LIBRARY
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University

 

 

 

This is to certify that the

dissertation entitled

Crossability studies & the isolation, culture
and regeneration of protoplasts of Petunia
alpicola

presented by

Jane Laverne Ford-Logan

has been accepted towards fulfillment
of the requirements for

PhD. degree in Horticulture

1 Major professor

Date March 12i1987

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

 

 

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GOSSABIIJTY STUDIES am: ISOLATION. CULTURE AND MANOR
a" ma‘mmammu

By
Jane Laverne Ford-Inga

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

WGPHIIDSOPBY

Department of Horticulture
1987

ABSTRACI’

CROSSABILITY STUDIES um: ISOLATION. CULTURE AND MANOR
I!" W W mm

By
Jane Laverne Ford-Logan

In order to expand the diversity of germplasm available for Improvement of
petunia. Maia “£15.91! was investigated for its use as a potential genetic resource
for introgression into the cultivated 2. mm Iiort This study was conducted to
determine the breeding behavior of E. alpicglnrith selected Mapscies. to
determine the stageIs) where failure occurs in the reproductive cycle between crosses
of 2. m and other m species. and to develop a procedure for the isolation.
culture and regeneration of plants of this species from protoplasts.

Self-pollination of different 2. shim]; plants and reciprocal cross-pollination
of 2. mm with 2. mm and 2. Mwere performed under greenhouse
conditions to assess self-compatibility and crossability relationships. The degree of
crosaability among the species was determined by the number of seeds set. seed
germination and the number of successful intercrosses between the species. A
fluorescence technique was used to monitor growth of pollen tubes in each of these
self- and cross-pollination. 2. Was found to be self-incompatible and all
interspecific cromes failed to produce hybrids. the failures being caused by pre-
and/or post-zygotic incompatibility.

Precamu'es were developed for the regeneration to plants of 2. mm from
callus and suspension culture protoplaats. Protoplasts were released from plasmolyzed
cells in a defined enzyme mixture. plated in liquid culture medium and plating
efficiency was determined. Growth of macroscopic colonies was enhanced by plating
cells between layers of semi-solid agar. 0n transfer of protoplast-derived calli to

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JANE LAVERNE HID-WAN

regeneration medium. numerous adventitious shoots were formed from which rooted
phntlets were regenerated.

-' incl 4! lliliél (1 'io‘ no:

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To My Mother and Oscar.

for their never-ending love. support and encouragement

ACKNO'LWENTS

I wish to express my sincere appreciation to I. C. Sink. maior professor. and to
members of my guidance committee. If. I. Adams. J. F. Hancock. J. I. Hanover. S.
lionma and D. E. Keathley for critically reviewing this dissertation.

i am indebted to the Michigan State University Competitive Doctoral Fellowship
Program which made all of this possible.

Thanks are given to all the very special friends I made while attending USU. who
gave continuous support and encouragement during the completion of the require-
ments for this degree. and finally ------ to my family who have made it all worthwhile.

--i ism.

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TABLEG‘CON'IINTS

us'ra'rm.......nun.....................
Lma'ncum.................................
may...”.............................
mamm.............................

11.

Ill.

MGIN. TAXONOIY ANDEVOLUI'ION (I MUNIA

1. main0.00IOOOOOOOIOCOOCO0.0.0.000...

2. Tmnomymdfivolufion eeeeeeeeeeeeeeeeeeeee
3. M'cncficsmmmeeeeeeeeeeeeeeeeee

INCOMPATIBILITY 1N MA

l. Incomp‘fibmty-Anml'vig'eeeeeeeeeeeeeeeeee
2. TheRoleofGlycoproteinsinIncompatibility . oo o o . o no .
3. TheCalloseResponseinIncompatibilitynone-nou-
4. Self-Incompatibilityinm eeeee’eeeeeeeeeeee
5. lnterspecificCross-Incompatibilityinm - . . . . . . . .

PROI'OPLAST ISOLATION. CULTURE AND REGENERATION

LProlopMImhfioneeeeeeeeeeeeeeeeeeeeeeee
2.Prompmclglmneeeeeeeaeeeeeeeeeeeeeeeee
3.Mlh‘cnenfioneeeeeeeeeeeeeeeeeeeeeeee
«l. RegenerationofEeflnigmeProtoplastso-Hon-no-

WWW

. Fusionofprotopm eeeeeeeeeeeeeeeeeeeeeee

ProtoplastsforStudiesofCellOrganelles - . . . . . . . . . . . .

EnuclafionofpmmpMeeeeeeeeeeeeeeeeeeeee

Plutpmtoplmrmsformon eeeeeeeeeeeeeeeee

MethodsofPlantProtoplast‘l‘ransformation - o . . . . . o . . .
infection (Co-cultivation of Protoplast-Derived Cells with
W eeeeeeeeeeeeeeeeeeeeeeeee
Chemically Stimuli“ Uptake of isolated DNA into Pmtopllsts
Fusion of Bacterial Spheroplasts with Plant Protoplasts- . o .
iiposome—EncapwiatedDeliveryofDNA - o o - . . . . . . . .

99»—

iv

Page
vi

vii

“WW

3:090

16
19

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srcnon I
mwuwmflwflmmmuflmul

AWeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
Introduction eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
Iaterialsanduethods
mm‘yfludiu eeeeeeeeeeeeeeeeeeeeeeeeeeeee
ucctmphoroflchybfidwnfific'fion eeeeeeeeeeeeeeeeeee
mumdmcuaioneeeeeeeeeeeeeeeeeeeeeeeeeeeeee
MflaMeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

SEIION 11

WNW. CULTURE AND PLANT WTION ll" PRUIOPLASTS
wnmmuummu.

suMOIOOOIOIOOOOOOOIOOOOIIIOOOCIOIOOOIOOO
Inmcuon0.0.0.0000...OOOOIOOIOOOIOOIOOOICOO

“rialsmdugthodseeeeeeeeeeeeeeeeeeeeeeeeeeeeee
Mlumdnhcmneeeeeeeeeeeeeeeeeeeeeeeeeeeeee

Raf°nncu.......0.00.0.0...OIOOOOIOOIOOOOOOOO

SWYmmNCLUSsteeeeeeeeeeeeeeeeeeeeeeeeee
”51mmeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

33
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Table

LISTG‘TADLES

REVIEW (E ”MATURE

. Procedures for culture and plant regeneration from protoplasts of Petunia

mm...’..............OOOOOCOOIOOOOOOOOOO

SETUONI

. Self- and reciprocal-crossability of 2. “pm with 2. mm and with

B,Meeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

Pollen-tube growth a hr alter self- and reciprocal cross-pollination of E.
M'nhn.mmd'ilhn.Meeeeeeeeeeeeeee

MON 11

. Mediausedinfi. MpmtopMculture and shootregeneration . . . .

vi

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LIST w FIGURES

Figure
SECIION I

l. Phntsandflowersofmalnimh.2.mmmandflhMcv.
“Min,“0.0...O'COOOCOOOOOOOOOOOOCCOCC

2. liaiate dehydrogenase electrophoretic pattern from 21m glpimlg. 2.
Mineral! mane will inhalant!!! mums-""-

3. IDEzymogramfromleafextractsofbfiemjgspecies - 0 o o o o o o o -

SMION ll
1. Division and formation of plants from protoplasts of 2. We . . . .

vii

35

:k

IO...

 

Guihnce Committee:

This dissertation is organized in journal style format in accordance with the
Department of Horticulture and Michigan State University requirements. Two papers
were prepared following the journal style formats of Wm and journal Elm
mm.

viii

 

 

 

!

INTRODUCTION

In 1803 A. I... Jussieu founded the genus 21mm to describe plants which he col-
lectedon the banksofthe PlataRiver. Be delineatedm Wand 2.
mm butthe formerspecies hadalready been decribedasmmtign Why
Lamarck in 1793. Itwas not until 1888 that the earliest trivial name. AIM was
associated with the generic name “when Britten, Sterns and Poggenburg listed
m “M (Lam) in their Preliminary Catalogue of Anthophyta and Pterido-
phyta At present there are approximately 30 recognised species of 21m (Sink.
1%). They are indigenous to Central and South America and extend north into
southern parts of the United States.

The precise genetic background of the cultivated m am has not been
established. although 2. Mend 2. 21911911 are considered to be progenitors
(Ferguson and Ottley. 1932; Gleason and Cronquist. 1963). Steers (1930). based on
cytological studies. suggested that 2. m as well as 2. m and 2. 1m have
contributed to the development of 2. m.

The species investigated in this research. 2. mm has generated interest since
it is the second “species now known to have a Zn - Zx - l8 chromosome number.
The other one is 2. m (Ferguson and Coolidge. 1932). Except for these two
species. the diploid chromosome number of all other species and cultivated types
reportedtodateiqu-Zg- 14.

The phylogenetic origin of 2. Wis unknown. although morphological ob-
servations suggest its closest affinity may be to 2. minors. in addition to sharing
the same chromosome number (211 - 18). they both have a prostrate or creeping

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2

growth habit. short-petioled succulent leaves and small magenta flavors. All of these
features are in distinct contrast to the Zn - 14 “species.

Since the first hybrid'mation of W species in the early lM‘s to create P_. u-
m Hort. there has been no further improvement in it based on wild species germ-
plasm. The bedding plant inustry. to which petunias are of considerable economic
importance. is presently experiencing a decrease in sales primarily due to increased
sales of other competing species. It is felt that improvement in botrytis "finance.
floral featum. growth forms and cultural management could renew the commercial
demand for petunias. 2. mm is a species which could serve as a potential germ-
plasm "source for these traits. but it is sexually incompatible with the cultivated
petunia (Sink and Power. l977). Thus. integration of desirable genes into 2. man
may require using novel tissue culture techniques such as protoplast fusion (Sink.
1980).

An objective of somatic hybridization is to combine species that exhibit incongru-
ity at the interspecific or intergeneric level in order to expand the diversity of germ-
plasm available for crop improvement. Based on research to date. it could be expected
that 2. glpimlg is also a potential genetic resource for introgresaion into 2. mm.
A prerequisite for somatic hybrid'mation would be to evaluate the type of incongruity
that exists between two potential species by identifying the stage where reproductive
failure occurs. and the methodology for regenerating plants from the protoplasts of at
least one of the species to be used in cell fusion. Thus. this investigation was con-
ducted to: l) determine the breeding behavior of 2. Wwith selected 21mm
species. 2) determine the stagels) where failure occurs in the reproductive cycle of
crosses between 2. aloicola and other m species. and 3) develop a procedure for
the isolation. culture and regeneration of plants of this species from protoplasts.

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mmumrm

Before Darwin called attention to the discontinuous intraspecific variation charac-
teristic of cultivated plants. taxonomists of the period often elevated these variants to
the rank of species. With the rediscovery of Mendelian genetics. many of these
species were correctly evaluated as lines differing in only one or a few major genes.
The development of genecology focused attention on the process of speciation. partic-
ularly the role of reprothctive isolation in the differentiation of species. This led to
the recognition of fewer. but more variable. species.

1. C161]. Tm mflflufllfll C mu

m: The species mm mm (Lam) BSD. and 2. MUM“. are con-
sidered to be the progenitors of the cultivated W Will Hort. According to
Ferguson and Ottley (I932). seeds of these two species were transported from their
indigenous South American countries to European countries about 1820-1830; subse-
quently. hybridhation between these species prohced the germplasm base for all
further breeding and selection. By 1937 a number of ornamental strains had been
developed. lather (1943) related a simihr occurrence in accounting for the origin of
2. man.

