5% llHlilUH“Ill”!lHllWlHlHlHHlHTIIIHHIIWHIIIIHI

lllflillll I mnmnnmnmwmmm

3 1293 00089 6625

__J..‘.¢

LIB R A R Y
Michigan State
University

,-.‘ --.......~ .._M

THESIS

This is to certify that the

thesis entitled

TISSUE CULTURE AND CALLUS PROTOPLAST REGENERATION
0F SALPIGLOSSIS SINUATA L.

presented by

Charlene Jan Boyes

has been accepted towards fulfillment
of the requirements for

M.S, #degreein H T ture

7/4/56. A...

Major professor

Date flM—rf/YI’CI
'77 //

0-7639

 

 

OVERDUE FINES:
25¢ per day per item
RETURNING LIBRARY MATERIALS:

Place in book return to remove
charge from circulation records

 

 

 

TISSUE CULTURE AND CALLUS PROTOPLAST REGENERATION
OF SALPIGLOSSIS SINUATA L.

 

BY

Charlene Jan Boyes

A THESIS

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

MASTER OF SCIENCE

Department of Horticulture

1980

ABSTRACT

TISSUE CULTURE AND CALLUS PROTOPLAST REGENERATION
OF SALPIGLOSSIS SINUATA L.

BY

Charlene Jan Boyes

Leaf explants were aseptically cultured on Murashige
and Skoog (MS) medium plus Zip, K, BA, NAA, IAA, and 2,u-n
singly or NAA and BA combinations (0.01, 0.1, 1.0, 5.0,
10.0 mg/l). Adventitious shoots arose at all MS + BA, Zip,
or K levels except at 0.01 mg/l. Callus and roots occurred
on MS + NAA or IAA except at 0.01 mg/l IAA. Optimum shoot
regeneration occurred from leaf callus plated on MS + 1.0
mg/l Zip. Protoplasts were enzymatically isolated from
friable callus. Continuous division to the macroscopic
callus stage was obtained in a liquid MS medium modified by
the deletion of ammonium ions and adding 250 mg/l glutamine,
0.1 mg/l serine, 2.0 mg/l thiamine’HCL, 0.5 mg/l IAA, 1.0
mg/l 2,u-D, 0.5 mg/l BA and 9% (w/v) mannitol. Small
colonies formed callus within 2.5 months and the latter
readily regenerated shoots on MS + 1.0 mg/l Zip. High

frequency rooting occurred on MS + 0.001 mg/l 2,u-D.

To Jim

ii

ACKNOWLEDGEMENTS

I extend my appreciation to Dr. Kenneth C. Sink for
his guidance in this research program and in editing this
thesis. I also wish to thank the other members of my
guidance committee, Dr. Robert C. Herner and Dr. Harry H.
Murakishi for their assistance in this study and in the
final preparation of this thesis.

Appreciation is also due to Dr. F. Javier Zapata, who
provided technical assistance and invaluable advice.

I also wish to extend my sincere thanks and apprecia-
tion to my family for all their love and encouragement

during my college education.

iii

TABLE OF CONTENTS

LIST OF TMLES.’O.......OOOOOOOOOOOOOO00.0.00... ...... .0 V

LIST OF FIGURESCOOOOOOOOOO00.000.000.00.0.0.0.0....0.... Vi

 

THE MORPHOGENETIC RESPONSE OF SALPIGLOSSIS
SINUATA L. LEAF EXPLANTS CULTURED IN_VITRO

INTRODUCTION. 0 O O O O O O O O O O O O O O I O O O O O O O O O O O O O O O O O O I O O ...... 1
MATE RIALS AND METHODS O O O O ..... O O ..... O ...... O O O O O O O O O C O O 2
“SULTS AND DISCUSSION. 0 O O O I~ O O I O O O O O O O O O O O O O O O O O O O O O O O O O a

SWRYOOOOOOOOOOOOCOOOOOO00.0.0... ........ ............O 13

 

ISOLATION, CULTURE AND REGENERATION
OF CALLUS PROTOPLASTS OF
SALPIGLOSSIS SINUATA L.

 

INTRODUCTION............................... ............. 1”
MATERIALS AND METHODS .................... . .............. 17
RESULTS AND DISCUSSION ......... ... ...... ...... .......... 23
RECOMMENDATIONS............. ......... ...... ............ . 39

SUMMARY ........ ........ ................................. 41

 

BIBLIOGRMHY ..... 0...... OOOOOOOOOOOOOO ......OOOOOOOOOOOO “2

iv

LIST OF TABLES

Table 1. Effect of cytokinins on morphogenesis of Sal-
piglossis sinuata leaf explants.............. 5

Table 2. Effect of auxins on morphogenesis of Salpig-
lossis sinuata L. leaf explants.............. 7

Table 3. Morphogenetic response of Sal i lossis sinu-
ata leaf explants cultured on BA ana NAA
cominationSOOOOO00............OOOOOOOOOOOOOO 9
Table 4. Root induction of regenerated shoots of Sal-
piglossis sinuata in MS basal medium with

different addenda........... ....... .......... 11

Table 5. Enzyme mixtures tested for the isolation of
Salpiglossis sinuata L. leaf protoplasts..... 19

 

Table 6. Enzyme mixtures tested for the isolation of
Salpiglossis sinuata L. protoplasts from
axenic shoot cultures ............ . ........... 19

Table 7. Enzymes tested for the isolation of Salpig-
lossis sinuata L. callus protoplasts ......... 21

Table 8. Test media for leaf protoplasts of Salpig-
lossis sinuata ......... ...... ...... .......... 27

Table 9. Test media for protoplasts from axenic shoot
cultures of Salpiglossis sinuata............. 27

 

Table 10. Test media for callus protoplasts of Salpig-
lossis sinuata............................... 31

Table 11. Test medium supporting the division of proto-
plasts from Salpiglossis sinuata callus,
(mg/1) .......... oooooooooo oooooooo ooooooooooo 32

Table 12. Media for the regeneration of shoots and
roots from callus derived from protoplasts
of Salpiglossis sinuata (mg/1) ..... .......... 36

 

LIST OF FIGURES

Figure 1. Regeneration of isolated callus protoplasts
of Salpiglossis sinuata...................... 35

 

vi

INTRODUCTION

The elicitation of morphogenetic responses through
manipulation of selected explants cultured in_zi§59 has
been demonstrated in many plant species of economic impor-
tance (Binding, 1973; Murshige, 197a; Kartha et al., 1976).
Likewise, organogenesis has been reported from a variety
of explants including stem pieces (Skoog, 19uu; Murashige
and Skoog, 1962; Rao et al., 1973) roots (Levine, 1950;
Norton and Bell, 195“) anthers (Cuba and Maheshwari, 1964;
Hughes et al., 1973) and embryos (Masheshwari and Baldev,
1962). Trends in somatic cell manipulation strongly suggest
the potential for crop improvement through protoplast tech-
nology. A favored source of protoplasts for conducting
cellular manipulation is the mesophyll tissue of leaves.
Leaf protoplasts can generally be obtained with relative
ease and in high yields with apparent uniformity. Regenera-
tion, from callus derived from either explants or proto-
plasts, may be determined through examination of hormonal
control of morphogenesis on leaf explants (Frearson et al.,
1973; Kartha et al., 1976). Leaf explants have been used-

for morphogenetic study in Nicotiana (Gupta et al., 1966)

 

and in Lycopersicon esculentum (Padmanabhan et al., 1974).

Anthers and leaf discs of Salpiglossis sinuata L. have been

 

cultured and plants regenerated from the resulting callus

1

2
(Hughes et al., 1973; Lee et al., 1977). Both reports
indicated difficulty in root initiation of the regenerated
shoots. However, a comprehensive study of the morphogenetic
response to phytohormones in various combinations and con-
centrations, especially concerning rooting, has not been con-
cluded. This inquiry was undertaken to determine 1) The

morphogenetic response of Salpiglossis sinuata L. leaf

 

explants to various phytohormones in vitro, both singly and
in combination; and 2) to determine an efficient method of
root initiation for regenerated shoots.

