THESIS

   
  

   

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thesis entitled
lg vitro Nutrient requirements and chlorophyll

precursor analysis of wild—type, nuclear and
cytoplasmic albino mutants of Petunia inflata.

presented by

Mr.Chumpol Borkird

has been accepted towards fulfillment
of the requirements for

Master's degwein Horticulture

Major professor

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£§.VITRO REQUIREMENTS
AND CHLOROPHYLL PRECURSOR ANALYSIS OF
WILD-TYPE, NUCLEAR AND CYTOPLASMIC ALBINO MUTANTS

OF PETUNIA INFLATA

 

BY

Chumpol Borkird

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
IN_VITRO NUTRIENT REQUIREMENTS AND
CHLOROPHYLL PRECURSOR ANALYSIS OF WILD-TYPE, NUCLEAR

AND CYTOPLASMIC ALBINO MUTANTS 0F PETUNIA INFLATA

 

by

Chumpol Borkird

The optimum in vitro nutrient requirements of three genetic strains

of Petunia inflate, the wild-type, nuclear and cytoplasmic albinos were

 

determined. Tests were done individually on iron, sucrose, thiamine-HCl,
indoleacetid acid (1AA), Kinetin (K), benzylaminopurine (BAP), variable
pH from 4-7 and complex grwoth promoting substances including coconut
milk, casein hydrolysate and plant extract added to Murashige and Skoog
(1962) medium. Optimum concentration of each compound were combined to
devise a new medium for each genetic strain. The chlorophyll precursor
analysis was analyzed from ammoniacal aqueous acetone extracts of plants
grown in different light regime with and without feeding withlb-Amino-
leuvulinic acid. Millimolar extinction coefficient and Arnon's equation
were used to calculate the quantities of chlorophyll and it's precursor
from the absorption spectra. Both albino mutants accumulate Protoporphy-

rin IX and the nuclear albino is more unstable.

ACKNOWLEDGEMENTS

I wish to extend my appreciation to D. K. C. Sink, for his
guidance during the course of this study. I also wish to thank the
other members of my guidance committee, Dr. M. J. Bukovac and Dr. W.
Tai, for valuable discussions, technical assistance and kindly making
available equipment necessary to perform experiments.

A special note of appreciation is given to my parents and my
grandmother for their encouragement and constant concern for my

education. To them, this and all future accomplishments are dedicated.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES........................................... v
LIST OF FIGURES ........ . ................ .. ............. ..viii
IN_ZII§9_NUTRIENT REQUIREMENTS OF
WILD-TYPE, CYTOPLASMIC AND NUCLEAR

ALBINOS OF PETUNIA INFLATA

 

INTRODUCTION. 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 ..... 0 0 0 1
MATERIALS AND METHODS 0 0 0 0 0 0 0 0 0 0 ..... 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ..... 3
RESULTS

Effect of BAP on Multiple Shoot Formation.......... 10

Effect of Complex Compounds on Fresh Weight

Increase..................... 10

Effect of Growth Regulators on Fresh Weight

Increase..................... 12

Effect of Thiamine-RC1 on Fresh Weight

Increase000000000000000 ..... 0 13

Effect of Iron, Sucrose and pH on Fresh

Weight Increase.............. 13
Combination Tests .......... . ..... . ................. l4
DISCUSSION0000000000 0.. 0.00.00.00.00... 00 0.0 000 000000000 32

SUMMARY000000000000.000.000.000...00000000000000.0000... 35

iii

CHLOROPHYLL PRECURSOR ANALYSES AND THEIR

MODIFICATION BY CHEMICALS IN CYTOPLASMIC

AND NUCLEAR ALBINO MUTANTS OF PETUNIA INFLATA

 

Page
INTRODUCTION............................. ..... ......... 36
MATERIALS AND METHODS
Plant Materials................................... 38
Chlorophyll and Precursor Analysis......... ...... . 39
Effect of Chemicals on Chlorophyll Formation...... 40
RESULTS

Chlorophyll and Chlorophyll Precursor Accumulation
in Normal Green, Nuclear and Cytoplasmic Albinos

Of Petunia inflata000000000.0000000000000000...0000 50

 

Effect of Iron Concentration and Sucrose on
Chlorophyll Formation in Nuclear and

Cytoplasmic Albinos................................ 52
DISCUSSION..................................... ...... ... 54
SWY00000000000000000000.000000.00000.0000000.0000000 6O

LITERATURE CITED0000000000000000 00000 000000.000 00000 .000 62

iv

LIST OF TABLES

Part I. In_Vitro Requirements of Wild-type, Cytoplasmic and Nuclear

Albinos of Petunia inflata

 

Table: Page

1. Concentrations of Complex Substances, Growth
Regulators, Vitamins, Iron, Sucrose and pH

Tested for Growth Promotion........................ 6

2. Combination Tests for Normal Green Wild-type

Petmia inflata00000000000000000.000.000.0000000000 7

 

3. Combination Tests for Nuclear Albino Petunia

inflata00000000..0000.0..0.00.00.00.00..000000.0000 8

4. Combination Tests for Cytoplasmic Albino

Petunia inflata0000...0....0.0000000000000000000000 9

 

5. Effect of Plant Extract on Fresh Weight Increase
of the Normal Green, Nuclear and Cytoplasmic

Albinos of Petunia inflata............. ..... ....... l9

 

Part II. Chlorophyll Precursor Analyses and Their Modification
by Chemicals in Cytoplasmic and Nuclear Albino

Mutants of Petunia inflata

 

Table:

l. Chlorophyll Accumulation in the Wild-type Petunia
inflata, Cultured in the Light, Light/Dark or Dark
and Fed with 0.1 N -ALA for O, 6 and 24 Hours..... 42

Table:

Chlorophyll Accumulation in the Cytoplasmic
Albino Petunia inflata, Cultured in the Light,

 

Light/Dark or Dark and Fed with 0.1 N -ALA

for O, 6 and 24 Hours.................. ..... ....

Chlorophyll Accumulation in the Nuclear

Albino Petunia inflata, Cultured in the Light,

 

Light/Dark or Dark and Fed with 0.1 N -ALA

for 0, 6 and 24 Hours............. ..... . ........

Chlorophyll Precursor Accumulation in the Normal

Green Wild-type Petunia inflata, Cultured in Light,

 

Light/Dark or Dark and Fed with 0.1 N -ALA for

O'6and 24 Hours...00.0.00.000000000000 00000000

Chlorophyll Precursor Accumulation in the

Cytoplasmic Albino Petunia inflata, Cultured in

 

the Light, Light/Dark or Dark and Fed with 0.1 N
-ALA for O, 6 and 24 Hours.............. .......

Chlorophyll Precursor Accumulation in the Nuclear

Albino Petunia inflata, Cultured in the Light,

 

Light/Dark or Dark and Fed with 0.1 N -ALA for

O, 6 and 24 Hours... ..... ......... ..... ..........

Visual Observation for Chlorophyll Formation 2

Days Following Feeding of Albino Petunia inflata

 

mutarlts With FeSO4000000000000000000 000000 0000.00

Visual Observation for Chlorophyll Formation

2 Days Following Feeding of Albino Petunia inflata

 

Mutants With FeC130000000000.00000000000000000000

vi

Page

43

44

45

46

47

48

48

Table:

9.

Visual Observation for Chlorophyll Formation

2 Days Following Feeding of Albino Petunia

inflata mutants with Na FeEDTA........... ........

2

vii

Page

49

In_Vitro Growth

Nuclear Albinos

Figure:

1.

Effect
of the
Albino

Effect

LIST OF FIGURES

Requirements of Normal Green, Cytoplasmic and

of Petunia inflata.

 

Page

of Coconut Milk on Fresh Weight Increase
Wild-type, Nuclear and Cytoplasmic

of Petunia inflata.................. ...... 17

of Casein Hydrolysate on Fresh weight

Increase of the Wild-type, Nuclear and Cytoplasmic

Albino

Effect

Of Petunia inflata........................ 18

 

of IAA on Fresh Weight Increase of the

Wild-type, Nuclear and Cytoplasmic Albino of

Petunia inflata000000000000000.0000000000000.0000 20

 

Effect

of Autoclaved versus Filter-Sterilized IAA

on Fresh Weight Increase of the Nuclear and

Cytoplasmic Albino of Petunia inflata............ 21

Effect

Normal

 

of Kinetin for Growth Promotion of the

Green, Nuclear and Cytoplasmic Albino

0f Petunia inflata000000000000000.00.00.000000000 22

 

Retest

of Kinetin on Fresh Weight Increase of

the Cytoplasmic Albino of Petunia inflata........ 23

Effect
of the

 

of Thiamine-HCl on Fresh Weight Increase

Wild-type, Nuclear and Cytoplasmic Albino

of Petunia inflata............................... 24

 

Retest
of the

of Thiamine-HCl on Fresh Weight Increase

Nuclear Albino of Petunia inflata......... 25

 

viii

Figure:

10.

ll.

12.

13.

14.

Page

Effect of Iron on Fresh Weight Increase of the
Wild-Type, Nuclear and Cytoplasmic Albino

of Petunia inflata............................... 26

 

Effect of Sucrose on Fresh Weight Increase of
the Normal Green, Nuclear and Cytoplasmic Albino

Of Petmia inflata....0000.000.00.0000.000.000.00 27

 

Test of Optimum pH on Fresh Weight Increase of
the Wild-type, Nuclear and Cytoplasmic Albino

Of Petunia inflata0000000000.000000000000000.0000 28

 

Combination Tests for Wild-type Petunia inflata.. 29

 

Combination Tests for the Nuclear Albino

Petunia inflata..00.0000000000000000000.00000.000 3O

 

Combination Tests for the Cytoplasmic Albino

Petunia inflata....0000000.00.0.0000000000 00000 00 31

 

ix

I NTRODUCTION

Plant protoplast cultures and their fusion to produce somatic
hybrid cell lines and plasts are two areas of tissue culture that
currently are receiving considerable research attention because of
their usefulness in genetic modification studies and potential for
plant improvement (Carlson and Pollacco 1975, Carlson 1973, Bajaj 1974).
A.major obstacle in protoplast fusion studies is the availability of a
sound selection system universally applicable to all plant species.

