5
53230835..

 

:43

653020 - b
_oeoquooxeoxfio

 

 

 

E5

353.525 28:36:

127

intramolecular rearrangement and removal of a hydroxyl group, and
(2) a glucosylation at position 3 (Kho et al., 1978). The enzyme
catalyzing these reactions is UDP-glucose:

cyanidin 3-0-glucosyltransferase, a dimer. A dominant_Ag3 allele
controls the synthesis of the anthocyanin precursor
dihydroflavonols (Kho et al., 1977).

Genes_Apl and An; regulate the amount of glucosyltransferase
activity present in the cell and thus are not structural genes (Kho
et al., 1978). .52; may affect the last stages of anthocyanin
biosynthesis by either (1) influencing the transcriptional activity
of the gene 35, which regulates the effect of gene 9;, etc., by
means of a sequential gene regulation (Britten and Davidson, 1969),
or (2) induction of the enzyme in relation to the amount of each
successive substrate product (Dooner and Nelson, 1977, Farcy and
Cornu, 1979).

Instability in anthocyanin alleles have been reported in P.
hybrida which involved red flecking on a white recessive
background, attributed to instabilities of Ag; and Ag; (Cornu,
1977, Farcy and Cornu, 1979, Bianchi et a1, 1978) and pale and dark
magenta sectoring, attributed to an unstable Hf; allele (Cornu,
1970, Maizonnier and Cornu, 1971). Reports of unstable anthocyanin
alleles in.P. violacea causing variegated phenotypes (Demerec,
1935, Malinowski, 1935) links the instability found in P. hybrida
to a possible instability in progenitor species.

The unstable alleles in Petunia have all been reported in
inbred lines or haploids displaying the floral variegation after
many generations of selfing or after direct mutagenic treatment.

The variegation described in this investigation is the first report

128

of the induction of instability in interspecific Petunia sexual or
somatic hybrids. It it possible that the cross compatibility
displayed between most Petunia species (Sink 1975, 1978, 1981),

with the exception of P. parviflora (2n=18) crossed to species

 

petunias, and the apparent cross compatibility and phenotypic
stability after in ziggg somatic hybridization of the unilaterally
prezygotic incompatible cross of _P. parodii 31:. inflata (Power et
a1, 1979), may reflect genetic homology and the maintanence of
stability under certain strictly determined in 23522 handling
procedures.

Recurrent off-types in inbred strains have been a difficult
problem for plant breeders. The frequent appearance of rogues in
well established lines have been attributed to seed contamination,
but may be due to chromosomal, mutational or linkage group
disruption factors (Burr and Burr, 1981). McClintock (1978)
suggested that wide crosses may provide a stress that could trigger
reorganization of parental genomes. Nicotiana hybrids provide well
documented evidence for such reorganization (Smith and Sand, 1957,
Gerstel and Burns, 1976).

Chromosomal instability has been linked to floral variegation
in Nicotiana hybrids (Gerstel and Burns, 1976). Unstable alleles
for carmine-coral locus in interspecific hybrids of_N. tabacum x_N.
otophora have been cytologically determined to result from
spontaneous breakage of large heterochromatic blocks of N. otophora
chromosomes in the N. tabacum nuclear background (Gerstel and
Burns, 1967, Burns and Gerstel, 1969). In addition to chromsome
breakage, the formation of large megachromosomes was occasionally

observed in these hybrids (Gerstel and Burns, 1976). It recently

129
has been shown that P, hybrid§_has dispersed heterochromatin and

not discrete heterochromatic blocks (Dietrich et a1, 1981).
Assuming the possible ancestral links of the species used in this
study to P, hybrida, it is likely that P: parodii and P: inflata
share the same dispersed heterochromatic patterns. Similar
reports on anthocyanin variegation in hybrids of the sexual cross
of N} tabacum x N, plumbaginifolia have also attributed the
instability to chromosomal aberrations (Ar-Rushdi, 1957, Moav,
1961).

The acyanic flecks and sectors appearing after protoplast
fusion of P, parodii ® 2, inflata may be due to hybrid dysgenesis
as reported in Drosophila (Engels, 1980, 1981, Kidwell, Kidwell and
Sved, 1977), or through the insertion of transposable elements at
or near the anthocyanin locus, causing suppression of the
structural gene. Insertion of such elements has been shown to
cause regular breaks at the locus of insertion. Alternatively, an
autonomous or nonautonomous controlling element system could have
been unmasked from the heterochromatin by the fusion process
itself.