MATERIALS AND METHODS

Salpiglossis sinuata L. seeds were sown on an artificial

 

planting mix (VSP Bay Houston Towing Co) and germinated at
22°C 1 2°C under 16 hr photoperiod at 7u-84 uEm-Zs-1 cool
white tubes (General Electric). In two to three weeks, the
seedlings were grown under a minimum temperature regime of
22°C night and 25°C day or higher, depending on the season.
Supplemental lighting of 550 uEm-zs-1, cool white fluores-
cent tubes (General Electric) for for 16 hr coinciding with
the natural daylight was used. Fertilizer solution was
applied to the seedlings at a rate of 200 ppm N twice weekly
using 20-20-20. Disease and insect measures were applied as
needed. Basal leaves for in yitgg culture were obtained

from the seedlings when they were 60-90 days old.

Leaves were surface sterilized in a 5% solution of

3

commercial sodium hypochlorite (Clorox) for 30 min followed
by six sterile distilled water rinses. Rectangular leaf
pieces, 1 x 8cm, were aseptically excised from the leaves;
the midrib and leaf margins were removed.

The leaf explants were cultured on Murashige and
Skoog (1962) macro- and micro-elements (MS), 3% sucrose,
0.8% agar (Sigma, Grade IV). Auxins added to the basal
medium included 2,4- dichlorophenoxyacetic acid (2,4-D),
indole acetic acid (IAA) and napthalene acetic acid (NAA)
and the cytokinins 6-benzylaminopurine (6-BAP), 6(YBY’
dimethylally amino) purine (21p), 6 furfurylamino purine
(K). In some experiments; the medium of Uchimiya and Mura-
shige (1974) (UM) was employed. The pH of all media was
adjusted to 5.8 with 0.2N NaOH or 0.1N HCL. All media were
autoclaved at 15 psi (1.46 Kg/cmz) for 20 minutes. The two
culture vessels employed were 60 ml glass jars with metal
screw caps or 100 x15 mm Petri dishes containing 25 and 20
ml of media respectively. One explant was placed into each
jar and four in each Petri dish. The culture vessels were
placed under 74 to 84 uEm-Zs-1, cool white fluorescent
tubes (General Electric) with a 16h photoperiod and a temé
perature range of 23°C i 2°. Each test medium was relicated
eight times and each experiment was repeated at least twice.
The leaf explants were routinely subcultured every four
weeks. Each leaf piece was visually evaluated five weeks
after initiated of the experiment for morphogenetic

response(s).

u

RESULTS AND DISCUSSION

The effect of cytokinins on morphogenesis - the
morphogenetic response of leaf explants plated on MS + BA,
K or 2ip'is presented in Table 1. The results on MS + K
and Zip were essentially the same although they occurred at
different concentrations. Shoots were observed at 1.0 mg/l
and 5.0 mg/I K and at 0.1 mg/1 and 1.0 mg/l Zip. The bio-
logical activity (stimulation of cell division) of Zip has
been reported as being ten times greater than K (Helgeson,
1968). Concentrations above or below these levels resulted
in either poor shoot development, spindly leaves or only
callus. Kartha et al. (1976) found similar results on

Lycopersicon esculentum cv Starfire, though no callus growth

 

occurred at the lowest cytokinin concentrations. Rao et a1.
(1973) obtained shoot development with BA, K or zeatin on

leaf explants of Petunia inflata. At the lowest levels of

 

2ip, K and BA, 0.01 mg/l, callus growth was induced, but it
was slow in developing; pale green or brown in color,
indicating suboptimal conditions. A purple pigment, prob-
ably anthocyanin, developed in the 0.01 mg/l K callus. Iso-
lated incidents of pink callus were also observed, but
profuse development of pink, purple callus indicated a
possible stress situation. The stress may have been due to
a suboptimum cytokinin level; one barely sufficient to
induce and support cell division. On MS + BA and MS + Zip
(0.01 mg/l) occasionally small roots developed. This

response may indicate a ratio of exogenous cytokinin to

5

Table 1. Effect of cytokinins on morphogenesis of
Salpiglossis leaf explants.

 

Cytokinin (mg/l) Response

 

BA 0.01 pale green callus occasional root
0.1 pale green callus occasional root
spindly leaves
0.1 callus shoots with spindly leaves
development slow
5.0 callus few shoots
10.0 callus shoot primorida only
K 0.10 callus, purple no shoots or roots
0.1 callus, green few shoots; develop-
ment slow
1.0 callus multiple shoots
5.0 callus multiple shoots
10.0 callus few shoots, develop-
ment suppressed
Zip 0.01 callus, pale green very few roots
0.1 callus multiple shoots,
development slow
1.0 callus multiple shoots
5.0 callus compact shoots
10.0 callus compact very few shoots,

development suppressed

 

6
endogenous auxin favorable to root initiation. High levels

of endogenous auxins have been reported in other Solanaceous

 

plants (Kartha et al., 1976); in the case of Salpiglossis

 

auxins may be low since a high level of cytokinin was not
necessary to initiate roots. MS + BA as compared to Ms +
K and MS + 21p was much less effective in eliciting morpho-
genetic responses. Shoot differentiation occurred, but
elongation and normal develOpment was inconsistent at the
1.0 mg/l level. BA is known to be less active than other
structurally related cytokinins in promoting cell division
(Haberlach et al., 1978), an important factor in callus O
growth and morphogenesis.

Effect of auxins on morphogenesis - all auxins tested

 

induced callus growth except for IAA at 0.01 mg/l indicating
it did not induce division in the leaf cells at as low a con-
centration as 2,4-D and NAA (Table 2). Callus induced at
this level 0.01 mg/l NAA and 2,4-D, grew slowly; NAA induced
callus was also quite compact. Callus induced on 0.01 mg/l
2,4-D was also compact in comparison to callus induced at
higher 2,4-D levels. However, 2,4-D induced callus was not
nearly as compact as that induced on NAA. Induction of cal-
lus involves the initiation of cell division and prolifera-
tion concomitant with dedifferentiation (Gresshoff, 1978).
Apparently IAA at 0.01 mg/l cannot activate leaf callus to
begin this process. Both NAA and 2,4-D are considered much ..
more potent auxins and are also longer lasting than IAA

(Murashige and Skoog, 1962). Thus, Salpiglossis shows

 

7

Table 2. Effect of auxins on morphogenesis of
Salpiglossis sinuata L. leaf explants

 

 

Auxin (mg/1) Response
IAA 0.01 No growth

0.1 callus, green very few roots

1.0 callus, green/pink very few roots

10.0 callus, green few roots
2,4-D 0.01 callus pale green

0.1 callus pale green

1.0 callus yellow soft

10.0 callus pale yellow
NAA 0.01 callus green very few roots

0.1 callus few roots

1.0 callus multiple roots

5.0 callus few roots one shoot

10.0 callus pale yellow few roots

 

8
specificity for auxin induced callus growth.

NAA at all levels and IAA at 10.0 mg/l resulted in
roots. Root initiation from leaf explants on MS basal
medium supplemented with IAA or NAA was also observed on
Petunia inflata and P. hybrida (Rao et al., 1973). Root ini-
tiation and elongation is generally promoted by high auxin
levels (Gresshoff, 1978). Rhizogenesis at lower concentra-
tions of NAA may be due to its higher level of activity as
compared to IAA. Vasil and Vasil (1974) reported root
formation at low NAA concentrations for Petunia callus.

All 2,4-D concentrations produced only callus. 2,4-D
is widely accepted to be a suppresser of organ differentia-
tion, especially at high concentrations (Sunderland, 1973;
Gresshoff, 1978). Strong morphogenetic suppression would
also result in the very slow callus growth that was evident
on the leaf explants. In Petunia (Rao et al., 1973), soft
friable callus formed from leaf explants and only occasional
development of roots occurred on MS + 1.0 mg/l 2,4-D; later
embryogenesis occurred but at a suppressed rate compared to
IAA or NAA.

Effect of Benzyladenine + Naphthaleneacetic acid on

Morphogenesis - Some callus formation occurred on all combi-

 

nations of MS + BA + NAA (Table 3). Shoot primordia devel-
oped on 10.0 mg/l BA + 1.0 mg/l NAA or 0.1 mg/l NAA; on 1.0
mg/l BA + 0.1 mg/l NAA resulted in multiple shoot develop-

ment. Salpiglossis leaf segments cultured at similar con-

 

centrations but substituting K for BA gave comparable

9

Table 3. Morphogenetic response of Salpiglossis sinuata
leaf explants cultured on BA and NAA combinations.

