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This is to certify that the

thesis entitled

RESPONSES OF PETUNIA TO SULFUR DIOXIDE OR

SODIUM SULFITE AT DIFFERENT LEVELS

OF BIOLOGICAL ORGANIZATION
presented by

Edward Paul Mikkelsen

has been accepted towards fulfillment
of the requirements for

Ph-D- degreein HORTICULTURE

 

 

</. C M

Major professor

J l 2
Date u y 1 , 1978

 

0-7639

 

 

RESPONSES OF PETUNIA CULTIVARS TO SULFUR DIOXIDE OR SODIUM

SULFITE AT DIFFERENT LEVELS OF BIOLOGICAL ORGANIZATION

By

Edward Paul Mikkelsen

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1978

ABSTRACT

RESPONSES OF PETUNIA CULTIVARS TO SULFUR.DIOXIDE OR SODIUM
SULFITE AT DIFFERENT LEVELS OF BIOLOGICAL ORGANIZATION

By

Edward Paul Mikkelsen

In order to evaluate a possible mutation-selection system for SO2
resistance in Petunia hybrida, the responses of five cultivars of

g, hybrida to treatments of SO2 or Nazso3 solutions at various levels of
plant organization were studied. Whole plants were fumigated with $02
at 5 ppm for 16 hours and rated according to the amount of necrotic and
chlorotic lesions on the leaves. Leaf discs were treated with Na2803
solutions at the concentrations of 2.5, 5.0, and 7.5 mM and evaluated
by measuring the absorbances at 665 nm of ethanol extracts of chloro-
phyll. Callus cultures were treated the same as leaf discs but were
evaluated by a triphenyl tetrazolium chloride (TTC) staining procedure
to determine viability.

At each level of organization studied there were significant dif-
ferences among the responses of the various cultivars. The trend of
the responses of whole plants was similar to that reported in the
literature. However, the rank of the cultivars according to their

responses as leaf discs was not the same as that of the whole plants.

Likewise, the rank of the cultivars according to the responses of

Edward Paul Mikkelsen

callus cultures grown on one medium was different from either the rank
of whole plants or of leaf discs. There were no significant differ-
ences among the responses of callus cultured on a second medium.
A significant correlation existed between the response of the callus
cultures and the viability determined by the TTC staining procedure.
Because of the lack of correlation among the responses of whole
plants, leaf discs, and callus cultures, it was concluded that responses
of g, hybrida at these various levels of organization to SO2 or its
ions in solution were not primarily determined by the physiochemical
systems directly affected by 802. Alternatively, the responses were
possibly determined by other factors that were unique for each level of
organization. Therefore, at this time, the most effective method of

selection of desirable phenotypes for resistance to SO2 in g, hybrida

is to screen whole plants by fumigation with $02.

ACKNOWLEDGEMENT

I wish to thank the members of my committee, Drs.
K. C. Sink, L. R. Baker, J. W. Hanover, R. C.
Herner, and P. S. Carlson, for their suggestions
concerning the research and for their constructive
criticisms of this text. I also wish to thank my
family for their encouragement and patience.

 

ii

TABLE OF CONTENTS

Page

LIST OF TABLES....00...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO v

LIST OF FIGURESoooooooooooooso...oooooooooooooooooooooooooooooooo Vii

 

INTRODUCTION...0.00.0000...00.0.0...0.0.0.000...

................. l

LITEMTUE REVIEWOOCOCOO0.00...0..00....OOOOOOOOCOOOOOOOOOO00.... 5

PhYSiOlogy Of $02 InjurYOOOOOO0.0...OOOOOOOOOOOOOOOOOOOOOOO 5
Mutation and Selection of Plant Cells in Tissue Culture.... 11

MATERIALS AND METHODS............................................ 18
Plant Material............................................. 18
Whole Plant Experiments.................................... 18
Whole Leaf Experiments..................................... 20
Leaf Discs................................................. 20
Lower Leaf Epidermal Strips................................ 21
Tissue Culture............................................. 22
Media................................................ 22
Callus culture initiation............................ 22
Viability staining................................... 23
Preliminary experiments.............................. 24
Cultivar by N32803 concentration factorial experi-
ments.......................................... 24
NaCl stress experiment............................... 26
NaCl, NazSO3 double stress experiments............... 26

RESULTS

Whole Plant Experiments.................................... 28
Whole Leaf Experiments..................................... 28
Leaf Disc Experiments...................................... 28
Epidermal Strips........................................... 30
Tissue Culture Experiments................................. 31
General growth of callus............................. 31
Correlation of growth and visual rating of TTC
staining....................................... 31

TABLE OF CONTENTS--continued Page

Effects of ionic strength and pH..................... 32

Duration of exposure to Na SO ....................... 32

Potency of Na2803 solutions.......................... 33

Effect of pH on Nazso damage........................ 34
Cultivar by N32803 concentration factorial experi-

ments.......................................... 34

Results of NaCl experiments.......................... 38

Results of double stress experiments................. 39

Dose Response of Leaf Discs and Callus Cultures to Na S0 41

smry Of Experiments.I.OOOOOOOOOOOOOOOOOOOOOIOO0...?OO?OO 41

DISCUSSIONCOOOOOOOOOOOOOOOOOOOOOOO0.00....OOOOOOOOOOOOOIOOOOOOOOO 44

RECOMNDATIONSOOOOOOOOO...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 52

APPENDIX: AOV TABLESOO.0.COCOOOOOOOOOOOOOOOOOOOO.OOOOOOOOOOOO... 53

BIBLIWRAPHYOOOOOOOOOOOJ'OOOOOOOOOOOOOOOOO.OOOOOOOOIOOOOOOOOOOOO. 57

iv-

LIST OF TABLES

TABLE

10.

ll.