Light sensitivity, nitrate reductase deficient and drug sensitive mutants
all have been successfully used (Power, et. al., 1977, Melchers and
Labib 1974, Glimelius, et. al., 1978). However, the most universally
applied selection system thus far has been the use of one or both fusion
parents as albino or chlorophyll deficient mutants (Gleba, et. al., 1975,
Krumbiegel and Schieder 1979, Schieder 1977, Belliard et. al., 1978,
Cocking, et. al., 1977, Dudits, et. al., 1977, Maliga, et. al., 1977).

Up to the present time, specific nutrient requirements and phenotypic
stability of chlorophyll deficient mutants of higher plants employed in
these protoplast studies have not been extensively studied.

In general, researchers have used either the basal medium formulated
by Murashige and Skoog (1962), Linsmaier and Skoog (1965), Gamborg et. a1.
(1968), Schenk and Hildebrandt (1971), Eriksson (1965), or Ohira et. a1.
(1973). Although there appears to be a high degree of constancy in the

major and minor salt requirements, in contrast the requirements for

organic constituents are quite variable (Murashige, 1973). In addition,
almost all previous studies on nutritional requirements used callus or
suspension cultures to test medium components (previous references)
but our experiments involved shoot tips.

Since albinos are auxotrophic, the experiments were designed to
test only the basic nutritional requirements of carbon source, iron
as Na2FeEDTA, pH, selected growth regulators and complex growth promoting
substances such as casein hydrolysate (CH), coconut milk (CM) and
plant extract (PE) and even for wild type plants, the growth increased

significantly in the modified medium as will be presented.

MATERIALS AND METHODS

Three genetic strains of Petunia inflata, the wild-type and two

 

mutants, a cytoplasmic and a nuclear-controlled albino, were used as
test organisms. The cytoplasmic albino originated as a mutant callus
culture derived from a wild type protoplast culture (J. 8. Power -
personal communication) and the nuclear albino by gamma irradiation of
seeds (K. C. Sink - personal communication). These three genetic strains
were propagated in_!itrg_by terminal and internode cuttings and
subcultured monthly in modified Murashige and Skoog (1962) medium
containing MS salts and iron with 0.8 mg/l Thiamine-HCl, 100 mg/l myo-
inositol, 3 mg/l IAA, 0.3 mg/l K and 3% sucrose. It was solidified with
9.0 qm/l agar and the pH was adjusted to 5.8 with 0.1 HCl or 0.2 N NaOH
prior to autoclaving at 15 psi for 20 minutes. This medium is herein
referred to as MS-C medium. IAA was initially filter sterilized but
further experiments indicated no effect of autoclaving IAA on the in_
vitrg responses of the P, inflata genetic strains.

The explants used for experiments were shoot tips approximately
2 cm. long with 3 - 4 expanding leaves, taken from one month old stock
plants in culture on MS-C. The shoot tops were weighed prior to and after
culture in the experimental medium for one month.

The experimental media were modified Murashige and Skoog (1962)
containing MS salts, 100 mg/l myoinositol and with various concentrations

of iron, sucrose, thiamine-HCl, idoleacetatic acid (IAA), kinetin (K),

benzylaminopurine (BAP); also, variable pH adjusted with 0.1 N HCl

or 0.2 N NaOH for the range between 4.0 to 7.0 was tested. Complex
growth promoting substances tested included coconut milk (CM), plant
extract (PE), and casein hydrolysate (CH) at different concentrations.

The coconut milk was obtained from the liquid endosperm of fresh
ripe coconuts. It was extracted and filtered through several layers of
cheesecloth. The pH was adjusted to 10.0 with 2.0 N NaOH and held
overnight at 4°C., and then the pH was readjusted to 7.0 with 5.0 N HCl
and stored at -20°C (H. Murakishi - personal communication).

The plant extract from wild-type P, inflata was prepared from
leaves of vegetative seedlings grown in the greenhouse. They were
homogenized in a commercial blender, the homogenate was filtered through
several layers of cheesecloth, heated to 70 - 80°C for 15 minutes and
refiltered. The extract was stored frozen at -20°C.

Each compound was tested individually and the optimum concentrations
of each test substance were combined to devise a new medium. The optimum
concentrations of growth regulators were bracketed and all possible
combinations were tested since the interaction of auxin and cytokinin
in plant tissue culture is well-established.

The concentration of tested compounds used singly in experiments is
indicated in Table I and for the combination tests in Tables II, III and
IV for the wild-type, cytoplasmic and nuclear albinos, respectively.

The stock plants as well as experimental plants were grown under
fluorescent light (Cool White G.E. F 40CW. RS. WM) at 19 Em"2s'1

16-hour photoperiod, 25* 1°C. Each treatment was replicated 5 times

and most were repeated at least once. The initial fresh weight of the
shoot explants was recorded and again 30 days later when an experiment
was terminated. The mean fresh weight of initial explants, final shoot

weights and increases in weight were calculated together with the

standard error.

Table 1. Concentrations of complex substances, growth regulators,

vitamin, iron, sucrose and pH tested for growth promotion.

 

 

 

Chemicals Concentrations
Complex substances
CM ( % ) 0 5 10 15 20 25 30 35
CH ( 9/1 ) 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
PE ( % ) 0 1 3 5 8 10 15 20
Growth regulators
BAP (mg/l) o 0.5 1 1.5 2 2.5 3 3.5
IAA ( mg/l ) 0 1 2 3 4 5 6 7
K ( mg/l ) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
K re-test 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Vitamin
Thiamine HCl 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
(re-test) 0 0.4 0.8 1.2 1.6 2.0 2.4 2.8
NazFeEDTA (mM) 0 .025 0.05 0.075 0.1 0.125 0.15 0.175
Sucrose ( % ) 0 1 2 2.5 3 3.5 4 5
pH 4 4.5 5 5.5 5.8 6 6.5 7
Note: Iron supplied as NazFeEDTA at 0.1 mM is the same concentration

as in MS medium (1962).

Table 2. Combination tests for normal green wild-type Petunia inflata.

 

 

 

Media CM CH IAA K Th-HCl pH Sucrose Fe PE
Code ml/l gm/l mg/l mg/l mg/l gm/l mM ml/l
G-l - - 1 0.2 0.6 5 40 0.1 -
G-2 — - l 0.2 - 5 40 0.1 -
G-3 - - l O 0.6 5 40 0.1 -
G-4 - - l 0.3 0.6 5 40 0.1 -
G-5 - - 2 0 0.6 5 40 0.1 -
G-6 - - 2 0.2 0.6 5 40 0.1 -
G-7 - - 2 0.3 0.6 5 40 0.1 -
G-8 - - 3 0 0.6 5 40 0.1 -
G—9 - — 3 0.2 0.6 5 40 0.1 -
G-lO - - 3 0.3 0.6 5 40 0.1 -
MS-C — - 3 0.3 0.8 5.8 30 0.1 -

 

G = Wild type Petunia inflata

 

Table 3. Combination tests for the nuclear albino Petunia inflata

 

 

 

Media CM CH IAA K Th-HCl pH Sucrose Fe PE
Code ml/l gm/l mg/l mg/l mg/l gm/l mM ml/l
N-l 20 - 5 0.1 1.5 6 3 5 0.1 1
N-2 - — 5 0.1 1.5 6 3 5 0.1 1
N-3 20 - 5 0.1 1.5 6 3.5 0.1 -
N-4 20 - 5 0.1 1.5 4 3.5 0.1 l
N-5 20 - 5 0.3 1.5 6 3.5 0.1 1
N—6 20 - 5 0.7 1.5 6 3.5 0.1 1
N-7 20 - 3 0.1 1.5 6 3.5 0.1 1
N-8 20 - 3 0.3 1.5 6 3.5 0.1 1
N-9 20 - 3 0.7 1.5 6 3.5 0.1 1
N-lO 20 - O 0.1 1.5 6 3.5 0.1 1
N-ll 20 - 0 0.3 1.5 6 3.5 0.1 1
N-12 20 - O 0.7 1.5 6 3.5 0.1 1
MS-C - - 3 0.3 0.8 5 8 3 0.1 -

 

N = nuclear albino Petunia inflata

 

Table 4. Combination tests for the cytoplasmic albino Petunia inflata

 

 

 

Media CM CH IAA K Th-HCl pH Sucrose Fe PE
Code ml/l gm/l mg/l mg/l mg/l gm/l mM ml/l
C-1 20 - 2 l 0.4 5 4 0.1 -
C-2 — - 2 1 0.4 5 4 0.1 -
C-3 20 0.5 2 l 0.4 5 4 0.1 -
C-4 20 - 2 l 0.4 5 4 0.1 3
C-5 20 - 2 0.5 0.4 5 4 0.1 -
C-6 20 - 2 2.5 0.4 5 4 0.1 -
C-7 20 - 1 0.5 0.4 5 4 0.1 -
C-8 20 - l l 0.4 5 4 0.1 -
C-9 20 - l 1.5 0.4 5 4 0.1 -
C-10 20 - 3 O 5 0.4 5 4 0.1 -
C-11 20 - 3 1.5 0.4 5 4 0.1 -
C-12 20 - 3 1.5 0.4 5 4 0.1 -
MS-C - - 3 0.3 0.8 5.8 3 0.1 -

 

C = cytoplasmic albino Petunia inflata

 

RESULTS

Effect of BAP on Multiple Shoot Formation

En MS-C medium, multiple shoot formation occurred sporadically for
the normal green and cytoplasmic albino plants and did not occur in the
nuclear albino. The multiple shoots originated from extensive adventitious
shoot formation on callus produced at the base of the Shoot tip cuttings
of the three genetic strains of P, inflata when grown in MS medium with
BAP from 0.5 - 5.0 mg/liter. The numerous, spindly shoots were not
suitable to use as explants for further experimentation. Also, the
growth of the main shoot seemed to be inhibited even at 0.5 mg/l BAP.
Therefore, medium MS-C was used to supply all explants and serve as the
control medium.

