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This is to certify that the

thesis entitled

ISOLATION, CULTURE AND REGENERATION
0F LEAF MESOPHYLL PROTOPLASTS
0F SELECTED ORNAMENTAL NICOTIANA SPECIES
presented by

JOAN ELIZABETH PASS I ATORE

has been accepted towards fulfil'lment
of the requirements for

M. S . degree in Horticulture

 

{/3 $25

Major professor

Date October 29, 1980

0-7639

 

 

LNG") 2 IQGI

 

OVERDUE FINES:
25¢ per du per item

RETURNING LIBRARY MATERIALS:

Place In book return to remove
charge from circulation records

 

 

ISOLATION, CULTURE AND REGENERATION
OF LEAF MESOPHYLL PROTOPLASTS

OF SELECTED ORNAMENTAL NICOTIANA SPECIES

 

BY

Joan Elizabeth Passiatore

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

ISOLATION, CULTURE AND REGENERATION OF LEAF MESOPHYLL
PROTOPLASTS OF SELECTED ORNAMENTAL NICOTIANA SPECIES

 

BY

Joan Elizabeth Passiatore

in vitro procedures are described for the regeneration to

whole plants of Nicotiana alata, y; sanderae and E; forgetiana

 

 

from leaf mesophyll protoplasts. Axenic shoot tip cultures

were established on semi-solid modified MS basal medium. A
one-step enzymatic isolation procedure released protoplasts
which were plated in media of various salts and growth regulator
concentrations to test for growth to callus. Cultures were
placed under 4 light regimes (dark, Gro-Lux fluorescent, cool
white fluorescent, dark for 3 days and transfer to Gro-Lux) at
plating densities of 2.5x104, 5.0x104 and 1.0x105 protoplasts/ml
and kept at 25i2°C. Macroscopic calluses were transferred to
regeneration media, where shoots formed. Plantlets were rooted
on MS basal medium lacking growth regulators or with 0.1 mg/liter
NAA. Regenerates were acclimated and transferred to greenhouse
conditions where they flowered. The results indicated that

genetic potential for growth in certain media may be transfer-

red from one or both parents (E; alata, E; forgetiana) to E;

 

sanderae, the hybrid, where it is expressed. Pollen viability
and chromosome number determinations were made. A high degree
of variation in chromosome number was found within root tip

cells of individual regenerated plants.

ACKNOWLEDGEMENTS

Grateful acknowledgement is due to my major professor,
K.C. Sink, and to members of my guidance committee, P.S.
Carlson, J.F. Fobes, and L.W. Mericle, for critically re-
viewing this thesis. I also wish to thank F.J. Zapata for his
many stimulating discussions. I extend thanks to my family
for their loving and unending support, and to Carl, my deep-
est appreciation for providing unfailing support and encourage-

ment in my work and for typing the final copy of the thesis.

ii

TABLE OF CONTENTS

page
List of Tables ..................... . .................... iv
List of Figures ........................................ . v
Introduction ............................................ 1
Materials and Methods
Source of Protoplasts ............................... 5
Preparation of Protoplasts. ............ ... .......... 6
Plating of Protoplasts ..... . ......... ... .......... .. 9
Estimation of Plating Efficiency ........ . ........... 11
Shoot Regeneration ........ . ......................... 12
Rooting and Transfer to the
Greenhouse..... .................................. . 12
Pollen Viability ........ . ........ . .................. 13
Chromosome Determinations ........................... 14
Results and Discussion. ............. . ................ ... 15
Summary ................................................. 33
Recommendations ................. .. ..................... . 35
List of References ...................................... 37

iii

LI ST OF TABLES

 

 

 

 

 

 

page
Enzymes tested for the release of protoplasts

from intact leaf tissue ............................... 7-8
Culture media screened for the support of cell

division and sustained growth of Nicotiana leaf

mesophyll protoplasts ................... . ............. 10
Experimental media tested for shoot regeneration ...... 13
Relative yields of mesophyll protoplasts obtained

from three Nicotiana species .......................... 18
Plating efficiencies (%) of protoplasts of 3 species

of Nicotiana plated at 2.5 x 104, 5.0 x 104 and

1.0 x 105/ml, 28-34 uEm'zs’1 Gro-Lux light ............ 22
Plating efficiencies (%) of 3 species of Nicotiana

in selected media under 4 light regimes; density

5.0 x 104 protoplasts/ml .............................. 25
Culture media promoting division and growth of
protoplasts of 3 Nicotiana species at 5.0 x 10
protoplasts/ml ........................................ 27
Media inducing shoot regeneration from calluses of

3 species of Nicotiana ................................ 28
Mean percent pollen viability as determined by

analine blue staining of pollen from regenerated

and seed-grown Nicotiana species ...................... 31

 

iv

1.

LIST OF FIGURES

Regeneration of leaf mesophyll protoplasts

of Nicotiana alata ............. ....

 

INTRODUCTION

"omnis cellula e cellula"
- Virchow, 1858

The utilization of plant protoplasts to study cell wall
biosynthesis, osmotic stress, virus infection, organelle iso-
lation, intromission of foreign genetic materials, somatic hy-
bridization and other biological phenomena began in the early
1960's (86). The concept of protoplast isolation, however, is
not a recent one. Klercker, in 1892 (85), released protoplasts

of Stratioides aloides by mechanical dissection of plasmolysed

 

cells. Cocking (13) first isolated quantities of protoplasts
from tomato root tips by dissolution of the cell walls with

snail (Helix pomatia) enzyme. Since then, combinations of vari-

 

ous commercial enzymes, primarily pectinases and cellulases,
for protoplast release have been refined (20,41). Hemicellu-
lases (26,41,62,70) and dextran sulfate have also aided in the
liberation of protoplasts from various plant tissues (54,63).
It is generally recognized that age and physiological condition
of the plant material are two of the key factors responsible
for success or failure in achieving a high yield of viable pro-
toplasts which will subsequently undergo division at high fre-
quencies. A systematic determination of enzyme combinations

and concentrations, pH and osmotic pressure of the solution,

2
rate of agitation, incubation period and light and temperature
regimes must be examined for each species studied. These para-
meters also govern the quality and quantity of protoplasts ob-
tained (80,82).