The appearance of acyanic (white) and p/m cyanic (pink and
dark magenta) flecks and sectors on the corolla limbs of several
somatic hybrids may be attributed to two causes. If the mechanism
of chromosome breakage is perpetuated by a process similar to the
breakage-fusion-bridge cycle reported in maize (McClintock, 1950,
1951, 1956) and Petunia (Maizonnier and Cornu, 1971), then
intensity differences may be due to breakage of the 531 or 53;
alleles at different points in the gene sequence. Since genes for

anthocyanin biosynthesis show quantitative effects (Kho et al.,

130

1978), then disruption by breakage at different positions in the
DNA sequence could result in variable anthocyanin expression and
thus intensity differences in the somatic sectors. Alternatively,
if the intensity differences were caused by instability of either
the Ag1_or An2_alleles, and assuming that each regulator element
has a dual nature composed of a mutator and expressor component
(Bianchi et al., 1978), then coloration changes observed could be
due to mutations, possibly deletions or insertions of small DNA
segments, into the £21 expressor component, causing varying degrees
of intensity changes.

Differences in flecking frequency and sector size among the
somatic hybrids may result from a second unlinked regulator,

analogous to fleck timer isolated in N. langsdorffii x.N. sanderae

 

hybrids (Sand, 1976). Further genetic analysis of progeny ratios
and identification of unlinked marker genes would be required to
substantiate a nonautonomous system as the source of the flecking
events. While controlling elements are directly involved in the
case of the recessive phenotype giving rise to sectors expressing
the dominant phenotype, the presence of controlling elements cannot
be excluded if only unstable mutant alleles are detected (Sand,
1976). However, the possibility exists that the genetic component
peculiar to the variegated phenotype could be viewed as an
independent dominant modifier of the sectoring allele.

An alternative explanation lies in the mutator component of the
presumed regulator element. Deletions and subsequent amplification
of the repetitive DNA assumed to comprise the regulator element
(Bianchi et al., 1978), could cause frequency changes in the

appearance of flecking through the course of successive cell

131

division cycles. The quantitative differences between_332 mutants
in_P. hybrida suggested that intermediate repetitive DNA may
contribute to the regulatory elements. Deletions in the mutator
would result in complete inhibition of the structural element for
the production of flower pigment. Anthocyanin biosynthesis would
resume after the deleted sequences were restored by amplification
of repetitive DNA during cell divisions of the growing flower bud.
The larger the deletion, the smaller the change of reparation and
the smaller the number of spots on the corolla. Thus, differences
in flecking frequency may be due to differences in the size of the
deletions in the mutator component which in turn may be influenced
by environmental conditions.

Maizonnier and Cornu (1971) characterized "striped" mutants in
_P. hybrida resulting after gamma radiation which bear strong
resemblence to the p/m cyanic sectoring phenotype described herein.
The instability resulted from breaks at the H£l_locus conditioning
petunidin synthesis. The resultant acentric fragment was found to
be translocated to the long arm of chromosome II. While this study
did not search for such translocated chromosomes, a similar
breakage at the Hfl allele may be causing the p/m cyanic sectoring
observed. Striped flowers were also correlated with corrugated
leaves. A correlation of variegated phenotypes with a phenotypic
condition labelled "ruffled" leaf was observed in this study. The
variegation and ruffled leaf characteristics may be linked.

The appearance of pink-flowered branches (15-3, 11-1, 15-2)
could have been caused by a mutation in the expressor which would
result in uniformly light pink flowers. Mutations in both the

mutator and expressor would result in white spotted pink flowers

132
which were not found.

The change in 15-2 from p/m cyanic sectors to uniformly
colored limbs with acyanic flecks may have been caused by a
chromosome translocation, if the mechanisms producing similar
phenotypes in P, hybrida are in operation in this plant. However,
such a change in the stability of the An_alleles and §f1_allele,
resulting in a shift of instability from Hfl_to an An_locus may
have been the result of a transposition of a mobile genetic
element, initiating instability at a second locus. Transposition
capacity is one of the features of maize nonautonomous controlling
element systems. SF2 progeny of 15-6 produced by self pollination
showed a similar change from p/m cyanic sectoring in the SFl to
acyanic flecks in the SF2, which may be indicative of transposition
of the instability from Hfl_locus to an Ag_locus.

Chimeric leaf patterns were found in this study to be
inherited independently from any of the variegated corolla
phenotypes. Furthermore, the distinct SFl plaStid variegation was
transmitted to the SF2 as a dispersed mosaic pattern.