W: The early literature concerning the taxonomic status
and nomenclature of 2. m and 2. m is confusing. Early taxonomic
workers on the genus used 2. m to describe much of the plant material under
mudy. including both species typesand cultivated types. Even today there exists an
unsettled taxonomic status with respect to 2. “and 2. m For many years.
2. mwureferredtoasn. WM. asliruillustrated bySims (1825).
although earlier it had been described by Lamarck (1793) as mm m. There

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4
is also the possibility that two other species. 2. m and 2. Mi, could have

entered into the evolution of the cultivated types. Steere (1930) described 2. M as
similar to 2. Mwith the exception of a longer corolla tube. smaller limb. pointed
lobes. and deep purple veining of the throat. Their similarities may have caused them
to he mislabeled due to both having awhite corolla. The characteristic long corolla
tube of 2. “Mil should have resolved the identity confusion because it would have
been transmitted to the offspring. as shown by Sink (lfll). By reason of the fact that
this obvious trait was not indicated in early figures describing 2. Wand the fact
that 2. Wm not taxonomically classified until 1930 by Steere. argues against it
playing an early role in the evolution of cultivated types (Sink. lfll). Using floristic
(hts plus flower color requirements and breeding behavior. Sink (lQl) excluded 2.
m as a possible ancestor.

M m which was described by Fries (1911). is quite similar morphologi-
cally to 2. 1m except for minor differences in corolla characteristics: it may
easily have been mistaken for the latter. Smith and Downs (1966) combined both 2.
mm and 2. in“ under 2. W (Book) Schinz and ‘l'ellung var. M
m. After comparing the taxonomic delineating characters of 2. m E. m
and 2. m Lamprecht (1933) considered them all to be 2. mm. His decision
was based on an evaluation of morphological characters as well as the genetics of
flower color which was determined by lather and Edwardes 0943).

Based on cytological studies. Steers (1930) reported that E. me be a
composite of the three species 2. m 2. Wand 2. 1m. Natarella and
Sink (1974). using thin-layer chromatographic analyses of phenolic leaf extracts.
concluded that 2. m and 2. mugwere most likely the immediate ancestors of
2. m. In contrast. analyses by electrophoresis (Natarella and Sink. 1975) sug-
gested that 2. m may have been a progenitor of 2. hybrid;

,
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5

W: The basic chromosome number in W is x - 7.
The diploid chromosome number for all m species and cultivated types reported to
date is 211 - 21 - 14. with the exception of 2. mm“. (Ferguson and Coolidge. 1932)
having Zn -2; -18. Chromosome counts ofE. aim (Zn - 14) were made by Steere
(1932). Ferguson and Coolidge (1932). and Sullivan (1947); on 2. M (211 - 14) by
Steere (1932) and Sullivan (1947); and on 2. 2m (2n - 14) by Koatoff and Kendall
(1931 ). Ferguson and Coolidge (1932). and Sullivan (1947).

Steere (1932) determined that chromosome pairing was synaptic in meiosis of 2.
M and likewise. Kostoff and Kendall (1931) found normal pairing of the 23 - Zx -
14 2. m chromosomes and about 2% abortive pollen. Dermen (1931) indicated a
Zn - 2; - 14 chromosome number for 2. Wand observed very loose pairing of the
chromosomes at diakinssia. so much so that some pairs showed almost no connection
between members. Rick (1971) found every posdble aneuploid in the progeny of a 31 x
2; 2. m cross; the plants were comparable in viability and had phenotypic mor-
phology almost identical to diploida. This observation was interpreted as an indication
of the large degree of genetic redundancy and tolerance to aneuploidy which exists in
2mm.

Skalinska and Ouchtma (1927) studied a number ofvarieties of m and report-
ed differences in chromosome size in different varieties. Halinowski (1928) also
reported chromosome size differences in a strain of variegated W and found on
average the chromosomes of hrge purple flowers were hrger than those of small lilac
ones. Contrary to this. Darmen (1931) found no significant dilference in size of chro-
mosomes between the small flowered and large flowered diploida that were used in
connection with his work on polyploidy in m

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6
ll. IICOIPATIIILITY 1' MIA

WM: Incompatibility m be defined u the failure.
following self- or cross-pollination. of a male and female gamete to achieve fertiliza-

tion. whereas each of them is capable of uniting with other gametes of the bmeding
group after similar mating or pollination. Pandey (1960) indicated that self-incom-
patibility has been found in 78 angiosperm families and occurs in every major
phylogenetic line (East. 1940: 1" ryxell, 1937; Brewbaker, 1957). Pandey has provided
excellent review articles on the evolution and description of self-incompatibility
(Pandey. 1960; Pandey. 1968; Pandey. 1977).

The operation of self-in compatibility centers on the fact that pollen that is incom-
patible with the ater tissue will either not germinate on the style or will produce very
slow pollen tuba growth.

Incompatibility. or compatibility, is dictated by a genetic system operating in
both the male and female. The functionality of the system is based on a physiological
interaction between the pollen (la) and the style (2;). A multiple allelic series.
designated 8. governs the system (Brewbaker. 1957). Traditionally. a single locus has
been proposed to control the mechanism. while Pandey (1977) indicated the possible
involvement of two loci in some species.

Incompatibility can be expressed in one of two systems. either gametophytic or
sporophytic. In each of these systems there are variations. particularly with regard
to the number of S loci. and the interactive relationship between the alleles at the
same or different loci (Pandey. 1937). The gametophytic system was proposed by Prell
(1921) with the first supporting data obtained by East and langelsdorf (1925. 1926) in
Whybrids and by Lehman (1926) in m. incompatibility reunite when a
pollen grain and the stigma have an allele in common. Thus. the incompatibility is
determined gametophytically by the particular allele in the pollen grain. Dominance

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7
among gametophytic S alleles cannot occur in the haploid pollen and is not known to

occur in the diploid style (Brewhaker. 1957). In contrast. 8 allele interactions do
occur in the heterogenic diploid pollen grain in autotetraploids (Brewbaker. 1957).
One of these is the competition interaction in which neither allele is fully active. with
the result thatsuch grains are not inhibited or are only partially inhibited and pro-
gress through pistillate tissue (llaheshwari. 1949). The second interaction observed
in tetraploids is that of dominance. in which one allele partially or completely sup-
presses the action of the other allele in the heterogenic pollen (Brewbaker. 1957).

The sporophytic system was first described with supporting data by Gerstel (1930).
The system contains a form of dominance in which 81 is dominant over all other alleles.
$2 is dominant over all but 51. and so on. In microsporogenesis all pollen. regardless
of genotype. retains the phenotypic response of the dominant allele in the male diploid
time (Brewbaker. 1957). There is also some evidence for dominance in the pistil of
plants having the sporophytic system (Brewbaher. 1957).

The difference between the gametophytic and sporophytic mechanisms is sug-
geatedtobebaeedon thetimeatwhichSallelesacttoproduceincompatibility
subdances or their precursors which hter change into incompatibility substances
(Lewis. 1936: Pandey. I938). Pandey (1958) mtggemed that the time of the 5 allele
action in the sporophytic system is after anaphase II in the pollen mother cell, before
the separation of the four microspores from the common cytoplasm. The specific
substances in the sporophytic system are already present in the cytoplasmic material
which forms the microapore wall. These substances produce the incompatibility
reaction on contact of the pollen grains with the incompatible stigma; thus. inhibi-
ting pollen germination (Pandey. 1960). In the gametophytic symem the time of 8
allele action is after cytokinesis. Therefore. the specific substances are prouced
internally within each microapore whose wall h free of specific substances. In
gametophytic species. pollen grains germinate and penetrate the incompatible style.

a .‘a

v . ’ 1
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8
but the growth of pollen tubes is stopped alter the specific substances of the pollen

tube and the corresponding substances of the style are brought together through
dimtsion or other procems (Pandey. 1960).

W: In the cmcifor Mn. the self-
incompatibility reaction is localized at the stigma surface. and occurs within minutes
alter the initial contact between the pollen and the papillar cells on the outer surface
of the stigma. In this genus. self-incompatibility is under the control of s single
genetic locus. the S locus. which is highly polymorphic. some 50 alleles having been
identified.

A molecular analysis of the genetic control of incompatibility may be performed

by detecting antigens specific to various S-locus alleles in stigma homogenates from
dill‘erent m strains (Nasrallah and Wallace. 1967). These antigens have been
shown to correspond to glycoproteins that may be resolved in various electrophoretic
systems (Nasrallah et al.. 1970; Nasrallah et al.. 1972; Nishio and flinats. 1977:
Nasrallah and Nasrallah. 1%). Several lines of evidence suggest that these glyco-
proteins play an important role in incompatibility. (l) The mobilities of these mole-
cules vary in stigma extracts derived from Mural” with dilTerent S-locus
alleles (Nam-allah and Nasrallah. 1%). (2) These molecules are found in the stigma
but not in stylar or seedling time (Nam-allah et al.. 1985a). (3) The increased rate of
synthesis of these S-locus-specil‘ic glycoproteins (SLSGs) in the developing stigma
correlates with the onset of the incompatibility reaction in the stigma (Nasrallah et al..
lflfia). (4) Mutations in genes unlinked to the S locus which remit in self-compati-
bility are also associated with reduced levels of these molecules (Nasrallah. I974).
(5) The inheritance of the various forms of $1.56 correlates with the segregation ol‘ 5
alleles in genetic crosses. indicating that the gene responsible for this polymorphism
must be genetically located at or closely linked to the S locus (Nasrallah et al.. 1972).

Nasrallah et al. (1%)” reported the isolation of a complementary DNA clone

. 1-'l- ',-a :IJ". -) unit 0. ~' .I,,‘ g ., .)

, a -'.
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9
containing sequences encoding an S-locus-specific glycoprotein from m

Win which they show that the spatial and temporal distribution of the mess-
enger RNA homologous to these sequences mirrors the appearance of the S—locus-
specific glycoprotein. Several fragments of the a. 9m genomic DNA. generaud
by reariction endonucleases. hybridize with the 5156 cDNA clone: polymorphisms in
certain of these fragments segregate precisely with alleles of the S locus.

111W: Evidonco that the callose moon» my
be used as an indicator of biocommunication between pollen and stigma. and a diag-
nostic tool for rapid assessment of the nature of a pollination in the biotechnology of
seed production has been reviewed by Dumas and Knox 0983). Callose. a cell wall
polysaccharids composed generally of 1.3 B-glucans. which can be localized by the
decoloriasd analine blue fluorescence (AH?) method (Currier. 1957; Linskens and
Esser. 1957 ). provides a useful phenotypic bioassay: to determine the site of rejection
of pollen tubes in incompatibility phenomena (Linskens and Esser. 1937): for esti-
mates of the dynamics of pollen tube growth in gametophytic competition (Hulcahy.
1973); and in Mg the viability of the pistil in determining the elfective
pollination period (Anvari and Stosser. 1978).

Callose. in appearance. is uncoloured and gelatinuous. amorphous and isotrophic
(Berth et al.. 1974). and is characterized by its solubility properties. The chemical
namieofcallosshasbeendiscussedbyaarke andStone (1%3). Calloseproducedin
pollen tubes of rye alter self-pollination proved to comprise a mixture of 1.3 and M B-
linhed glucans in the proportion of 9:77 (Vithanage et al.. 1%). Reynolds and Dashek
0976) found that lily pollen tube callose stained with the A! method. but not fol-
lowing protease treat-ism. suggesting the callose may be a glycoprotein. Dickinson
and Lewis (1973) could not detect any protein-staining of callose in stigmas of
Manna.

in several differentiation programmes in plant tissues. callose is rapidly

1!.

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10
synthem. especially after wounding and during plant host/ parasite interactions.

particularly during pollination (Aist. 1976; Beslop-Barrison. 1978). The callose

response (hiring pollination may be highly specific. occurring in stigma cells in

contact with incompatible. but not compatible pollen in genera such as m

M m and thich have well-developed sporophytic self-

incompatibility systems (do Nettancourt. 1977). Wall-held pollen proteins elicit the

response (Mop-Harrison et al.. 1974. 1973) and its specificity has been explored
using cell surface probes (Kerhoas et al.. 1983). Sood et a1. (1982) found that the
response may be induced not only by pollen grains. but by macerates of somatic
times.

There are numerous hypotheses on the role of callose since it is so strategi-

cally sited at the pollen-stigma interface.