Petunia hybrida cultivars used to study the response of

various levels of biological organization to treatments of

80 gas or Na SO solutions.................................
2 2 3

Mean R values of undetached petunia leaves after 16 hours

exposure tosppm $02 gas.0..00.000.00.00...00.00.000.000...

Mean percent damage to whole detached leaves after 24 hours
exposure to 10 mM Na2803 of initial pH of 5.0 to 5.5........

Normalized absorbances of chlorophyll extracts of leaf discs
of 5 petunia cultivars averaged over Na S0 concentrations
after 3 hours exposure to 2.5, 5.0 or 7.5 Na 80 at

initial pH Of 500 to 5.5.0.0000...O...OOOOOOOOO?IO?OOOOOOOOO

The effect of Na 80 concentration on mean normalized absorb-
ances of chloropfile extracts of petunia leaf discs, all
cultivars, after 3 hours exposure to solutions at initial pH

Of 500 to SOSOOOOOOOOOOOCOOOOOOOOOOOOO0.00000000000000000000

Callus growth rates of 5 petunia cultivars on two media as
determined by the ratio of the fresh weight after 12 days
to the initial freShweightOOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Effect of ionic strength and pH on TTC viability ratings of
petunia callus cultures after 3 hours exposure..............

Mean TTC viability ratings of petunia callus cultures after
1, 6, 24 and 49 hour exposure to 1000 ppm of Na S0 at

initial pH Of 5.5....COO...00.0.0000....0...‘.O?OO?OOCOOO0..

Mean TTC viability ratings of petunia callus after 2 hours
exposure to 1500 ppm.NaZSO starting 1, 4, 24, and 48 hours
after preparation of the salution...........................

The effect of pH on mean TTC viability ratings of petunia
callus after 24 hours exposure to 500 ppm NaZSOB............
The effect of Na SO concentrations on mean TTC viability
ratings of petun a gallus, all cultivars, after 3 hours
exposure to solutions at initial pH of 5.0 to 5.5...........

Page

19

29

29

29

29

31

32

33

33

34

36

LIST OF TABLES--continued

TABLE

12.

l3.

14.

15.

16.

17.

A3.
A4.
A5.
A6.

A7.

Mean TTC viability ratings of the callus of 5 petunia culti-

vars after 3 hours exposure to 2.5, 5.0 or 7.5 mM Na 80 at

2

initial pH of 5.0 to 5.5. ADV showed no significant differ-

ences among the cultivars on PM medium......................

Mean TTC viability ratings of the callus of 5 petunia culti-

vars after 3 hours exposure to 2.5, 5.0, and 7.5 mM Na2S03.
Analysis by pairs of blocks treated simultaneously.........

Correlation coefficient within cultivars of mean TTC via-
bility ratings of controls with the sum of the normalized
TTC viability ratings of the Na2803 treatments.............

Mean TTC viability ratings of callus of cultivar 1006 after
24 hours exposure to 5% NaCl and/or 24 hours exposure to
5mNastBOOOOOOO.O...COOOOOOCOOOOOOOOOOOOOOO00.0.0...O...

TTC viability ratings of callus of cultivar 1006 after 21
hours exposure to NaCl solutions and 3 hours exposure to

Nast3 salutionSo000000000osocoo.oooooooo000.000.000.000...

Rank of 5 petunia cultivars to SD or Na 80 susceptibility
at different levels of biological organizat on.............

ADV: Whole P1ants.........................................
ADV: Whole Leaves.........................................
ADV: Leaf Discs...........................................
ADV: MSNP Medium..........................................
ADV: PM Medium............................................
ADV: NaCl Stress..........................................

AOV: Second Double stIBSSoooooooooooooooo0.000000000000000

vi

Page

37

37

38

40

40

43
59
59
60
60
61
61

62

FIGURE

1.

2.

LIST OF FIGURES
Page
The effect of pH on mean TTC viability ratings of petunia
callus after 24 hours exposure to 500 ppm N32803°"°'°'°°°° 35
The effect of Na SO concentration on the mean normalized
absorbance of chIorgphyll extracts of petunia leaf discs

and on the mean normalized TTC viability ratings of
petunia callus cultures. Exposures were for 3 hours....... 42

vii

INTRODUCTION

The ability to culture and regenerate plant cells and protoplasts
ig_vi££g_give the plant breeder a potential method to improve crop
species by mutation induction and selection of specific traits at the
cell level. Since mutation rates are as low as 10'.6 to 10.7 (10,11,38,
49,64,65), large populations must be screened to find desirable mutants;
therefore, selection of cells in culture is more manageable than selec-
tion of whole plants in greenhouses or fields. However, four biological
criteria must be met in order for this method to be effective for the
plant breeder. Firstly, mutation of cells in culture must be possible
or natural genetic variants must exist. Secondly, the desired charac-
teristic must be expressed in culture in such a way that variants with
the desired characteristic can be selected. Thirdly, whole plants must
be capable of being regenerated from the selected variant cells. And
fourthly, the regenerated plants must express the desired trait that was
selected in culture.