 

 

Phytohormones (mg/l) Response

BA + NAA

10 10 nodulated callus, no shoots or roots
10 1.0 callus shoot primordia

10 0.1 callus shoot primordia
1.0 10 callus no shoots or roots
1.0 1.0 callus multiple shoots
1.0 0.1 callus multiple shoots
0.1 10 nodulated callus few roots

0.1 1.0 callus few shoot primordia
0.1 0.1 callus no shoots or root

 

10

results (Lee et al., 1977). The development of shoot
primordia and shoots at these phytohormone levels may be
attributed to the relatively high cytokinin to auxin ratio.

At other levels of both hormones, either roots or
callus alone were produced, resulting in different responses
from low to high concentrations. Callus induced on the com-
binations of MS + BA + NAA.was not superior in growth rate
or quality to that induced on MS + BA or MS + NAA alone.
Engvild (1973) found similar results in Datura. Synergism
between auxin and cytokinin has been observed in tobacco
tissue cultures (Murashige and Skoog, 1962). In this study,
interaction between auxin and cytokinin was not an absolute
requirement for shoot initiation; shoots occurred on the
NAA + BA combinations and also on BA alone (Table 1). Roots
and shoots did not develop on the same concentration of BA
+ NAA. The greatest obstacle in obtaining regenerated

plants from Salpiglossis was in the rooting of shoots. To

 

achieve efficient rooting ten MS based media were tested
(Table 4). In addition, temperature fluctuations from 20°C
to 25°C, rooting directly in an artificial planting mix and

28-1 were utilized.

varying light levels from 11 to 80 uEm-
In Datura the spontaneous tendency for shoot formation
is very pronounced; root formation is difficult (Engvild,

1973). Salpiglossis behaved in a similar manner. Kartha et

 

al. (1976) using Lycgpersicon esculentum shoots found root-

 

ing to occur on MS basal medium without phytohormones. With

Salpiglossis however, rooting did not exceed 40% on this

 

11

Table 4. Root induction of regenerated shoots of Salpiglossis
sinuata in MS basal medium with different addenda.

 

 

 

Medium (mg/l) Light (mm-'23.1 % Rooting
NAA (1.0) Agar (0.35%) 11 20
NAA (0.1) Agar ().35%) 20 30
NAA (0.1) Agar (0.35%) 80 30
No hormones 20°C 3 days to 24°C 20 0
No hormones 20 20
No hormones 80 40
No hormones 1.2% Sucrose 20 33
No hormones 0.5% Sucrose 20 33
No hormones 0.4% Agar 74 33
No hormones 0.4% Agar

20°C, 3 days to 24°C 20 no
2,4-D (5.0) BA (2.5) 15
2.4-0 (5.0) BA (2.5) 7a
Charcoal (1%) NAA (1.0) 20 15
Charcoal (2%) NAA (1.0) 20
Charcoal (1%) 20 0
2,4-D (0.001) 15 25
2,4-D (0.001) 20 40
2,4-D (0.001) 80 75
Soil 80 25

 

The mean percentage rooting values are from 8 plates; all
experiments repeated at least twice.

12

medium (Table 4). With Solanum xanthocarpum, roots

 

developed on levels of 0.2 mg/l and 0.1 mg/l 2,4-D (Rao
and Narayanaswami, 1968). Low levels of 2,4-D (0.001 mg/l)
gave the best rooting (75%) of any test treatment of the
Salpiglossis shoots.

Leopold (1964) suggested root initiation results
when auxin levels increase at potential active meristematic
sites on stems. The inability to readily form roots may be
related in part to some type of abnormal auxin metabolism

or transport. Ahuja and Hagen (1966) cultured Nicotiana

 

tumor tissue and found it unable to root, although growth
was not impaired. They suggested an inability to mobilize
the auxin preperly for the initiation of roots as the
probable cause. Inability to perceive the exogenous auxin
at the auxin receptor site could also be a cause for lack of
rooting.

Generally speaking, the more vigorous Salpiglossis

 

shoots rooted with more ease than less vigorous ones.

There is much heterogeneity in Salpiglossis as is evidenced

 

in the variable flower color and pollination habit (Lee et
al., 1978); this situation may have confounded the rooting

response(s).

13

SUMMARY

Callus and shoots were readily initiated on leaf
explants of Salpiglossis sinuata cultured in_yi§£g. Of
the cytokinins tested, Zip at 1.0 mg/l was most effective
in initiating shoot regeneration on leaf sections and on
callus. Culturing leaf explants on MS with combinations
of BA and NAA did not enhance shoot initiation or develop-
ment above that obtained with BA, Zip or K alone. Rooting
occurred with difficulty which may be attributed to:

1) an inability to mobilize endogenous auxins to the site
of root initiation, or

2) lack of recognition at the receptor site for auxin.

Regenerated plants were grown to flowering in the green-

house and were phenotypically identical to the parental

line.

INTRODUCTION

The potential for crop improvement through protoplast
technology has been widely discussed (Evans and Cocking,
1975; Vasil, 1976; Thomas et al., 1979) and significant
advances have been made in the last decade (Takebe et al.,
1971; Carlson et al., 1973; Hess et al., 1976; Vasil and
Vasil, 1979). These advances in in zitrg methodology and
their eventual application are intended to make significant
contributions in plant breeding. An essential requirement
for the use of protoplast technology is uniform, large
populations of isolated protoplasts which can be cultured
to callus and subsequently regenerated into plants. Leaves
(Durand et al., 1973; Kartha et al., 1976; Shepard and
Totten, 1977; Sink and Power, 1977) and callus (Power and
Berry, 1979; Simmonds et al., 1979; Vasil and Vasil, 1979)
and cell suspension cultures (Grambow et al., 1972; Keller
et al., 1973; Kartha et al., 1974) have all proven to be
good cell sources for protoplasts and have also yielded
regenerated plants.

To date the vast majority of plant species that have
been successfully regenerated from isolated protoplasts are

food, drug and ornamental crops in the Solanaceae family.

 

In Petunia, five species have been regenerated from proto-
plasts (Frearson et al., 1973; Binding, 1974; Hayward and

14

15
Power, 1975; Sink and Power, 1977). Regenerating systems
have also been developed for Atropa (Gosch et al., 1975:

Krumbeigel and Schieder, 1979) Browallia, (Power and Berry,

 

1979) Datura, (Krumbeigel and Schieder, 1979) Lycopersicon,
(Zapata et al., 1976) and Solanum (Binding and Nehls, 1977;
Shepard and Totten, 1977).

A review of the methodologies used in achieving
regeneration with these species clearly indicates that
several prime factors regulate the in gitrg responses.

One of the most important factors in obtaining uniform,
large populations of isolated protoplasts is the physio-
logical condition of the source tissue or plant. The sus-
ceptibility to digestion of the cell wall and the stability
of the protoplasts are highly affected by the physiological
condition prior to isolation (Uchimiya and Murashige, 1974;
Shepard and Totten, 1975). Therefore, temperature and
illumination as well as age and nutrition are parameters
that must be determined experimentally for the species under
study. Enzymatic methods of isolation, while eliminating
the disadvantages inherent in mechanical methods, however,
leave the protoplasts exposed for long periods of time to
a variety of exogenous and endogenous enzymes. Osmotic
potential can have a great deal of influence over the
recovery of viable or nonviable protOplasts (Schenk and
Hildebrandt, 1969). Thus, an assessment of the plasmolysis

2+

as well as pH, presence of Ca , temperature and duration

of the enzyme treatment is essential (Vasil, 1976).

16

Studies on protoplasts isolated from roots, fruits
and leaves have indicated a strong and direct effect of
auxins. Increased uptake of water, expansion and bursting
when auxin is added indicate an effect on the membrane
permeability (Gregory and Cocking, 1965; Vasil, 1976).
Phytohormones, especially cytokinins and auxins, influence
the ability of protoplasts to undergo division (Bui Dang Ha
and MacKenzie, 1973; Evans and Cocking, 1975; Gamborg et al.,
1975). Auxins are exceptional for rhizogenesis, whereas
cytokinins influence initiation of shoots and have some
control of cell division (Haberlach et al., 1978). Media
components, organic and inorganic, also exert control over
the behavior of the cell (Gamborg, 1970; Bui Dang Ha and
_ MacKenzie, 1973; Upadhya, 1975; Shepard and Totten, 1977).
The morphogenetic potential of leaf explants of

Salpiglossis sinuata has been determined. Hughes et al.