- that it prevents tissue dehydration through control of cell wall equilibrium by the
intervention of calcium and potassium ions. Calcium ions block water molecules
on the surface of callose: potassium ions liberate these water molecules (Vithan-
age at al.. 1%0).

- it mobilizes reserve carbohydrate. according to the transitory nature of callose
deposits (Currier. 1937).

- thatittakespartin defense reactions. Callose playsboth an active and passive
role in incompatibility: is related to stress responses. both trauma and envi-
ronment (Vithanage and Knox. 1977; Aist. 1976: Lewis. 130). by isolating or
sealing pollen from the stigma (Mp-Harrison. 1975; de Nettancourt. 1977).

- it has a trophic role. Calloae formation utilizes substrate that would otherwise be
avaihble for tube growth (Sedgley. 1977).

- aphysiological role in pollen tube growth: fixingrowth activated by 1.3 B-
glucanases (Reynolds and Dashek. 1976). These enzymes may act during growth
1111!! to maintain tip growth through control of balance of wall-synthesizing

“'UJ

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1 1
and degrading enzymes. Callose accumulation in incompatible tubes could be due

to a change in balance.

W: The self-incompatibility reaction in Maia is
gametophytically controlled by one locus with a series of S alleles (Lewis. 1944). All 2.
W accessions studied to date exhibit self-incompatibility. and sib-matings are
required for seed production. Only on rare occasions have seedling derived plants
been found that set a very low quantity of seed from a few self-pollinmd flowers (Sink.
1%1). Physiological smdies of self-incompatibility in 2. m have been conducted
by Brewbaker and liaiumder (1961).

Both 2. m and 2. W are self-fertile and fruit abundantly. both in the
greenhouse and in open culture (Ferguson and Ottley. 1932). in general. more than
9) percent of the flowers form large capsules. A given capsule may contain from :00
to 1G!) or more seeds. 2. mm“, another self-compatible species. produces
approximately one-hundred seeds per capsule which are naller but otherwise similar
to the seeds of 2. m (Ferguson and Ottley. 1932). B. Macedon; while
not readily pmthtcing the self-seed quantity per capsule or per plant as 2. m and
2. m3. does set seed following self-pollination (Sink and Power. 1978).

2. m exhibits a functional self-incompatible system when selfed. but
Flaschenriem and Ascher (1979) found plants which produced varying amounts of seed
when used as the seed parent in crosses with unrelated individlals homozygous for the
same 5 allele. This phenomenon has been termed pseudo-self-compatibility (P512) and
is attributed to the action of non-allelic genes which affect normal S-gene activity and
result in self-seed (lather. 1943). Takahashi (1973) found the P50 in 2. m to be
the result of a stylar reaction which resulted in faster pollen tube growth in styles of
phnts which expressed higher levels of P86 and also to the increased vitality of some
pollen.

4

lilfit‘

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12

Wain: 2mm axillatis. 2- inflate. 2.
m and 2. :19“ have all been readily intercrossed with 2. hybrid; (Sink.
1975). Intercrosees among these selected m species have been successful using
standard pollination techniques with the exception of the cross between 2. m1 and
2. m (Sink et al.. 1978). Small quantities of hybrid seed were obtained by bud-
pollination of 2. “it although the reciprocal cross failed. it was later shown that
these two species exhibit a unilateral cross-incompatibility with e pro—zygotic mode of
nprothnctive isolation preventing hybrid‘netions with B. inflate as the maternal
parent (Sink et al.. 1978). The interspecific hybrih obtained from all of these crosses
set abundant seed by self-fertilization and backcroseed readily with each of the
parental species (Sink. 1975).

Because only the 2. M by 2. Whybrid‘mtion failed using standard pol-
lination techniques and fertile Fl offspring can be obtained which cross easily with
each parental species. a high degree of genetic homology between both species is
indicated (Sink. 1975). It is suspected that a minor portion of the chromosome comple-
ment of these two species is responsible for the reproductive isolation.

Sink and Power (1978) reported reproductive isolation between 2. minor; and
the four previously mentioned m species plus 2. mm cv. Comanche. using
standard and bud-pollination procedures. Reciprocal attempts at the crosses were also
not succemful. The incongruity of 2. Wwith the 5 m species was
established by the failure of approximately 1M pollinations. A later study showed
thatthefraction 1 protein patternsoffi. miflpndiffersin havingesingle small
subunit polypeptide located between the two polypeptides found in the other species
and cultivars (Gatenby and Cocking. I977b). The small subunit composition may
represent the point of divergence of 2. min“: from the other petunias. This lends
supportto the theory thatalthough 2. mend 2. Wm” have given rise to
the fourteen chromosome petunias by allopolyploidy. they were probably not

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13
immediately related to 2. minor; or its progenitor. This is further suppomd by the
diminct growth habit. plant and flower morphology of 2. mimic! (2n - 21 - 18)
compared to the aforementioned petunias (Ferguson and Ottley. 1932) and its record of
no intercrossing (Sink and Power. 1978). Subsequently. Power et al. (1980) reported
the prohction of somatic hybrid plants between 2. m and 2. mm in an
attempt to affect morphological change in ornamental petunias by the transfer of the
different growth habit of minor; to 2. m.

11]. m 15011110., cums}: m "AUDI

Plant protoplasts are routinely isolated through the use of cell wall degrading
enzymes. With the appropriate enzyme treatment it is possible to isolate protoplasts
from virtually any plant species or any type of plant time. However. the ability to
isolate protoplasts capable of sustained cell division with subsequent callus or plant
regeneration is limited to a small. but increasing. list of plant species.

W: Protoplasts were first isolated using mechanical methods
(Klercker. 1892). In mod cases. the yield was small. and only large and highly
vacuolated cells could be used for isolation. The use of cell wall degrading enzymes
(Cocking. 1960) was soon recogn'med as the preferred method to release large numbers
of uniform plant protoplasts.

Enzymes for protoplast isolation are dissolved in an osmoticum which usually
conaims of a sugar such as glucose or sucrose or a sugar alcohol such as mannitol or
sorbitol. Isnnitol and sorbitol. separately or in combination. have been used most
ollen with mannitol preferred for the isolation of leaf mesophyll protoplasts. Glucose
has been used successflilly as an alternative to these hexitols for cultured cells (Kao and
Iichsyluk. 1974). In some cases mineral salts. particularly K01 and CsClz. are added

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14
to increase protoplast membrane stability (Gamborg et al.. 1975; Rose. 1980). Magne-

sium chloride has also had a positive effect on the release of stable protoplasts. The
effective osmotic concentration depends on the cell osmotic pressures at the time of
isolation. Endogenous cell osmotic pressures are influenced by environmental
conditions (Shepard and Tetten. 1975) and can be manipulated by dark pretreatment of
plants. use of young leaf tissue. etc. Agents such as potassium dextran sulfate (Takebe
et al.. I968; Passiatore and Sink. 1981) and polyamines (Galsten et al.. 1978) have been
added to counteract the effect of toxic substances which are present as contaminants or
released by the cells during protoplast isolation. Minimal enzyme concentrations are
used to obtain viable protoplasts. depending on factors such as enzyme type. protoplast
source. and incubation temperature. Enzyme preparations also exhibit specific pH and
temperature optima but these parameters must be adjusted to levels that are not
deleterious to the plant cells. The pH of the enzyme isolation solution has been varied.
usually between 5.4-62. It has been suggested that higher pH. 6.0-7.0. is most favor-
able to release mesophyll protoplasts of M (Pelcher et al.. 1974). However. a
lower pH. 5.8. has been used to release mesophyll protoplasts of Glycine, (Schwenk et
al.. lQl ). a closely related seed legume. In some cases buffering agents such as a
phosphate or MES lZ-(N-morpbelinochanesulfonic acid] are added for pli stabil’ma-
tion (lee and Iichayluk. 1975). These compounds minimize the shift to acidic pll that
may occur (baring protoplast isolation (Gamborg. 1976). Incubation temperatures of
20-27% are commonly employed but extremes such as 2°C (De LaRoche et al.. 1977) and
36°C (Othman and Paranietby. 1%0) have been used. The time required for isolation
can range from 30 min (Nagata and lshii. 1979) to 24 h (Kae et al.. 1974) depending on
protoplast source. enzymes. pH. and temperature. While the effect of light on
isolation of protoplasts has not been studied in detail. protoplasts are usually isolated in
the dark (Gill et al.. 1981). or in low-light intensity (Chellappan et al.. lull).

15
Pretoplasts may be isolated from awide range of tissues or cell types (Vasil and

Vssil. 1980). Leaf tissue and cell suspension cultures have been used as protoplast
sources in many studies because of their availability and the satisfactory yields that
can be obtained from them. Leaf protoplasts have been obtained by a two-step method
involving treatment with pectinase to release cells from the mesophyll tissue followed
by treatment with cellulase to convert the cells into protoplasts (Takebe et al.. 1968). A
single step system involving the use of mixed enzyme solutions is more frequently used
in protoplast isolation. Solutions of dilTerent enzyme combinations may be used in
sequence (Eartha et al.. 1974; Gamborg et al.. 1975) or the initial enzyme solution is
discarded along with cellular debris and dead protoplasts which are often released
during the early period of incubation (Gresshoff. 1980). Tissues derived directly from
plants generally require surface sterilhatien. although a procedure for obtaining
sterile protoplast preparations from non-sterile leaves has been described (Wilson et
al.. 1900). Leaf tissue can be mixed with the enzymes or floated on the surface of the
enzyme solution. in the case of suspension cultures. specific volumes of cells in liquid
medium are mixed with the enzymes or the medium is discarded alter centrifiagation
and replaced by the enzyme solution.

Pmcetharal modifications can facilitate protoplast isolation. These include peeling
the lower epidermal layer (Power et al.. 1976; Zapata et al.. 1977) or brushing the leaf
with carborundum to expose the mesophyll cells (Hughes et al.. 1978). slicing leaf
tissues into thin strips to facilitate enzyme entry (Chin and Scott. 1979). drawing the
enzyme into intercellular spaces through vacuum treatment (Chin and Scott. 1979) and
agitating the enzyme system (Chin and Scott. 1979).

Several factors or conditions influence the rate of release. final yield obtained.
and stability of the isolated protoplasts. The physiological condition of the donor tissue
prior to enzyme treatment as well as the isolation process are significant factors. The
growth conditions of the donor plants critically affect both yield and stability of leaf

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16
protoplasts. In many instsncos shoots or plantlots to bo uaod for protoplast isolation

havo boon grown asoptically mm in ordor to control growth paranotors noro
ol‘l‘octivoly (Dursnd. 1979; Schonk and Bollnan. 1979).

Following isolation. protoplast proparations must bo washod to ronovo tho hydro-
lytic onzynos. coll dobris and toxic products roloasod from tho donor tissuo. lost
purification procoduros includo polloting yin contrimgation followod by rosusponsion
in wash or culturo nodiun. ln sono audios. protoplasts hsvo boon washod by
flotation in concontratod osmotica such as nannitol (Gatonby and Cocking. 1977a).
aucroao (Shopnrd snd ‘l‘otton. 1977) or ficoll (Larkin. 1976). In addition. discontinuous
gradiont contrilugation and two phaso aoparation tochniquos hm boon vory ofl‘octivo
in ro-oving coll dobris and contaminating orgonollos (Pivowarczyk. 1979: Slabas ot
al.. 1%). Collulsr dobris has also boon ronovod by binding to an anti-galnctan-
sopharooo conjugato (Kollor snd Stono. 1978).

W: Following isolation and purification. protoplasts aro suspond—
od in nodiun for cumin. A minimal donsity in u» ordor of 1041.: is gonorally
roquirod l‘or culturing protoplasts. Viablo protoplasts will rospond by rogonorau'ng a
coll wall and undorgoing coll division (Vasil and Vasil. 180). Maximizing plating
omcioncy is an important goal in protoplast culturo. Many factors inl'luonco tho
viability and ultimato plating ol‘l'icioncy. Thoso includo tho physiological condition of
tho donor colls prior to protoplast isolation. tho procoduros uaod in tho iaohtion
procoas. tho conposition of tho culturo sodium. and tho onvironinontal conditions
ostablishod for culturo naintonanco.