Leaf mesophyll cells offer an ideal cell source of pro-
toplasts for biological investigations since large, homogeneous
populations of identical genetic composition are readily and
inexpensively obtained (10). Mesophyll protoplasts can be
plated at high efficiencies and are very uniform with respect
to chromosome number at the time of isolation. In contrast,
protoplasts derived from callus cells often have gross chromo-
somal aberrations (4,57).

To date, species of the Solanaceae are most amenable to

 

protoplast isolation and subsequent culture. Nagata and Takebe
(56) first reported cell division in cultured tobacco protoplasts;

henceforth, Nicotiana protoplast systems have been extensively

 

studied and refined (l,5,32,54,57,58,74,84), and regeneration
to whole plants has been accomplished in many species. Petunia

(24,35,76), Salpiglossis (8), Lycopersicon (89), Browallia (65),

 

 

 

Solanum (75), Datura (68,69) and Atropa (33,46) are other
Solanaceous crops which have been cultured to plants from iso-
lated protoplasts.

Aside from the favored use of greenhouse-grown seed-
lings for isolating tobacco mesophyll protoplasts, a number of
other cell sources have been found satisfactory. Protoplasts
have been isolated from axenic shoot tips or nodal cultures

(4,17,22,31,32,72) as well as from callus and cell suspension

3
cultures (8,47,62,65,82). The advantages of sterile shoot
cultures are based on the physiological uniformity of the
leaves as a consequence of culture in a controlled environment
and growth medium, of the rejuvenation at each transfer, and by
absence of contaminating organisms and, thus, the need to dis-
infect material by exposure to toxic agents (4).

Several techniques are used for the culture of isolated
protoplasts. Most commonly, protoplasts are diluted to an op-
timal plating density in several standard or modified liquid
media. Plating protoplasts in a low concentration of agar
nutrient medium (soft agar) has also proven successful (6,79,88).
An overlay of liquid medium containing the protoplasts on agar-
solidified medium is reported to promote division and subsequent
colony formation of several species (31,57,79). Microdroplets,
where single or few protoplasts are plated in very small vol-
umes of liquid medium, allow the observation of cell colony
formation from single cells and , thus, allow the establishment
of clonal plants from single protoplasts (32). Different nu-
trient media have been successfully employed for protoplast cul—
ture. Specific requirements for vitamins, salt concentration
and organic addenda are observed among species; however, an
absolute requirement of an exogenous auxin and cytokinin for in-
ducing and sustaining cell division exists (49,56,83,88).

Few protoplasts are found to divide in the absence of nitrogen.
Cell wall and cleavage divisions occur when NHZ, high urea con—

centrations, and N03 + glutamine are included in the culture

medium as the nitrogen source (50).

4
Much documentation of plant regeneration from isolated

protoplasts of various Nicotiana species now exists. Vasil,

 

gt al., (85) list some species which have been regenerated

including haploid E; alata (6), E; otophora, E; sylvestris x

 

E; otophora, and N; tabacum x‘fl; otophora (1), y; debneyi (32),

E; sylvestris (1,7,58), haploid E; sylvestris (17,22), E;

 

 

tabacum (57,79), haploid g; tabacum (61), E; rustica (31), g;

plumbaginifolia (30) and E; acuminata, diploid N; alata, E;

 

 

glauca, E; langsdorffii, E; longiflora, g; paniculata and E;

 

 

 

suaveolens (5).

 

Successful regeneration of protoplasts into whole plants
free from genetic aberrations is prerequisite for determining
the consequences of transferring genetic materials, foreign
particles, or use in somatic hybridization. The investigation
reported herein describes regeneration systems for three dip-

loid species of Nicotiana:

 

1) N; alata (2n=18) (Link and Otto)

2) E; forgetiana (2n=18) (Hort, ex. Hemsley)

 

and their hybrid,
3) fl; sanderae (2n=18).

N; alata and E; forgetiana are believed to have evolved

 

through aneuploidy and amphiploidy following chromosome dele-
tion from earlier species possessing 12 chromosome pairs (78).
These species hold potential as parents in future somatic hybrid-

izations between other Nicotiana species and ornamental genera

 

within the Solanaceae, thereby extending genetic variability as

 

a means toward improvement of these ornamental species.

MATERIALS AND METHODS

Source of Protoplasts

 

Seeds of N; alata cv 'Sensation Mixed' were obtained from
the Joseph Harris Co., Inc., Rochester , NY; E; sanderae and

E; forgetiana were supplied by L.G. Burk, USDA-SEA Tobacco

 

Research Laboratory, Oxford, NC. Seeds were surface sterilized
by soaking in a 5% solution of diluted commercial Clorox