Experiments have been conducted which demonstrate the feasibility
of mutation and selection of specific mutants ig;3§£323 Mutants resis-
tant to various amino acid analogs (ll,l7,49,64,65,66), pyrimidine base
analogs (33,37,42), and streptomycin (5,38) have been successfully
selected. Also, some "leaky" auxotrophic mutants have been isolated

(10). However, whole plants could not be regenerated from.most of the

selected cell lines; therefore, it is not known whether those selected
traits would have been expressed by whole plants.

The criterion that the desired trait be expressed by cells in_
vitrg and by whole plants is paramount. Research studies have been
done which indicate that certain whole plant traits can be expressed by
cells in culture. For example, the single dominant gene conferring

resistance to Phytophthora infestans race 0 in Nicotiana tabacum was

 

expressed by cells in callus culture under very defined and stringent
conditions (24). Correlation of responses between whole plant and cells
in gi££g_has been reported for other host-pathogen systems (20,22,28,62).
Recently, the unique physiological characteristic of low photo-respira-
tion of C4 plants was expressed by cells in zi££g_and was, therefore,

not dependent on the unique Kranz anatomy of C plants (31). The

4
resistance and susceptibility of four lines of N, tabacum to ozone
injury were also expressed when cells grown in gi££g_were exposed to
ozone (T. Rice and P. Carlson, personal communication).

There are relatively few examples where traits selected at the
cellular level in zi££g_were expressed by their regenerated plants.
Cells of gggug§y§_L. from Texas male-sterile cytoplasm lines were
selected for resistance to Helminthosporium maydis Race T pathotoxin
(C. Green, personal communication). Regenerated whole plants were
resistant to the disease, but the plants had lost their male-sterile
cytoplasmic characteristic. Haploid E, tabacum.cells that were resist-

ant to the methionine analog, methionine sulfoximide were selected

in_vitro (ll). Diploidized regenerated plants were not only resistant

to the analog but were also resistant to Pseudomonas tabaci. The toxin
of P, tabaci is also an analog of methionine. Haploid N, tabacum cells
resistant to streptomycin have also been selected in zi££2_(38).
Diploidized regenerated plants were not tested directly for resistance,
but leaf discs of resistant and control plants were placed on a medium
containing streptomycin. Discs of the control plant produced some white
callus, while discs of the mutant produced large amounts of green callus.
Reciprocal crosses of the mutant plant with control plants showed that
the resistance was maternally inherited.

The project reported herein was undertaken to study the feasibility
of an in vi££g_cultural system.for developing sulfur dioxide, $02,
resistance in Petunia hybrida. P, hybrida was chosen as the experi-
mental plant species since tissue culture techniques have been previous-
ly reported. Callus induction and subsequent plant regeneration has
been accomplished for both diploid and haploid types (3,4,18,54).
Protoplast isolation and whole plant regeneration was reported for
diploid (18) and haploid Petunia (4). Most recently, protoplasts of
P. hybrida and 1:. parodii were fused to produce somatic hybrids (51).
Furthermore, much of the cytology, genetics, breeding, and taxonomy of
Petunia has been established (7,8,16,17,21,29,40,44,45,50,52,58).

The problem of SO resistance was selected since Petunia hybrida

2

cultivars exhibit susceptibility to ambient air pollution, of which 802
is the main component worlddwide (6). Also, numerous cultivars have
been evaluated with respect to 802 susceptibility on a whole plant

basis (15). Likewise, some of the detrimental modes of action of SO2

on plant cell physiology have been examined. Reported effects include

direct interference with photosynthetic CO2 fixation and energy metabol-

ism via inhibition by 8032, inhibition of various enzymes by redox

products of 8052 and cellular metabolites, and interference with
membrane function and ultrastructure (37). Despite the many possible

modes of action for 802, single gene mutations for SO resistance might

2

be detected because 1) certain processes could be more sensitive to
SO2 than others; and, a mutation of that system to resistance would

increase overall resistance, and 2) genetic differences for SO2 resist-

ance have been reported on a whole plant basis (15). There are,
however, potential limitations in this type of research. Correlations

between whole plant and in_vitro cell responses, with respect to 802

damage, may not exist since the stomata may play an important role on a
whole plant basis. Obviously, stomata play no role in unorganized cells
cultured i§_vitro. Likewise, since cultures are grown in the dark,

photosynthetic fixation of CD will not be a factor in conferring SO

2 2

susceptibility.ig.vitro.

LITERATURE REVIEW

Physiology of SO6 Injury

Probably no plant species is immune to SD injury, but there is

2
great variability amongst species relative to the concentrations neces—
sary to produce phytotoxicity. For example, the eastern white pine
(Pinus strobus) showed damage after exposure to 100 parts per billion

(ppb) SO2 for eight hours (14). In contrast, duckweed (Lemma minor L.)

 

was not affected by $0 at levels as high as 300 to 600 ppb (9).

2

In fact, the variable response to SD damage even extends to the culti—

2
var level. Certain petunia (Petunia hybrida) cultivars sustained
minimal damage (about 1%) while others suffered extensive damage (19%)
when exposed to the identical treatment of 2.5 parts per million (ppm)
SO2 for 1 hour (15).