‘l'ho composition of protoplast culturo nodia varioswith tho plant spocios studiod.
Dotailod doscriptions of tho conpononts ol' protoplast culturo nodia hsvo boon pub-
lishod (Go-borg. 19W; Eriksson. 1977). As tho nutritional roquiro-onts of culturod
plant colls and protoplasts aro yory similar. protoplast nodin aro usually modifications
of l'roquontly usod coll culturo nodis. Ganborg's BS (Ganborg ot sl.. 1968) and

I7
Murashigo and Shoog (I962) coll culturo modia aro most commonly usod as a basis for

protophst modia Altorations in thoso and othor coll culturo modia havo boon usod for
optimum growth of protoplasts.

It has boon proposod that concontrations of iron. zinc. and ammonium in tho
standard coll culturo modium may bo too high for somo protoplasts (Von Arnold and
Eriksson. 1977). Ammonium has boon found to bo dotrimontal to protoplast survival.
and modia hays boon dovisod for many spocios. such as tomato (Zapata ot al.. 1981).
that aro dsvoid of ammonium. Calcium concontration is incroasod 2-4 timos oyor tho
concontrations normally usod for coll culturos (Eriksson. I977).

'hilo glucoso may bo tho proforrod carbon sourco for most protoplasts (Gamborg.
I977). othor carbon sourcos. including sucroso. may bo proforrod or nocossary for
somo spocios. Uchimiya and Murashigo ( I976) havo shown that tobacco protoplasts
grow oqually woll on sucroso. collobioso. or glucoso. Most protoplast modia contain a
mixturo of carbon sourcos. For tomato. microso and glucoso aro misod in a 2:1 ratio
(Zapata ot al.. IQI). Kao and Michayluk (I974) showod that tho proforrod carbon
sourco (in this caso. glucoso) can also bo tho proforrod osmoticum. 0n tho othor hand.
in somo casos a nonmotaboliziblo osmoticum may bo nocossary. such as for poa moso-
phyll protoplasts whoro only mannitol and sorbitol could bo usod as osmotica (Von
Arnold and Eriksson. I977).

Humorous organic nutrionts hays boon addod to protoplast culturo modia. In most
casos. vitamin roquiromonts aro tho samo for plant colls and protoplasts. [so and
Michayluk (I974) hays suggostod that addition of sovoral vitamins. organic acids.
sugar. sugar alcohols. and undofinod nutrionts such as casamino acids and coconut
watsr for culturo of protoplasts in vory low donsitios. Moro olton than not. many of
thoso compononts aro unnocossary for culturo of protoplasts. as no bonofit can bo
attributod from thoir uso.

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18
Typos and concontrations of growth rogulators aro tho modia compononts that

havo boon variod most froquontly. Changos in growth rogulators havo boon shown to
havo dramatic offocts on culturod colls. Noarly all modia contain an auxin and for
somo spocios tho addition of a cytokinin may bo nocossary. 2.44) is tho growth rogula-
tor most commonly usod in protoplast modia: howovor. in somo spocios. othor growth
rogulators aro proforrod. l-‘or tobacco protoplasts. Uchimiya and Murashigo (I976)
obsorvod a highor rato of coll division in culturos with NAA than in culturos with 2.4-D
or 1AA. Also. in tobacco. cytokinin is unnocossary to induco coll division in culturod
protoplasts. Von Arnold and Eriksson (I977) roportod tho roquiromont for both auxin
(2.44)) and a cytokinin (Zip) to induco coll division in pan mosophyll protoplasts.

In somo instancos conditionod modium obtainod from coll susponsion culturos has
boon utilizod to supplomont protoplast culturo modia (Durand. 1979). Nurso tissuo
tochniquos including culturo on an undorlayor of irradiatod colls (Colla and Galun.
1980) or co-culturo with albino coils (Monml ot al.. 1978; Evans. I979) havo boon usod
to incroaso plating officioncy in low donsity culturos. Globa (1978) and Cabocho (I980)
woro also ablo to achiovo high plating officioncios in low donsity protoplast popula-
tions aftor an initial culturo poriod at high donsitios.

‘I‘ho physical aspocts of protoplast culturo can inl‘luonco plating ofi'icioncy and a
numbor of tochniquos for ostsblishing culturos havo thoroforo boon dovolopod. Proto-
plasts aro commonly suspondod in liquid modium and platod oithor as droplots or thin
layors in potri dishos. llicrodrop tochniquos havo boon dsvolopod to pormit tho
culturo of small numbors of protoplasts (Globa. 1978) and multiplo drop arrays havo
boon usod to tost largo numbors of modia modifications (Ilarms ot al.. 1979). Proto-
plastshavoalsoboon omboddodinagarandinsomocasossustsinoddivision couldonly
bo obtainod in solid modium (Gill ot al.. I979). Pipotting protoplast susponsions onto
filtor papor placod on agar modium has Iod to improvod plating officioncy in somo
spocios (Partanon. lfll ). Othor modifications havo includod transfor from liquid to

I9
agar modia altor short culturo poriods (Li ot al.. 1980) and uso of rosorvoir modia in

quadrant dishos (Bidnoy and Shopard. I980). Aftor succod'ul culturo ostsblishmont.
tho dividing colls roquiro tho addition of frosh modium. During such foodings tho
concontration of tho osmoticum is gonoraliy roducod in a aoquontial mannor.

W: Tho rogonoration of plants from protoplasts has boon
achiovod in a numbor of spocios with tho groatost auccoss obtainod with mombors of
tho Solanacoao. Thoso includo m spocios. 29mph spocios. and m
spocios. Unfortunatoly. ovon among tho Solanacoao whoro most offort on protoplast
rogonoration has boon diroctod. an oconomic food crop. W m
cannot bo officiontly rogonoratod from protoplasts. Tomato doos not soom to bo as
amonablo to protoplast rogonoration as othor solanacoous spocios (Niodz ot al.. 1%)).

Protoplasts isolatod from callus. coll msponsion. loaf. and flowor potsl havo all
boon rogonoratod. Most of tho mothods for protoplast rogonoration vary botwoon
spocios and donor tissuo. Rogonoration is gonorally achiovod through organogonosis
(Powor ot al.. 1976; Bourgin ot al.. 1979). although somatic ombryogonosis has boon
inducod in protoplasts of a fow spocios (Dudits at al.. 1976; Zapata and Sink. lfll).

Sovoral probloms romain unrosolvod in tho aroa of protoplast culturo. ono of
which is tho gonoral lack of auccoss in coroal protoplast culturo (Potrykus. I980). Coll
division has boon obsorvod in protoplast culturos of somo spocios. but plating officion-
cios havo gonorally romainod low and morphogonosis is still vory limitod. Logumo
protoplast culturos havo boon of limiud valuo for tho induction of morphogonosis. In
tho sood logumos such as poas and soyboans. protoplast-dorivod calli havo olton boon
obtainod (Gamborg at al.. 1975; Oolck ot al.. 133). but plants havo thus far not boon
consistontly rogonoratod. In tho caso of forago logumos succossos in plant rogon-
oration havo boon roportod for alfalfa (Dos Santos ot al.. 1980; Kao and lichayluk.
1”; Johnson at al.. I981) and whito clovor (Grosshoff. I980).

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20
Protoplast culturos of a numbor of spocios lack morphogonic capacity although

plants can bo rogonoratod from callus. coll susponsions. or culturod oxplants. ln
thoso spocios. plants havo proviously boon rogonoratod from tho samo tissuos utilizod
for protoplast isolation. It is not cloar whothor roducod morphogonic capacity is
rolatod to oxposuro to tho onzymos. disruption of tissuo organhation during protoplast
isolation or to irrovorsiblo offocts inducod by tho protoplast culturo conditions.

Tho list of spocios othor than solanacoous capablo of plant rogonoration from
protoplasts has boon stoadily oxpsnding to includo both monocots and dicots. and a
numbor of oconomically important crops. As this list incroasos. it is anticipatod that
tho uso of protoplasts in somatic hybridization and gonotic manipulation oxporimonts
will bo oxtondod to includo othor oconomically important crops such as tho logumos and
coroals.

WW: Bu- m! Point“! (1972) 0550”“ di-
vision of isolatod m m protoplasts and Potrykus and Durand inducod callus
formation in I972. In l973. Durand otal. rocovorod intact plants of potunia - thus
comploting tho ontiro soquonco from isolatod protoplasts to wholo plants. Sinco that
timo. within tho gonus m othor spocios and brooding linos havo boon found to
bo amonablo for rogonoration into plants from isolatod protoplasts (Binding and
Krumbogol-Schrooron. 1%4. Thoro aro sovoral roports of rogonoration in both
haploid (Binding. I974) and diploid (I-‘roarson ot al.. 1973; Vasil and Vasil. I974)
protoplast systoms of 2. m Hort. and for somo othor spocios of m 2.
mm (Powor ot al.. 1976). 2. m (Powor ot al.. l976). 2. Mi (Hayward and
Powor. 1973; Malt ot al.. IQI). 2. minim: (Sink and Powor. 1977) and 2.
mm (Powor ot al.. 1976).

All tho abovo citod roports. with tho oxcoption of 2. mm (Sin! and Powor.
I977). havo utilisod 21mm spocios posaossing an a - 7 haploid or 2a - 2; - l4 diploid
chromosomo numbor. 2. mm is a spocios documontod to havo a 2n - 2; - 18

 

 

14

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21
chromosomo numbor. Evon though tho m spocios studiod to dato all havo an x - 7

baso numbor. thoy havo variod with rospoct to protoplast isolation procoduros. compo-
sition of protoplast culturo modia. and shoot and root rogonoration modia (Tablo I).
This distinct variability in cultural roquiromonts indicatos that taxonomic difforoncos
aro rofloctod in mm culturo systoms.

IV. MOPLAST WOW!

W: Ono of tho most important usos of protoplast culturo is for
somatic hybridization. Somatic coll fusion loading to tho formation of viablo coll
hybrids has boon dovolopod primarily as a mothod for tho gonotic manipulation of
plant colls. This tochniquo onablos tho construction of hybrids botwoon taxonomically
distant plant spocios boyond tho limits of soxual crossability. and also croatos colls with
now gonotic. nucloar as woll as cytoplasmic. constitutions that othorwiso aro
unobtainablo. Tho oxporimontal mblishmont of now combinations of nucloi.
chloroplasts. and mitochondria providos a novol and potont tool to study tho gonotic
and physiological intoraction botwoon thoso organollos.

Tho spontanoous fusion of mochanically isolatod protoplasts was first obsorvod by
Kustor as oarly as 1909. Tho first inducod protoplast fusion was producod by Cooking
and collaborators using sodium nitrato as tho Ihsogon (Powor ot al.. I970). Bowovor.
tho officioncy of this tochniquo was found to bo low. During subsoquont soarchos for a
moro suitablo fusogon. troatmont with golatin (Kamoya. I973). concanavalin
(Bartmann ot al.. I973: Glimolius ot al.. I974). and dilToront salt solutions (Eriksson.
I971; Kamoya and Takahashi. I972) woro triod. Also. Kamoya (I975. I979. I982:
Kamoya ot al.. IQI) found that high molocular woight doxtrans in tho prosonco of
high concontrations of inorganic salts causo protoplast aggrogation and fusion. which
aro onhancod by NaOH or by oloctrical troatmont.

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24
Keller and Melchers (1973) introduced an effective fusion technique based on the

treatment of protoplasts with Caz‘ ions. The ability of Caz‘ to induce fusions could be
increased by incubating the mama in media containing c22* ions in high
temperature (37°C) and at the highly alkaline pH of 10.3.