In addition to variegation phenomenon, wide crosses have also
been shown to cause morphological abnormalities attributable to
chromosomal-cytoplasmic incompatibilities. Disruptions in
nucleo-cytoplasmic interaction resulting in split corollas was
observed by Clayton (1955) in the first backcross generation of the

amphiploid N} debneyi-tabacum to N: tabacum as the male parent.

 

Such disruptions took the form of male sterility and corolla
splitting with associated anther abnormalities. Further
backcrosses substantiated that N: debneyi cytoplasm interacting

with the N: tabacum genome resulted in morpholgical aberrants (Sand

133

and Christoff, 1973), as if regulatory circuitry for developmental
and structural features of the corolla had been upset by some type
of incompatible reaction between cytoplasmic factors and nuclear
chromosomal elements leading to altered differentiation.

The interaction of genic, chromosmoal and cytoplasmic factors
in the induction of unstable mutations in interspecific crosses
parallels the phenomenon of hybrid dysgenesis in Drosophil .

Hybrid dysgenesis results from chromosomal-cytoplasmic interactions
in certain interstrain crosses, and produces several aberrant
traits including temperature sensitive sterility, segregation
distortion, sex ratio distortion, and high frequencies of mutation,
male recombination, chromosome rearrangement and nondisjunction
(Engels, 1981, Engels and Preston, 1980, Bingham, 1981, Kidwell,
Kidwell and Sved, 1977). These traits are not observed in
homozygous strains, but are limited to specific hybrid combinations
and are conditioned by the direction of the cross.

The dysgenic phenomenon was observed only if a portion of the
wild strain chromosome in a laboratory strain background was used
in the cross (Engels, 1981). Strains were further classified as
maternal (m) or paternal (p) in their contribution to any dysgenic

trait. Many D. melanogaster genomes contain p strains that appear

 

to carry multiple copies of the mobile element P_which has been
determined to consist of discrete segments 300 to 3000 nucleotides
long, whose insertion at unstable loci initiates chromosome
breakage (Engels, 1981, Burr and Burr, 1981). Some dysgenic males
produce chromosome inversions and complex multibreak rearrangements
at frequencies near 10Z.

A model involving spatial organization of chromosomes has been

134

suggested to explain this phenomenon (Sved, 1976). Normal nuclear
functioning may require a specific spatial organization of
chromosomes which could involve chromosome-membrane associations.
The chromsomes inherited from the female parent retain their
,spatial distribution by virtue of the mechanics of fertilization,
while spatial ordering from the male parent is not necessarily
maintained. Paternally derived chromosomes may carry information
coding for correct positioning of chromosomes within the nucleus of
the zygote. Hybrid dysgenesis results when the correct
organization information is lacking from the male strain.

That an analogous dysgenic mechanism is functioning in the
incompatible reactions in plants has been suggested by some authors
(Burr and Burr, 1981). The sexual cross of_P. parodii x_P. inflata
can be accomplished only if_P. parodii is used as the female parent
for bud pollination. The incompatible reaction in this cross is
prezygotic (Sink et al., 1979), indicating direct fusion of
maternal-paternal gametes with the onset of dysgenic behavior
cannot occur. However, the possibility of dysgenic behavior exists
with protoplast fusion, especially since constraints exist
requiring absolute co-functioning of both the two parental
cytoplasms and nuclei. Disruption of nuclear organization upon
fusion of a cell in 61 phase with cells actively synthesizing DNA,
each of which is assumed to have specific organizational
relationships with the nuclear membrane and to each other, may
result in the induction of mutability or chromosomal
rearrangements. Such rearrangements could cause structural genes
to be inserted near heterochromatin, resulting in partial

suppression of gene activity, or may result in the regular

135

induction of chromosomal breaks allowing the exposure of regulator
and receptor systems of controlling elements heretofore hidden in
heterochromatin. McClintock (1950) proposed that disturbances in
the quantity and organization of heterochromatic elements could
give rise to a series of changes in chromosome structure, behavior,
and genic reactions that could drastically alter phenotypic
expression.

The physical structure of heterochromatin is dominated by the
presence of highly repeated DNA sequences (Hilliker and Appels,
1980). These highly repeated sequences may affect gene expression
by their association with certain classes of proteins which
influence the chromatin structure of adjacently transcribed
sequences or by influencing the three-dimensional structure of the
interphase nucleus with respect to nearest neighbor relationships
between regions of different chromosomes and chromosome-nuclear
membrane associations (Hilliker and Appels, 1980). Repeated DNA
sequences in heterochromatin are often AT-rich, and thus are
inherently weaker and provide likely points of breakage upon stress
conditions such as alkaline pH and the high temperatures used
during protoplast fusion. Changes in gene expression following
hybrid dysgenesis implicate the three-dimensional structure of the
nucleus: heterochromatin may similarly influence gene expression
through three-dimensional effects. Disruption of the
three-dimensional structure of the nucleus by fusion therefore may
have resulted in altered gene expression through dysgenic
mechanisms or involvement with heterochromatin.