 

(1973) derived callus from Salpiglossis anthers which dif-

 

ferentiated into plantlets. Lee et al. (1977), utilizing
Salpiglossis leaf discs and a more extensive range of hor-
mones, reported callus, shoot and root initiation on NAA
and K in various combinations. It was also reported that
leaf age seemed to be a limiting factor in obtaining organo-
genesis. Neither of these studies reported greater than
60% rooting of the regenerated shoots.

Salpiglossis is a promising species for somatic
hybridization; therefore, this investigation was undertaken

to determine the potential for regeneration of protoplasts,

17
isolated from leaves or from callus, through manipulation
of phytohormones, medium constituents and environmental

factors.

MATERIALS AND METHODS

Plants for providing leaf explants as a source of
callus cells for protoplast isolation were obtained from
seeds sown at weekly intervals on artificial planting mix
(V.S.P., Bay Houston Towing Co.) and maintained at 21°C
:ZOC, 64 uEm-25-1 cool white fluorescent tubes (General
Electric) for 16 hr per day. After 2.5 weeks growth the
seedlings were transplanted to artificial planting mix in
plastic cell packs and placed in the greenhouse. Vegetative
growth continued under natural photoperiod supplemented
with a total of 550 UEm-ZS-1 for 16 hr each day. A minimum
night temperature of 22°C:2°C was maintained; daytime tem-
perature fluctuated with the season. The plants were
fertilized three times weekly at a rate of 200 ppm N from
20-20-20. Control of diseases and insects was according to
standard cultural procedures.

Experimental plant material was also obtained from
aseptically germinated seed. Shoots, 7 mm in height, from
axenic cultures were excised from two-week old seedlings
and cultured on Murashige and Skoog (1962) medium (MS) and
Gamborg's BS (Gamborg et al., 1968) in 60x15 mm Petri
dishes. Axenic, single stem shoot cultures were maintained

at 27°C with a 16h photoperiod, Z4 uEmuzs-l1 from cool white

18
fluorescent tubes (General Electric).

Fully expanded leaves were obtained from the lower
portion of seedlings 60 to 90 days after germination. They
were surface sterilized with a 7% commercial sodium hypo-
chlorite solution (5.25% NaOCl) for 30 min followed by six
separate rinses with sterile distilled water. Seeds for
axenic culture were surface sterilized with a 10% sodium
hypochlorite solution for 30 min followed by eight separate
sterile distilled water rinses. These seeds were then
aseptically transferred to modified Murashige and Skoog
(1962) medium in 60x15 mm Petri dishes. Germination pro-
ceeded at 27°C, 24 (mm-'23"1 cool white fluorescent tubes
(General Electric).

Leaves were allowed to become flaccid after sterili-
zation, and the lower epidermis was removed by peeling with
the aid of jewelers' forceps. The exposed surface was
placed down in a 100x15 mm Petri dish containing a cell/
protoplast wash solution (Frearson et al., 1973) of 13%
mannitol at a pH of 5.8. After 0.5 hr the CPW solution was
removed and replaced with a solution of CPW salts and proto-
plast enzymes in various combinations and concentrations
(Table 5). Axenic leaf tissue was removed, aseptically
placed in a 60x15 mm Petri dish; sliced; then covered with
an enzyme solution (Table 6).

Callus for protoplast isolation was gently pressed
through a 35 u sieve while rinsing with a CPW 8% mannitol

solution. The cells were pelleted by centrifuging this

19

Table 5. Enzyme mixtures.tested for the isolation of
Salpiglossis sinuata L. leaf protoplasts.

 

 

 

 

Code Enzyme Components
. v w
LA 2% Meicelase P, 0.1% Macerozyme ,
13% Mannitol, CPW
LC 2% Meicelase P, 0.08% Macerozyme,
13% Mannitol, CPW
LD 2% Meicelase P, 0.06% Macerozyme,
13% Mannitol, CPW
LE 2% Meicelase P, 0.05% Macerozyme,
13% Mannitol, CPW
LB 3% Meicelase P, 0.1% Macerozyme,
13% Mannitol, CPW
LBA 3% Meicelase P, 0.1% Macerozyme,
40mg Penicillianx, lmg Tetracy-
clineY, 1mg Gentamycinz,
13% Mannitol, CPW
v Meiji Seika Kaisha
w Cal Biochem
x Sigma
y Sigma
2 Sigma

Table 6. Enzyme mixtures tested for the isolation of
Salpiglossis sinuata L. protoplasts from axenic

 

shoot cultures.

 

 

Code Enzyme Components
A1 2.5% Driselasey, 4.5% Mannitol,
CPW 2
A2 2.5% Driselase, 0.2% Pectinase ,

4.5% Mannitol, CPW

 

y Kyowa Hakko Kogyo
2 Sigma

20

slurry at 80xg for 2 min. The supernatant was removed and

the cells were resuspended in a test enzyme and CPW solu-

tion for protoplast release (Table 7).

The pH of all enzyme solutions was adjusted to 5.8

and they were sterilized by filtration through a .45 u filter

(Sybron or Satorius).

1)

Z)

3)

Incubation and isolation proceeded in three ways:
Leaf pieces from greenhouse grown material were held
in the enzyme for 16 hr at 27°C in the dark. The enzyme
was removed and replaced with a 21% (w/v) sucrose in
CPW solution, placed in a 125x16 mm screw capped tube
and centrifuged at-100xg for 10 min. The band of proto-
plasts that floated to the surface during centrifugation
were resuspended in test medium.
The leaf pieces from axenic shoot cultures were incu-
bated for 5 hr at 27°C in the dark. The released proto-
plasts were gently pelleted at 80xg for 2 min, the
supernatant removed and the pellet resuspended in a
15% (w/v) sucrose CPW solution. The protoplasts were
separated from undigested cellular material by cen-
trifuging at 100 xg for 10 min. The resultant band
at the solution surface was removed and placed in test
culture medium.
Friable callus was incubated for 5 hr at 27°C on a
rotary shaker at 50 rpm. The enzyme mixture plus
released protoplasts was centrifuged at 80 xg for 2

min, the supernatant removed and replaced with 15%

21

Table 7. Enzymes tested for the isolation of
Salpiglossis sinuata L. callus protoplasts.

 

 

Code Enzyme Components
C 2% Cellulase R-1oz, 1% Macerozyme,
1% Driselase, 8% Mannitol,
CPW
CM 2% Cellulase R-10, 1.4% Macerozyme,
1% Driselase, 8% Mannitol,
CPW
4 1% Driselase, 0.5% Pectinase,
8% Mannitol, CPW
5 1% Driselase, 0.75% Pectinase,
8% Mannitol, CPW
CP 2% Cellulase R-10, 0.75% Pectinase,

1% Driselase, 8% Mannitol,
CPW

 

zKinki Yakult

22
(w/v) sucrose in CPW. Centrifugation proceeded at
100 xg for 10 min; the meniscus of protoplasts was
collected and placed in test culture medium. All
freshly isolated protoplasts were subsequently counted
and diluted to a final density of 2.5x1o‘l to 2.0x105/ml.
The protoplasts were plated as 4 ml in 60x15 mm Petri
dishes at 27°C, 28 ilEm-Zs"1 cool white tubes (General
Electric) under a 16 hr photoperiod.

Media tested for protOplast division included Murashige
and Skoog (1962), Uchimiya and Murashige (1974), a modified
Durand's (1973), Nagata and Takebe (1971), Kao and Michayluk
(1975), and Gamborg's BS (Gamborg et al., 1968). Various
combinations and concentrations of auxins and cytokinins
were added to these media. Mannitol at 9% (w/v) was added
to all liquid medium as the osmotic stabilizer. All water
was double distilled in a Pyrex glass still and all media
were autoclaved at 15 psi for 20 min. Four replicates of
each test media were conducted.

Shoot tips from regenerated protoplasts were dipped
in 1000 ppm indole-butyric acid (IBA) and placed in perlite.
Under intermittent mist in the greenhouse, the shoot tips
rooted in 12 to 14 days with 75% efficiency. Roots 1 to 2
cm long were removed and pre-fixed in a 0.1% solution of
colchicine for 2.5 hr at 23°C in the dark. Fixing occurred
overnight in a 3:1 absolute alcoholzglacial acetic acid

solution, the tips were then stored in 70% ethanol at 4°C.

Hydrolysis was in 1N HCl at 60°C for 11 min. The root tip

23
was smeared and stained with a 1% aceto-carmine solution
(Darlington and LaCour, 1975). The number of root tip cells

counted to establish the chromosome number was 8 to 10.