Another very successful and more popular method for the fusion of protoplasts
was developed by [so and associates (Kao and Michayluk. 1974; Constabel and Kao.
1974) and by 'allin u a. (1974); also based on the use of c.” ions but with ioni-
concentrations This method involves the agglutination of protoplasts with the aid of
high molecular weight (1") polyethylene glycol (PEG. 1" cm 60%). Protoplasts
treated with PEG solutions containing Caz’ fuse during the elution and/or dilution of
PEG in the presence of. or by eluting with solutions containing high Caz’ at high pH
and high temperature (Burgess and Fleming. 1974; [so et al.. 1974; Vallin et al.. 1974;
Schieder. 1977).

Zimmermann and Scheurich (1981a. b: Zimmerman. 1%) described a completely
new approach to min. the application of an electric field for protoplast aggluti-
nation and fusion. Protoplasts from different tissues and species have been fused via
this method which has also been utilized for the production of viable hybrids of animal
cells and of yeast. There are no reports of the application of this method for the
production of somatic hybrids of higher plants. This technique has also been used to
release individual chloroplasts from mesophyll protoplasts of Anna m (Zimmer
man et al.. 132) and may prove suitable for the isolation of small numbers of pure
plastids.

in addition to the above mentioned techniques. a wide range of additives such as
poly-L-ornithine. poly-D-lysine. poly-L-lysine. cytocholasin B and protamine sulfate
(Grout and Coutts. I974). lysuyme (Potrykus. 1971). glycerols and dimethyl sulfoxide
(Ahkong et al.. 1975) have been employed.

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25
The existing techniques of protoplast fusion are suitably efficient and appropriate

for most applications in parasexual plant hybridhation. These and developing tech-
nologies may in the future become an efficient complement to the classical methods of
plant breeding.

WWW: Phat 91‘0pr provide a unique
system for studying the structure. chemistry, and function of cell organelles. Organ-
elles can be isolated without harsh mechanical methods necessary for disrupting plant
cells. Isolation of many organelles has been achieved using plant protoplasts (Fovke
and Gamborg. 1%0; Galun. 1981). Experiments using isolated cell organelles such as
nuclei (Lou and Potrykus. 1978). chloroplasts (Potrykus. 1973). or mitochondria have
been described and have also been successfisl with respect to physical uptake of the
organelles into protoplasts.

By means of isolated chromosomes. a promising new scheme for genetic manipu-
lation called chromosome-mediated gene transfer has been developed (Klobutcher and
Ruddle. lfll). The use of plant material for such studies has been hindered. until
recently, by the lack of reliable procedures for mass isolation of plant chromosomes.
Recent developments in protoplast and cell culture of plants may soon change this
situation. Although considerable efforts have been made in this field (Ilalmberg and
Griedach. 1%0; Sssbados et al.. 1981; Griesbach et al.. 1982). the isolation of plant
chromosomes is still not vell developed.

Badlaczky et al.. (1982, 1983) have developed a procedure for mass isolation of
plant chromosomes. in milligram quantities. from protoplasts. Plant chromosomes
isolated by this method exhibit excellent preservation of morphology. and the purity of
the chromosomes has made them suitable for structural and biochemical studies.

In studies on somatic genetics, there is interest in transplantation of chloroplasts
and their extrachromosomal genetic information into protoplasts. Such studies are of
importance in understanding developmental biology and how the development of the

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26
chloroplast is controlled through nuclear versus chloroplastic DNA. Such procedures

are of interest when considering the potential for improving photosynthetic effi-
ciency within or between species. A first step in developing the transfer of chloro-
plasts from one species into protoplasts of another is to isolate pure. intact. functional
chlorophsts.

Until the early I970s. the only established means for isolation of chloroplasts was
to disrupt the plant cell wall by mechanically grinding the leaf material. This was a
limitation. since most species are very resistant to mechanical grinding. However. in
the 1970s. the procedures for isolation of protoplasts from various species became well
established. it was found that chloroplasts could be efficiently isolated from proto-
plasts by mild lysis of the plasmalemma (Gutierrez et al.. 1975). Since then. the list of
species from which intact. functional chloroplasts can be isolated has grown dramati-
cally. but the full potential has not been realized.

Protoplast isolation allows a much wider range of species from which intact chlo-
roplasts can be isolated. including C4 and Cramulacean acid metabolism (CAM) plants
(Huber and Edwards. 1975; Edwards et al.. 1978). However. among the leaf materials
of species examined. not all are susceptible to digestion by the commonly used
commercial cellulase and pectinase. Also. isolation of protoplasts is a more difficult
and time-consuming process. and the yields are often relatively low. Nonetheless.
protoplast isolation is an excellent procedure by which intact chloroplasts can be
isolated from many species and has allowed a number of studies. including intracellu-
lar compartmentation of enzymes. metabolite transport. metabolic activity. and the
isolation and study of the properties of chloroplast envelopes (Robinson et al.. 1979).

Protoplasts have also been utilized for studies of cytoskelehl elements of plant
cells. Emphasis has primarily focused on microtubules. particularly regarding their
relationship to cell wall formation and cell shaping (Lloyd et al.. 1980; Gunning and
flardham. lQZ; Robinson and Ouader. 1932). information concerning other

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27
cytoskeletal components such as contractile elements and intermediate filaments is still

very limited. The work of Yamaguchi and Nagai (1981) illustrates the potential of
protoplasts for microfilament isolation and identification.

Plant protoplasts also provide an excellent system to probe the plant plasma mem-
brane which normally is inaccessible due to the presence of a cell wall and provides
direct access to this vital cellular component and emaciated cell organelles (Fowke et
al.. lfl3).

Coated vesicles and pits are numerous in cells which are involved in active cell
wall formation. Very little is known about the function of coated vesicles in plants.
The idea that they are exocytotic and are responsible for contributing material to the
growing cell wall has received wide support (Fowke etal.. 1983). Until recently it has
not been possible to determine the direction of movement of coated vesicles and
arguments for exocytosis rather than endocytosis have been based on circumstantial
evidence. Protoplasts derived from rapidly growing cultured plant cells contain
numerous coated vesicles and thus are particularly well suited to studies of this cell
organelle (Mersey et al.. lQZ). Ultrastructural investigations of thin sections of
protoplasts (Van der Valk and Fowke. 1981) and isolated plasma membrane fragments
(Doohan and Palevitz. 1980; Van der Valk and fowke. lfil) have provided valuable
information regarding the distribution and morphology of plant coated vesicles.
Protoplasts also offer advantages for the isolation of these (Fowke et al.. 1983).
Fractions highly enriched in coated vesicles have been obtained from soybean proto-
plasts and biochemical character'ustion of these organelles is being pursued (Mersey et
al.. 1%3).

Research with plant protoplasts has provided the only clear demonstration of the
direction of movement of coated vesicles in plant cells. The experiments with soybean
protoplals indicate that endocytosis of cation'med ferritin (0") can occur via coated pits
and coated vesicles (Tanchak etal.. lQ3. 1%). Further research is required to

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28
characterize the process and to determine whether such a mechanism operates in

intact plant cells.

W: The analysis of somatic hybridization products with
respect to nuclear-cytoplasmic interaction is rather complicated. To avoid some of the
problems associated with the combination of both nuclear genomes and mixed cyto-
plasmic material. aubprotoplaats (protoplast fragments) can be used to replace one or
even both of the fusion partners. Subprotoplasts can experimentally be prepared by
the fragmentation of isolated protoplasts into miniprotoplasts and enucleated cytoplasts
('allin et al.. 1978; Lorz et al.. 1981; Bradley. l983). In general. protoplasts without
green chloroplasts isolated from cell suspension or callus cultures are more suitable for
enucleation than mesophyll protoplasts.

The fragmentation of protoplasts is achieved by centrifugal forces during centri-
fugation. Different specific densities of the cellular components (nuclei versus
cytoplasmic material) allow the enucleation of protoplasts into iso—osmotic density
gradients (Lon et al.. 1981). Additional expomnre of isolated protoplasts to cytochalasin
B in combination with centrifugation was also found to be beneficial for enucleation
('allin et al.. 1978). Suitable components for establishing gradients for protoplast
centrifugation are inorganic salts. sugars. and modified silica gels such as Percoll
(Harms and Potrykus. 1978; Lou et al.. lfll; Lesney et al.. 133).

Cytoplasts are very fragile structures and are metabolically less active than
nucleated protoplasts (Lon et al.. lfll ). More important. miniprotoplasts and enu-
cleated protoplasts are suitable for fusion experiments. and cytoplasts are especially
useful experimental tools for transfer of chloroplasts and mitochondria (Brecha and
Sher. lQl; Haliga et al.. 1932).

W: Conventional plant breeding programs have
introduced numerous improvements in agronomic crops during the past centuries.

However. plant breeders may have reached a limit in the ability to introduce new

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29
genetic information into plants and to create new plant varieties through conventional

plant breeding techniques. The introduction of exogenous DNA into cells can result in
a stable and heritable change in phenotype. This process known as transformation. is
well established in several bacterial genera. In higher plants numerous transfor-
mation experiments have been reported.

Protoplasts are often the material of choice in genetic transformation studies.
because the absence of the cell wall should presumably remove one barrier to DNA
entry. Protoplasts are being used as a single-cell crown gall transformation system
instead of the traditional wound infection procedure of whole plants. seedlings. or
different parts of a plant. The advantages of a single-cell transformation system are in
facilitating controlled conditions. and also in the possibility of obtaining a large
number of simultaneously transformed cells (cell lines) derived from individual
transformation events. which can be used in comparative studies (Ooms et al.. l982).

Rapid advances in recombinant DNA technology have permitted the transfer.
integration. and expression of foreign genes in plants. Much of the success. to date.
has resulted from the use of the tumor-inheing (Ti) plasmid of W
W a soil bacterium. as a gene vector (Chilton et al.. 1980; Thomashow et al..
lQO). The transformation of protoplasts by foreign DNA necessitates a balance
between maximizing the transformation frequency and maintaining an acceptable
level of protoplast viability. Methods of DNA delivery to protoplasts include (1) infec-
tion (co-cultivation) of protoplast—derived cells with intact agrobacteria. (2) chemi-
cally stimulated uptake of isolated DNA into protoplasts. (3) fusion of bacterial
spheroplasts with protoplasts. and (4) fusion and/or uptake of liposomes carrying DNA
into protoplasts (Power et al.. 1986).

Crown gall transformation of protoplasts requires the selection of transformants.
Transformants can be selected by the tumorous character of growth substance inde-
pendence. or the antibiotic redstance conferred by foreign genes (e.g.. kanamycin

 

 

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30
resistance). An indication of transformation to the tumorous condition is opine

syntheds since the production of opines is encoded by the integrated sequence of
Ti-planids in the plant cells. Unambigious evidence for the presence of foreign DNA
in transformants is integrated Ti— plasmid sequences found in opine-negative clones by
DNA-DNA hybridization techniques. as shown by Thomashow et al. (1980) and Ooms et
al. (192).

Methods of Plant Protoplast Transformation

1. Infection (Co-cultivation of Protoplast-Derived Cells with W - The
coculture technique has become a procedure of general use in the molecular biology of
the crown gall transformation of plant cells (Merton et al.. I979; 'ullems et al.. 1981;
Come et al.. 1982). This transformation procedure is less labor intensive than methods
involving uptake of isolated plasmids. liposome delivery. or fusion of plant protoplasts
with bacterial spheroplasts. It has been used with different strains of Wu
and various plant species (Basesawa et al.. 1981; 'ullems et al.. 1%1).

The high transformation frequency in cocultures and selection at the cultured cell
level made possible significant progress in the field of plant cell genetic engineering.
Achievements using the coculture techniques include the expression in plants of
Ti- plasmids carrying chimeric resistance genes. thereby conferring drug resistance
on the plant cells in culture (Caplan et al.. 1983). The possibility of selection based on
drug resistance of transformed plant cells allows the elimination of those genes from
the “Ii-plasmids that cause the tumorous growth of transformants alter integration.