0n the other hand, cross incompatibility in the form of

corolla splitting may have resulted from a genetic mechanism

136

unrelated to dysgenesis. In progeny tests in Nicotiana, corolla
splitting was interpreted as an effect of quantitative genes for
broader floral tubes present in the chromsomes of the recurrent
parent (Sand, 1968, Sand and Christoff, 1973). In this study,
corolla modifications were observed without the appearance of
stigmoid anthers leading to male sterility as described in
Nicotiana. Conversely, the number of ovaries and styles were found
to vary from the normal single ovary and style to bipartate stigma
with fasciated styles and then to double styles and double ovaries.
The occurrence of double, and in one case triple, styles and
ovaries in somatic hybrids may be the result of activation and
transcriptional read-off from duplicate sets of female reproductive
structural genes, amplified in blocks or tandem arrays in the
somatic hybrid genome. Such duplications may have resulted from
the translocation of female reproductive structural genes from one
parent and integration into the chromosomal location of the other,
but based on style lengths closer to P: inflata (0.8 cm for P:
inflata, 5.4 cm for P: parodii and 1.46 cm for double styled
somatic hybrids), it is more likely that DNA amplification caused
by inadvertant selection pressure during the in_vi£52_culture
period possibly resulted in tandem repeats of pistil structural
gene families and duplicate sets during development.

Sand and Christoff (1973) were able to separate genetically
the range of morphological alterations found in the tobacco
corollas, lending further support to the view that the factors for
male sterility residing in the cytoplasm were hyperstatic to the
morphological abnormalities conditioned by the nucleus. Similar

conclusions were reached by Belliard et al. (1978) after the

137

somatic reconstruction of this cross and analysis of chNA
restriction patterns. Results presented for somatic hybrid corolla
splitting here also support the contention of nuclear gene
involvement in conditioning split flowers.

Flowers of 15-2 separated into an ordered series of corolla
splitting. This phenotypic array suggests a series of related
effects, increasing quantitatively toward normal, conditioned by
elements found in the phenotypes, where floral class 1<2<3<4.

There is also a correlation between the degree of corolla splitting
and the particular pistil and stamen modification observed in the
flower. The reduction in chromosome number occurring in the more
modified 15-2 flower categories supports the contention that
nucleo-cytoplasmic incompatibilities are produced as one of the
parental genomes is preferentially lost with successive mitotic
divisions of the apical meristem and the adverse reaction of the
remaining chromososmes in the hybrid cytoplasm. Cytoplasmic
factors appear to disrupt normal nucleo-cytOplasmic interactions by
eliminating chromosomes carrying either incompatibility suppressors
or compensating factors, which result in functional tetraploid
derivatives with "normal" phenotypes, or by enhancing the
expression of incompatibility factors present on the remaining
chromosomes. Gradual chromosome elimination may create a dilution
in the amount of incompatibility suppressor by reducing the copy
number. This hypothesis is supported by the progression of
significantly (P=0.01) distinct split blossom phenotypes.

The findings of Potter et al. (1979) that tissue culture
provides a permissive environment for the transposition of mobile

elements in the Drosophila genome may relate to the induction of

138

instability seen in Petunia somatic hybrids. If the instability is
due to an insertion of a transposable genetic element into or near
the gene conditioning anthocyanin biosynthesis in the hybrids, then
a blocked pathway which has been shown to condition the recessive
white phenotype in P. axillaris and_§. hybrida could possibly
result in the flecking phenotype. Conversely, the regularity of
the formation of bridges in meiotic anaphase may indicate
instability due to the presence of a transposable genetic element
near the anthocyanin locus which conditions chromatid breaks,
unmasking the recessive phenotype carried by the chromosomes
contributed by‘g. parodii. The timing of the breaks in floral
development would account for differences in sector size,
corresponding to the situation described by Sand (1976) of
nonautonomous two element systems in hybrids of N. langsdorffii x
N, sanderae. The regulator, fleck timer, presumably provides some
excision function. In analogy to bacterial systems, perhaps the
excision function is an exonuclease providing staggered cuts,
coupled with sequences coding for a repressor to block gene
activity (Calos and Miller, 1980).