RESULTS AND DISCUSSION

Of the enzymes tested (Table 5) for protoplast
release from leaf material, LA gave the lowest yield and
concomitant low quality protoplasts as evidenced by their
shrunken condition and emerging vacuoles. Variations of
this enzyme mixture, LC, LD, LE gave better yields yet
digestion was not always complete and the protoplasts were
often damaged. The protoplasts were released but their
vacuoles emerged through the plasma membrane and bursting
occurred frequently.

Utilizing the same level of pre plasmolysis, LB and
LEA gave infinitely better results. LB efficiently con-
verted cells to protoplasts that were spherical with the
chlor0plasts evenly distributed about the cell periphery.
However, bacterial contamination necessitated the use of
antibiotics; enzyme LBA. Protoplasts obtained from this
enzyme treatment were also spherical with even distribution
of the chloroplasts. Within three days chloroplasts had
moved to the center of the cell, cytoplasmic strands were
evident. The antibiotics employed were not sufficient to
overcome the apparent endogenous contamination of green-
house grown leaf cultures. The bacterial contamination

was persistent and could not be controlled by a standard

Z4
antibiotic regime.

Axenic shoot and callus cultures circumvented the
contamination problem, but the former gave very poor proto-
plast yields. Enzyme A1 released a fair amount of spheri-
cal protoPlasts. The yield was much greater though with
enzyme AZ; however, the vacuoles consistently emerged after
24 hr culture, apparently due to pectinase damage of the
protoplasts. Pectinase is known to contain toxic basic
proteins that could cause this problem (Evans and Cocking,
1975).

Callus was subsequently employed as the protoplast
source. Enzyme C (Table 7) yielded spherical protoplasts
with active cytoplasmic streaming; systrophy occurred within
14 hr. However, the released number of protoplasts from
callus was very low. CM improved the yields only slightly.
Enzymes 4 and 5 gave increased yields, but many were dead
(brown in color) and shrunken. The protoplasts that sur-
vived displayed cytoplasmic strands and were spherical. A
combined form of other test enzymes, CP converted callus to
protoplasts at a rate of 6.0x105 from 1 g of callus tissue.

Testing of enzymes resulted in a combination (CP)
that would allow the release of spherical intact proto-
plasts.

The effectiveness of an enzyme mixture may be attrib-
uted to three causes:

1) The callus may superficially appear to be uniform

though it is not necessarily so. Timing of callus

2)

3)

25
subculture has an influence on the effectiveness of the
enzyme (Uchimiya and Murashige, 1974). There are gen-
erally higher yields from rapidly growing cells (Con-
stabel, 1975). Cell walls of many cultured lines may
be resistant to crude cellulase preparations, though
the use of Driselase and Macerozyme often eliminates
this problem (Evans and Cocking, 1975).
The shape of the cell itself and its physical arrange-
ment may also be a factor in the ability of the enzyme(s)
to convert cells to protoplasts. In maize and wheat it
has been suggested that the cells have an interlocking
structure (Evans and Cocking, 1975). It is conceivable
that cells would be specific for types of enzymes as if
it were a key to these cells and others would not be
quite specific enough to work (Raj and Herr, 1970).
The enzyme must be highly specific; without specificity
isolations may be carried out, but at the expense of

the cell. Pectinase is effective in.Lycopersicon fruit

 

(Gregory and Cocking, 1965) and Solanum nigrum (Raj and

 

Herr, 1970). This study indicated the effectiveness of
pectinase on Salpiglossis. Specifically, it carries
polygalaturonase which attacks the middle lamella and
facilitates the separation of the cells. However, it
may also weaken the plasmalemma destroying the integrity
of the cell.

After isolation, the protoplasts were spherical with

peripherally situated chloroplasts. They were cultured in

26
various media (Table 8) that resulted in changes in their
shape, size and organelle distribution. With leaf proto-
plasts, a spherical shape was often retained for several
days. In one medium, P1, the cells remained alive and
spherical for seven days. However, in the majority of the
test media the chloroplasts became polarized, the vacuoles
emerged and burst. Occasional budding did occur; when
observed, the protoplasts did not divide.

Axenic derived leaf protoplasts lived no longer than
four days in any of the test media (Table 9). Within 24 hr
after plating, the chloroplasts became polarized. Shortly
thereafter the cells became brown and shrunken.

Callus protoplasts were tested in 24 media (Table 10).
The majority of these media maintained the protoplasts in a
spherical shape, with even distribution of the chloroplasts.
Withhathree or four days after plating budding often
occurred and the protOplasts rapidly declined. In PM, low
in ammonium and containing organic nitrates, the proto-
plasts clustered together and became reniform. However, in
less than two weeks they had become brown. Only occasionally
did cytoplasmic streaming and systrophy take place. One
test medium, MSG4, did invoke profuse systrophy and cyto-
plasmic streaming (Table 11).

In MSG4 the protoplasts aggregated and became reniform
in two to three days. During this time the cells also
expanded. The extent of this enlargement appeared to depend

upon physiological condition of the callus prior to isolation

27

 

 

 

Table 8. Test media for leaf protoplasts of
Salpiglossis sinuata

Code Hormones

MS basal
P1 2.0 mg/l NAA, 0.5mg/l BA

1.0 mg/l BA

35 5.0 mg/l 2,4-D, 2.0 mg/l BA
A 3.0 mg/l NAA, 0.1 mg/l BA

Gamborg basal

B/S

1.0 mg/l NAA, 1.0 mg/l 2,4-D,
2 mg/l BA

Modified Durand

 

 

 

F/S 2.0 mg/l NAA, 1.0 mg/l BA
Table 9. Test media for protoplasts from axenic shoot
cultures of Salpiglossis sinuata
Code Hormones

 

Modified MS basal medium

GI
GII
GIII

3.0 mg/l NAA, 1.0 mg/l BA
2.0 mg/l NAA, 0.5 mg/l BA
2.0 mg/l IAA, 1.0 mg/l BA

 

zMinus NHuNO plus 250 mg L glutamine, 0.1 mg/l serine,
2.0 mg/l thiamine.

28
and the plasmolyticum used (Bui Dang Ha and MacKenzie, 1973).
The change in shape also indicated the presence of a new
cell wall (Grambow et al., 1972).
The changes in organelle distribution and protoplast

size and shape have been documented in Pisum sativum

 

(Constabel et al., 1973) and many other species (Nagata and
Takebe, 1970; Vasil and Vasil, 1979). Budding and cell wall
regeneration are also well documented (Evans and Cocking,
1975; Vasil, 1976).

Several different combinations of growth hormones were
tested to determine the optimal medium constituents for sus-
tained division of Salpiglossis protoplasts. In no case
did first division occur although the cells remained viable
for up to seven days. With the addition of L-glutamine (250
mg/l) and L-serine (0.1 mg/l) and the deletion of ammonium
nitrate, division did occur and was sustained. The reduc-
tion or deletion of NHuNO3 has proven beneficial in the cul-
ture of potato protoplasts (Upadhya, 1975; Shepard and
Totten, 1977). In asparagus, the addition of L-glutamine
was advantageous to division of protoplasts (Bui Dang Ha
and MacKenzie, 1973). Bayley et al. (1972) demonstrated
that the addition of L-glutamine eliminated the need for
reduced nitrogen in soybean callus. Joshi and Ball (1968)
determined that glutamine and casein hydrolysate in the
medium resulted in substantial increases in growth. How-
ever, both of these studies (Bayley et al., 1972; Joshi and

Ball, 1968) showed that ammonium ions would satisfy any

29

requirement for reduced nitrogen the soybean or peanut cell
required. This need for ammonium ions and the failure to
use nitrate successfully was attributed to a lack of an
efficient nitrate reductase or an interchangeable factor
with glutamine (Bui Dang Ha and MacKenzie, 1973; Bayley et
al., 1972; Joshi and Ball, 1963).

Interchangeability may be questionable. Solanum
tuberosum protoplasts did not divide until the ammonium
ions were toally deleted. This was found to be the case

herein with Salpiglossis. Utilization of the division

 

medium (Table 11) with the addition of NHuNO3 did not result
in division. Further, deletion of glutamine and serine once
NHaNO3 was added did not result in division of the proto-
plasts. Halperin and Witherell (1965) reported ammonium
ions and glutamine not to be interchangeable in embryo
formation.