2. Chemically Stimulated Uptake of Isolated DNA into Protoplasts - Detailed
procedures have been published for the isolation of WTI- plasmid by
buoyant density centrifugation (Davey et al.. 1980: Draper et al.. IQZ). One of these
involves a mechanical shearing step to fragment the bacterial chromosomal DNA
(Davey et al.. 1%0; Draper et al.. lm); the other utilizes a high pl! to denature the
chromosomal DNA. Theoretically. the use of isolated Ti-plasmid should overcome any

 

 

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31
host range limitations which may arise when attempting to transform plant cells with

intact mm. The methodology for transformation of protoplasts by isolated
Ti-plasmid is based upon the use of chemical agents originally employed to stimulate
virus uptake into protoplasts. e.g.. poly-L-ornithine (PLO). or those used to induce
protoplast fusion. e.g.. polyethylene glycol (PI-26). Krens et al. (1982) reported
transformation of mesophyll protoplasts prepared from shoot cultures of W
Mv. Petit Havana $111 by Ti-plasmid using PEG to stimulate uptake. A significant
detail of the technique is the addition of calf thymus DNA to act as a carrier for the
plasmid DNA.

3. Fusion of Bacterial Spheroplasts with Plant Protoplasts - The second approach
to overcome host range limitations involves the fusion of W spheroplasts
with plant protoplasts. Treatment of spheroplast-protoplast mixtures with a polyvinyl-
alcohol resulted in the uptake of W spheroplasts into m m cell
suspension protoplasts. and expression of T-DNA in (Ll—02% of protoplast-derived cell
colonies (Basesawa et al.. 1981 ). Since it is most convenient to perform genetic manip-
ulations in E. 9911. it is useful to be able to transfer genes directly from E. £911 to higher
plant protoplads. This has been achieved by fusing E. Elli spheroplasts with tobacco
mesophyll protoplasts. giving a transformation frequency of 2.0 in 103.

4. liposome-Encapsulated Delivery of DNA - liposome-mediated delivery is a
promising new technique for introducing macromolecules into plant protoplasts.
These are small artificial lipid vesicles prepared (Uchimiya and Heath. 1981) for
phosphatidyl choline and stearylamine by a process known as reverse phase evapo-
ration (REV). Nucleic acid entrapped in such liposomes renders it highly tolerant to
attack by nucleases. A number of studies established that incubation of liposomes with
plant protoplasts resulted in their association with plant cells (Matthews et al.. I979:
Lurquin and Sheehy. 1%2; Fraley and Papahadiopoulos. l982). It has been demon-
strated by several laboratories that plant viral RNAs encapsulated in liposomes can be

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32
used to infect protoplasts at high efficiency (Nagata et al.. 1981; Vatsnabe et al.. 1982;

Fraley. l983; Christen and Lurquin. 1983). It is likely that this method will also have
application to DNA delivery experiments in studies on stable plant cell transformation
or in short-term transient expression assays.

Protoplasts used in liposome studies have been prepared from avariety of plant
species including carrot (Matthews et al.. 1979). tobacco (Fraley et al.. 1982). petunia
(Fraley. 1983). and cowpea (Lurquin. 1979). using relatively standard enzymatic
isolation methods. Complete removal of the cell wall is essential for maximum uptake
(Nagata et al.. 1981; 'etanabe et al.. 1982).

Optimal conditions for the uptake of nucleic acids into plant protoplasts have been
reviewed (Ohgawara et al.. 1983). In general. optimum delivery of plasmid DNA
encapsulated in liposomes is achieved with negatively charged liposomes in the
presence of 15% w/v me 6000. Maximum infection by TMV-RNA occurs using the same
conditions. Garrently. reports of transformation of plant cells by liposome-encap-
sulated Ti-plasmid exist. but are unsubstantiated. The transformation frequency is
impartially low. probably reflecting the problems inherent in encapsulating such a
large plasmid («l-150 MDa).

tilt

at.

 

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”5”le W m mm

W. crossability. fluorescence technique. germplasm

Am. Self-pollinations of different 2. mm plants and reciprocal cross-
pollinations of 2. mean 2. “man and 2. mm were performed under
greenhouse conditions to assess self-compatibility and crossability relationships. The
fluorescence technique was used to monitor growth of pollen tubes in each of these
self- and cross-pollinations. 2. alnimla was found to be self-incompatible and caused
by pre-zygotic incompatibility preventing the pollen tubes from growing beyond the
stigmatic region. All interspecific crosses failed to produce hybrids. 2. M
pollen germinated on the 2. m1; stigma. but there was no subsequent tube growth.
In the reciprocal. nongerminating seeds were produced from this cross even though
pollen tubes were only observed to extend into the lower half of the style without
penetrating the embryo sac: thus. indicating the occurrence of pre- and/or post-
zygotic incompatibility. Likewise. reciprocal pollinations between 2. with“ and
2. Mwere incompatible as confirmed by the inability of pollen tubes to grow past
the stigmatic region of the style.

At present there are approximately 30 recognized species of Petunia (16. 19). They
are indigenous to Central and South America and extend north into southern parts of
the United States. Since the first hybridization of Wspecies in the early 1800's.
which created the cultivated mm mm Hort. there has been no further
bneding endesvors based on wild species germplasm. The bedding plant industry. of
which petunias are of considerable economic importance. is presently experiencing a
decrease in sales of petunia primarily due to increased sales of competing species such
as impatiens and geranium. Improvement in botrytis resistance (7). floral features.

and growth forms could renew the commercial demand for petunias.

33

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The potential value of a wild 21mm species. Petunia miflm loss. which
could serve as such a germplasm resource for these traits was recognized by Sink and
Power (17). However. these authors reported 2. mm to be sexually incompatible
with the cultivated petunia (18); thus. Sink (15) proposed protoplast fusion to integrate
desirable genes into E. m. m glpjggh is another wild species which might
also serve as a potential genetic resource for 2. m. It possesses small magenta
flowers and a highly branched. prostrate growth habit very similar to that of 2. pg:-
zmgn. These two species. with 2n - 2; - 18 chromosomes. are distinctly different
from all other Emmi; species and at present are the only available sources for
potential genetic changes in cultivated petunias.

Before somatic hybrid'wation is attempted. knowledge of the cmbility and
breeding behavior in selected interspecific Petunia crosses should first be assessed

along with a determination of the stage(s) where failure occurs in the reproductive
cycle between 2. “219211 and other m species.

Materials and Methods

W. Plants of m Mwen obtained from Maureen Han—
son. Cornell University. and subsequently taxonomically verified by Lyman B. Smith.
Smithsonian Institute. Seeds of 2. miflmand 2. mm 'Red Joy lmproved' were
germinated and plants grown to flowering (Fig. l) in the greenhouse using standard
cultural. disease and insect control practices. The greenhouse was maintained at
Zl-Z7°C with a 16 hour photoperiod provided by incandescent lamps. At flowering.
percent pollen viability was assemd by staining freshly dehisced pollen grains in
analine blue. Pollen grains that exhibited a sharp and uniform stain were considered
normal and viable. The number and percentage of normal and defective pollen grains
were calculated from three replications. filly fields per replication.

Self-pollinations were performed at anthesis on different 2. 11219.91! plants and

 

 

 

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35

Fig. l. Plantsand flowersoffigmmalpimlfla. b). E. mflhtflc, d) andE.
barman. Red Joy improved (e , f).

 

 

 

37
reciprocal cross-pollinations between glpigolg and the other Maia species were made

using the standard procedure for emasculation and pollination. The immature corolla
tube was slit open 24 hours prior to antbesis and pollination was performed the next
day or when the stigmatic exudate appeared. Bud-pollinations were also carried out on
B. glpimlg by slitting open the corolla tube at various bud lengths prior to anthesis
and emasculating anthers followed by immediate pollination. The degm of cross-
ability among the species was determined by the number of seeds set. seed germination
and the number of succemlul intercrosses between the species. Pollen grain
germination and tube penetration in the style was observed in standard self- and
reciprocal cross-pollinations of E. Mwith 2. miflmand B. m 48
hours after pollination by use of the analine blue fluorescence technique (9). Pollen
tube growth was rated using the following numerical system: 1) pollen grains present.
but no germination; 2) pollen tubes in the stigmatic region; 3) tubes in upper half of
style; 4) tubes in lower half of style; 5) tubes penetrated to the style base.

WWW. Axenic shoot cultures of 2. 11219911 and
inbred lines of 2. mm 2. m1!“ Joy lmp.', 2. “117.05., 2. mm
Fries and B. m (Lam) BS. P. were maintained on Linsmaier and Skoog (LS) salts
(10) supplemented with the following (mg/liter): myo-inositol. 1w: nicotinic acid.
0.5; pyridoxine 1101. 0.5; thiamine 1101, 0.1; glycine. 2; sucrose. 30M and agar. 8000.
Culttm conditions were 28°C under 16 hours of cool white fluorucent light of 32
uEm'zs' 1. Leaf extracts for malate dehydrogenase (MDH) electrophoresis were
prepared from the parental species and others by grinding approximately 1/2 g of leaf
material in 10 drops of the extraction buffer (1.0 M Tris-citrate buffer. p]! 7.0) plus 2
drops of cold mercaptoethanol between two plastic weighing dishes. The extract was
absorbed into 6x8 mm filter paper wicks.

Horizontal slab starch gels were prepared using the modified system described by
Meisel and Markert (12) and poured into a gel form to set. The gel was covered with

I
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a

38
plastic wrap. refrigerated overnight. and trimmed of excess starch the next morning.

The paper wicks were inseer along a cut in the gel 5 cm from the cathodal end.
The gel was placed between the electrode buffer trays and the electrode reservoirs
filled with goo ml of 5% 1.0 M Tris-citrate buffer (pH 7.0). Vinyl sheets over the wicks
and thin sponges between the gel and buffer trays were umd to establish contact. The
wicks were removed after 1 hour (300 V/fimA); afterwhich. the run continued at 300
V at 4’0 until the front moved about 8 cm from the origin (ca. 5 hours).

Upon termination of the run. the trimmed gel was cut horizontally into 3 slices
and assayed with the substrate stain. The gels were stained at room temperature over-
night. in the dark. with 50 mg B-nicotinamide adenine dinucleotide (NAD). 20 mg
nitro blue tetrazolium (NET) and 5 mg phenazine methosulfate (PM) dissolved into 50 ml
of 02 M Na-malate (pH 7.0) plus 50 ml of 02 M Tris-citrate buffer (pH 8.3) just prior to
use. Three replications for each species was repeated four times with similar results.

Results and Discussion

2. M99]; had a pollen viability of 93.1%. butwas found to be self-incompatible
as shown by the inability to set med following standard or bud-pollinations (Table 1).
Pollen readily germinated on the surface of the stigma but the tubes only grew into the
stigmatic region of the style with a mean growth rating of 2.6, (8 hours alter polli-
nation (Table 2). (Ilservations of pollen grain germination and pollen tube growth
suggest the self-incompatibility as probably pre-zygotic in nature. Previous studies
have shown that the self-incompatibility reaction in 29131111! is gametophytically
controlled by one locus with a series of S alleles (2. 3. 8). Gametophytic self-
incompatibility functions by regulating pollen tube growth in the style. Recognition
between the pollen and style is mediated by the S gene. which has many allelic forms.

-|}en

39

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40

Table 2. Pollen-tube growth 48 hr after self- and reciprocal cross-pollinations of
B. Mwith B. m and with 2. hybrids.

 

 

Mean Pollen
No.of Tube Growth
Pollination Pollinations Ratingz
2. 51212911 (9 11 2.6
34421221! x 10 2.0
E. miflora
E. miflm x 10 2.0
12. mm;
2. 11219211 x 10 1.0
B. may.
B. mm: x 14 4.2
2. slnissna

 

zMicroscopic observation with 1 - pollen but no pollen tubes present; 2 - tubes in the
stigmatic region; 3 - tubes in upper half of style; 4 - tubes in lower half of style; 5 -
tubes penetrated to the style base.

41
When the pollen S allele matches an S allele in the style. as occurs in all self-

pollinations. normal growth of pollen tubes is prevented. The action of incompatibil-
ity (S) genes has been shown to be a result of the interaction of the proteins released
by u» pollen with those of the stigma. similar to the antigen-antibody reaction. and
is supported by evidence that specific glucoproteins form in the pistil following
incompatible matings (6, 13).