Somatic hybrid plants treated with griseofulvin which induces
chromosome loss in cell cultures (LoSchiavo, NutiRonch, and Terzi,
1980), resulted in all the flecking variegation patterns observed
in the untreated somatic hybrids but the process appeared to occur
at increased rates (R.J. Griesbach, unpublished results).

The presence of twin spots an 21-1 and 21-5 may indicate that
somatic crossing-over or nondisjunction is occuring in somatic
cells. Twin spots occurred with regularity in these plants. Twin

spots were noted by Stern (1936) in conjunction with yellow body

139

color and singed bristle phenotype, a hypermutable allele in

Drosophila. Twin spots have also been associated with controlling

 

elements. Transposition of modulator (Mp) away from the stable red
site (PRR) resulted in twin spots in maize (Greenblatt and Brink,
1962). In heterozygous genotypes, twin spots can be found as a
result of somatic crossing-over and segregation of homologous
chromosomes so that twin spots develop as homozygous sectors
derived from heterozygous cells. Alternatively, twin spots may
also occur in homozygous cells and result from a crossover in a
pericentric inversion giving rise to sectors of cells with
duplications and deficiences of the two color genes (Barrow, 1973).

The possibility that twin spots result from chromosome
addition and elimination in sectors of somatic cells cannot be
excluded (Mulder, 1981). Trisomic segregation resulting in
triplicate doses of anthocyanin genes could produce dark magenta
sectors, while the hemizygous condition resulting from the loss of
3 or less of the 4 chromosomes bearing the anthbcyanin genes would
result in pink flecks. White flecks would occur after loss of all
4 chromosomes. Mulder (1981) found an independently segregating
factor which was dosage sensitive, that influenced the formation of
twin sectors of variegation in Petunia.

The tetraploid sexual hybrid, 77-17, produced an occasional
acyanic fleck on an otherwise uniformly magenta corolla after shoot
culture prepagation and prolonged maintenance in the greenhouse.
Also, several of the shoot culture propagules had very light pink
flowers, which would be explained if a mutation in the expressor
component of the regulator had occurred and the resulting mutant

sector propagated during culture.

140

The callus phase of the tissue culture process appears to
result in increased chromosomal instability as well as genic
instability. The fusion process may contribute to the instability
by causing chromosomal breaks due to either the high temperature or
high pH conditions given during fusion, which are subsequently
perpetuated in the permissive tissue culture environment.

Abnormalities attributed to the tissue culture process such as
ruffled leaves, petal, pistil, and anther number increases, anther
adnation and degeneration, and the formation of petaloid structures
on the corolla limb have been described for regenerated Nicotiana
plants (Skirvin, 1978). Since the alleles .221 and_222 have been
found to cause increased numbers of floral organ structures in.§.
.hybniga (Wiering, 1979), it is possible that tissue culture allowed
for amplification of the_Dg gene block and the retention of
amplified genes through some type of selection pressure
inadvertently applied during culture.

The fusion process itself involves the use of conditions which
are conducive to DNA strand separation, thus leaving the strand
vulnerable to breakage. At alkaline pHs, double stranded DNA
(dsDNA) denatures to single stranded DNA (ssDNA), but the molecule
remains intact. The denaturation of dsDNA by excessive alkali
coupled with excessive heat and rapid cooling results in strand
mismatch and thus sequence errors and hairpin loop formation
(Britten and Kohne, 1968). In addition, excessive heat induces
heat clips in the strands. AT-rich regions, found frequently in
heterochromatic areas, would be affected first. Such breakage may
initially induce the formation of a breakage-fusion-

bridge cycle by activation of silent receptor systems lying in the

141

heterochromatic sequences or by allowing the incorporation of a
foreign genome present in the cytoplasm or nuclear region to insert
in the broken areas.

High pH also negates hydrogen bonding through interference
with counter cation concentrations which shield the negative
phosphate groups on the double helix. Low concentrations of Na+, K+,
and NH4+ lead to helix destabilization by disruption of
phosphate-phosphate bridges formed ip yiyg by divalent cations.

At pHs above 9.0, nonhistone chromosomal proteins would have a
negative charge, resulting in possible interaction with divalent
counter ions and with the calcium ions supplied in the fusion
solution. Once the nonhistone chromosomal proteins are effectively
removed, coding regions would be exposed to a variety of shear
forces and/or accessibility to nucleases, resulting in alteration
of the DNA.