Ammonium salts may only be inhibitory in the respect
that an organic compound is needed for division to commence.
Gamborg (1970) suggested a reduced organic nitrogen source
such as glutamine can be much more desirable than ammonium
salts. Glutamine may in fact provide a necessary carbon
skeleton in addition to supplying nitrogen (Gamborg, 1970).

The elimination of ammonium ions and addition of

glutamine to the test medium for the Salpiglossis proto-

 

plasts may have enabled the cell to prepare for division by
rapidly synthesizing new proteins and nucleic acids.

The actual stimulation of division is probably

30
facilitated by the phytohormones. In studies on bindweed
(Calystegia sepium) a definite requirement was found for
both auxins and cytokinins for division to begin. However,

work on Nicotiana suggested that only the presence of a

 

cytokinin is necessary for cell division. Added auxin did
increase the rate of division (Evans and Cocking, 1975).

The division medium for Salpiglossis (Table 11) con-

 

tains both an auxin source and a cytokinin. This medium,
duplicated exactly, but substituting NAA for IAA (Table 10)
did not result in division. NAA is generally considered a
much stronger and longer lasting auxin than IAA (Murashige
and Skoog, 1962). NAA in combination with 2,4-D may act
synergestically to completely inactivate the process of cell
division. This may be especially so if there is a high
level of endogenous auxin. High levels of endogenous

auxins have been reported in Solanaceous plants (Kartha et

 

al., 1976). The possibility of a specific auxin receptor
with a higher affinity for IAA than NAA or 2,4-D also exists.
Vreugdenhil et al. (1979) demonstrated the occurrence of a
particle-bound auxin receptor in tobacco pith. They sug-
gested that a similar receptor exists on the outer side of
the plasma membrane of tobacco leaf protoplasts. Auxin
receptors show affinity for different growth regulators
(Vreugenhil et al., 1979). Tobacco, however, has a higher
affinity for IAA than 2,4-D. It is entirely possible that
Salpiglossis callus protoplasts have a higher affinity for

IAA than NAA; thus, stimulating division where NAA did not.

Table 10.

31

Salpiglossis sinuata.

Test media for callus protoplasts of

 

 

Basal medium Hormones
MS basal
P1 2.0 mg/l NAA, 0.5 mg/l BA
PM1 3.0 mg/l NAA, 1.0 mg/l BA
PM3 2.0 mg/l NAA, 1.0 mg/l BA
PM3 3.0 mg/l NAA, 0.5 mg/l BA
35 5.0 mg/l 2,4-D, 0.5 mg/l BA
PD 1.5 mg/l NAA, 0.5 mg/l 2,4-D,
1.0 mg/l BA
CG4 0.5 mg/l IAA, 1.0 mg/l 2,4-D,
0.5 mg/l BA
N26 0.5 mg/l NAA, 1.0 mg/l 2,4-D,
0.5 mg/l BA
Modified MS
GI 3.0 mg/l NAA, 1.0 mg/l BA
GII 2.0 mg/l NAA, 0.5 mg/l BA
GIII 2.0 mg/l IAA, 1.0 mg/l BA
GIV 0.5 mg/l IAA, 1.0 mg/l 2,4-D,
0.5 mg/l BA
GV 0.5 mg/l NAA, 1.0 mg/l 2,4-D,
0.5 mg/l BA
PM 3.0 mg/l NAA, 1.0 mg/l BA
Uchimiya and Murashige
UM 2.0 mg/l 2,4-D, 0.25 mg/l K
UMP 0.6 mg/l NAA, 0.1 mg/l K
Gamborg
BS 2.0 mg/l NAA, 2.0 mg/l 2,4-D,
2.0 mg/l BA
Modified Durand
F5 2.0 mg/l NAA, 1.0 mg/l BA
Kao and Michayluk
KM 1.0 mg/l NAA, 0.2 mg/l 2,4-D,
0.5 mg/l z
Nagata and Takebe
NT 3.0 mg/l NAA, 1.0 mg/l BA

 

32

Table 11. Test medium supporting the division of proto-

plasts from Salpiglossis callus, (mg/l).

 

 

Basal medium

a)

b)

c)
d)
e)

f)

9)

salts;

KNO3 (1700), Mg 304' 7H20 (370), MN sou“ 4H20 (17),
Cusou'SHZO (0.025), 2nsou‘7n 0 (8.6), CaC1 ‘ 2H20 (440).
KI (0.83), CoC12 (0.025), KH

2
PO (170), H BO (6.2),
NazMoOu'ZHZO (0.25)

Iron in a solution of 3.724 g/l of the disodium salt

2

2 4 3 3

of ethylene diaminetetraacetic acid, and 2.784 g/l of

FeSOu°7H20 (10 ml/l)

thiamine HC1 (2.0)

myoinositol (1.0g)

organic nitrate sources
L-glutamine (250) L-serine (0.1)
Hormones

IAA (0.5), 2,4-D (1.0), BA (0.5)
carbon sources

sucrose (30 g/l)

Osmoticum

Mannitol (90 g/l)

 

 

33

During the initial period (48 hr) of culture, approxi-
mately 25% of the isolated protoplasts died. The remaining
protOplasts tended to aggregate and become reniform in
shape and within five days entered first division. The
initial period of incubation prior to division appeared to
vary depending upon the physiological condition of the
callus. In some cases first division was not initiated
until the fourteenth day of culture. Density was also
important in the initiation of division (Evans and Cocking,
1975). Protoplasts plated at 5 x 104 /ml divided much
earlier than those at 2/5 x 10“ /ml. At a density of
2.5 x 104' protoplasts showed cytoplasmic streaming and
systrophy; yet only occasionally divided after ten to
twelve days. At densities of 1.0 x 105 /ml and 2.0 x 105 /ml
the protoplasts remained spherical with even distribution
of the chloroplasts, finally becoming brown and dying. The
plating efficiency of 5.0 x 104 /ml was determined to be
28% whereas 2.5 x 104 /ml was 12%.

Once first division had commenced, second division
was observed in about six days (Figure 1). The cells
became light green in color indicating chlorophyll synthesis
and specialization of the plastids by the twenty-sixth day
of the culture. At this time micro-colonies had formed
(Figure 1).

After 2.5 months growth, colonies of 2 to 3 mm in
diameter were aseptically transferred to shoot regeneration

medium (Table 12) where they continued to grow. Colonies

34

Figure 1. Regeneration of isolated callus protoplasts of
Salpiglossis sinuata. a. Freshly isolated proto-
plasts from 7 day-old callus subcultures. x 200.
b. First division of isolated protoplast, 6 days
after plating. x 125. c. Second division 14 days
after plating. x 125. d. Two-month old cell colony.
x 200. e. Shoots produced from callus on MS +
1.0 mg/l Zip. actual size. f. Adventitious root
initiation on shoots cultured on MS + 0.001 mg/l,
2,4-D. actual size.

35

 

Figure l

36

Table 12. Media for the regeneration of shoots and roots

from callus derived from protoplasts of
Salpiglossis sinuata (mg/l).

 

 

Inorganic salts:

NHuNO3 (1650), KNO3 (1900), mgSOu 7H20 (370), MNSO

4H20 (22.3), BNSOu 4H20 (8.6), CuSOu QHZO (0.025),

CaC1 20 (440), KI (0.83), COC12 6H20 (0.023),

KHzPOu 3 3 2 Noon 2H2
Iron in a solution of 3.724 g/l of EDTA NA
g/l FeSOu 7H20
Organic components:

Nicotinic acid (0.5) pyridoxine HC1 (0.5) thiamine

HC1 (0.1) glycine (2.0) mYo-inositol (100)

u

2 2H

(170), H BO (6.2) NA 0 (0.25)

2 and 2.784

Carbon source
Sucrose 30 g/l
Agar 8 g/l

Hormones 3

Shoots : Zip (1.0)
Roots: 2,4-D (0.001)

 

37

less than 1 mm in diameter generally failed to grow on this
medium when removed from liquid culture. The callus
rapidly increased in size and in twenty-one days shoot
primordia were observed with the aid of a stereoscope.
Growth slowed abruptly at this point; the callus remained
green and friable. Shoots elongated and could be excised
in fourteen more days (Figure 1). Shoots of separate callus
origin were transferred to the root induction medium
(Table 12). This transfer was preceded by a quick dip of
each shoot in 1000 ppm IBA. After placing in the root
induction medium, roots were evident fourteen days in 75%
of the cultures (Figure 1).