Green sectors were occasionally observed on adventitious shoots of

the cytoplasmic albino when grown in MS medium + BAP.

Effect of Complex Compounds on Fresh Weight Increase

 

Each genetic strain of P, inflata had different growth factor
requirements. The wild-type plants did not respond to CM, CH or PE. In
contrast, some of the compounds inhibited the growth of normal green
plants even at the lowest concentration tested, 5% CM and 0.5 gm/l CH
(Appendix I, II and Table V). PE was inhibitory at 5% and above and was

without effect at lower concentrations (Table V). CH even at 0.5 gm/l

10

11

to 3.5 gm/l inhibited the growth of all three genetic strains

(Appendix II). Even though the fresh weight of the cytoplasmic albino
in medium with 0.5 gm/l CH is higher than the control medium, the fresh
weight increase was due mostly to callus formed at the base of cuttings.

The cytoplasmic albino grown on media with CH changed color from
pale yellow to almost white. This pigment change was consistently
observed. CH seems, therefore, to inhibit chlorophyll synthesis in the
cytoplasmic albino; however, no effect was observed in the normal green
or the nuclear albino.

Even though CM inhibited the growth of normal green plants, it had
some stimulatory effect on the nuclear and cytoplasmic albinos at 20%
whereas higher concentrations were inhibitory and lower concentrations
were not adequate (Appendix I).

PE did not have an obvious promotive effect on the growth of the
normal green plants at 3% or below and was inhibitory at higher
concentrations. A similar trend was found for cytoplasmic albino plants
where 5% PE was inhibitory and lower concentrations had no effect. In
contrast, low level of PE at l - 3% stimulated the growth of the nuclear
albino (Table V). The best concentration tested for fresh weight increase
was 1%. Higher concentrations inhibited growth and also appeared to
inhibit chlorophyll formation especially at levels above 10%. The nuclear
albino is unstable and plants showed considerable chlorophyll formation
which is in contrast to the cytoplasmic albino which was quite stable in
the MS-C medium. Addition of PE above 10% resulted in less chlorophyll
formation in the nuclear albino which may be the consequence of

growth inhibition.

12

Effect of Growth Regulators on Fresh Weight Increase

 

The three genetic strains of P, inflata had a lower IAA requirement
for growth than the level in MS—C (3 mg/l). The nuclear albino also grew
in MS basal medium without IAA, which produced the highest fresh weight
after one month in culture, but the increase was primarily from
extensive callusing at the base of cuttings. The nuclear albino also
had the highest fresh weight at the highest level of IAA (5 and 7 mg/l).
Therefore, 0, 3.0 and 5.0 mg/l IAA were used in combination tests with
different concentrations of K at all combinations. The best IAA
concentration tested for the normal green and cytoplasmic albino was
1.0 and 2.0 mg/l respectively (Appendix III).

There were no differences between autoclave and filter sterilized
IAA in growth of nuclear or cytoplasmic albinos but autoclaved IAA was
superior for the normal green plants at 3.0 mg/l (Appendix IV).

The optimum concentrations of K for normal green and nuclear albino
plants were 0.2 and 0.1 mg/l, respectively, which is lower than the
0.3 mg/l used in MS-C. The cytoplasmic albino responded quite differently
from the other two genetic strains. Maximum fresh weight growth was
obtained at lO-fold higher concentration than for the nuclear albino and
5—fold higher than for the normal green plants (Appendix V and VI).

Moreover, K also had a similar stimulatory effect on green sector
formation as did BAP on the cytoplasmic albino. Green sectors on leaves
were formed using basal medium with K from 0.4 to 1.0 mg/l. No green
sectoring was observed below 0.4 or higher than 1.2 mg/l K. Actually,

green sectors were sometimes noticed in leaves of adventitious shoots

13

of the cytoplasmic albino stock cultures but they occurred very
infrequently. The high levels of cytokinins, BAP and K showed obvious
effects on promoting green sectors and they occurred consistently

and were repeatable.

Effect of Thiamine-HCl on Fresh Green Weight Increase

 

Thiamine—HCl appeared to be the only vitamin requirement for
the three genetic strains of P, inflata, since the other vitamins in
MS medium did not show any promotive effect when compared to MS-C, which
contained only thiamine-HCl. The optimum levels of thiamine-HC1 for the
normal green and cytoplasmic albino plants were found to be 9.6 and
0.4 mg/l, whereas that for the nuclear albino was higher. The greatest
fresh weight obtained from the nuclear albino was in MS + 1.4 thiamine-
HCl and was still increasing, suggesting that the optimum was higher
(Appendix VII). Therefore, experiments with higher concentrations of
thiamine-HCl were conducted and the greater fresh weight obtained was
at 0.8 mg/l (Appendix VIII). However, of 1.2 and 1.6 mg/l thiamine-HCl,
the 1.6 level resulted in the best overall growth. The intermediate

concentration of 1.5 mg/l was selected for use in the combination test.

Effect of Iron, Sucrose and pH on Fresh Weight Increase

 

Iron supplied at 0.1 mM as in MS medium proved to be optimum for
maximum growth of all genetic strains of P, inflata (Appendix IX).
The three genetic strains all had a rather high requirement for

sucrose when provided as the sole carbon source. A level of 3.5% and

14

4.0% were optimal for the nuclear albino and both the normal green and
the cytoplasmic albino, respectively (Appendix X). All plants grown in
media with low sucrose (1%) or in the absence of sucrose had very low
fresh weight and many died.

With respect to optimum pH, the three genetic strains preferred
low pH medium. Routinely the pH of media were adjusted to 5.6 - 6.0
but pH 5.0 was found optimum for the maximum growth of the nuclear
albino. 0f more interest, the highest fresh weight of the nuclear albino
occurred at pH 4.0. Also, the growth of the other genetic strains was
not inhibited at pH 4.0 while above pH 6.0, growth inhibition of the

three genetic strains occurred (Appendix XI).

Combination Tests

 

The new formulated media yielded 2, 2.5 and 4-fold increases in the
final fresh weight when compared to the original MS-C medium for the
nuclear albino, wild-type and the cytoplasmic albino plants (Appendices
XII, XIII, XIV). The media composed of the optimum concentrations
observed from the individual tests of each substance did not result in
the highest fresh weight and some modification is required for the best
growth of each genetic strain. CM was not necessary when the hormone
level is optimum (media N-2 and C-2, Tables 3 and 4, Appendices XIII,
XIV). In contrast, 3% PE resulted in the greatest fresh weight of the
cytoplasmic albino (medium C-4, Table 4, Appendix XIV). However, it was
not essential for the nuclear albino (medium N-3, Table 3, Appendix XIII).

CH and thiamine-HCl were also found to be unnecessary for the cytoplasmic

15

albino and the normal green plants (media G-2, C-3, Tables 2 and 4,
Appendices XII, XIV). Interestingly, the nuclear albino had the second
best growth in medium N-4 which had pH 4.0 (Table 3, Appendix XIII).
From the results on different hormonal combinations, it can be
concluded that K is necessary for growth of the normal green plants
and the optimum concentration in the combination test was 0.3 mg/l,
whereas the optimum concentration of IAA was 1.0 mg/l, which is lower
than the MS-C medium by three-fold (media G-3 through G—10, Table 3,
Appendix XIII). Both K and IAA were also found necessary for the growth
of the nuclear albino, especially at high concentrations, 0.7 and
5 mg/l, respectively (media N-S through N-12, Table 3, Appendix XIII).
For the cytoplasmic albino, the fresh weight increase seems to depend
upon the concentrations of K more than that of IAA (media C-5, C-1, C-6;
C-7, C-8, C-9; and C-10, C-ll, C-12, Table 4, Appendix XIV). However,

the optimum concentrations are 2.0 mg/l IAA and 1.0 mg/l K.

16

SF ( G ) - Standard error of the final shoot weight of the normal
green 2, inflata

SE ( C ) - Standard error of the final shoot weight of the cyto-
plasmic albino

SE ( N ) - Standard error of the final shoot weight of the nuclear
albino

08W : - Original shoot weight

SE ( 08W ) - Standard error of the original shoot weight

o——-o Normal green, wild-type g. inflata
0""‘D Cytoplasmic albino g, inflata

o——-O Nuclear albino 1:. inflata

17

Figure 1. Effect of coconut milk on fresh weight increase of the wild-

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. f
5:13“)

 

type, nuclear and cytoplasmic albino of Petunia inflata.