Symptoms of SO2 phytotoxicity range from reduced growth to cellu-
lar and whole plant death. With broadleaf species the chronic symptoms
are interveinal chlorosis. Acute injury first appears as water—soaking
and loss of turgor followed later by dead areas, bleached ivory to tan
in color. If other pigments are present which were not concurrently
destroyed by the 802, then the necrotic areas may appear red or black.
Monocots usually deve10p necrotic streaks along the midveins. If the
leaf blade has a bend, often the necrosis begins at the bend. Injury

to species with needles begins at the tips of the needles and extends

downward. Mild exposure often results in chlorosis and premature drop

of older needles. A high dose results in water—soaking which later
changes from green to reddish brown. A succession of exposures results
in distinct reddish brown banding of the needles (30).

Since 802 enters through the stomata, the leaf is most susceptible
when the stomata are wide open. This generally occurs at high light
intensities from 10 AM to 2 PM together with high relative humidity,
adequate plant moisture and moderate temperatures. Therefore, plants
are most sensitive to SO2 in late spring and early summer. Middle-aged
leaves are most sensitive and older leaves are most resistant to injury
probably because of variation in the number, size, and activity of the
stomata (26).

Due to the high solubility of SO

in water, H is first pro-

2 2303
duced in the cell before reacting with other chemical components (37).
The pKa's of H2803 are 1.8 and 7.2 (32); therefore, the predominant ions
in the cell sap are sulfite (SOEZ) and hydrogen sulfite (H803).

Most plants are able to withstand and actually utilize a certain
level of $02 in the atmosphere. Alfalfa (Medicago sativa) exposed to
chronic, non-phytotoxic concentrations of $02 converted the sulfite ions
predominantly to sulfate ions (61). Some sulfur was actually incorpor-
ated into organic compounds. Furthermore, if sulfate in the soil was
low, the fumigated alfalfa had more organic sulfur than the non—
fumigated plants. IMitochondria of etiolated oat seedlings (£122é_

sativa) had an enzyme, sulfite oxidase, capable of converting sulfite to

sulfate (60). Similar utilization of SO in plant growth was shown to

2

occur with duckweed, Lemna minor L. (9). Sulfur fromSO2 was converted

to sulfate and then incorporated into amino acids and sulfoquinovose of
sulfolipids. Duckweed had an efficient sulfite oxidizing system, and

tolerated a relatively high concentration of SO in the atmosphere.

2

Trees can also incorporate SO in normal metabolism (55). White

2
birch (Betula papxrifera Marsh), aspecies sensitive to 802, and pin oak
(ngrcus palustris Muenchh.), a species resistant to 802’ were planted
at two locations in Ohio. One location was low in SO2 (less than 4 ppb)
and the other was high in 802 (27 ppb) but below injury levels. The

sensitive species, birch, grew better in the high SO environment, while

2
the resistant species, pin oak, grew worse in the high SO2 environment.
The author thinks that perhaps the 80 environment acted as a fertilizer

2
for the birch, but actually was deleterious to the pin oak growth since

the 802 stimulated stomatal closure.

If SO2 in excess of plant requirements enters, no matter how

slowly, chronic injury marked by chlorosis will occur (37). This
chlorosis is due to excess sulfate--oxidized sulfite--producing an

excess ion effect (30). If the excess SO2 enters faster than the cell

can convert it to sulfate, then the damage is the direct toxic effect
of sulfite. Mesophyll cells are primarily affected, and the chloro-
plasts become plasmolyzed or bleached.

Various effects of SO damage to chlorophyll, chloroplasts, and

2
photosynthetic capabilities have been reported. Rao (53) showed that

a 24—hour exposure to 5 ppm SO degraded chlorophyll a of lichen.

2
Chlorophyll a of lichen was affected more than chlorophyll b (52).

Chloroplasts of Vicig faba underwent a reversible swelling when exposed

to 250 ppb SO2 for one hour (63). Sulfur dioxide, even at levels indi-
cating no visible signs of injury, reduced the amount of chlorophyll of
cotyledons and primary needles and the dry weight of seedlings of Pinus
resinosa (l3). Chloroplast structure as well as photosynthetic activ-
ity, as measured by the Hill reaction, was impaired by $02 (36).
Chloroplast integrity correlated well with the photosynthetic activity
of Pinus contorta Dougl. needles. Chloroplasts from the basal portion
of the needle were affected the least and showed the most photosynthetic
activity; the opposite was true for chloroplasts from the tip of the
needle.

Aside from affecting chloroplast integrity and chlorophyll con-
tent, SO2 also disrupted enzymes involved in photosynthesis. Sulfite
ions affected the activity of ribulose-1,5-diphosphate carboxylase,
which fixes CO2 during photosynthesis, of isolated spinach (Spinacia
oleracea) chloroplasts (67). so;2 was a non-competitive inhibitor with
the substrate ribulose diphosphate with R1 of 14 mM 8032. Sulfite was

a competitive inhibitor with the substrate hydrogen carbonate ion with

3 Ri of 3 mM 3032. Finally, sulfite acted as a non-competitive inhibi-
-2

tor with the cofactor Mg+2 with a K1 of 9.5 mM SO3 .

first substrate to be affected by sulfite in the system‘was dissolved

Therefore, the

C02.

Sulfur dioxide or so;2 reacts with other cellular constituents.