One method of circumventing self-incompatibility is through the use of pseudo-
self-compatibility (PSC). which has been reported in most species studied (1). PSC can
be defined as limited or occasionally full seed set following incompatible pollination of
a plant known to possess self-incompatibility. Bud-pollination is another form of P50
which is commonly used to inbreed plants in mm W and . i
mm but does not work on all individuals within a species or population.
Sink and Power (18) were successful in producing a large quantity of self-”ed from
W m which does not set seed following standard self-pollination due to the
one-locus gametophytic type of incompatibility (5). Similarly. they produced
abundant seed following bud-pollination of m m which also exhibits a
gametophytic type of incompatibility (3). Unfortunately. similar success was not
realbed in trying to achieve self-pollination of 2. £219.91! by sib matings.

The most common form of P50 is the regular production of a few seeds from a small
percentage of self-pollinations. Ascher (1) suggested that this form of P50 might
explain the apparent self-compatibility of some individuals in Petunia. This probably
explains the very limited seed production from 2. 11215.91! bud-pollinations obtained by
Jane Smith at Harvard University (Personal Communication to K. C. Sink). When seed
was sown on moistened filter paper in petri dishes. viable seedlings were never
recovered in this study. Susceptibility to environmental interaction and failure to
respond to selection for the PSC character suggests quantitative inheritance (11. 20). ’

Temperature is a major component of the environment and environmental interaction

. 9"“

\ "
g 1.1

42
with genetic factors appears to be the common explanation for PSC (4).

Crossability data for reciprocally crossed m species are presenwd in Table l.
The term crossability. as used herein. denotes the relative ease with which hybrid seed
could be obtained from a cross between two Petunia species. the germination ability of
this hybrid seed. and the percentage of confirmed hybrids. Usually. lower frequency
of crossability indicates a more distant genetic relationship and. in general. the more
distantly related the parents. the more difficult it is to produce a hybrid between them.
All interspecific crosses failed to produce seed with the exception of 2. hybrid; as the
female parent crossed to 2. “pink. Out of )0 flowers pollinated. a total of 74 seeds
were obtained from 3 capsules having 10. 2. and 62 seeds. respectively. Attempwd
germination of 65 of these ”eds failed to produce viable seedlings. From the
remaining 47 pollinated flowers. ovaries were harvested which contained dried chaff
inside. Examination of 2. hybrid; styles pollinabd with 2. M21221! pollen resulted in
a mean pollen tube growth rating of 42 (Table 2). Most tubes extended into the lower
half of the style while a few penetrated as far as the bottom. but none were observed
which had entered the embryo sac. In as much as entry into the ovules was not
observed using the analine blue fluorescence technique. it is still uncertain whether
fertil'aation occurred in obtaining the 74 nongerminating seeds from this interspecific
cross. Pollen germination and pollen tube growth must occur for timely delivery of
the male gamete to allow successful fertilization. and endosperm and embryo
development must follow to produce viable hybrids capable of gene transfer. These
results indicate pre-zygotic and/or post-zygotic barriers may be in effect. Conversely.
in the reciprocal cross 2. hybrid; pollen. with 91% viability. germinated profhsely on
the 2. Matias but subsequent pollen tube growth did not occur.

The interspecific crom between 2. m (98% viability) as female and 2.
mm as the pollen parent was initially believed to be a compatible mating. as
indicated by the number of seeds set and the percentage of seeds that germinated

 

l

43
(Table 1). However. cross-incompatibility was confirmed since the plants were not

hybrids. The plants from this mating were also electrophoretically examined and
identified as being contaminants of 2. mm by the identical malate dehydro-
genase (MPH) isosymo pattern ofthe parental plant material (Fig. 2b). The migration
didance and bonding intensities of all isosymes found in the species examined are
shown in Fig. 3. Similarly. the reciprocal cross proved to be cross-incompatible.

The answer to producing 2. m interspecific clumes may ultimately involve
using techniquessuch asshortening thestyle lengthsothatpollen tubescanreach
the ovules. hm pollination and fertilisation might also be attempted to circumvent
thou pre- and post-zygotic incompatibilities. However. hm fertilization tech-
niques (14) are used primarily to overcome pro-zygotic self- and cross-incompatibility
factors resulting from pollen-style interactions. and may not be the solution for the
peat-zygotic incompatibilities. Therefore. somatic hybridisation may be a potential
means to overcome both pre- and postaygotic type incompatibility barriers existing
between 2. Wand othermspecies. 'hilemoetmspecies can be
readily regenerated to plants from protoplasts. somatic hybridisation appears to be a
viable alternative.

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f. . 34" "'1’ all" 0"‘ J19" 04‘.’ 1:1.13. J ‘0’, "Js' J. 'thh - ;.' . - 1) hI, 2' 4”,.ll ‘1 ”PJ’s"

pr... “nix-.1 . 5 '."is.m-n1--n‘.' “unis 04!). 91:. 638111.50 and} mm} drink; 1.1? .cbndvd
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2 Iimmvm. shins:

44

Fig. 2. Malate dehydrogenase electrophoretic pattern from mm 11219211“). 2.
Quintin“). E. hm“). 2. 9.8429411“). 13- minimum 2. M“)-

45

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Fig. 3. MDH zymogram from leaf extracts of 6 Petugig species.

47

 

 

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Moderately Stained

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48

literature Cited

Ascher. PD. 1976. Self-incompatibility systems in floriculture crops. Acta Hort.
63205-215.

Ascher. PD. 1984. Self-incompatibility. pp. 92-110. In: LC. Sink (ed.). Petunia.
ginographs on Theoretical and Applied Genetics 9. Springer-Verlag. Berlin
idelberg.

Brewbaker. Jl. and SI. Majumder. 1961. Cultural studies of the pollen population
effect and the self-compatibility inhibition. Amer. J. Bot. 48:457-464.

East. EM. 1923. Genetical aspects of self- and cross-sterility. Amer. J. Bot.
10:468-473.

. East. EM. 1928. The genetics of the genus W. Bibliogr. Genetica

4243-320.

. Heslop—Harrison. J. 1975. Incompatibility and the pollen-stigma interaction.

Ann. Rev. Plant Physiol. 26:403-425.

Laemmlen. 1" 1’ . and KC. Sink. 1978. Evaluation of petunia cultivars for botrytis
resistance. Plant Dis. Reptr. 62:361-365.

Lewis. D. 1944. Incompatibility in plants. its genetical and physiological
synthesis. Nature 153:575-578.

Linskens. HT. and K. Esser. 1957. Uber eine spesifische Anfarbung der
Pollenschlauche im Griffel und die Zahl der Kalloapfropfen nach Selbstung und
l-‘remdung. Naturwissenschaflen 44:16.

Linsmaier. EM. and F. Skoog. 1965. Organic growth factor requirements of
tobacco tissue cultures. Physiol. Plant. 18:100-127.

Mather. K. 1943. Specific differencesinmig. I. Incompatibility. J. Genet.
45115-235.

Meizel. S. and C1..Markert. 1967. Malate dehydrogenase isozymes of the marine
snail 111mm 9.123212! Arch. Biochem. Biophys. 122:753-765.

Nasrallah. ME. J.T.hrber and DH. Wallace. 1970. Self-incompatibility proteins
in plants: detection. genetics. and possible mode of action. Heredity 2523-27.

Rangaswamy. NS. 1977. Applications of in vitro pollination and in vitro
fertilization . p. 412-424. In: J. Reinert and YPS. Baiai (eds). Applied and
fundamental aspects of plant. cell. tissue. and organ culture. Springer-Verlag.
Berlin.

Sink. KC. 1980. Plant protoplasts - a novel method for producing new hybrids of
horticultural crops. Part I. Flor. Rev. 167 (432922; 57-58. Part II. Flor. Rev.
167 (4324):”; 62.

16.

17.

18.

19.

49

Sink. KC. 1984. Taxonomy. pp. 3-9. In: KC. Sink (ed.). Petunia. Monographs
on Theoretical and Applied Genetics 9. Springer-Verlag. Berlin Heidelberg.

Sink. I ..C and JB. Power. 1977. The isolation. culture and regeneration of leaf
protoplasts of mm mm. Plant Sci. Lett. 10: 335-340.

Sink. KC. and JD. Power. 1978. lncongruity of interspecific and intergeneric
crosses involving Rigging and m species that exhibit potential for somatic
hybridization. Euphytica 27:725-730.

Smith. LB. andR..J Downs. 1966. Solanacea. p. 321. In. PR. Reitz (ed..) Flora
illustradaCatarinense.1taiai S. Catarina. Brazil.

. Takahashi. H. 1973. Genetical and physiological analysis of pseudo-self-

compatibility in 29.111111! hybrids. Jap. J. Genet. 4827-33.

'I'III ISOLATIOI. CULTURE AID WHOM W PROTOPLASTS
(I? m “.2199“

Summary

Emma 81219211 is recognized as a potential germplasm resource for enhancing
the cultivated 29.111111! MHort. 2. flpimlg is sexually incompatible with the
other species and thus somatic hybridization may be a viable alternative. The isolation
and culture of protoplasts isolated from callus and suspension cultures of this species
was achieved as a prerequisite to cell fusion. Efficient shoot regeneration was
achieved from protoplast-derived calli on MS o zeatin (1.0 mg/l) and mating on either
MS 0 NAA (0.01 mg/l) or IBA (1.0 mg/l).
Key Words: 29.121111 . germplasm. protoplasts. regeneration.

Introduction

Regeneration of plants from protoplasts has been reporwd for many members of
the Solanaceae family and for different species within the genus Petunia. Comparative
studies on protoplast regeneration have been performed with different genotypes.
species. and hybrids in regard to protoplast technology. taxonomic relationships. and
somatic hybridization (Frearson et al.. 1973; Power et al.. 1976; Izhar and Power.
1977). Earlier protoplast work on Petunia has involved species possessing a 2n - 21 - 14
diploid chromosome number. with one exception; that being 2. mm Juss.. the
first species documented to have a 2n - 2; - 18 chromosome number (Ferguson and
Coolidge. 1932). The species investigated herein. 291111111 unison. has the same
chromosome number (211 - l8). and. although its phylogenetic origin is uncertain.
morphological observations suggest its cloust affinity may be to 2. minor;
(Ford-Logan. unpublished). They both have a prostrate or creeping growth habit.

short-petioled succulent leaves and small magenta flowers. All of these features are in

50

51
distinct contrast to the 2p - 14 Maia species. The two 18-chromomme species have

been recognized as potential germplasm resources for the integration of desirable
genes into the cultivated 2. hybrid; Hort. but are known to be sexually incompatible
with the cultivated petunia (Sink and Power. 1978; Ford-Logan. unpublished). Thus.
novel tissue culture techniques such as protoplast fusion may be required before
further exploitation can be realized. This study was conducted to develop a procedure
for the isolation. culture and regeneration of plants of B. glpigoig from protoplasts.
based upon established protocols of plant protoplast isolation which have been either
developed for Maia. adapwd to this species or utilized for it (Binding and
Krumbegel-Schroeren. 1984).

Materials and Methods

Plants of 2. gipigqigwere obtained from Maumen Hanson. Cornell University.
Because 2. 81215218 is self-incompatible (Ford-Logan. unpublished) and does not set
selfed seed. a micropropagation system was developed in order to maintain a constant
supply of plant material. Excised shoot-tips. approximately 1 cm in length. were
cultured on Murashige and Skoog (MS) medium (Murashige and Skoog. 1962) with 1.0
mg/l zeatin at 27°C for 16 h with 17 uern'zs’l from cool-white fluorescent tubes (6. E.
F40CV-RS-UM). Every four weeks the multiple shoots that occurred were subcultured
on the same medium with single stem growing plants maintained for experimental use
as shoot-tip cultures on MS medium without growth regulators.