(5.25% NaOCl) for 20-25 minutes, followed by 3 sterile distilled
water rinses. They were sown on Murashige and Skoog(53) basal
medium (MS) supplemented with (mg/liter): folic acid 0.001;
kinetin (6-furfurylaminopurine) (K) 0.03; indole-B—acetic acid
(IAA) 0.00875; sucrose 3.0%; agar 0.8%, pH 5.8 (before auto-
claving) in 60x15 mm Petri dishes and wrapped with Parafilm(R)
for germination. The dishes were kept at 25:2°C under 28-34
uEm-zs-1 (400-700 nm) cool white fluorescent light on a 16 hr
photoperiod (without supplemental incandescent lighting). Ger-
mination occurred in 10-12 days and in 1 month the seedlings

were transferred to modified MS medium in 100x80 mm Petri dishes
for single stem growth and leaf expansion. Seedlings of N; alata
were placed into MS + (mg/l) IAA 2.0; 6-benzylaminopurine (6-BAP)
1.0; agar 0.8% in 60x15 mm Petri dishes and kept under 15 uEm"Zs-1
at 2512°C on a 16 hr photoperiod. Three seedlings each of E;

sanderae and E; forgetiana were placed into modified MS, half

 

nitrate concentration, sucrose 2.5%, agar 1.0%, pH 5.8 in 100x80

5

6

2s“1(4oo-7oo nm) Gro-

mm Petri dishes and kept under 28-34 uEm'
Lux fluorescent lamps. Seedling leaves were of sufficient

size (2.5-3.0x1.0-2.0 cm) for protoplast isolation approximately
45 days after transfer. After the leaves were excised, the shoot
tips were recultured. These recultured shoot tips produced
adequate size leaves for protoplast isolations after 30 days.

Three such reinnoculations of the shoot tips were possible within

the same vessel before the nutrients became depleted.

Preparation of Protoplasts

 

Table 1 lists the enzyme combinations that were tested
for optimal release of viable leaf mesophyll protoplasts from
the shoot tip cultures. Enzyme solutions were adjusted to pH
5.8 with 0.2N NaOH or 0.1N HCl before being sterilized (0.45um
Nalgene [Sybron] disposable filters). Leaves were excised and
placed in 100x15 mm plastic Petri dishes containing the test
enzyme (1 gm leaf tissue per 10 ml solution). Leaves were
sliced outwardly from the midrib to their margin at 1 mm in-
crements with a scalpel blade while holding the entire leaf in

(R)

the enzyme solution. Each dish was sealed with Parafilm and
statically incubated for a 3-5 hr period at 26-30°C. Gentle
teasing with a Pasteur pipette aided the release of protoplasts
from the digested leaf tissue. Protoplasts suspended in the
enzyme solution were passed through a 61um sieve to remove large

pieces of debris. The protoplast mixture was transferred to

16x125 mm culture tubes and gently pelleted by centrifugation

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m.m o.m o.N m.m m.N m.m m.N m.N

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powwow

mmfihncm .H magma

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<mD ..ou HMUHEmnu msmwm I mmmcwuoom

amass ..cuq ..oo .mmz pasxmw excflx I oHIm .mxsuoco.ummmasaflmo
<mD .Eonooflnamu I calmnmESNoumomz

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"moousom HmoHEmsu 0cm mfimucme

 

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mnemasm
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.I.o.ucoo. H magma

9
(100 x g; 8 min). The supernatant was decanted and replaced
with CPW salts + 15% sucrose (24) and protoplasts were separated
by centrifuging (80 x g; 5 min).
The procedures utilized for E; alata protoplast isolation

were slightly modified for E; sanderae and E; forgetiana. Re-

 

leased protoplasts of E; sanderae and E; forgetiana were pelleted

 

at 80 x g for 7 minutes, the supernatant was decanted and the
protoplasts were resuspended in CPW salts + 15% sucrose. Cen-
trifugation at 80 x g for 7 minutes produced a band of viable
protoplasts at the solution surface. Routinely, 2 washes with
CPW salts were used to further remove traces of the enzyme
solution. Finally, protoplasts were collected at the surface
with a Pasteur pipette and suspended in 10 ml of test culture
medium. A small aliquot was removed and counted on a haemo-
cytometer. Subsequently, the protoplast solution was diluted

4 4 S

to test densities of 2.5x10 , 5.0x10 and 1.0x10 protoplasts/m1

and dispensed as 3 ml per 60x15 mm plastic Petri dish and wrapped

with Parafilm(R).

Plating of Protoplasts

 

Growth regulators (Table 2) were tested in combination with
5 basal salt and vitamin media (24,27,53,57,82) employed for sup-
port of protoplast division and sustained growth. Growth regu-
lators were autoclaved in the medium (pH 5.8) at a pressure of

1.46 kg/cm2

(121°C), for 20 minutes. Osmotic pressure was main-
tained with 4.5% (0.25M) mannitol. In all eXperiments the proto-
plasts were plated in liquid test media; in some they were also

plated in media + agar. Plating in soft agar was done by using

10

Table 2. Culture media screened for the support of cell
division and sustained growth of Nicotiana leaf
mesophyll protoplasts.

 

 

 

Basal medium Growth regulators (mg/liter)
K IAA NAA** 2,4-D** 6-BAP

MS (53)

p1 — - 2.0 - 0.5

pD - - 1.5 0.5 1.0

35 - — 5.0 - 0.5
MS-G*

I - - 3.0 - l 0

II - — 2.0 - 0.5

III - 2.0 - - 1.0

IV - 0.5 - 1.0 0.5

V - - 0.5 1.0 0.5
F5 (24) - - 2.0 - 1.0
NT (57) - - 3.0 - 1.0
UM (82) 0.1 - 0.6 - -
8-5 (27)

p1 - - 2.0 - 0-5

R5 - - — 1.0 0.5

 

*MS-G = MS salts + 1.09 meso-inositol; 2.0 mg/l thiamine-HCl;
250.0 mg/l L-glutamine; 0.1 mg/l L-serine; 3.0% sucrose,
pH 5.7

**NAA = alpha-napthaleneacetic acid

***2,4-D = 2,4-dichlorophenoxyaceticacid

11

culture medium containing 1.0% agar held at 45°C until plating.
Isolated protoplasts were suspended at twice the desired final
plating density in 10 ml of the same liquid culture medium and
the two phases were mixed at equal volumes to obtain a final
density of 5.0x104 or 2.5x104 protoplasts/ml. After plating, the
cultures were placed under 4 test light regimes at 2512°C:

1) dark (D)

2) cool white fluorescent light, 18-22 uEmmzs-1 (R)

(without supplemental incandescent)
3) Gro-Lux fluorescent light, 28-34 uEm-zs-1 (G)

4) dark, transferred after 3 days to Gro-Lux
fluorescent light, 28-34 uEm-Zs-l (D to G).