Bailey £2 21. (1) showed that .3 M SO

3
hydryl groups of proteins. Even though high concentrations of cellular

was capable of breaking sulf-

sulfite is unlikely, such reactions at lower concentrations are possible,

perhaps to a lesser, but still damaging extent. More recently, sulfite
was established to react mostly with interchain disulfide bonds rather

than with intra-chain disulfide bonds (12).

—2
3

demonstrated (2). Bean (Phaseolus vulgaris) cotyledon and maize

Inhibition of ATP formation in plant mitochondria by SD was

3
at 77 and 70% of the controls, respectively. At 10 mM 8032, production

(gea_ggy§) coleoptile mitochondria exposed to 3 mM 80 produced ATP
of ATP was reduced to 56 and 44%. Even though bean was more sensitive
to $02 in gigg_than maize, the responses of the mitochondria ig;gi££2_
were not significantly different from each other. Therefore, this inhi-
bition did not account for all of the plant sensitivity to SO2 air
pollution.

Mkudt and Werner (41) showed that sulfite affects indole-3—acetic
acid (IAA) oxidation. Sodium metabisulfite at a concentration of .05
mM stimulated the initial reactions of IAA oxidation but inhibited the
subsequent formation of the other intermediates. They hypothesized
that the sulfite ions were scavenging the free radicals necessary for
the further reactions. They also found that the higher concentration

of 1 mM HSO3 inhibited even the initial reactions of IAA oxidation.
Using polyuridine (poly U), Shapiro (56) showed that 1 M sodium
bisulfite produced sulfonated residues on the uracil moeity. When such
residues were present, poly U would not base-pair with poly A.
Likewise, the ability of polyribouracil to code for phenylalanine

incorporation into protein was increasingly reduced as the number of

sulfonated residues was increased. At 2.6% of saturation, there was

10

only 54% incorporation of phenylalanine in comparison to the control;
and at 10.5% of saturation, there was only 8% incorporation. Shapiro
suggested that a slight degree of modification of mRNA (perhaps a single
uracil being sulfonated) could block translation.

Along the same lines, treatment with l to 10 mM bisulfite degraded
the double stranded DNA of the phage T7 (22). Manganese ion was neces-

sary, presumably to catalyze the autoxidation of SD with water to

3
form the radicals SD; and HZOE° These radicals were responsible for
the DNA breakdown. Detectable damage occurred after only 5 minutes of
exposure to 8032.

In addition to causing damage by direct chemical reaction with
vital cellular components, sulfite was able to react with cellular
constituents to form other inhibitor substances. Alpha-hydroxy-Z-
pyridinemethanesulphonate (aHPMS), glyoxal bisulfite, as well as sodium
bisulfite, were shown to inhibit CO2 fixation in the light by Atriplex
spongiosa, a 04 plant. However, there was no light-CO -fixation

2

inhibition on the 03 plant Atriplex hastata. These components also
inhibited the enzymes glycolate oxidase and phosphoenolpyruvate carboxyl-
ase of both species (48).

Murray and Bradbeer (43) found that aHPMS inhibited 002 fixation
by spinach chloroplasts by 50% at a concentration of 7 mM. They also
found that phosphoenolpyruvate carboxylase was competitively inhibited
at a concentration of 1 mM aHPMS. Hydroxysulfonates also inhibited

NADH:malate dehydrogenase in the mitochondria and peroxisomes.

11

In summary, $02 when in solution as bisulfite (H803) or sulfite
($032) interfered in a number of ways in cellular systems. It reacted
with membranes as evidenced by swelling and plasmolysis of chloroplasts;
it acted as a competitive inhibitor of a number of enzymes such as
ribulose-1,5-diphosphate carboxylase and those concerned with ATP pro-
duction in the mitochondria; it denatured protein by breaking sulfhydral
bonds; it disrupted protein synthesis by reacting with uracil residues
of mRNA's; it disrupted genetic information by degrading DNA; and it
formed a number of secondary compounds that disrupted normal cellular
functions. At this time, however, investigations are unable to defin-
itely establish which specific reaction(s) is(are) the most important in

determining SO damage of plants.

2

Mutation and Selection of Plant Cells in
Tissue Culture

 

A breeding system using mutation and selection in xigrg must have
several characteristics if it is to be effective in crop improvement.
1) Mutants must exist, either by occurring spontaneously or by induction
with a mutagenic agent, 2) the desired characteristic must be expressed
at the cellular level so that the desired cell types can be selected,
3) the mutant cells must remain totipotent so that whole plants can be
regenerated from them, and 4) the characteristic must be expressed in
the regenerated whole plant. The following literature review reveals
that certain characteristics at the whole plant level are expressed at
the cellular level, that mutation and selection of specific variant cell

types ig_vitro are possible, and that in some systems, characteristics

12

mutated and selected at the cellular level are expressed in plants
regenerated from the selected cells.