The progressive intensity changes of anthocyanin in the
corollas of 5-4 mimics the case of rabbit ear rogue in garden pea,
where upright stipules in F1 hybrids become the predominant
condition as development proceeds in the rogue pea (Burr and Burr,
1981). It would be interesting to relate progeny coloration and
intensity changes in our plants as was done with the rabbit ear
rogue, but unfortunately, 5-4 was effectively sterile. The
situation appears to be the reverse of the anthocyanin repression
described for variegated phenotypes in the other somatic hybrids in
which the changes was directed from wild-type magenta to the
recessive white condition. Flowers of 5-4 initially flowered as
the recessive white phenotype with only blushes of magenta at the

limb margins. As development proceeded, increased magenta

142

coloration was produced. Restoration of anthocyanin synthesis may
have resulted through DNA amplification after an initial deletion
in the expressor component of the regulator.

Similar phenomenon have been attributed to paramutable alleles,
where interaction between alleles leads to a directed heritable
change at the locus at high rates (Brink, 1973). Paramutable
alleles are reversible and appear to result from insertion and
excision of labile elements, initially exogenous in origin or
latent in heterochromatin of the genome. Such shifts in phenotypes
are unidirectional and occur with regularity under certain
conditions (Brink, 1973).

The progression from split corolla through ruffled sectored
corollas in 15-2 along with occurrence of distinct hairless
branches of flowers with tobacco-like corollas has been correlated
with chromosome elimination in this study. Flowers resulting in
the phenotype consisting of half-sectored and ruffled,
half-tobacco-like flowers indicate that the progression of
elimination can occur early in floral primordia development,
specifically at the first few cell divisions in the floral
meristem.

The deviant testcross ratios and the apparently simple
inheritance of acyanic and cyanic variegation suggests that two
unstable alleles condition variegated corollas in Petunia somatic
hybrids. This study also indicates that the expectation of
palingenesis in somatic hybrids after protoplast fusion may be
unfounded in some cases.

Segregation ratios were confounded by tetraploidy and

flecking. Testcross populations likewise reflected the expression

143

of the dominant gene controlling color formation with its diluted
expression as pale magenta. Allotetraploid gene segregation is
complicated by many factors not found in diploid gene segregation.
Instead of one type of heterozygote at a locus, there are three:
AAA'a' (triplex), AAa'a' (duplex) and Aa'a'a' (simplex).
Homozygotes are AAA'A' (quadriplex) and aaa'a' (nulliplex). In
addition, there are many aneuploid genotypes present in the
population that do not breed true for chromosome number. Each of
the seven chromosomes may also be added individually to each
heterozygote creating an array of genotypes, each with its own
segregation ratio. Ultimate ratios will depend on the frequency of
double reduction, the relative rates of formation and transmission
of polysomic chromosomes, and the relative viabilities of the
resulting euploid and aneuploid zygotes. In addition, they depend
on environmental conditions, methods of pollination, and assume
random chromosome pairing, equal viability of all phenotypic
classes, and somatic stability of chromosome number (Doyle, 1973).

For highly unstable genes, mutational and segregation products
may be confounded in the progeny arrays, so that the observed
results are genetically ambiguous without the aid of simultaneously
segregating stable marker alleles (Sand, 1976). The search for
stable markers co-segregating with the anthocyanin alleles showed
slight correlation between variegated phenotypes and another gene
producing a ruffled leaf phenotype.

In contrast to the somatic hybrids of Power et al. (1978), and
Cocking et al. (1976) , where stable somatic hybrids segregated as
their 43 sexual counterparts, hybrids from this study exhibited

corolla flecking, streaking, aberrant flower colors, and irregular

144
segregation ratios. Preculture conditions may have been of
sufficient pressure to physiologically predispose the plants
involved to enhanced chromosomal instability during the fusion
process, since fusion is very sensitive to factors such as plant
age and preconditioning (Butenko, 1979). Also, differences in the
enzymes used for protoplast isolation may have enhanced
chromosomally related abnormalities. Minor biological disruption
of segregation ratios in the case of one 3. hybrida®§. parodii
somatic hybrid which had a slight excess in the bluish white class
and deficiences in the pale purple class of test crossed prageny
while displaying excessive numbers of red/purple classes in the
backcrossed progeny was suggested to be a consequence of the fusion
treatment or the 33 ziggg culture period (Power et al., 1979).
Alternatively, P. parodii was thought to be conditioning the minor
variation due to the fact that many individuals used for the
mesophyll protoplast isolations are produced by seed massing
self-pollinated plants.

Experiments designed to further elucidate the cause of the
instability would contribute to information on gene regulation.
Further crossing is necessary to pinpoint the exact nature of the
inheritance of unstable alleles. Precise identification of the
gene product of the unstable alleles is required before molecular
characterization of the mutants can begin. Evidence from this
study points to Ag; and Ag; alleles causing anthocyanin
instabilities and_§§1 allele causing unstable petunidin synthesis.