Leaf pieces (1-2 mmz) from inziim shoots were used to initiate friable callus when
placed on MS o 1.0 mg/12.4-dichlorophenoxyacetic acid (Z.4~D). Dark green callus
developed over the entire surface of the leaf explants and was subcultured after 5-6
weeks. These cultures were held at 28°C and 58 uEm‘Zs'1 for 16 h from mol-white
fluorescent tubes (G. E. F96-T12-C' ) and routinely subcultured every 21 days to
maintain actively dividing callus. Suspension cultures were subsequently established

52
from this callus in liquid MS + 1.0 mg/12.4-D and maintained by subculturing every

10-14 days in the same liquid medium. These cultures were maintained in 125-ml flasks
on a gyratory shaker at 90 rpm at 27°C in diffuse light.

Protoplasts were isolated from both callus and cell suspension cultures. With the
aid of aspatula. callus (approx. 3 g) was gently separated by passage through a coarse
sieve (35 list) placed in a 1m x 15 mm plastic Petri dish and rinsed with a cell protoplast
washing (CPU) solution (Frearson et al.. 1973) containing 8% (w/v) mannitol (8M).
The cell suspension cultures were handled differently in that the liquid medium was
removed by transferring the cells to 16 x 125 mm culture tubes and pelleting them by
centrifugation (80 x g; 5 min). The medium was replaced by CPI 8M afterwhich the
cell slurry was pipetted onto a coarse sieve (35 um) and separated in the same manner
as callus. Hereafter. all cells. regardless of media source. were handled in the same
manner. The cells were plasmolyzed in the CPU 8M solution for 1 h at room temper-
ature in the light. without agitation. The plasmolyzed cell slurry was transferred to
screw-capped culture tubes and pelleted by centrifugation (80 x g; 5 min). The
supernatant was replaced by a filter sterilized enzyme solution which consisted of 2%
Cellulysin. 2% Macerase. 2% Driselase and 8% (w/v) mannitol dissolved in CPU salt
solution. pH 5.8. The cells suspended in the enzyme solution (approx. 5 m1 packed; 20
ml) were transferred to 1m x 15 mm Petri dishes. which were wrapped with Paral‘ilm®
and incubated 17-19 h in dark at 27°C on a gyratory shaker (35 rpm). Following
incubation. the cells were gently teased with a Pasteur pipette to release any proto-
plasts not liberated by enzyme action alone. The enzyme-protoplast mixture was
passed through a fine sieve (61 um) and collected in 100 x 15 mm Petri dishes before
being transferred to culture tubes. The protoplasts were pelleted by centrifugation
(11!) x g; 5 min). and the supernatantwas removed. The protoplasts were washed free
of enzyme by remispension in CPU 8M and centrifuging (100 x g; 5 min). The

supernatant was replaced by 6 ml of a CPU solution containing 25% sucrose and

53
centrifuged (1m x g; 15 min). A second wash in CPU 255 removed any carryover of

enzyme and debris. Protoplasts were removed with a Pasteur pipette to culture tubes
and resuspended in liquid culture medium. counted and plated at test densities in 60 x
15 mm plastic Petri dishes. The components of the plating medium are listed in Table 1.
Four m1 of protoplasts were added to each dish. which were wrapped with Parafilm®
and incubated at 25°C with constant illumination of 15 M434 provided by cool-white
fluorescent tubes (6. E. F20-T12-CV ). The effectiveness of the isolation procedure for
obtaining high yields of viable protoplasts was monitored by fluorescein diacetate
(FDA) (Widholm. 1972).

The dishes were replenished with the same medium with the mannitol level held
constant during feeding at 2 and 4 week intervals after plating. The viability and
division of the protoplast-derived cells were monitomd by examining the culture dishes
periodically under an inverted microscope. Plating efficiency (Pl-I.) was determined
by the percentage of viable protoplasts 24 h after isolation that produced cell colonies
after 7 days. After approximately 6 weeks in culture. the dishes were decanted of old
culture medium and the macro-colonies were plated between two layers of semi-solid
agar. This was done by adding the cell colonies plus 1 ml of fresh MS culture medium
to a dish containing 2 ml of 0.4% agar cooled to 45°C and layering an additional 1 ml of
cooled agar on top of the cells and swirling gently. When the proliferating cell
colonies were of sufficient size. after 2 to 6 weeks. they were transferred to semi-

solidified regeneration medium (Table l) to produce shoots.

Results and Discussion

Leaves of 2. glpigqig proved to be technically unsuitable for protoplast isolation
due to their small size and the difficulty involved in handling. Callus and suspension
cultures proved excellent sources because these cells were already conditioned to

growth in culture. and the requirements for their continued growth. differentiation

54
Table 1. Media used in E. glpigglg protoplast culture and shoot regeneration.

 

 

Liquid Culture Shoot Regeneration

Medium Medium
Compound (mg/1) (mg/l)
m mlts 4627.93 4627.93
lye-lnositol 100. 100.
Thiamine-HG 0.1 0.1
Glycine 2.0 2.0
Nicotinic acid 0.5 0.5
Pyridoxine—HCI 0.5 0.5
2.4-D 1.0 -
NM 20 -
6-BAP 0.5 -
Zeatin - 1.0
Coconut water 20 ml -
Sucrose 30 ill). 30 (m
Mannitol 130 000. -
Agar - 0.4
9H 58 53

 

 

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and organogonosis were established. There was no difference in the suitability or

response of either of these cell sources when isolating and culturing protoplasts.

Protoplast yielrb were consimently 2-3 x 10° per gram of callus or nrspension
culture cells. These yieb were considerably lower. approximately 6 x 105. when
protephmswere inlatedfrom cuburesnearing the timeofsubculture. Protoplasts
isolatedbythismethodwere initiallysphericalinshapemg. la)andmostexhibited
normal ultrastructures when tested for their ability to hydrolyse fluorescein diacetate.
Afterarhyin culture. theybecame ovalshaped. indicating cellwallsynthesisflaoet
al.. 1970). with active cytoplasmic dreaming. Soon after protophets are cultured in
an appropriate medium. an increase in cell organelles indicates a general activation of
themetebolicactlvityofthe protoplamsferthe regenerationofacellwall. With the
increase in cell size. new cytoplasmic mrands are formed. and mestof the cell orga-
nelles. particuhrly the chloroplams. aggregate around the nucleus (Vasil. 1976).
Fir: division occurred within 48-72 h (Fig. 1b). 'ith repeated divisions (Fig. 1c) the
number of chloroplasts per cell visibly decreased. and the cells became more vacu-
elntedwith micro-colony formation observed alter 4 days (Fig. 1d). Pale green. visible
macro-colonies (0.3-1 mm diam.) were formed in 3-4 weeks. A phting efficiency of
85% wasrecordedwhen protoplamswere platedatadensityof l x 103 protoplasts/mun
theliquidculturemedium. Priortohveleplngthe mediumwhich “noddivision of
2. m protoplam-derived cells. several modil'icationsof the B culture medium
wereteeted. TheMSmedium (fmmTable l)withthesamelevelofmannitol.
naphthaleneacetlc acid (IAA) and 6-benzylaminopurine (6-BAP). hm lacking 2.4-D
andcecenuwateflCI). amnmgmmnuro’m; first
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MOM. termed preteplambuddlng. occurs when pectin is not incorporated into
the new cellwall (Hankeandlorthcete. 1974)andisthereeultofweakenedareasin
the newly synthesbd cell wall (Tanks and Camber-g. 1’0). In the same culture

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56

Fig. 1. Division and formation of plants from protoplasts of B. ginigqig: Freshly
isolated protoplasts suspenud in culture medium. x 400 (a). first division in cell
regenerated from protoplast. x 400 (b). protoplast-derived cell following the second
mitotic division with non-dividing cell in immediate vicinity. x 400 (c). multicellular
colony. x 400 (d). macro-colonies upon further plating in soft agar (actual size) (e).
differentiation of shoots on protoplast-derived callus (actual size) (f). and adven-
titious roots produced on regenerated shoots on MS medium with 1.0 mgll IBA(actual

size) (g).

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58
medium (Table 1) locking only coconut water. protoplasts were observed dividing with

a plating efficiency of 60%. Oulurre medium containing 20% coconut water. 2.4-D (1.0
mgll). IAA (2.0 mgll). and BAP (0.5 mgll) increased the plating efficiency to 85%.
The 25% increase might be he to the stimuhting synergistic effect of coconut water
and 2.4-Daeseen byStewardandCaplin (1951)th the culture ofpotato tuber cells.
Coconutwater is generally believed to contain cytokinin-like substances aswell as
rerhlced nitrogen and possesses detoxifying properties. all of which may have value
for certain tisnre cultures (Pollard etal.. 1961; Tulecke etal.. 1961).

In initialexperiments. the cultureflsheswere replenishedevery twoweeksalter
phting with 0.5-ml aliquots of the appropriate culture medium containing rerhlced
mannitolhveleof ll. 9. 6. 3 and0$. Thisprecehre mailed in browning andevent-
uallythedeathofallviable cell colonies.

In liquidculturemediumtherewaslimitedgrowtbaftercoloniesreachedthe
multicelbhr stage (Fig. 1c) unless tranderred to the softagar. Transferring cells at
the muhicellular dogs to interfacing layers of semi-solid agar allowed further growth
anddevelepmentefthe green. vhible colonies (Fig. 1e). which reunited in the growth
ofcellsin compactand discrete clumers. Thismothodwasamodification ofthe plating
technique used by Iagataand Takebs (1971) for culturing isolated tobacco mesophyll
W.

Cslliwereofufficientabo (3-4 mm)tobe transferredtodoetregeneration
mebm (Fig. 1f) appdeximatoly8-12weeksalterplating the pretephis. Atthisstage
the calli were moved a a higher um intensity 0158 uEm’zs“ (e. E.F96-T12-CI) roi- 16
h at28°C. Onceacalhleinitiatedsheetprlmerdiaitcontinuedtoproduce norhrlated
callusand prolific shoots. Shoot-tipeof2 cmor longerwere separated singly from the
shoot regeneration cultures and transferred to rooting media. either MS with 0.01 mgll
NAA or 1.0 mgll IBA (indolebutryic acid) (Fig. 1g). Root primordia generally emerged
between the Mud second week. although afew shoots had1-3 mm roots alter 6

 

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59
(hys. Both auxins produced lmt rooting of shoots. In either medium. the regener-

ates were grown for four weeks to allow roots to develop. Shoot-tips were also trans-
ferred to an MS medium with auxins and cytokinins completely eliminated (M80). but
only few or no reotoworo initiated.

Enablhhmont of the regenerates directly into soil or a soil-loos planting medium
was unauccomful. A mixture of peat. perlite and vermiculite (V. S. P. — Bay Houston
Towing Co.) was found to cause necrosis ofthe roots and all plants were highly
susceptible to fungal attack. The 111111.11 rooted regenerates continued to grow when
they were trensforred to cell packs containing morilisod perlite. The regenerated
plants. after growing in the highly promoted. artificial culturo environment were
foundtobovoryoonsitivotomoisurrostrosoandsuscoptibloto pathogen attackduoto
the water retention capacity of the initial planting medium. The grarhral opening of
polyethylonobags. uoodtoprovidoahighhumidity. wasattomptodtoacclimato
plantlots but dehydration repeatedly occurred. To date. all efforts to successfully
acclimatethorogonoratodplantstothooutoidoonvirenmonthovofailod. Sonsitivityto
mromhringacclimationioapparontlyboin parttolackofcuticloontholoavos
(Groutand Ashton. 1977; Suttor and Longhano. 1979). In aflition. plantlots were
highly sensitive to bhydretion because their momatos may not have been functioning
effectively (Brainord and Fuchigami. lfll ).

Thiomudyindicatodthatplontscaabo rogonoratod from protoplastsoffi. gipimig
andpreviboaaoxporimontalbaoisforfuturoworkin oomoticcoll goneticswlth this
species.

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.11 {:1'J111a a: 1d M11131 ‘l'--’l.l‘1 laminruvne 9111;111:1196101almiq11311313111331 am 91111111141.
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cm 1913:».

60
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