Most of the work that demonstrates expression of whole plant
characteristics at the cellular level have been host-pathogen inter-
action systems. Helgeson gt §l° (25) used callus culture of two similar
lines of Nicotiana tabacum L. in their research on the pathogen
Phytophthora infestans par. As whole plants, one was resistant and the
other susceptible to race 0 of the pathogen, and both were susceptible
to race 1. The resistance was conferred by a single dominant gene.
Under proper combinations of hormones and temperature treatment, the
resistance and susceptibility of the two lines to race 0 was expressed
in culture. Both lines in culture were susceptible to race 1. On a
medium with another hormonal composition, both lines were susceptible
to race 0; however, freshly excised pith also showed no differences in
resistance to race 0. Likewise, at temperatures above 27°C the
resistant line became susceptible. However, seedlings of the resistant
line also became susceptible at temperatures above 32°C. Because of
the specificities of the lines to the races of the pathogen and because
of the similar temperature responses, the differences in susceptibility
were apparently due to the genetic expression of resistance in callus
culture. Furthermore, plants regenerated from the two cell lines
retained their race specific resistance-susceptibility. Further experi-
ments were done with essentially genetically identical lines except
for resistance of one line to race 0 (24). F1, F and F generations

2 3

and testcrosses of homozygous recessive (susceptible) lines with the F1

13

were tested as whole plants and as callus. There was (near) perfect
correlation between the response of each of the whole plants and its
respective callus to both race 0 and race 1. This further substantiated
the claim that the dominant gene for resistance to race 0 was expressed
at the cellular level.

An earlier and less comprehensive study was done by Ingram and
Robertson (28) on the interaction of Phytophthora infestans and tissue
cultures of Solanum tuberosum. Resistance-susceptibility of two cul-
tures was expressed by large cell aggregates in culture, and, to a
lesser extent, by cells in suspension culture. Also, living cells were
necessary to inhibit stimulation of the pathogen; that is, dead cells
of the resistant cultivar stimulated P, infestans growth. Further
research using two races of the pathogen revealed that the resistance-
susceptibility in culture to both races correlated with the whole plant
responses (27).

A similar study was done by Warren and Routley (62) on
Phytophthora infestans race 0 and Lycopersicon esculentum. Again,
resistance-susceptibility was expressed in tissue culture.

Resistance of nonsterile cytoplasm Egg may§_and the susceptible

Texas male-sterile cytoplasm Zea mays to Helminthosporium‘maydis race T

 

pathotoxin was expressed in callus culture (20). Furthermore, variants
were selected within the susceptible Texas male-sterile cytoplasm maize
cultures that were resistant to the toxin. Analysis of mitochondrial

activity from both normal cytoplasm.cells and from the selected variant

cells showed that the mitochondria were not affected by the toxin, but

l4

mitochondria from the Texas male-sterile cytoplasm cells were affected
by the toxin. No plants could be regenerated from these variants, due
to their age in culture. However, in a later experiment (C. Green,
personal communication), plants were regenerated from selected variants;
they were resistant to the toxin, but no longer had male-sterile
cytoplasm.

A most interesting correlation between whole plant response and
cellular response was reported by Kennedy (31) on the photorespiration
of Portulaca oleracea, a C4 plant, and Streptanthus tortuosus, a C3
plant. It was formerly believed that the reduced level of photo-
respiration of 04 plants was related to their unique "Kranz anatomy" of
the leaves. However, C4 suspension cultures had about a 1:1 light:dark
photorespiration rate, like the whole plant, and the 03 cultures had
about a 3:1 light:dark photorespiration rate, like the whole plant.
Furthermore, photorespiration of P, oleracea callus was little affected
by 02 concentration, while photorespiration of S, tortuosus callus in-
creased with increased 02 concentration. Again, these responses are
typical of C4 and 03 whole plant responses. The dark respiration of
plants and callus of both species were the same at all 0 concentra-

2

tions. He concluded that the 04 reduced photorespiration rate was a
cellular based phenomenon and was not due to the "Kranz anatomy".

Four cultivars of Nicotiana tabacum were assayed for ozone damage
in gitrg, As whole plants, two were resistant to damage by ozone and
two were susceptible. Growth and cell viability of the resistant

cultivars in culture were reduced less by ozone than the susceptible

ones (T. Rice and P. Carlson, personal communication).

15

The previously mentioned experiments, except those of Gegenbach
and Green, utilized existing differential responses of whole plants to
demonstrate similar responses in tissue culture. There have been a
number of instances where cells in gi££g_have been mutated and selected
for certain characteristics, but in many instances whole plants could
not be regenerated from them. In some cases, mutagens such as ethyl
methanesulphonate (EMS) (10,11) or Ndmethyl-N-nitro-N-nitrosoguanidine
(NTG) have been used to induce mutations (23,33,47); in other instances
(5,19,38,39,49,64,65,66) naturally occurring or spontaneous mutations
have been selected. The predominant selection technique involved grow-
ing callus or suspension cultures on media that contained toxic chemi-
cals. Only those mutants that were resistant to the toxic chemicals
continued to grow. Thus, those resistant mutants were selected. Cells
resistant to amino acid analogs (ll,l9,49,64,65,66), base analogs (33,
39,47), pathotoxins (20), and streptomycin (5,38) have been selected by
using this technique.

Another scheme was used by Carlson (10) to isolate auxotrophic
mutants. Single haploid cells of Nicotiana tabacum were mutated by a
treatment of 0.25% EMS for 1 hr. Survival of the treated cells was 47
to 68%. The cells were grown in the dark on a minimal medium containing
5-bromodeoxyuridine (BUdR) for 36 hours. The growing (autotrophic)
cells incorporated BUdR. Then the cells were plated-out on a supple-
mental medium and exposed to light, thus killing the autotrophs.
Presumptive auxotrophs grew on the supplemented medium in the light,

since they had not incorporated BUdR.while on the minimal medium.