Hughes et al. (1973) and Lee et al. (1977) have
shown that phytohormone manipulation in the culture medium

can result in morphogenesis and plantlets in Salpiglossis

 

callus. Regeneration of plants from protoplasts has been
reported in several Solanaceous plants (Frearson et al.,
1973; Binding and Nehls, 1977; Zapata et al., 1946; Shep-
ard and Totten, 1977; Hayward and Power, 1975; Sink and
Power, 1977; Binding, 1974; Nehls, 1978). Regeneration
of plants from protoplasts derived from callus has also
been reported (Bui Dang Ha and MacKenzie, 1973; Power and
Berry, 1979; Vasil and Vasil, 1979).

Regenerated plantlets examined cytologically were
found to have a somatic chromosome number of 2n=44 except
in one case where the chromosome number was determined to

be 88. A normal diploid count is 44 (Darlington and

38
wylie, 1955; Hughes et al., 1973). Instability of callus
in culture has been demonstrated (Evans and Cocking, 1975).
Chromosome aberrations in in yitrg cell cultures are known

to occur, the frequency of which increases with the time

in culture (Sunderland, 1973).

39

RECOMMENDATIONS

The results herein indicate that Salpiglossis sinuata

 

could be a potential parental species in somatic hybridiza-
tion attempts. The success of such experiments could be

improved threefold with respect to Salpiglossis: 1) increas-

 

ing the yield of protoplasts, 2) increasing the plating
efficiency, and 3) minimizing genetic aberration in the
protoplast callus source. The yield of protoplasts may be
increased by testing further combinations of enzymes used in
the isolation step. The use of new enzyme combinations may
unintentionally lead to increased plating efficiency since
protoplast viability is dependent to some extent on the
concentration of enzymes and the length of incubation time.
More extensive use of the initial callus derived from leaf
sections and their early subcultures may serve to Optimize
the results obtained in increasing yield and plating effi-
ciency. Furthermore, this practice should aid in maintain-
ing the genetic stability of the callus cultures and thus
the resultant protoplasts and regenerated plants.

Selection systems are readily apparent for the recov-

ery of heterokaryons using Salpiglossis in fusion experiments

 

based on its limited or non-division in several culture
media. This in gitrg response may be linked to the use

of the alternate parent as a chlorophyll deficient type that
divides. Thus, the ability for protoplast division may be
transferred during fusion to the heterokaryon cells from

the albino parent. The heterokaryon cells would also be

40
assumed to carry chlorophyll synthesis competency from
Salpiglossis and thus be distinguished from the albino
parent cells by their green pigmentation.

Several closely related Solanaceous genera merit

 

consideration for fusion studies with Salpiglossis. These

 

include Browallia, Nicotiana and Petunia, all of which can
presently be regenerated from protoplasts. For some of

them, chlorophyll deficient mutants are also available.

41

SUMMARY

Protoplasts were successfully isolated from leaves and

callus of Salpiglossis sinuata. Callus provided a sterile

 

source of protoplasts and isolation techniques were pursued.
Enzyme containing pectinase was found to be most effective
in converting callus to high quality protoplasts in large
quantities. Effectiveness of the enzyme is attributed to
physiological condition of the callus, specificity of the
cells for certain enzymes and complete degradation of the
middle lamella. Division occurred when protoplasts were
cultured in a modified MS medium containing 250 mg/l
glutamine, 0.1 mg/l serine, 2.0 mg/l thiamine and deleting
NHuNO3. The enhancement of division with glutamine is
well documented (Bayley et al., 1972; Bui Dang Ha and
MacKenzie, 1973), as are the possible toxic effects of
ammonium ions (Shepard and Totten, 1977; Upadhya, 1975).
The division medium also contained IAA, 2,4-D and BA; sub-
stitution of NAA for IAA did not result in division. This
suggests a possible affinity for IAA at the auxin receptor
site. Callus and regenerated shoots occurred within three
months after isolation. The shoots were rooted and the
plants were examined cytologically. One aberrant plant
was found of the ten examined. Instability of callus, from
which the protoplasts were derived; genetic and epigenetic
has been demonstrated (vasil, 1976; Ogihara and Tsunewaki,

1979).

BIBLIOGRAPHY

BIBLIOGRAPHY

Ahuja, M. R., and G. L. Hagen. 1966. Morphogenesis in
Nicotiana debneyitabacum, N. longiflora and their
tumor-forming hybrid derivatives in vitro. 22!.
Biol. 13: 408-423.

 

Baylay, J., J. King and O. L. Gamborg. 1972. The ability
of amino compounds and conditioned medium to alleviate
the reduced nitrogen requirement of soybean cells
grown in suspension culture. Planta 105: 25-32.

Binding, H. 1974. Cell cluster formation of leaf proto-
plasts from axenic cultures of haploid Petunia hybrida
L. Plant Sci. Letters 2: 185-188.

 

Binding, H. and R. Nehls. 1977. Regeneration of Isolated
Protoplasts to plants in Solanum dulcamara L.
z. Pflanzenphysiol 85: 279-280.

 

Bui-Dang-Ha, D. and J. A. MacKenzie. 1973. The division
of protoplasts from Asparagus officinalis L. and their
growth and differentiation. Protoplasma 78: 215-221.

 

Carlson, R. S., H. H. Smith, and R. D. Dearing. 1972.
Parasexual interspecific plant hybridization. Proc.
Nat. Acad. Sci. (USA) 69: 2292-2294.

Constabel, F., J. W. Kirkpatrick and O. L. Gamborg. 1973.
Callus formation from mesophyll protoplasts of Pisum
sativum. Can. J. Bot. 51: 2105-2107.

Constabel, F. 1975. Isolation and culture of Plant Proto-
plasts pp. 19. In: 0. L. Gamborg and L. R. Wetter
(editor), Plant Tissue Culture Methods, National
Research Council of Canada, Saskatoon.

Darlington, C. D. and L. F. La Cour. 6th ed. 1975. The
Handling of Chromosomes, John Wiley and Sons, New York.

Darlington, C. D. and A. P. wylie, 2nd ed. 1955. Chromo-
some Atlas of Flowering Plants. George Allen and

Unwin L. T. D., London.

Durand, J., I. Potrykus, G. Donn, 1973. Plantes issues de
protoplasts de Petunia. Z. Pflanzenphysiol 69: 26-34.

 

42

 

43

Engvild, D. C. 1973. Shoot differentiation in callus
cultures of Datura innoxia. Physiol. Plant. 28:
155-159.

Evans, P. K. and E. C. Cocking. 1975. The techniques of
plant cell culture and somatic cell hybridization.
pp. 127-159. In: H. H. Smith (Editor), New Tech-
niques in Biophysics and Cell Biology vol. 2. The
Garden City Press L. T. D., Letchworth.

Frearson, E. M., J. B. Power and E. C. Cocking. 1973. The
isolation, culture and regeneration of Petunia leaf
protOplasts. Dev. Biol. 33: 130-137.

 

Gamborg, O. L., R. Miller, and K. Ojima, 1968. Nutrient
requirements of suspension cultures of soybean root
cells. Exp. Cell Res. 50: 151-158.

 

Gamborg. O. L. 1970. The effect of amino acids and
ammonium on the growth of plant cells in suspension
cultures. Plant Physiol 45: 373-375.

 

Gamborg, O. L., J. Shyluk, and K. K. Kartha, 1975. Factors
affecting the isolation and callus formation in proto-
plasts from the shoot apices of Pisum sativum L. Plant
Sci. Letters 4 285-292.

 

Gosch, G., Y. P. Bajaj, and J. Reinert, 1975. Isolation,
culture and induction of embryogenesis in protoplasts
from cell suspension of Atropa belladonna. Proto-
plasma 86: 405-410.

 

Grambow, H. J., K. N. Kao, R. A. Miller, and O. L. Gamborg,
1972. Cell division and plant development from proto-

plasts of carrot cell suspension cultures. Planta
103: 348-355.

Gregory, D. W. and E. C. Cocking, 1965. The large scale
isolation of protoplasts from immature tomato fruit.
J. Cell Biol. 24: 143-146.

 

Gresshoff, P., 1978. Phytohormones and growth and differ-
entiation of cells and tissue cultured in vitro pp: 1-
23. In: L. Letham, Goodwin, and S. Higgins (E Editors)
Phytohormones and Related Compounds - A Comprehensive
Treatise. Elsivier /*North-Holland Biomedical Press.