Biochemical characterization of anthocyanin alleles would
enable the search to begin for mRNA from wild-type cells so that

cDNA probes would be available for hybridization screening of

145

Petunia total genomic DNA from normal and mutant lines. Such
screening would provide the appropriate sequence information to
detect insertions or deletions in the mutants. Molecular analysis
has important implications for genetic engineering. Information
gained would be benefical in terms of understanding eucaryotic gene
regulation as well as in understanding the conditions which promote
alterations through_i§_viggg manipulations. The ability of
transposable elements to recombine nonhomologous DNA segments would
greatly enhance efforts to clone specific genes and transfer the
desired genotypes efficiently, perhaps by creating more
sophisticated cloning vectors. Incorporation of isolated genes
into plant cells by vectors is now restricted to dsDNA plant
viruses and_gi plasmid of_Agrohagtgrium. Once the mechanisms for
nonhomologous recombination are understood, perhaps systems
analogous to transposable elements can be designed to promote
expression of existing or inserted cloned genes by inducing
recombination in the absence of extended genetic homology between
the isolated genes and the recipient DNA.

The possibility of recombination between homoeologous
chromosomes in interspecific somatic hybrids has lead researchers
to speculate that even with the selective elminination of one
parental species, introgression could be readily achieved by using
in 31552 manipulations. It becomes evident that such possibilities
for overcoming barriers to wide crosses may be confronted with the
introduction of new obstacles to hybridization at the cellular
level. Although the concept of creating sexually incompatible
generic combinations in the Solanaceae through somatic

hybridization has been proposed (Sink and Power, 1978), this study

146
has shown that nucleo—cytoplasmic incompatibilities may, under some
conditions, limit the types of plant combinations possible by
protoplast fusion. In addition, the fusion process itself or
subsequent callus culture period may induce heritable genetic
changes which can be viewed as either deleterious during the
propagation of stable genomes or beneficial from the standpoint of

studies in eucaryotic gene regulation.

147

LITERATURE CITED

Ar-Rushdi, A.H. (1957): The cytogenetics of variegation in a
species hybrid in Nicotiana. Genetics 425312-325

Barrow, J.B.: Chaudhari, H.: Dunford, M.P. (1973): Twin spots on
leaves of homozygous cotton plants. g, Hered. 645222-226

Belliard, G. et al. (1978): Morphological characteristics and
chloroplast DNA distribution in different cytoplasmic
parasexual hybrids of Nicotiana tabacum. Mol. Gen. Genet.
1653231-257

 

 

Bianchi, F. et al. (1978): Regulation of gene action in Petunia
hybrida: unstable alleles of a gene for flower color.
Theor. Appl. Genet. 533157-167

 

Bingham, P.M. (1981): A novel dominant mutant allele at the
white locus of Drosophila melanogaster is mutable. Cold
Spring Harbor Symp, Quant. Biol. 553519-525

 

Brink, R.A. (1973): Paramutation. Ann. Rev. Genet. 23129-152

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SUMMARY AND CONCLUSIONS

SUMMARY AND CONCLUSIONS

1.5Z Meicelase-P was found to release maximum yields of P.
parodii wild-type leaf mesophyll protoplasts at 25i2°C after 16
hours in conjunction with 0.15Z Macerase, 9Z mannitol, 40 ug/ml
ampicillin, 10 ug/ml tetracycline, and 10 ug/ml gentamicin in CPW
salts at pH 5.8. A rapid 2 hour isolation procedure was designed
which released adequate yields of_P. parodii (wt-m) protoplasts
using 0.5Z Driselase, 0.52 Macerase, 9Z mannitol, 40 ug/ml
ampicillin, 10 pg/ml tetracycline, 10 ug/ml gentamicin, and 0.5Z
postasium dextran sulfate in CPW salts at pH 5.8. While Cellulase
"Onozuka” R10 released adequate numbers of.P. parodii (wt-m)
protoplasts in conjunction with Driselase and Macerase, it had an
inhibitory effect which was expressed as delayed or suppressed cell
division. ’

Immediate placement of freshly isolated_P. parodii (wt-m)
protoplasts under a photon fluence flux of 18-24 uEmfzs-l
(400-700 nm) at 25i2°C resulted in delayed cell division in
standard medium supporting P. parodii (wt-m) protoplast growth. A
24 hour dark period given prior to placement in the light hastened
the onset of cell division.