16
From an original cell population of about 1.75x106, 119 calli were
selected. Only six were found to be auxotrophs requiring either nucleic
acids, vitamins, or amino acids. Although the auxotrophs were haploids,
they grew slowly on unsupplemented media and have been called "leaky"
auxotrophs. The explanation put forth for these incomplete auxotrophs
was that N, tabacum is an allopolyploid, and hence, the haploid cells
may still have multiple genes for some (many) traits.

There are three reported instances where mutated cells have been
selected for certain traits, whole plants have been regenerated from
these selected cells, and the selected traits have been expressed by
the whole plant. The work of Green (personal communication) has already
been mentioned. Carlson (ll) mutated cells of haploid N, tabacum by
treatment with EMS. He then selected mutants resistant to the
methionine analog, methionine sulfoximide (MSO). Three selected cell
lines were diploidized, and whole plants were regenerated. Plants were

found to be resistant to M80 and to the pathogen Pseudomonas tabaci.

 

The toxin of P, tabaci is also an analog of methionine. The plants of
two lines had increased levels of free methionine. The resistance was
due to a single semi-dominant gene, as crosses with susceptible lines
gave a 1:2:1 F2 ratio. The third mutant was thought due to possibly
recessive genes at two loci with additive effects, as the F1 progeny

gave about a 9:3:3:1 F segregating ratio.

2
The third instance of regenerated plants expressing a trait
selected for in_vitro was reported by Maliga, Sz.-Breznovits, and

Merton (38). They selected haploid N, tabacum.cells in callus culture

17

that were resistant to streptomycin. Plants were regenerated from
spontaneously diploidized cells. Leaves of the resistant mutant and

a susceptible line were placed on a medium with streptomycin. The leaf
discs from the resistant plant produced green callus; whereas, leaf
discs from the susceptible plant produced very little white callus.

(It may be debated as to whether or not this demonstrated that the
intact plant is resistant to streptomycin.) They performed crosses
between resistant and susceptible lines and found that the resistance
was only inherited when the resistant plant was the female parent. This
suggested that resistance to streptomycin was determined by cytoplasmic
gene(s).

The above reviews reveal that cells can be mutated and selected
ignzigrg, and desired traits can be expressed by whole plants regener-
ated from the selected cells. The purpose of this project was to study
the feasibility of an in zitrg cultural system for developing SO2
resistance in Petunia hybrida. This was accomplished by studying the
response to 802 (or its ions in solution) of whole plants, leaf discs,
and in zitrg_callus cultures to determine if the resistance and suscepti-
bility reactions were similar at the different levels of plant organiza-
tion. Of particular interest was whether or not the responses of callus
cultures correlated with whole plant responses because mutation and

selection could be carried out on callus cultures.

MATERIALS AND METHODS

Plant Material
Seven cultivars of Petunia hybrida were used in these experiments.
The Michigan State University accession numbers, cultivar names, flower

types (G - grandiflora, M - multiflora), SO responses according to

2
Feder gt El: (15), and the sources of seeds are given in Table 1.
Cultivars 990, 993, 997, and 1000 were used in preliminary experiments
for establishing procedures for tissue culturing. Cultivars 1003, 1006,
1012, 1015, and 1016 were used for whole plant, leaf, leaf disc, and

tissue culture experiments.

Whole Plant Experiments
Eight month old plants, with flowers removed, were exposed to a

steady stream of air with 5 ppm SD for 16 hours under artificial light

2
of 1.5 mwatts/cm2 (ca. 1000 ft-c). Exposures were conducted by Dr. R.
Bressan in the exposure chamber of Dr. P. Filner of the Michigan State
University Plant Biology Laboratories. One plant each of 1003, 1006,
1012, 1015, and 1016 were repotted from a 4" clay pot to a 4" plastic
pot, and the tops of the pots were wrapped in "Parafilm" in order to

prevent absorption of 80 by the soil. Injury consisted of wilting,

2
chlorosis, and necrosis. Twenty-four hours after the end of the
exposure, the plants were rated as follows: 1) the number of leaves

with no injury, 0-25% injury, 25-50% injury, and greater than 50%

18

19

Table l. Petunia hybrida cultivars used to study the response of vari-

ous levels of biological organization to treatments of SO

 

 

 

gas or NaZSO3 solutions. 2
Accession Flower 80
Number Cultivar Names Type1 Response2 Source
990 Victory M Susceptible Harris Seed Co.
993 warrior G Susceptible Harris Seed Co.
997 Blue Danube Double G Resistant Pan Am Seed Co.
1000 Comanche M Not tested Dr. J. B. Power
1003 Calypso G Resistant Pan Am Seed Co.
1006 Cherry Blossom G Resistant Pan Am Seed Co.
1012 Victory M Susceptible Harris Seed Co.
1015 warrior G Susceptible Harris Seed Co.
1016 Lilac Time G Resistant Harris Seed Co.

 

lM - multiflora, G - grandiflora.

2A8 determined by Feder gtngl, (15).

20

injury were counted, 2) these numbers were normalized on the basis of
100 leaves per plant, and 3) an R value was calculated using the
formula
R = (la + 2b + 3c)/3
where

a - normalized number of leaves with 0-25% injury

b - normalized number of leaves with 25-50% injury

c = normalized number of leaves with 50-100% injury.