 

 

Guha, S., and S. C. Maheshwari, 1964. In vitro production
of embryos from anthers of Datura. Nature 204: 497.

Gupta, G. R., S. Guha, and S. C. Maheshwari, 1966. Differ-
entiation of buds from leaves of Nicotiana tabacum L.
sterile culture. Phytomorphology 16: 175-182.

 

nu

Haberlach, G., A. D. Budde, L. Sequeira, J. P. Helgeson,
1978. Modification of disease resistance of tobacco
callus tissues by cytokinins. Plant Physiol 62: 522-
525.

 

Halperin, W. and D. F. Witherell, 1964. Adventive embryony
in tissue cultures of the wild carrot, Daucus carota.
Amer. J. Bot. 51: 274-283.

 

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

 

Helgeson, J., 1968. The cytokinins. Science 161: 974-981.

Hess, D., G. Schneider, H. Lorz, and G. Blaick, 1976.
Investigations on the tumor induction in Nicotiana
glauca by pollen transfer of DNA isolated from
N1cot1ana langsdorfii. Z. Pflanzenphysiol. 77:
147-254.

 

Hughes, H., S. Lam and J. Janick, 1973. In vitro culture

of Salpiglossis sinuata L. HortScieHEe 8: 335-336.

 

Joshi, P. and E. Ball, 1968. (Growth of isolated palisade
cells of Arachis hypogaea ig_vitro. Dev. Biol.
17: 308-313.

Kao, K. and M. Michayluk, 1975. Nutritional requirements
for growth of Vicia Hajastana cells and protoplasts
at a very low population density in liquid media.
Planta 126: 105-110.

Kartha, K. K., O. L. Gamborg, J. P. Shyluk, and F. Constabel,
1976. Morphogenetic investigation on in vitro leaf
culture of tomato Lycopersicon esculentum. Mill cv.
Starfire. Z. Pflanphysiol. 77: 292-301.

 

 

Keller, W., B. Harvey, K. Kao, R. Miller, and D. L. Gamborg,
1973. Determination of the frequency of interspecific
protoplast fusion by differential staining. pp. 456-
463. In: J. Tempe (Editor) Protoplast gt Fusion de
Cellules somatique vegetables. Collag. Int. Cent.
Nat. Rech. Sci. 212.

Krumbiegel, G. and A. Schieder, 1979. Selection of somatic
hybrids after fusion of protoplasts from Datura innoxia

Mill. and Atropa belladonna L. Planta 145: 1-5.

 

Lee, C., R. Skirvin, A. Solters, and J. Janick, 1977.
Tissue culture of Salpiglossis sinuata L. from leaf
discs. HortScience 12: 547-549.

 

 

45

Leopold, A. C., 1964. Plant Growth and Development, pp. 466.
McGraw-Hill, New York.

Levine, J., 1950. The growth of normal plant tissue in vitro
as affected by chemical carcinogens and plant SEEstances.
I. The culture of the carrot tap-root meristem. Amer.
J. Botany 37: 445-4491

Maheshwari, P., and B. Baldev, 1962. In vitro induction
of adventive buds from embryos of Cuscuta reflexa
Rosb. pp. 129-138. Plant Embryotogy, a Symposium.
C.S.I.R., New Delhi.

 

 

Murashige, T., 1974. Plant propagation through tissue
cultures. Ann Rev Plant Physiol 25: 135-166.

 

Murashige, T. and F. Skoog, 1962. A revised medium for
rapid growth and bioassays with tobacco tissue culture.
Physiologia Plantarum 15: 473-497.

 

Nagata, T., and I. Takebe, 1970. Cell wall regeneration
and cell division in isolated tobacco mesophyll proto-
plasts on agar medium. Planta 92: 301-308.

Nagata, T., and I. Takebe, 1971. Plating of isolated
tobacco mesophyll protoplasts on agar medium. Planta
99: 12-20.

Nehls, R., 1978. Isolation and regeneration of protoplasts
from Solunum niguim L. Plant Sci. Letters. 12-183-187.

 

Norton, J. P., and W. G. Boll, 1954. Callus and shoot
formation from tobacco roots in vitro. Science 119:
220-221.

~ Ogihara, Y., and K. Tsunewaki, 1979. Tissue culture in
Haworthia. III. Occurrence of callus variants
during subcultures and its mechanism. Japanese
‘g. g: Genetics 54: 271-293.

 

Padmanabhan, V., E. F. Paddock, and W. R. Sharp, 1974.
Plantlet formation Lycopersicon esculentum leaf
callus. Can. J. Bot. 52: 1429-1432.

 

Power, J. B. and S. F. Berry, 1979. Plant regeneration
from protoplasts of Browallia viscosa. Z. Pflanzen-
physiol. 94: 469-471.

 

 

Raj. B. and J. M. Herr, 1970. The isolation of protoplasts
from the placental cells of Solanum nigrum L. '
Protoplasma 69: 291-300.

 

 

46

Rao, P. S., and S. Narayanaswami, 1968. Induced morpho-
genecis in tissue cultures of Solanum xanthocarpum.
Planta 81: 312-378.

 

Rao, P. S., W. Handro, and H. Harada, 1973. Hormonal control
of differentiation of shoots, roots and embryos in
leaf and stem cultures of Petunia inflata and Petunia
hybrida. Physiol. Plantarum. 28: 458-463.

 

 

Schenk, R., and A. Hildebrandt, 1969. Production of proto-
plasts frOm plant cells in liquid culture using puri-
fied commercial cellulases. Crop Sci. 9: 629-631.

Shepard, J., and R. Totten, 1975. Isolation and regenera-
tion of tobacco mesophyll cell protoplasts under low
osmotic conditions. Plant Physiol. 55: 689-694.

Shepard, J., and R. Totten, 1977. Mesophyll cell proto-
plasts of potato, isolation, proliferation and plant
regeneration. Plant Physiol 60: 313-316.

Skoog, F., 1944. Growth and organ formation in tobacco
tissue cultures. Amer. J. Botany 31: 19-22.

Simmonds, J., D. H. Simmonds, and B. G. Cummings, 1979.
Isolation and cultivation of protoplasts from morpho-
genetic callus cultures of Lillium. Can. J. Botany.
57: 512-516.

Sink, K. C., and J. B. Power, 1979. The isolation, culture
and regeneration of leaf protoplasts of Petunia
parviflora Juss. Plant Sci. Letters 10: 335-340.

 

 

Sunderland, N., 1973. Nuclear cytology. _pp. 177-205. In:
H. E. Street (Editor) Plant Tissue and Cell Culture.
Blackwell Scientific, Oxford.

 

 

Takebe, I., G. Labib, and G. Melchers, 1971. Regeneration
of whole plants from isolated mesophyll protoplasts
of tobacco. Naturwissenschaften. 38: 318-320.

 

Thomas, E., P. J. King, and I. Potrykus, 1979. Improvement
of crop plants via single cells in vitro - an assess-
ment. Z. Pflamzunuchtg 82: 1-30.

 

 

Uchimiya, H., and T. Murashige, 1974. Evaluation of para-
meters in isolation of viable protoplasts from cul-
tured tobacco cells. Plant Physiol 54: 936-944.

 

Upadhya, M., 1975. Isolation and culture of mesophyll
protoplasts in potato (Solanum tuberosum L.) Potato
Res 18: 438.445.

 

47

Vasil, I., 1976. The progress, problems and prospects of
plant protoplast research. Advances in Agronomy
28: 119-160. .

Vasil, I., and V. Vasil, 1974. Regeneration of tobacco and
petunia plants from protoplasts and culture of corn
protoplasts. In Vitro 10: 83-96.'

Vasil, V., and I. Vasil, 1979. Isolation and culture of
cereal protoplasts. I. Callus formation from pearl
millet (Pennisetum americum) protoplasts. Z.
Pflanzenphysiol 92: 397-38 .

 

 

Vreugdenhil, D., A. Burgess,-and K. R. Libbenga, 1979.
A particle bound auxin receptor from tobacco pith
callus. Plant Sci. Letters 16: 115-121.

Zapata, F. J., P. K. Evans, J. B. Power, and E. C. Cocking,
1976. The effect of temperature on the division of
leaf protoplasts of Lyc0pe£§icon esculentum and
Lycgpersicon peruvianum. Plant Sci. Letters
8: 119-124. '