A Licor 185 (sensor LI-190) was used to measure uEm-Zs-1 (400—

700nm) values. Cultures were examined daily under a Nikon invert-
ed micrOSCOpe and the viability and division assessed. Reduction
of osmoticum from 4.5% mannitol was accomplished by adding 1 ml
of culture medium containing 2.25% or 0% mannitol to each Petri
dish every 2 weeks in the case of N; 31333 and E; sanderae and

1 to 1% weeks for E; forgetiana. For exceptionally high plating

 

efficiencies, half of the protoplast solution was transferred
to a separate Petri dish after 3 weeks. Experiments were dupli-
cated at least 2 times, with a minimum of 6 Petri dishes per

experiment.

Estimation of Plating Efficiency

 

In order to compare the efficacy of each test culture
medium on the promotion of cell division and growth to callus,

approximate plating efficiencies were calculated. Plating

12
efficiency (P.E.) was defined as the percentage of viable
protoplasts which formed cell colonies after 21 days in culture.
Three days after intial plating, the number of viable cells was
counted. Small cell colonies were observed after 21 days; thereby
enabling the percentage of viable cells forming colonies to be

determined:

_ number of cell colonies

P'E' - number of colonies + number of live cells

x 100.

 

In some cases, protoplasts were plated in soft agar to permit a

more accurate determination of the plating efficiency.

Shoot Regeneration

 

Macroscopic calluses, approximately 2 mm in diameter, were
transferred to solidified shoot regeneration media. Table 3
lists the media tested for shoot proliferation of the 3 species.
All cultures were maintained at a constant 25i2°C and 15-20
uEm-Zs-1 (400-700 nm) cool white fluorescent lamps on a 16 hr

photoperiod. Regenerated plantlets 1 cm or taller were separated

from the callus and transferred to medium for rhizogenic induction.

Rooting and Transfer to the Greenhouse

 

Regenerated shoots were transferred to MS medium plus
3.0% sucrose, 0.8% agar, pH 5.8 or 3.0% sucrose, 0.1 mg/l NAA
and 0.35% agar to induce root formation. Rooted plantlets were
transferred to VSP soil-less planting medium and covered with

clear polyethylene to maintain humidity. Plantlets were kept

13

Table 3. Experimental media tested for shoot regeneration.

 

 

Species Growth regulators (mg/liter)*

IAA NAA 6-BAP Zeatin
(a,s,f) 2.0 - 1.0 -
(a) - 2.0 0.5 -
(a,s,f) - - - 1.0
(a,f) - 1.0 1.0 -
(a) - 0.1 2.0 -

 

*Basal medium = MS salts + vitamins, 3.0% sucrose, 0.8% agar

 

 

and pH 5.8.
a — N_ alata
s - N; sanderae
f - N_ forgetiana
under 74-84 uEm-Zs"1 (400-700 nm) cool white fluorescent lamps

on a 16 hr photoperiod and gradually exposed to ambient relative
humidity. Plantlets were acclimated in 10-20 days and transferred
to the greenhouse where, under standard cultural and fertilization

practices, they flowered.

Pollen Viability

 

The percentage of viable pollen was established for the
regenerated plants and compared to that of seed-grown plants

of the three Nicotiana species. Pollen from flowers at an-

 

thesis was collected and placed on a 3x1 inch glass microscope
slide and a drop of analine blue stain was added. After 20

minutes, examination under a Nikon Labophot microscope allowed an

14
accurate count of viable pollen grains. A minimum of 300

pollen grains from each regenerated plant was counted.

Chromosome Determinations

 

Cuttings of the 3 species were rooted in sand under
intermittent mist. Root tips were collected and treated for
16 hours with 0.1% colchicine at 25°C in the dark. Subsequent-
ly, root tips were washed, fixed in 3:1 absolute alcohol to
glacial acetic acid (v/v) overnight at 4°C, washed again and
stored in 70% ethanol. The tips were squashed in 1.0% aceto-

orcein for cytological observation (16).

RESULTS AND DISCUSSION

The isolation and culture methods developed in this
study enabled the liberation of large quantities of intact
mesophyll protoplasts, and their subsequent cell division and
regeneration to whole plants (Figure 1). Of the enzyme combina-
tions tested for protOplast release (Table 1), A, B, C and D all
containing 2.5% Driselase + 4.0 to 4.7% mannitol or sorbitol

5 6

in CPW salts solution released between 8.0x10 and 1.5x10

protoplasts per gram fresh weight of leaf tissue from E;.§l§£§'
Enzyme mixture L containing 1.0% Driselase, 1.0% Macerozyme-R-IO,
1.0% Cellulase 'Onozuka" R-10, 0.5% potassium dextran sulfate and
4.0% mannitol in CPW salts solution was found most effective,

5 5 5

releasing 7.6x10 to 8.5x10 and 4.0x10 to 1.0x106 protoplasts

per gram of leaf tissue from N; sanderae and N; forgetiana,

 

respectively (Table 4). Since shoot tips were grown under
sterile culture conditions and common isolation procedures were
followed, the range of protoplast yields found within each species
may be attributed to the variability that exists among the tissues
(48). The majority of liberated protoplasts were derived from
palisade and mesophyll cells and some from epidermal cells of
the leaf tissue directly exposed to the enzyme solution.