 

 

 

P
- o..D.. '0
O. 0.
qua o.
.0
.0
...
O. 0
w
O~“‘~“‘ "".—e ..
‘V’
I l l L l l 1 I
'0 5 10 15 20 25 30 35
% Coconut milk

SE ( c )E .442 .086 .0844 .2041 .1477 .1344
OSW .0147 .0095 .0102 .0091 .0114 .0115
SE ( osw ) .008 .0064 .005 .0057 .007 .0013
SE ( c ) .1566 .1645 .2885 .1799 .1593 .3481
03w .0223 .02 .0364 .0487 .023 .0184
sE ( osw ) .0051 .0181 .0153 _.0408 .0125 .0076
SE ( N ) .0666 .0354 .0545 .1039 .1781 .0819
nsw .0146 .0102 .0141 .0125 .0142 .0134
SE ( 05w ) .0071 .0034 .007 .0033 .0036 .0034

.0841
.0165
.0081

.2142
.0151
.0076

.0555
.0113'
.0031

.0789
.0139
.0055

.1543
.0111
.0019

.1065
.0152
.0107

18

Figure 2. Effect of casein hydrolysate on fresh weight increase of the

wild-type, nuclear and cytoplasmic albino of Petunia inflata.

 

 

 

 

o r
’-
8.
1.5 -
V
...—o
J:
U)
ma
4)
II
1; '1.0 _
<3
.2
U)
.—..
en
.5
LLOQS -
0 0.5 1 1.5 2 2.5 3 3.5
gm/l casein hydrolysate
SE ( c ) .3752 .4667 .1431 .0815 .0888 .0344 .1291 .1005
osw .0259 .0148 .021 .0159 .0171 .0202 .0225 .0228
SE ( osw ) .0102 .006 .0157 .0117 .0129 .0079 .0076 .0093
SE ( c ) .0527 .4551 .0711 .0566 .0979 .0986 .0266 .1254
osw’ .0279 .0209 .0195 .0231 .0231 .0333 .0371 .0171
SE ( 08w ) .0147 .01 .0142 .0143 .0078 .011 .0137 .006
SE ( N ) .092 .1481 .1587 .0358 .0337 .0814 .0686 .0526
08W .0135 .0121 .0141 .0143 .0181 .021 .0241 .0163

SE ( OSW ) .0052 .0029 .0038 .0025 .0115 .0054 .0119 .0038

19

Table 5. Effect of Plant Extract on Fresh Weight Increase of
the Normal Green, Nuclear and Cytoplasmic albino of

Petunia inflata.

 

 

Genetic Strains.

 

 

 

% Extract
Normal green Nuclear albino Cytoplasmic albino
0 ++++ + ++++
1 ++++ ++++ ++++
3 ++++ +++ ++++
5 ++ ++ ++4+
8 ++ ++ +++
10 ++ + +++
15 ++ + ' ++
20 ++ _ + ++
Key:
+ = Poor
++ = Fairly good
+++ = Good

++++ Excellent

20

Figure 3. Effect of IAA on fresh weight increase of the wild—type,

nuclear and cytoplasmic albino of Petunia inflata .

Final shoot weight ( gm )

 

 

 

1.5 b
L I l l l L l 1
mg/l IAA

SE ( G ) .1995 .2814 .1098 .2276 .1954 .2054 .1025 .1125
03w .0232 .0206 .0182 .0259 .0269 .0264 .0226 .0326
SF ( OSW ) .0077 .0046 .0062 .0113 .0102 .0071 .0095 .0084
SE ( C ) .1006 .3193 .3245 .0619 .2238 .1541 .1286 .2704
03w .024 .0128 .019 .0184 .0184 .0301 .0252 .0217
SE ( OSW ) .01 .0073 .0146 .0161 .0101 .012 .0151 .0196
SE ( N ) .0854 .0613 .1167 .1487 .1821 .1857 .1611 .1857
05w .0168 .0173 .0163 .0227 .0198 .0223 .0222 .0218

SE ( OSW ) .0105 .0089 .0142 .0086 .0062 .0025 .006 .0085

21

Figure 4. Effect of autoclaved versus filter—sterilized IAA on fresh
weight increase of the wild-type, nuclear and cytoplasmic

albino of Petunia inflata.

 

 

 

 

 

2.0r
1.5F
1.0" r-
0.5- ~
535 P10
::: 11'
::°: III
::: Il'l
--. 11
FA FA FA
Green CYtoplasmic Nuclear
SE 4 .442 .2161 .1566 .1081 .1199 .0152
05w .0147 .0115 .0223 .0218 .0118 .0145

SE ( OSW ) .008 .0067 .0051 .0079 .0019 .0029
A - Autoclaved

F - Filter-sterilized

Final shoot weight ( gm )

Figure 5. Effect of kinetin on fresh weight increase of the wild-type
nuclear and cytoplasmic albino of Petunia inflata.
1.5 ’
(J 5 - c%b 1’
o O O
o o
O. .0
38'. ..
/ \\ ... _ "‘
l l l n 1 1 n L
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
mg/l Kinetin

SE ( G ) .1753 .1322 .2643 .4599 .3201 .1458 .2287
08w .0191 .0182 .0275 .0213 .0184 .0201 .0222
SE ( 08w ) .0042 .0085 .0116 .0085 .0053 .0057 .0083
SE ( C ) .1884 .1827 .1521 .3415 .1524 .3156 .18
05w .0253 .0263 .0183 .0301 .0256 .0247 .0322
SE ( 08w ) .0109 .0094 .0061 .0078 .0088 .004 .0074
SE ( N ) .0271 .1093 .0577 .1111 .1545 .0179 .0433
05w .0109 .0103 .0114 .0139 .0098 .0127 .013
SF ( 08W ) .005 .0035 .0018 .0086 .0048 .0073 .0041

 

22

 

 

 

.2514
.0185
.0113

.3068
.0233
.0082

.1075
.0115
.0039

Final shoot weight ( gm )

23

Figure 6. Retest of kinetin for fresh weight increase of the cytoplasmic

albino of Petunia inflata.

 

 

2.0 r
‘1.5 b If: ”‘5‘.
:' '.‘=
1.0 " .“5
.5"

0.5 -

0 0.2 0.4 0.6 0.8 1.0 1.5 2.0

mg/l Kinetin

SE ( C ) .1574 .2221 .3679 .4925 1.0028 .6964 .9896 .2343
OSW .116 .0107 .0098 .0123 .011 .0156 .0103 .004

SE ( OSW ) .0066 .0021 .0028 .0102 .0026 .0121 .0036 .001

24

Figure 7. Effect of thiamine-RC1 on fresh weight increase of the wild-

2.0
A
E!
a, 105 '
v
con!
.2
U)
ma
d)
:3
4". 100
O
O
.C
a:
u--.
(U
£5
u. 0.5
SE ( G )
OSW
5E ( osw )
SE ( C )
OSW
SE ( osw )
SE ( N )
OSW

SE ( osw )

type, nuclear and cytoplasmic albino of Petunia inflata.

 

 

 

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
mg/l Thiamine-HCl

.2317 .2058 .1486 .4381 .1621 .3751 .2378 .4385
.0118 .0099 .0154 .0136 .0119 .0142 .0175 .0216
.0054 .0028 .0111 .0027 .0034 .0139 .0062 .024

.2291 .3539 .3524 .4015 .2194 .4997 .1662 .2641
.0155 .0244 .0338 ..0196 .0171 .016 .0259 .0257
.0044 .0152 .0201 .0074 .0122 .0076 .0198 .0129
.0617 .089 ,1386 .0669 .0765 .0866 .3326 .2157
.0214 .0109 .0154‘ .0133 .0174 .0147 .0117 .0196
.0052 .0074 .0076 .007 .0088 .0055 .0041 .0084

25

Figure 8. Retest of thiamine-H01 for fresh weight increase of the

 

nuclear albino of Petunia inflata.

 

 

2.0 -
A
3‘ 1.5 -
v
...:
.c
U)
--0
GO ,
z:
‘1; 1.(1 '
O
.C
on
0—0
on
.5.
u- 0.5 '
IN
Q‘s-c." \‘o—"‘—V———‘——o
L 1 L l l l I
0 0.4 0.8 1.2 1.6 2.0 2.4 2.8
ng/l Thiamine-HCI
SE ( N ) .0154 .0265 .1862 .055 .092 .0412' .0653 .0365
osw .014 .0097 .0164 .0142 .0117 .0122 .0119 .0101
SE ( osw ) .0056 .0052 .0032 .0037 .0033 .0024 .0038 ..0058

26

Figure 9. Effect of iron on fresh weight increase of the wild—type

Final shoot weight ( gm )

SE (
osw
SE (

SE (
osw
SE (

SE (
0511
SE (

no
0
CD

.2
U1

...8
o
O

0
U1

 

G)
OSW)
C)
0511)
N)

0514 )

nuclear and cytoplasmic albino of Petunia inflata.

 

 

 

L L l l l I 1 I
0 .025 .05 .075 .1 .125 .15 .175
mM Fe

.0387 .0246 .2704 .2782 .1196 .0383‘ - -
.0221 .0238 .0262 .0102 .0208 .0202 - -
.0124 .0102 .007 .0058 .0057 .015 - -
.0279 .0102 .4038 .2414 .1757 .0642 - -
.0153 .0102 .0143 .0122 .0137 .0122 - -
.0063 .0032 .0033 .0034 .0016 .0058 - -
.0548 .0668 .0954 .182 .1195 .1151 .0233 .0545
.0102 .0114 .0119 .0125 .0152 .0176 .0133 .0134
.0047 .0047 .0044 .0035 .0065 .0071 .003 -90104

Final shoot weight ( gm )

27

Figure 10. Effect of sucrose on fresh weight increase of the wild-type

nuclear and cytoplasmic albino of Petunia inflata.