Reliable recovery of high yields of P. inflata cytoplasmic
determined chlorOphyll deficient protoplasts from liquid suspension
cultures were obtained using ZZ Cellulase "Onozuka" R10, lZ
Driselase, lZ Macerase, and 8Z mannitol in CPW salts at pH 5.8.
Lower concentrations of Cellulase "Onozuka" R10 concentrations

resulted in inadequate cell wall degradation, while higher

153

154
concentrations did not provide further increases in protoplast
yield and resulted in lower protoplast viability.

Growth dynamics of_P. inflata cells in liquid suspension
culture expressed by compacted cell volume and number of
aggregates/ml showed a linear increase.over time. Protoplast yield
was not dependent on the compacted cell volume or the number of
aggregates/ml. Maximum yields of 25.8 x 106 protoplasts/ml were
obtained by the 7th day in culture.

Prot0plast isolation from ip;yip£p_shoot cultures of P,
parodii and P. inflata nuclear and cytoplasmic determined
chlorophyll deficient mutants resulted in low yields and impaired
protoplast viability when performed using O-3Z Extractase, 2.52
Driselase, and 4Z mannitol in CPW salts at pH 5.8. Nuclear
chlorophyll deficient mutants of both P. parodii and P. inflata
were not affected by any Extractase concentration.

Isolation of protoplasts from_P. inflata cytoplasmic
determined chlorophyll deficient callus cultures was optimal using
lZ Cellulase "Onozuka" R10, lZ Driselase, lZ Macerase, and 7Z
mannitol in CPW salts at pH 5.8. Yields obtained using callus
cultures were inferior to those obtained from liquid suspension
cultures.

Inheritance of chlorophyll deficiency in_P. parodii line PD
IR-4O and_§. inflata line INF IR-61 obtained after gamma irradiation
treatments gave a good fit to a 3:1 ratio expected for the
segregation of a single recessive gene. Petunia parodii line
P-1043 approximated a 4:1 ratio which may reflect the interaction
of lethal modifiers in producing the recessive phenotype. P-1043

and PD IR-40 were determined to be distinct nuclear mutations in P.

155

_pgrodii. Petunia parviflora line P1048 gave a good fit to a 15:1

 

 

ratio (P=0.01), indicating the recessive phenotype was conditioned
by two interacting genes.

Fusion of_P. parodii (wt-m) with_P. inflata (ca-susp) using
calcium-high pH resulted in the selection of 155 green calli based
on complementation to chlorophyll synthesis in the hybrids and
restricted growth of the green-P. parodii parent. Fusion success
rate was 31Z. Fourteen point four percent of the green, putative
hybrid calli regenerated into plants, and 5.7Z proved to be somatic
hybrids upon flowering.

At a fusion rate of 0.0049Z for experiment P80-1, selection
efficiency was 14.282. The frequency of somatic hybrid plant
production was 5.3 plants per 106 parental protoplasts used.
Parental regeneration resulted in a 10.2Z reversion rate.

Somatic hybrid plants displayed various degrees of aberrant
phenotypes including variation in leaf shape and size, reproductive
characteristics, and leaf and corolla variegation. Chromsome
number varied from 20-36 (4p=28). Pollen viability ranged from
complete sterility in one plant, and from a low of 16.5Z to a high
of 71.7Z in other hybrid plants.

Developmental anomalies observed in somatic hybrid plants
included double styles and ovaries, fasciated bipartate styles,
acyanic petaloid structures on corolla limbs, and ear-shaped
projections at corolla lobe junctions.

Anthocyanin variegation in corolla limbs could be separated
into 5 classes based on the size and color of flecks and sectors.
Twin spotting was encountered in two somatic hybrids. Segregation

ratios in self, sib-mate, and testcross progeny suggested

156
variegation was the result of instability in two independent
anthocyanin alleles. Such disruptions in anthocyanin biosynthesis
in other plant species have been attributed to the insertion of
segments of DNA at or near the structural locus resulting in
suppressed gene activity.

Corolla splitting and floristic characters observed in one
somatic hybrid followed a significant linear progression from the
narrow split blossom to ”restored" phenotypes. Corolla splitting
may result from chromosomal-cytoplasmic incompatibility upon the
elimination of parental chromosomes in a hybrid cytoplasm.

The high alkaline and temperature conditions used during the
fusion process are conducive to DNA strand dissociation and
breakage. Strands may have become mismatched after ligation and
reannealing, which may relate to the induction of instability
observed in the anthocyanin alleles. An alternative hypothesis of
chromosomal loss in somatic cells resulting in sectors of
hemizygous cells is also a viable alternative to explain
anthocyanin instability. Instability could also result from hybrid
dysgenesis due to the inherent incompatibiity of this interspecific

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"‘111mm