The majority of protoplasts were spherical and their

chloroplasts were evenly distributed at the periphery of the

15

16

Figure 1. Regeneration of leaf mesophyll
protoplasts of Nicotiana alata.
a. Freshly isolated protoplasts (390x).
b. First cell division, 3 days after
plating (1500x). c. Second division,
approx. 10 days (1500x). d. Small cell
colony stage after 21 days (352x). e.
Callus obtained after 4-5 weeks (320x).
f. Shoot regeneration from callus ob-
tained after 2 months, actual size. 9.
Rooted plantlet, actual size. h. Small
plants ready for transfer to greenhouse
conditions after approx. 4 months, actual
Size.

 

18

Table 4. Relative yields of mesophyll protoplasts obtained
from three Nicotiana species.

 

 

 

Species Enzyme Average protoplast yield
solution per g fresh leaf tissue
5 6
N; alata A-D 8.0x10 - 1.5x10
N; sanderae L 7.6x105 - 8.5x10S
N; forgetiana L 4.0x105 - 1.0x106

 

 

plasma membrane. By staining with fluorescein diacetate (FDA)
the integrity of the plasmalemma was confirmed, and thus, pro-
toplast viability (42). Damaged protoplasts appeared broken,
collapsed and had irregular or severely polarized chloroplast
configurations. They also did not fluoresce with FDA but
rather stained red.

A 3-5 hr leaf incubation period in a solution of high
enzyme concentrations resulted in less damage to protoplasts,
as determined by visual assessment, than did tissue incubated
for longer durations using an enzyme mixture of lower concen-
trations. Thus, a short incubation period was preferred, de-
creasing the toxic effects often resultant after long periods
of enzyme exposure (11).

The polyanionic structure of potassium dextran sulfate
in mixture L exerted a protective effect on the plasmalemmae
and aided in increasing the number of intact protoplasts of

higher quality as revealed by FDA staining (14,63). Takebe

19

and coworkers (1968) suggested that Macerozyme contains some
basic protein(s) toxic to tobacco cells and that dextran
sulfate protects the protoplasts from damage by blocking their
action. Polyanions have been found to electrostatically bind
to proteins and thus inhibit the enzymatic activity of various
basic proteins (3).

Plasmolysing of leaf tissue prior to enzyme treatment
was found not to influence the quality or quantity of proto-
plasts released and was, therefore, discontinued. Tobacco
and soybean mesophyll cells isolated from greenhouse-grown
leaf tissue were obtained without preplasmolysis but 0.6 or
0.3 M sorbitol was included in the maceration solution (12,73).
High photosynthetic rates were measured and thus, exposure to
a plasmolysing agent appears not to be a general requirement
for the isolation of metabolically active mesophyll cells.

A common developmental sequence was observed in proto-
plasts following isolation. Similar patterns have been re-
ported in other plant species (2,22,24,28,29,39,56,61,64,71).
Twenty-four hours after plating, protoplasts became slightly
enlarged and chloroplasts were polarized toward one end or
rearranged unevenly throughout the cytoplasm. Subsequently,
cells became ovoid and chloroplasts appeared to decrease in
number. Changes in shape were indicative of cell wall refor-
mation (64) and evidence of cytOplasmic streaming and chloro-
plast degradation suggested the beginnings of cellular dedif-
ferentiation. Seventy-two hours after plating, systrophy, a

rosetting of chloroplasts around the nucleus occurred, cells

20
became reniform and first divisions occurred.

Precise determination of plating efficiency was
complicated by strong.aggregation afgnon-dissociable proto-
plast clumps in the culture medium; thus, plating efficien-
cies could only be approximated (38,64,74). Clumping was
possibly due to the presence of calcium ions from CaC12°2HZO
which neutralizes the net negative surface charge on proto—
plasts by obliterating the electrostatic repulsion. Thus,
aggregation occurred through attractive and constant van
der Waals forces (55). The least aggregation was observed

in B5 medium (150 mg/l CaCl ~2HZO) and the greatest amount

2
was observed in MS, MS-G, UM (440 mg/l) and F5 (850 mg/l)
media.

Protoplast survival ranged from 47-78% 3 days after
plating, depending on the culture medium employed. Visual
observations indicated a gradual decline in viability until
the time plating efficiencies were determined. Budding and
release of cytoplasm occurred in many cells followed by
cytolysis. Starch accumulation was evident in aging leaf
protoplasts which failed to divide. Dividing cells rapidly
formed small colonies (20—30 cells) in approximately 21 days.
Colonies subsequently produced calluses. A small percentage
of colonies suffered osmotic shock upon mannitol reduction,

became necrotic and died. Shepard and Totten (74) suggested

that an unreasonably high uptake of organic and inorganic

21

substances resulting from the reduced osmoticum produced a
toxic effect on potato colonies. Ribonuclease levels within
the cells are increased when cells suffer osmotic shock and
thus, RNA degradation is thought to occur under conditions
of lower mannitol concentrations (43); hence, injuring
colonies. A priori, plating efficiencies of the 3 species
at various densities (Table 5) indicated a slight increase
4 5

in percent plating efficiency from 5.0x10 to 1.0x10 pro-

toplasts/ml for N; sanderae and N; forgetiana. Plating

 

efficiency of N; alata, however, decreased at the higher
density. The diffusion of beneficial chemical substances
throughout the medium, released by the protoplasts and pre-
sumed to be necessary for division and growth, possibly be-
came deficient when cell densities were low (32,57). At
2.5x104 protoplasts/ml, salts concentration may be sufficient-
ly high such that toxic effects to the protoplasts occur (74).
Plating protoplasts at a high density (1.0x105) had a posi-
tive effect on growth. Initial divisions occurred at higher
frequencies, approximately 15% greater, but division rate
decreased once cells reached a critical density. Beneficial
metabolites are rapidly produced and diffuse throughout the
medium promoting cell division, but cell density soon becomes
the limiting factor. In three weeks, protoplast cultures
were divided in half and fresh medium with a reduced osmo-
ticum added in order to maintain the growth rate and mini-
mize the loss of small colonies. Gleba (32) reported that

preculturing protoplasts for 1-3 days in high density

22

Table 5. Plating efficiencies (%) of protoplasts of 3 species
of Nicotiana plated at 2.5 x 104, 5.0 x 104 and
1.0 x 105/ml, 28-34 uEm-Zs-l Gro-Lux light.