 

 

 

 

1.5 '

1.0 '

0.5 t

0 1 2 2.5 3 3.5 4 S
’ % Sucrose

SE ( C ) - .0473 .229 .0378 .1667 .3095 .3164 .2402
OSW - .0228 .0248 .0306 .0245 .025 .0258 .024
SF ( 08W ) - .0096 .0116 .0137 .0066 .0105 .0088 .0098
SE ( C ) - - .2085 .1986 .2273 .5865 .2584 .1536
05w - - .03 .037 .0344 .0348 .029 ,0314
SE ( OSW ) - - .0042 .01 .0061 .0142 .0097 .0089
SE ( N ) - .0372 .0526 .0624 .1787 .3476 .1693 .2894
osw - .015 .0212 .0212 .0228 .0246 .021 .0298

SE ( osw ) - .0014 .0067 .0115 .0082 .0144 .0057 .0126

28

Figure 11. Effect of pH on fresh weight increase of the wild-type, nuclear
I
and cytoplasmic albino of Petunia. inflata.

 

   

    

 

 

A
S: 1-
9" ‘1'5 ‘
...w
.5
U)
wd
m v
) h 9.....0.. q
.3 100 g. ...0...
C) o
.. A
U) 0. \
—4 \ 1'
(U 0
C K\ ...]
ad _ ‘0’
u- 0.5
\.-._,
l l J l L l 1 L
4 4.5 5 5.55.86 6.5 7
SF ( c ) .4557 .3878 .3008 .3327 .6045 .1229 .2149 .0923
08W .021 .0268 .0222 .0316 .0234 .026 .0328 .0263
SE ( osw ) .0143 .0056 .0091 .0062 .0027 .0066 .0052 .0087
SE ( C ) .6168 .2883 .4051 .5081 .5766 .4609 .3082 .1562
080 .0079 .0127 .0168 .0165 .0135 .0141 .0131 .0096
SE ( OSW ) .0044 .0028 .0023 .0046 .0043 .0099 .0097 .0065
SE ( N ) .0078 .2175 .171 .1566 .0651 .3055 .1723 .071
05w .0111 .0148 .0092 .0176 .021 .0204 ..0135 .014
SE ( 08W ) .004 .0098 .0025 .0076 .0041 .011 .005 .0025

29

@500.
womo.
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QoND. come. came. «one. mmwo. m~mo. mmmc. Nmo. ammo.
~mmm.. mnom. Nome. been. Mmqm. «mum. mdmm. Amen. docs.

mam: ceno mum mac sum osw mum «no mno mum ens

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mucamcfi masouom :mouw HmEuo: pom mumou cowumafibsoo umfi ouawfim

 

2mo
Mm

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mac. mmfio. Mbfio. memo. mqfio. No. wmo. mega. mmmo. mmmc. qqmo. Bmo
mmso. bwmo. mang. «mn~. dmgm. meflm. mafia. mmd. mmmd. aged. emmm. Mm

gum: $122.12 212 mnz muz 512 012 msz v12 muz .muz _...z

 

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31

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mmmc. .omuc. mags. come. oNNo. ammo. bmmo. mmo. bNo. mafia. pogo. memo.
mc.~. new. ammo. wuom.~ nmom. gumm. aama. wmmm. mew. bmow. mmmw. Now.

a-mz NF-UFF-U 04-8 a-a a-a 2-8 a.a m-a 4-8. m-a N-a

A 2mo v mm

 

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F

 

 

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( wfi ) 105188 10009 IPUIJ

1
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3m:
Mm

DISCUSSION

Marked increases in fresh weight of three genetic strains of
P. inflata can be achieved by modification of a few components in
MS medium without any change in the mineral composition. The fresh
weight increases are significant, from 2 up to 4 times higher than in
the previous standard medium, MS—C. The requirements for growth factors
such as CM and PE are optional but still helpful for the nuclear and
cytoplasmic albinos whereas CH was found to be inhibitory. The role
and composition of these complex factors have been suggested by
Tulecke 1961, Steward and Caplin 1952, but up to now they are still
obscure. In this research, growth regulators in the complex substances
might not be the major contributors to the fresh weight increase since
they were also tested both individually and in combinations and did not
stimulate such increases. However, the growth regulators are still
required for optimum growth. The hormonal requirements of albino plants
were studied in the tobacco chlorophyll-deficient mutant by Schaeffer
and Menser (1975) and high IAA (up to 10 mg/l) was found beneficial for
growth which is in contrast to our work, in which high levels of IAA
were inhibitory. All genetic strains of P, inflata in these studies
required a rather high concentration of sucrose, even for the normal
green plants, and they did not survive at the low level of sucrose (1%)
although the green plants were autotrophic. The sucrose requirement is

probably due to the lower light intensity which is inadequate for the

32

33

normal green plant to photosynthesize properly as compared to the
greenhouse growing conditions. This high sucrose might also serve as
a carbon source and partly as an osmotic stabilizer as proposed by
Brown, et. a1, 1979, and Coffin, et. al., 1976. Also, the optimum
pH for maximum growth was found to be lower than in other media
proposed (Murashige and Skoog 1962, Linsmaier and Skoog 1965, Gamborg
et. al., 1968, Schenk and Hildebrandt 1972, Eriksson 1965, Ohira, et.
al., 1973).

It is obvious that new media formulations with different salt
concentrations and combinations might not be necessary to obtain
optimum growth of chlorophyll deficient mutants. Actually, minor changes
in the concentrations of the ingredients previously used in MS-C were
found to increase the growth of three genetic strains of P. inflata from
two up to four-fold. Apart from that, the quality and manner of
incorporating the ingredients into media, e.g., autoclaved versus
filter sterilized, were also found to be important, as had been suggested
by Romberger and Tabor (1971). In order to obtain optimal growth,
experiments have to be run systematically since interaction of ingredients,
especially growth regulators, might be the key factor that regulates
growth and differentiation as has been proposed in numerous publications.
However, the minor point as stated above about integrating the ingredients
and their quality must not be overlooked.

Tissue culture has great potential in industrial uses, agriculture
and scientific studies. (Murashige 1974, Puhan, et. al., 1971, Reinert
and Bajaj 1977, Thorpe 1978). Especially in plant propagation, the maximum

growth in_vitro is desirable to reduce operating and high labor cost.

34

This probably can be accomplished by simply some minor changes in the
media ingredients or concentrations which can be tested quite easily

in commercial laboratories. For scientific research, albinos interest
scientists working in photosynthesis and also plant protoplast culture
and.scmatic hybridization. However, very little work is done with albinos,
especially the cytoplasmic albino because of the unavailability of
plant materials. Optimum growth 12.31EEQ is required to overcome this
problem. Regularly, somatic hybridization using albino or albino
complementation systems utilize albino suspension cultures as cell
sources of protoplasts. It is well-established that genetic instability
exists in the calls state of tissue culture and also occurs in albino
tissue culture in our experiments as well as regreening in albino

in suspension culture of P, hybrida (Ad Kool - personal communication).
The use of axenic culture of albino plants might have the advantage over
the conventional suspension culture. Also, the regeneration capacity can

also be retained.

SUMMARY

1. BAP was found non-beneficial for multiple shoot induction
of three genetic strains of P, inflata. Green sectors were formed on
leaves of cytoplasmic albino when grown in MS medium with BAP or K.

2. Growth factors, CM and PE stimulate growth in the albinos but
inhibit in normal green plants. CH is inhibitory to all genetic strains
tested as well as inhibiting chlorophyll formation in the cytoplasmic
albino.

3. Growth regulators, IAA and K, are required for maximum growth
and an antagonistic effect of auxin and cytokinin was observed.

4. Thiamine-HCl is required for the nuclear and cytoplasmic albino
but not for the normal green plants.

5. Iron at 0.1 mM as in MS medium is optimum whereas higher sucrose
and low pH are required for all genetic strains of P, inflata. The
nuclear albino can perform quite well at pH 4.

6. Combination tests are necessary to identify the best combination

for maximum growth because of the antagonistic effect of each ingredient.

35

ABSTRACT

CHLOROPHYLL PRECURSOR ANALYSIS AND THEIR MODIFICATION

BY CHEMICALS IN CYTOPLASMIC AND NUCLEAR ALBINO MUTANTS

OF PETUNIA INFLATA

 

by

Chumpol Borkird

A nuclear albino and a cytoplasmic albino of Petunia inflata

 

were analyzed to determine the site of blockage in the chlorophyll
biosynthetic pathway. Precursor accumulation was studied by ammoniacal
aqueous acetone extracts of plants grown in different light regimes with
and without feeding of 19- aminoleuvulinic acid. Millimolar extinction
coefficient and Arnon's equation were used to calculate the quantity of
chlorophyll and its precursors from absorption spectra. Wild-type plants
were used as control. Total chlorophyll of wild-type plant is higher
than either the nuclear or cytoplasmic albinos. The Chlorophyll a/b ratio
increased when the normal green plants were grown in the dark and
decreased in the albinos. The chlorophyll biosynthetic pathway of both
albinos was blocked at Protoporphyrin IX. The nuclear albino can be
considered less stable than the cytoplasmic albino. Plant extract and
casein hydrolysate were found to inhibit chlorophyll synthesis in the

nuclear and cytoplasmic albinos respectively. FeCl partially overcame

3
albinism in both the nuclear and cytoplasmic albinos whereas FeSO4 and

NazFeEDTA only maintained traces of chlorophyll. Anthocyanin pigmentation

was observed after incubating leaves in sucrose solutions containing iron.