 

 

 

 

 

 

 

Species Medium Density
2.5 x 104 5.0 x 104 1.0 x 105

meal—ate

MS P1 0 32 31

NT 0 37 34
N; sanderae

MS P1 0 32 36

NT 0 30 32
N; forgetiana

MS Pl 0 13 17

NT 0 33 34

 

suspensions of 1.0 x 104 - 1.0 x 105 protoplasts/ml signifi-
cantly improved the plating efficiency of tobacco protoplasts.
Preculturing protoplasts in a "conditioned" medium could sup-
plant the need for nutrient-enriched medium. A density of
5.0 x 104 protoplasts/ml permitted a constant growth rate
throughout the early stages of development and could be con-
sidered the standard recommendation for these 3 species.

A high propensity toward budding and cell lysis with
concomitant extrusion of cytoplasm occurred with protOplasts
plated in nutrient agar medium under all light regimes. Takebe,

33 al., (79) found that most mesophyll protoplasts

23

deteriorated within several days when plated in agar. Frequency
of first division decreased compared to those plated in liquid
medium (61). Caboche (9) suggested that the agar source be con-
sidered as an important factor in the successful growth of pro-
toplasts. Unpurified agar, the type used in this study, may
adsorb certain toxic components of the medium and thereby be
unsuited for this use, which may account for the inhibited
growth response. Protoplasts survived for several days during
which they followed a similar developmental pattern as described
for those in liquid medium. Success was obtained by plating in
agar in only one experiment. N; sanderae plated in G-II medium
+ 0.5% agar rapidly entered first division under D and G light
conditions. The most marked growth occurred after dark cultures
were placed under G conditions; within 15 days after plating,
visible colonies had formed. Meyer and Abel (49,50) found de-
velopment of colonies to be more frequent if the medium was
solidified with agar. Cells grown in agar medium tended to
radiate laterally from the callus mass; whereas cells in liquid
culture were more compact. The slower diffusion of beneficial
compounds among the protoplasts plated in agar may explain the
overall failure to obtain protoplast growth in soft agar. Agar
concentrations employed may act as a physical constraint on the
capability of cells to divide and inhibit sufficient oxygenation
which may also influence their ability to grow. Agar purity is
another factor which should be considered when culturing in
semi-solid media.

Dark—grown protoplast cultures were precocious in first

division in all cases; however, light-grown cultures soon at-

24
tained equal division rates. Cell wall regeneration of

Convolvulus protoplasts was reported to occur more rapidly under

 

dark conditions (36). The fact that cell wall regeneration is a
prerequisite for cell cleavage divisions (49,71) may explain the
more rapid induction of division in the dark. Cultures kept in
the dark retained their bright green color approximately 3-5 days
longer than those exposed to light due to accelerated chlorophyll
degradation.

Cultures exposed to Gro-Lux irradience were found to di-
vide 1-2 days earlier than cultures exposed to the other light
sources. Rapid growth ensued after first division and plating
efficiencies were slightly increased over most D and R con-
ditions when cultures were kept under Gro-Lux fluorescent light
(Table 6). Beneficial responses have been reported for tobacco
protoplast cultures using Gro-Lux light at a constant exposure
of 1000 lux (83). Cool white fluorescent light at 18-22 uEm-Zs-1
(R) generally gave poorer responses in terms of division rate

and frequencies, over D and G light regimes, a priori (Table 6).

However, R was found promotive for N; forgetiana protoplasts

 

cultured in NT medium a slightly higher plating efficiency was
observed (Table 6).

Transferring cultures from the dark to Gro-Lux light con-
ditions (G) after 3 days was tried with N; sanderae based on
previous findings (6,46,48,58). Exposure to dim illumination for
48 hr after isolation has been shown to increase plating ef-

ficiency of Nicotiana tabacum cv 'Samsun' (18). A priori, the

 

D to G transfer regime increased plating efficiency above R

conditions; however, it did not increase plating efficiency above

25

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26
either D or G conditions.
Of the 13 salts and growth regulator combinations tested
for support of cell division and growth (Table 2), only 4 were

successful (Table 7). All 3 Nicotiana species divided and pro-

 

duced callus in NT and MS P1 media. MS G-I only supported the
growth to callus of E; alata; MS G—II promoted division and
growth of E; sanderae.

MS G-I medium contained the same growth regulator levels

as in NT medium and the same salts as MS P differing from

1’

MS P by including 1.0g meso-inositol, 2.0 mg/l thiamine~HCl,

1
250.0 mg/l L-glutamine and 0.1 mg/l L-serine and lacking NH4NO3.

E; sanderae and g; forgetiana remained viable for 7-10 days in

 

MS G-I, but did not divide.
MS G—II contains the same salts and vitamins as MS G-I

and the growth regulators of MS P MS G-II permitted cell

1.
division of E; sanderae; E; alata and E; forgetiana protoplasts

 

remained healthy in MS G-II but failed to divide. The cells
declined in vigor and eventually died following strong aggrega-
tion which began 2 weeks after plating. Thus, it appears that
the ability to tolerate lower ammonium levels and the amino
acids, glutamine and serine, in MS G-I medium was contributed to
the hybrid, E; sanderae, by E; alata. The capability of the hy—
brid, E; sanderae, to grow in MS G-II medium whereas neither of
its parents did, suggests that complementation occurred in the
hybrid which permitted division and growth under those condi-
tions. Izhar and Power (37) proposed that several genes are

responsible for and exert control over differential responses

27

Table 7. Culture media promoting division and growth of
protoplasts of 3 Nicotiana species at 5.0x10