INTRODUCTION

Chloroplast development and chlorophyll biosynthesis have been
under intensive investigation for a number of years but, due to their
complex control by both nuclear and cytoplasmic genomes (Akoyunoglou
and Argyroudi-Akoyounoglou 1978), the genetic and biochemical mechanisms
have not been fully elucidated. Many attempts and approaches have been
used to study the chloroplast complex system including those on greening
of ethiolated plants (Bengston, et. al., 1977, Shlyk, et. al., 1970),
biochemical, physiological and genetic studies of mutants defecttive in
chlorophyll biosynthesis (Gough 1972, Mascia 1978, Kahn, et. al., 1977,
Nielso and Kahn 1973). Most of the mutants studied have been nuclear-
controlled chlorophy-l dificiencies, especially in higher plants, since
sytoplasmic-controlled mutant cells generally appear less frequently and
mostly as small sectors of white tissue among green cells (Kirk and
Tilney—Bassett 1978). Thus, they are not readily available as experimental
materials. Cytoplasmic mutants of lower organisms, single cell algae

Chlamydomonas, have been studied extensively (Sager 1972, Sager 1970,

 

Sager and Ramanis 1971, Chiang 1971, Gilham 1969) but their chlorophyll
biosynthetic mechanism differs significantly from higher plants.
Therefore, we used tissue culture techniques to provide plantlets of
both nuclear and cytoplasmic inherited albinos of P, inflata for
analysis of chlorophyll precursors which accumulated under controlled

environmental conditions following feeding with b -aminoleuvulinic acid

36

37

(Granick 1959, Mascia 1978). Also, the effect of selected chemicals

on the stability of albinism was investigated since chlorophyll
formation has previously been found to be regulated to some degree by.
the chemica ingredients in the culture medium in lower organisms

(Horrum and Schwartzbach 1980, Ellis, et. al., 1975, Aoki, et. al., 1965)

and iron has also been found to correct albinism in maize (Bell, et. al.,

1958, Bell, et. al., 1962).

 

MATERIALS AND METHODS

Plant Materials

 

Three genetic strains of P, inflata, the wild-type plants, a
cytoplasmic and a nuclear inherited albino were used in these studies.
All are diploid 2n = 2x = 14. The cytoplasmic albino originated as a
mutant callus culture derived from wild-type protoplasts (J. B. Power -
personal communication) and the nuclear albino by gamma irradiation of
seeds (K. C. Sink - personal communication). These three stocks were
propagated in ziE£9_monthly by terminal and internode cuttings plated in
modified Murashige and Skoog (1962) medium (MS-C). The modified MS
medium contained MS salts and iron with 0.8 mg/l thiamine-HC1, 100 mg/l
myoinositol, 3.0 mg/l indoleacetic acid (IAA), 0.3 mg/l kinetin (K)
and 3% sucrose. It was solidified with 9.0 mg/l agar and the pH was
adjusted with 0.1 N HCl or 0.2 N NaOH to 5.8 prior to autoclaving. All
leaves of the cuttings were removed to prevent any carry-over chlorophyll
contamination to the etioplasts. Subcultures one month old were used for
all studies. The three genotypes were grown under 1) light Em"zs'l
16-hour photoperiod 25* 2°C for 30 days; 2) 15 days in the same light
condition and then transferred to the dark for another 15 days;

3) in the dark by wrapping the culture vessels with aluminum foil and

kept in the dark for 30 days.

38

39

Chlorophyll and Precursor Analysis

 

Plant tissue, leaves and stems above the agar line of the light,
half-light, half-dark (light/dark) and the dark-grown plants were
removed and some were incubated with 0.01 N ti-aminoleuvulinic acid
(3 -ALA) (MaScia 1978) . There were three classes of b -ALA feeding:

1) no feeding; 2) feeding for 6 hours; 3) feeding for 24 hours. All
steps were performed in the dark under a dim green safe light. The
green safe light was constructed using a fluorescent tube wrapped with
several layers of green cellophane. The spectrum of the light emitted
was measured with a spectroradiometer.

The leaves were homogenized with ammoniacal aqueous acetone (1 ml
of 25% NH4OH:9 ml H20:90 m1 acetone) at 4°C under the green safe light,
centrifuged at 10,000 rpm and the supernatant was collected. The detailed
procedures of Kahn, et. al., 1976, were followed. The absorbance of the
extracts was measured on a Beckman spectophotometer Model 25 and the
pigment concentrations were calculated from the millimolar extinction
coefficient by Kahn, et. al., 1976. The equations of Arnon (1949) were

used to determine the amount of chlorophyll a and b:

Millimolar extinction coefficient

Pchlide = 0.0426 00628 + 0.0072 00590 — 0.0343 00575
Proto IX = 0.1962 013575 + 0.0466 OD590 - 0.0585 OD628
Mg-Proto = 0.0618 00590 + 0-0238 09575 ‘ 0°°°35 OD628

4O

Millimolar extinction coefficient (continued)

(/A-moles/ml extract)

C .

a 0.0127 OD663 - 0.00269 OD

645

ch 0.0229 00645 ~ 0.00468 00663

Ctotal = Ca+Cb or OD652 X 1000
34.5

 

All mg/l extract experiments were conducted with five replicates
for all plants grown in an experimental environmental condition and
feeding with 3 -ALA. The mean values were used to calculate the
precursor quantity, chlorophyll accumulation and likewise to establish

absorbance curves.

Effect of Chemicals on Chlorophyll Formation

In the previous section of this thesis, some of the tested chemicals,
casein hydrolysate (CH) and plant extract (PE), showed an inhibitory
effect on chlorophyll formation by visual evaluation in the cytoplasmic
and nuclear albino plants. In addition, certain forms of iron have
previously been found to correct albinism in maize (Bell, et. al., 1958
and Bell, et. al., 1962) and were included in our studies to elucidate
the influence of these chemicals on chlorophyll formation.

Nuclear and cytoplasmic albino plants were grown in modified MS
medium as previously described. The light was 19 Em'zs'1 16-hour

photoperiod at 25* 2°C for one month after which the leaves were cut

41

into small pieces (0.2 - 0.5 cm2) and incubated in the chemical
solutions. The aqueous test solutions had 3% sucrose with 0, 0.5,

l, 2, 5, 10, 100 and 500 ppm iron from FeSO4, 7H20, FeCl3, or

NazFeEDTA. The incubation proceeded for 2 - 3 days in plastic petri
dishes placed in an environmental chamber under 54 - 60 Em"zs-1 light
(GE F40CW, RS, WM), at a temperature of 251 2°C. Changes in pigmentation

were evaluated visually.

Table 1. Chlorophyll Accumulation in the Wild-type Petunia
inflata, Cultured in the Light, Light/Dark or Dark

42

and Fed with 0.1 N A-ALA for 0, 6 and 24 hours.

 

Time of

Chlorophyll Accumulation (mg/1)

 

 

Environment
-ALA Feeding
Chl a Chl b Chl (total) Chl a/b ratio
Light 0 hr. 31.1980 29.0130 60.2100 1.0753
6 hr. 30.7200 18.1500 48.8670 1.6926
24 hr. 30.8940 20.3090 51.2030 1.5212
Light/Dark 0 hr. 22.1500 6.6748 22.8170 3.3185
6 hr. 23.1200 6.3914 29.5110 3.6174
24 hr. 19.9820 5.9211 25.9040 3.3741
Dark 0 hr. 0.1864 0.0891 0.2755 2.0920
6 hr. 0.5734 0.0891 0.2755 2.3047
24 hr. 0.3363 0.2397 0.5760 1.4030

 

 

43

Table 2. Chlorophyll Accumulation in the Cytoplasmic albino
Petunia inflata, Cultured in the Light, Light/Dark

 

or Dark and Fed with 0.1 N 3-ALA for 0, 6 and 24

Hours.

 

Chlorophyll Accumulation (mg/l)

. Time of
Env1ronment

 

b-ALA Feeding

 

Chl a Chl b Chl (total) Chl a/b ratio
Light 0 hr. 1.3562 0.4533 1.8095 2.9918
6 hr. 1.1433 0.6522 1.7955 1.7530
24 hr. 1.5320 1.3152 2.8472 1.1648
Light/Dark 0 hr. 0.5215 0.2460 0.7675 2.1199
6 hr. 0.3343 0.1647 0.4990 2.0298
24 hr. 0.3597 0.3823 0.7420 0.9409
Dark 0 hr 0.0087 0.0297 0.0384 0.2929
6 hr. 0.0863 0.1654 0.2517 0.5218
24 hr. 0.0374 0.0958 0.1332 0.3904

 

44

Table 3. Chlorophyll Accumulation in the Nuclear Albino Petunia

inflata Cultured in the Light, Light/Dark or Dark and

Fed with 0.1 N b -ALA for 0, 6 and 24 Hours.

 

Environment

Time of

A -ALA Feeding

Chlorophyll Accumulation (mg/l)

 

 

Chl a Chl b Chl (total) Chl a/b ratio
Light 0 hr. 7.5495 3.0676 10.6170 2.4610
6 hr. 6.2590 3.0480 9.3070 2.0535
24 hr. 6.3070 3.3194 9.6264 1.9000
Light/Dark 0 hr. 1.4417 0.6562 2.0979 2.1970
6 hr. 0.9931 0.5235 1.5166 1.8970
24 hr. 0.8492 0.6839 1.5331 1.2417
Dark 0 hr. 0.1169 0.1015 0.2184 1.1517
6 hr. 0.1440 0.0675 0.2115 2.1333
24 hr. 0.1556 0.1442 0.2998 1.0791

 

“firm 11“ “vb.
1 ,

45

Table 4. Chlorophyll Precursor Accumulation in the Wild-type Petunia
inflata, Cultured in the Light, Light/Dark or Dark and Fed
with 0.1 N b-ALA for o, 6 and 24 Hours.