 

 

 

 

protoplasts/ml.
Medium Species (% P.E.)
N; alata N; sanderae N; forgetiana
MS P1 32 32 13
MS G-I 15 - -
NT 37 30 33
MS G-II - 32 -

 

to growth factors; that is, the progressive stages of proto-
plast development in vitro could be under control of separate
genes. Genetic complementation of the parental species in the
hybrid could activate all these required genes and allow a posi-
tive growth response. Izhar and Power (37) found that in all
cases, F1 hybrids were superior to the parental lines of Petunia
with regard to the range of growth regulator combinations that
permitted growth. The hybrid species, N; sanderae, was respon-
sive in the greatest number of test media and also exhibited the
highest plating efficiency. The fact that neither N; alata nor

N; forgetiana protoplasts divided in G-II medium, yet they

 

retained the potential to complement when the hybrid grew on
G-II, implies that a separate genetic control for each stage of
protoplast development exists. Possibly, by overcoming the meta-
bolic block for first division, genetic potential for continued

division is expressed.

28

Table 8. Media inducing shoot regeneration from calluses of
3 species of Nicotiana.

 

 

 

 

Medium Species
N; alata N; sanderae N; forgetiana
A. MS + +++ +++ +++

1.0 mg/l zeatin

B. Ms + 2.0 mg/l +++ +++ , +
IAA; 1.0 mg/l
6-BAP

C. MS + 2.0 mg/l ++
NAA; 0.5 mg/l
6-BAP

D. MS + 1.0 mg/l ++ +
NAA: 1.0 mg/l
6-BAP

E. MS + 0.1 mg/l +
NAA; 2.0 mg/l
6-BAP

 

+++ prolific shoot regeneration, 5-10+ shoots/callus
++ sparce shooting, 3-5 shoots/callus
+ very sparce shooting, 1-2 shoots/callus

Regeneration media (Table 8) were selected on the basis
of results obtained from earlier experiments employing callus

derived from leaf sections of the 3 Nicotiana species. Shoot

 

regeneration of protoplast-derived callus occurred for the 3
species on media A and B (Table 8). fig priori, calluses de-
rived from protoplasts were more vigorous and regenerated
shoots sooner than those originating from leaf sections. Pro-

lific shoots from callus were observed for all species except

29

N; forgetiana (medium B). N; alata calluses produced sparce

 

shoots on media C and D while infrequent regeneration occurred

on medium E for N; alata and medium E and D for N; forgetiana

 

calluses. No variations were noted regarding the propensity
of shoot regeneration as related to callus originating from
different culture media. Anlagen appeared 3-4 weeks after
calluses were transferred to regeneration media; shoot for—
mation was observed 1-2 weeks later. Shoots formed slightly
faster from callus of E; sanderae (3-4 weeks), perhaps demon-
strating increased genetic potential for growth on the test
media due to heterosis.

Observations from several laboratories indicated that
exogenous auxin and cytokinin sources are required for in yitgg

morphogenetic responses of Nicotiana callus tissues (44,45,

 

52,59). Exogenous auxin was not required for shoot induction

of the 3 Nicotiana species studied herein. Calluses readily

 

produced shoots on medium containing only 1.0 mg/l zeatin.
Auxin generally exerts its effect in proportion to the concen-
tration of other growth regulators present in the medium (77);
therefore, it seems that endogenous auxin levels were adequate
to induce prolific bud differentiation on zeatin medium. It

has been reported (23) that strains of Nicotiana have been

 

selected which require neither an auxin nor a cytokinin, and
suggested that variations in the chromosome complement resulting
from in vitro culturing were responsible for the increased
biosynthetic ability of the callus tissue. Thus, the production

of higher levels of growth substances within the cells caused a

30
reduced need for exogenous hormones for organogenesis. A simi-
lar occurrence may have allowed autonomous production of auxin
in these 3 species.

Zeatin, which has the highest biological activity of any
of the natural cytokinins, is the preferred cytokinin for induc-
ing organogenetic activity at meristemoid regions; 6—BAP, in
comparison, gives a less striking response (34). Levels of
6-BAP in combination with IAA or NAA were less effective in pro-
moting shoot regeneration. Decreasing the levels of both 6-BAP
and NAA proportionately reduced the frequency of shoots and did
not induce a substantial amount of shoots in the case of N;

sanderae and N; forgetiana. A priori, IAA seemed to be more

 

effective than NAA as an auxin in shoot formation in N; alata
and N; sanderae, but produced only infrequent shoots from N;

forgetiana callus. IAA is perhaps more potent than NAA due to

 

its natural occurrence in 3139. Secondary products derived
from IAA breakdown are apparently the active substances in
tissue cultures, since photo-oxidation of IAA readily occurs
(21,25,87).

Rooting (100%) occurred in 6—10 days on the regenerated
shoots placed in media containing MS salts and vitamins + 3.0%
sucrose, 0.8% agar or 3.0% sucrose, 0.1 mg/l NAA and 0.35% agar.
Sufficient levels of naturally occurring auxin may have been
present in the regenerated shoots as exemplified by the high
rate and efficiency of rhizogenesis. High levels of endogenous
auxin have also been found in tomato explants (40).

Within 5 months of intial protoplast isolation, regener-

ated plants at anthesis were assessed for pollen viability and

31
seed set. Seeds were produced on N; alata through sib-polli-
nations among the regenerated plants. Regenerated plants dis-
played a slight decrease in percent viable pollen when compared

with seed-grown plants (Table 9). Pollen counts for N; forgetiana

 

were not available since plants did not reach anthesis at the
time the study was concluded.
Table 9. Mean percent pollen viability as determined by

analine blue staining of pollen from regenerated
and seed-grown Nicotiana species.

 

 

 

 

Species Regenerates Seed-grown
N; alata 75* 89
N. sanderae 67** 73
N; forgetiana not available 64

 

 

*probability of a larger X2 value 0.10 between regenerates
and seed—grown plants.

**probability of a larger X value 0.05 between regenerates
and seed-grown plants.

Chromosomal variants are a common occurrence in plants
regenerated from protoplast—derived callus cultures (1,6,59,67,
76). Overall, regenerated plants appeared morphologically iden-
tical to seed-grown plants. Upon careful examination, however,
split corollas, flower size variations or slight flecking in the
petals was apparent through parts of the population, indicating
that genetic changes may have occurred. Variations in chromosome
number (from the normal 2n=18) from 7-27 were observed in 6 plants
counted of N; alata and in 3 plants of N; sanderae. Number vari-
ations occurred within a single root tip as well as between root

tip cells of different plants tested. The high levels of

32

aneuploidy and euploidy often found in tobacco cell cultures (6,
66,67) can be explained by unequal or asynchronous divisions,
multinucleate protoplasts formed through spontaneous fusion or
chimeral associations of abnormal cells that had aggregated and
produced callus of mixed ploidy. Reports of regeneration in an-
euploid, polyploid, hypotriploid and hypotetraploid cells have
been made (67), thus, restoration of whole plants through di-
plontic selection as suggested by Nishiyama and Taira (59) is
not necessarily favored. These marked differences in chromosome
number can account for the changes in morphology, decreased fer-
tility and capability of autotrophic production of growth regula-
tors for differentiation normally supplied exogenously.

Multinucleate cells have been found in cell cultures re-
sulting from spontaneous fusion events during the isolation pro-
cess or through endomitosis (51,80). Under low osmotic levels
during isolation, a higher percentage of multinucleate protoplasts
have been found (19). Plasmolysing the leaf tissue prior to the
enzyme treatment has been shown to reduce the occurrence of multi-
nucleate protoplasts by severing plasmodemata, thereby preventing
the fusion of adjacent cells (24). Since no preplasmolysing
treatment was used, a greater number of multinucleate cells may
have been anticipated. Ogura (60) reported a wide range of chro-

mosome numbers from regenerates of Nicotiana tabacum tissue cul-

 

tures; such plants were regarded as cytologically chimeric or
mixoploid in the root tips. D' Amato (15) reported evidence of
polyploidy, aneuploidy and chromosome number mosaicism (chimerism)
in cultured cells and regenerated plants of tobacco and other

species, indicating that these are relatively frequent phenomena.

SUMMARY

The study conducted herein established the in vitro

protocol for three species of Nicotiana: N; alata, N; sanderae

 

and N; forgetiana from leaf mesophyll protoplasts to whole

 

plants. Several points emerge from the results obtained:
1. Optimal protoplast release from leaf tissue of N;
alata occurred using an enzyme mixture containing 2.5%
Driselase and 4.0-4.7% mannitol in CPW salts solution.

N; sanderae and N; forgetiana leaf tissue responded best

 

to a solution of 1.0% Driselase, 1.0% Macerozyme R-10,
1.0% Cellulase 'Onozuka' R-10, 0.5% potassium dextran
sulfate and 4.0% mannitol in CPW salts solution for

release of protoplasts.

2. Generally, a plating density of 5.0x104 protoplasts/ml
permitted a constant growth rate throughout the early
stages of protoplast development for all 3 species.

3. Gro-Lux light at 28—34 uEm-Zs-l (400-700 nm) was,
overall, the best of test light regimes in promoting

early first division of the 3 species.

33

34

4. It appears that a complementation of accumulated
genes in N; sanderae, the hybrid, from the parental
species may be responsible for its positive growth
response in media in which the parents were non-respon—
sive. Protoplasts of N; sanderae develOped into callus
in the greatest number of test media as well as exhibit-

ing the highest plating efficiency of the 3 species.

5. Variations in chromosome number within root tip
cells of N; alata and N; sanderae suggest chromosome
instabilities of the developing tissues, in vitro,
perhaps attributable to spontaneous fusion of the
protoplasts, unequal mitoses or chimeral associations

of abnormal cells, or a combination of these events.

RECOMMENDATIONS

Further investigations are warranted in light of the
successful results obtained from the preceeding study. A
more thorough understanding of the cytogenetics of cultured
cells derived from protoplasts is necessary to explain the
variations in chromosome number found in the regenerated
plants. To reduce the possibility of multinucleated proto-
plasts formed from spontaneous fusion during the isolation
procedure, a more complete study of the effects of pre-plasmo-
lysis should be conducted. In addition, experiments testing
agars of different purity may improve methods of plating in soft
agar to increase plating efficiencies. Improvement in the
semi-solid agar culture method may eliminate or lessen the
degree of chimeral association of the cultured cells forming
callus.

Selection systems for the identification of callus derived
from somatic cell hybrids are necessary before attempts at
parasexual hybridizations are made. Albino seedling of N;
sanderae were found during the course of this study and
shoot tip cultures were established and maintained. Cells of
the albino mutant may serve as potential parents in protoplast
fusion studies utilizing albino complementation as a selection
system.

The somatic hybridization between N; alata and

35

36

N; forgetiana to produce N; sanderae can be attempted in order
to characterize morphological or biochemical differences, if
any, in the somatic hybrid versus the sexually-produced hybrid.
Careful attention to the cytogenetics may reveal gross chromo-
somal aberrations which may account for any differences ob-
served.

Since regenerating systems have now been established

for N; alata, N; sanderae and N; forgetiana, somatic hybridi—

 

zation with other Solanaceous species, especially those pos-
sessing a diploid chromosome number of 18 (e.g., Petunia

parviflora, N; bonariensis, N; langsdorffii) should be

 

 

attempted for the purpose of combining desirable character-

istics and, consequently, creating new germplasm.

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10.

11.

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