 

Precursor Accumulation (n moles/m1)

 

 

Environment Time Of
b - Feeding Proto-IX Mg-Proto Pchlide
Light 0 hr. 107.2900 60.0130 28.3340
6 hr. 76.8690 43.4030 21.0000
24 hr. 85.6300 47.3900 23.4200
Light/Dark 0 hr. 43.5800 23.4330 10.4640
6 hr. 41.6400 22.9500 10.9170
24 hr. 40.0100 21.4200 9.5008
Dark 0 hr. 0.9678 0.4206 0.1080
6 hr. 2.8800 1.2670 0.3052
24 hr. 3.2940 1.3430 0.2461

 

46

Table 5. Chlorophyll Precursor Accumulation in the Cytoplasmic Albino
Petunia inflata, Cultured in the Light, Light/Dark or Dark

 

and Fed with 0.1 N 3-ALA for 0, 6 and 24 Hours.

 

Precursors Accumulation (n moles/m1)

 

 

Time of
Environment
b_' Feeding Proto-IX Mg-Proto Pchlide
Light 0 hr. 3.5529 1.7705 0.6478
6 hr. 7.8882 3.7804 1.3802
24 hr. 16.3600 7.3261 2.4088
Light/Dark 0 hr. 2.2097 1.0421 0.3049
6 hr. 2.3327 0.9980 0.3394
24 hr. 4.8383 2.0773 0.6525
Dark 0 hr. 0.4209 0.1969 0.0133
6 hr. 2.1100 0.8705 0.1126
24 hr. 1.9118 0.7662 0.0626

 

47

Table 6. Chlorophyll Precursor Accumulation in the Nuclear Albino

Petunia inflata, Cultured in the Light, Light/Dark or Dark

 

and Fed with 0.1 N A-ALA for 0, 6 and 24 Hours.

 

Precursors Accumulation (n moles/ml)

 

 

. Time of
EnVironment
b 41in Feeding Proto-IX Mg-Proto Pchlide
Light 0 hr. 14.7500 8.1225 3.7160
6 hr. 17.9400 9.8018 5.4683
24 hr. 39.5370 12.2225 7.5250
Light/Dark 0 hr. 3.6914 1.8740 0.7309
6 hr. 4.4682 2.0166 0.6251
24 hr. 10.5030 3.6156 0.9086
Dark 0 hr. 0.9261 0.4275 0.1249
6 hr. 1.2321 0.5063 0.0546
24 hr. 1.5778 0.6706 0.2125

 

0

48

Table 7. Visual Observation for Chlorophyll Formation 2 Days

Following Feeding of Albino Petunia inflata mutants

 

 

 

 

FeSO4 (ppm) + 3% Sucrose
Mutants
0 0.5 l 2 5 10 100 500
Nuclear W Y+ANT Y+ANT Y+ANT Y Y Toxic Toxic
Cytoplasmic W Y Y Y Y+ANT Y+ANT Toxic Toxic

 

W - White (loses the traces of chlorophyll)
A — Greenish—yellow color (with traces of chlorophyll)

ANT - Anthocyanin Formation

Table 8. Visual Observation for Chlorophyll Formation 2 Days

Following Feeding of Albino Petunia inflata mutants with

 

 

 

 

F8Cl3
FeCl3 (ppm) + 3% Sucrose
Mutants
O 0.5 1 2 5 10 100 500
Nuclear W Y Y+ANT Y+ANT Y+ANT Y+ANT Toxic Toxic

Cytoplasmic W Y Y Y Y Y Toxic Toxic

 

49

Table 9. Visual Observation for Chlorophyll Formation 2 Days

Following Feeding Albino Petunia inflata Mutants

 

with NazEDTA

 

NazEDTA (PPm) + 3% Sucrose

Mutants

 

0 0.5 1 2 5 10 100 500

 

Nuclear W Y+ANT Y+ANT Y+ANT Y+ANT Y+ANT Toxic Toxic

Cytoplasmic W Y Y Y Y Y Toxic Toxic

 

RESULTS

Chlorophyll and Chlorophyll-Precursor Accumulation in Normal Green,

 

Nuclear and Cytoplasmic Albinos of P. inflata

 

Normal green plants, with or without feeding with 3 -ALA,
accumulated considerably greater amounts of chlorophyll a, b and total
chlorophyll in comparison to the albinos (Tables 1, 2 and 3). The
nuclear albino accumulated higher quantities of chlorophyll than the
cytoplasmic albino. The total chlorophyll content of the nuclear albino
was about six-fold less than that of the normal green plants when grown
in the light, irrespective of the duration of b-ALA feeding periods.

The chlorophyll content of the cytoplasmic albino was 20 to over 30 times
lower than that of normal green plants under similar conditions. In the
light/dark regime, the chlorophyll content of the normal green plants

was only 2 - 3 times lower than those under light grown conditions. But,
when compared to the albinos, the chlorophyll content of the nuclear
albino was five to six times lower than the light grown condition and

10 - 20 times lower than the normal green plants grown under the same
condition. For the cytoplasmic albino, under the light/dark growing
condition, the chlorophyll content decreased two to 4.5 times in comparison
to the light grown plants. This response is similar to the decrease in
the normal green plants and when compared to the normal green plants in

the same growing condition, is about ten- to twenty-fold lower. In the

50

51

dark, the chlorophyll content of the green plants was reduced sixty
to ZOO-fold, significantly lower than that for light grown plants. In
contrast to the normal green plants, the chlorophyll content of the
dark grown nuclear albino decreased less than fifty-fold, whereas it
was much lower for the cytoplasmic albino. The comparison was made for
all durations of feeding with 3 -ALA.

Even though there were contrasting differences in the chlorophyll
content between the normal green and albinos, the chlorophyll a/b ratio
was similar. However, the chlorophyll a/b ratio tended to increase when
the normal green plants were exposed to dark conditions. A reverse
response occurred in the albinos, for plants grown in light/dark or
total darkness.

The chlorophyll precursor analyses are summarized in Tables 4, 5
and 6. The level of precursors accumulated in normal green P, inflata
were higher than those of the nuclear albino. In turn, the nuclear albino
accumulated higher levels of precursors than the cytoplasmic albino. The
nuclear albino had obvious traces of chlorophyll on the leaves, especially
at the tips and the mid-rib regions while the cytoplasmic albino had a
uniform pale yellow color. The amount of precursors accumulation is known
to be controlled by the feedback inhibition of the precursors themselves;
therefore, plants were externally fed withb-ALA in order to more
precisely determine accumulation. The resulting data were compared by
computing the ratio between Proto IX to Mg-Proto and Pchlide, by setting
Pchlide at 1.0 because accumulation is a relative value.

The normal green plants did not accumulate a significantly higher

amount of any precursor upon feeding with b-ALA. Only dark grown plants

52

had a small increase in Proto IX. However, the Pchlide of the dark
grown normal green 2, inflata did not increase.

Unlike the normal green plants, the precursors present in albinos
accumulated significantly higher quantities following feeding with
13-ALA. This increased precursor accumulation occurred not only in
plants under the dark grown condition but also for those under
light/dark and light grown conditions. When Proto Ix and Mg-Proto were
compared to the Pchlide, the quantity of Mg-Proto did not increase but
Proto Ix increased three to 4.5-fold under dark grown conditions and
1.2 to 2-fold in the light/dark grown condition. The increases in the
precursors were more obvious in the cytoplasmic as compared to the

nuclear albino.

Effect of Iron Concentration and Sucrose on Chlorophyll Formation

 

in Nuclear and Cytoplasmic albinos

 

Iron, in all forms tested, maintained traces of chlorophyll content
in both the nuclear and cytoplasmic albinos. Leaf pieces floated in the
sugar solution without iron turned white. Leaf pieces incubated in the
sucrose solution with iron ions maintained traces of chlorophyll. The
nuclear albino leaf pieces did not exhibit traces of chlorophyll in the
sucrose solution alone. However, chlorophyll content was present on
pieces incubated with sucrose solution plus 0.5 — 1.0 ppm iron from
FeCl3, FeSO4 and NaZEDTA. The iron sources were found to be toxic at

100 and 500 ppm. No obvious differences were found in the degree of

greening of top leaves incubated in sucrose solution containing

 

53

0.5 - 10 ppm iron from NaZEDTA or FeSO However, with FeCl3 the

4.
quantity of the chlorophyll for the nuclear albino was more intense in
solutions with one to five ppm. Less greening occurred in the 0.5 and

10 ppm FeC13 solutions.

Anthocyanin pigment accumulated when the leaf pieces of the nuclear
albino were incubated in the solution containing certain forms and
levels of iron. FeC13 between one to ten ppm, NazEDTA at 0.5 to ten ppm
and FeSO4 at 0.5 to two ppm all induced anthocyanin accumulation in
nuclear albino leaves. The anthocyanin pigmentation occurred as purple
sectors on the otherwise pale, greenish-yellow leaves.

The cytoplasmic albino responded similarly to the nuclear albino
in that traces of chlorophyll were not retained in the sucrose solution
without iron. The greenish-yellow color was only retained in sucrose
solutions with iron in the form of FeC13 at 0.5 to ten ppm or at 0.5
to ten and at 0.5 to 1.0 ppm of FeSO4 or NazEDTA, respectively. Iron
from all forms was toxic at one hundred and five hundred ppm. For
FeCl3, increases in the degree of greening was correlated with changes
in concentrations. Greening was not pronounced in solutions with 0.5 ppm
FeC13 and as the concentration increased from one to ten ppm, the
greening decreased. The occurrence of anthocyanin pigmentation in the

cytoplasmic albino was rare. A few sectors that contained faint

anthocyanin were observed only at five to ten ppm FeSO4.

DISCUSSION

This research indicated that the nuclear controlled Petunia
inflata albino is unstable with respect to the small amount of
chlorophyll accumulation. Conversely, the cytoplasmic albino mutant
was quite stable. These observations were not entirely unexpected since
visual observations on the degree of greening of stock cultures of
these two mutant lines revealed the same pattern. The phenotypic
variability of a mutant defective in chlorophyll biosynthesis was

previously studied in Nicotiana tabacum var. "Samson" and was believed

 

to be controlled genetically (Deshayes 1979). Regreening as a result
of a back mutation may be observed more frequently in the nuclear
albino which is inherited as a recessive trait, because one back
mutation in the nuclear genome could directly correct the albinism in
all the progeny cells. Since there are many chloroplasts per cell, the
cytoplasmic albino would have to undergo the sorting-out process following
a back mutation prior to the green pigmentation being readily observed.
That chlorophyll content decreased more in etiolated wild-type
plants in comparison to the albinos was also expected, since it contains
a much higher amount of chlorophyll. However, the observation that the
chlorophyll a/b ratio increased in normal green plants but decreased in
the albinos when grown in the dark needs more study to reach a

definitive interpretation. Some chlorophyll inhibitors have been

54

55

found to change the a/b ratio. The antibiotic terramycin, digitoxin and
plant growth regulators such as BAP and K altered the chlorophyll a/b
ratio (Wolf 1977). BAP and K, which decrease the chlorophyll a/b ratio
and change chlorophyll content (Parthier 1979) may stimulate back
mutation in suspension cultures of albino P, hybrida Comanche (Ad Kool -
personal communication). Thus, they might be responsible for this
phenomenon as observed herein by green sector formation on leaves of

the cytoplasmic albino P, inflata. The albinism might cause removal

or accumulations of the inhibition; therefore, the chlorophyll a/b

ratio decreased, whereas it increased in normal green plants. However,
added cytokinins were not able to overcome a genetically-caused defect
in chloroplast formation nor were they necessary for the regreening
process in N-deficient leaves of plants (Parthier 1979). Changes in
chlorophyll a/b ratio have also been observed in mutants of pearl millet
where the ratio increased, decreased or remained constant (Kodurn

and Kao 1980).

The discovery by Granick (1959) that plants accumulate more
chlorophyll precursors upon exogenous feeding with b -ALA has enabled
studies in chlorophyll defective mutants to be investigated extensively.
Both maize and barley have been studied relative to where blockage
occurred in the chlorophyll biosynthetic pathway by determining
chlorophyll precursor accumulation (Mascia 1978, Gough 1972). However,
there are few reports on how to bypass the blocked steps (Bell, et. al.,
1958, Bell, et. al., 1962).

The cytoplasmic and nuclear albinos were both found to be blocked

56

at the Proto IX synthesis step since it was accumulated when externally
fed with 3 -ALA. These Petunia mutants are thus similar to the mutants

1* Blandy-4, OY-1040 and I-13 in maize (Mascia 1978).and several other
mutants as reviewed by Gough (1972). There are several possible reasons
for the block in chlorophyll biosynthesis pathway. First, a change in

a structural gene causing inhibition of enzyme production or alteration
of the enzyme conformation so that the pathway cannot proceed. A second
reason could be changes in the chloroplast membrane. Since the
biosynthetic reactions following Proto IX are membrane-bound (Smith and
Rebeiz 1979), mutations affecting conformation of the chloroplast
membrane may result in defective chlorophyll biosynthesis. Bell, et. al.,
(1958) and Bell, et. al., (1962) also proposed that albinism can be
caused by the inability of plants to take up iron for chlorophyll synthesis
and can be corrected by external feeding with the propor iron form.

Iron is known to be required for chlorophyll biosynthesis and chloroplast
development (Machold and Stephan 1969 and Stocking 1978). The last
possibility may be operative in the Petunia albino mutants. Traces of
chlorophyll in albino leaves could be maintained by feeding with sucrose
solutions containing iron, but the regreening response was only partial
since the leaves did not turn completely green. Further efforts could be
made by culturing the leaves in zitrg_on medium containing FeCl3 instead
of NazFeEDTA. However, the involvement of growth regulators, especially
cytokinins, which are known to alter chlorophyll content and the optimal
requirement of iron for growth, which does not relate to its optimal
concentration for growing, have to be taken into account.

The cytoplasmic albino was observed to form green sectors when

57

grown in_vit£9_with high levels of cytokinins (K or BAP). This
phenomenon was not observed with the nuclear albino. The green sectors
are believed to be formed by back mutation of the chloroplasts to
wild-type. The progenitor heteroplastic cells divide and are accompanied
by the sorting-out process which eventually leads to certain cells
containing only normal green chloroplasts (Kirk and Tilney-Bassett 1978).
Since the high cytokinin level in the culture medium induces multiple
shoots, the sorting-out process may occur more frequently in the newly-
formed shoots. Or, if the shoots are derived from callus at the base of
terminal shoot cuttings, the green sectors are more liekly to be caused
by back mutation and not by albinism correction since the other leaves
remain phenotypically albino.

The previous experiments also showed that casein hydrolysate and
plant extract supplied in the culture medium inhibited chlorophyll
biosynthesis in the cytoplasmic and nuclear albino, respectively,
since the usual traces of chlorophyll disappeared. The nutritional
regulation of chlorophyll formation has previously been studied in
algae (Horwen and Schwartzbach 1980, Ellis, et. al., 1975). The
ratio of nitrogen to carbon is believed to control the quantity of
chlorophyll in Euglena and elevated concentrations of acetate,
especially combined with a low level of nitrogen, inhibit chlorophyll

synthesis in Goclenkinia. More recently, McCarty and Rebeiz (1980)

 

found that an extract from cucumber cotyledons inhibited chlorophyll
biosynthesis and accumulation in the cotyledon itself. This response is
similar to our experiment wherein the wild-type plant extract inhibited

chlorophyll biosynthesis of the nuclear albino. As stated previously,

58

the mutants accumulated Proto Ix; however, the block is incomplete.
Plant extract may act as a secondary block for the bypass chlorophyll
at Pchlide. Plant extract was found effective only for the nuclear
albino. On the other hand, casein hydrolysate inhibited traces of
chlorophyll formation only in the cytoplasmic albino. The mode of
action of casein hydrolysate on chlorophyll biosynthesis has not been
reported but can be considered to be different from plant extract

since it differentially affected the nuclear and cytoplasmic controlled
albinos.

In experiments on external feeding of iron in three different forms
to albino leaf pieces, it was observed that anthocyanin pigment was
formed when the cytoplasmic albino was fed with FeSO4, but only faintly
in a few sectors. In contrast, anthocyanin pigmentation occurred in
abundance in the nuclear albino when supplied with any form of iron
tested. Anthocyanin has been reported to accumulate in cell cultures of

Happlopappus gracilis when grown in low auxin (Constabel, et. al., 1971)

 

and Onslow (1925) reviewed the effect of sugar, physical and chemical
factors, reaction with iron and mechanical injuries influence anthocyanin
pigmentation. The formation of anthocyanin pigment in the albino leaves
might occur through a combination of the above effects since sugar
feeding alone was not stimulatory.

It is obvious that the nuclear albino is physiologically quite
different from the cytoplasmic albino and these differences are
quantitative. Nuclear genes controlling albinism have been studied
extensively. Conversely, cytoplasmic albinos have been studied much less

extensively, especially in higher plants, because of the unavailability

59

of plant materials. However, since both types of mutants result in
the same phenotype expression, the inability to produce a normal
quantity of chlorophyll, the background physiological processes can
be assumed to be alike.

Kirk and Tilney-Bassett (1978) have suggested that tissue culture
would be a useful tool to study the genetics and physiology of
albinism in plants. This is obviously true, especially for the cytoplasmic
determined albinos which need more intensive investigation in order to
understand their role in chlorophyll biosynthesis and their interaction

with nuclear genes.

SUMMARY

1. The nuclear albino Petunia inflata was more unstable than the

 

cytoplasmic albino and accumulated total chlorophyll six times lower
than the normal green plants. The nuclear albino accumulated twenty to
thirty times less than the normal green wild—type plants.
2. Chlorophyll content of normal green plants decreased more than
in either albino mutant when grown in the dark as compared to plants
grown in the light.
3. The chlorophyll a/b ratio increased when normal green plants
received the dark grown condition but hte situation was reversed for
both albino mutants.
4. Etiolated normal green plants accumulated a small amount of
Proto IX and the accumulation was not increased significantly when the
plants were fed with 3 -ALA. Pchlide was not observed to show any increase.
5. Both the cytoplasmic and the nuclear albinos accumulated
Proto IX at a significantly higher amount when grown in the dark. The
cytoplasmic albino accumulated relatively higher levels of Proto IX
when compared to the nuclear albino.
6. The albino mutants retained traces of chlorophyll upon external
feeding with FeSO4, FeCl3 or NazEDTA. FeC13 showed a shift effect on the
greening level of the albino leaf pieces incubated in the sucrose solution

with FeCl3 at certain concentrations.

60

61

7. Anthocyanin pigmentation accompanied chlorophyll retention
in the albino mutants. The nuclear albino, upon feeding with certain
concentrations of FeCl3, FeSO4 or NazFeEDTA, consistently showed

anthocyanin formation, but it was very rare with the cytoplasmic albino.

LITERATURE CITED

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