Two exposures were conducted and the data were analyzed using analysis

of variance (AOV) for a randomized complete block design.

Whole Leaf Experiments

The third or fourth fully expanded leaves of 1003, 1006, 1012,
1015, and 1016 were floated in petri dishes containing 50 cc of either
a control solution of 20 mM NaCl at pH 4—5 or 10 mM NaZSO3 at an ini-
tial pH of 5.0-5.4. After 24 hours, the leaves were rinsed and placed
on moist filter paper in petri dishes for another 24 hours to allow
symptoms to develop. Symptoms were water-soaking with loss of turgor
and yellow or necrotic lesions. Each rating was the visual determina-
tion of the approximate percent of the leaf area that was injured.
Four leaves were floated together and their values were averaged. Four

treatments were conducted and ADV was calculated for a randomized com-

plete block design.

Leaf Discs
Leaf discs of the 2nd through 5th fully expanded leaves of 1003,

1006, 1012, 1015, and 1016 were made using a 5 mm cork borer. All the

21

leaf discs of each cultivar were put in a beaker of water and stirred
in order to randomize them. Ten discs of each cultivar were placed in
a petri dish containing 30 cc of either a control solution of 20 mM
NaCl at pH 4.0-4.5 or a treatment solution of either 2.5, 5.0, or 7.5
mM Na2803 at pH 5.0-5.5. After 3 hours the solutions were removed.

The leaf discs were rinsed with distilled water and returned to their
respective petri dishes with 10 cc of distilled water. The dishes were
placed under 1000 ft-c for an additional 16 to 20 hours. Chlorophyll
was extracted by placing the 10 leaf discs in a test tube containing
80% ethanol. The tubes were placed in a boiling water bath and the
ethanol was boiled until the volume was reduced to a few cc's. A 2nd
and 3rd extraction were carried out using 5 cc each time. The three
extracts were combined and brought to a volume of 10 cc by adding 80%
ethanol. The absorbance at 665 nm was determined using a Beckman Model
25 SpectrOphotometer. The raw data was normalized by dividing the
values of the treatments by the value of the control for each cultivar.
Three treatments were conducted, and the ADV was computed for a random?
ized complete block design with 15 treatments, 5 cultivars and 3 Na SO

2 3

concentrations.

Lower Leaf Epidermal Strips

Epidermal strips were peeled from the under side of fully expanded
young leaves. Three stains were used in an attempt to determine via-
bility of freshly prepared strips: 1) triphenyl tetrazolium chloride

(TTC) at a concentration of 0.5%--viable cells become purple within 24

22

hours, 2) phenosafranin at a concentration of 0.1%--nonviable cells are
stained red after a few minutes, and 3) neutral red at a concentration
of 0.0l%--viab1e cells take up the stain and are red after 20 minutes.
Neutral red was also used to stain epidermal strips treated concurrently

with leaf discs.

Tissue Culture

 

H9222: Culture media contained the salts of Murashige and Skoog
(42), the vitamins of Nitsch and Nitsch (46) for anther culture of
N, tabacum, 30 g/liter sucrose and 8 g/liter agar. Medium designated
MSNP had 2 ppm naphthalene acetic acid (NAA) and 0.5 ppmN6 benzyl-
aminopurine (6-BAP) as hormones added to the previously stated salts
and vitamins. The medium designated PM had 1 ppm.2,4-dichlorophenoxy—
acetic acid (2,4-D) as the sole hormonal component.

Callus culture initiation. 1) Stem sections of 990, 993, 997,
and 1000 were washed in 95% ethanol for about 5 minutes, rinsed with
water, and surface disinfested in 0.5% NaOC1 with a few drops of Tween
20 added. After 10 minutes the tissue was rinsed twice with sterile
water. With the damaged ends removed, the stem sections were placed on
medium PM. Callus that was produced was excised and transferred to
fresh medium. 2) Seed of 1003, 1006, 1012, 1015, and 1016 were surface
disinfested with 0.5% NaOC1 for l to 2 minutes, rinsed with sterile
water and placed on media MSNP and PM. Seeds were germinated under 300
ft-c. Seeds germinated as normal seedlings or directly produced callus.

Only seedling stems and leaves were cut-up and returned to the callus-

inducing media since the origin of callus emerging directly from the

23

seed could have been from maternal tissue. Once callus was induced in
the dark, it was subcultured about twice per month. All cultures were
maintained at prevailing room temperature. This method of callus
initiation was used in order to avoid contamination problems encountered
when using explants of greenhouse grown plants.

Viability staining, Triphenyl tetrazolium chloride (TTC) at a
concentration of 0.5% was used as a vital stain. The solution was
filter-sterilized using a Millipore filter of 0.22 micron pore size;

3 to 5 cc were added to each 100x15 mm petri dish. In the preliminary
experiments the stain was added immediately after the treatment solu-
tions were removed. Viability was scored between one and 24 hours
later. For the later experiments, TTC was added 24 hours after removal
of the treatment solutions. Scoring was done 24 hours later. In order
to determine the effectiveness of staining in measuring viability, a
correlation was determined between the growth rate and TTC scoring of
control and treated tissue. First, a correlation between fresh weight
and dry weight of the callus tissue was determined and found to be very
highly significant (r - 0.981***) so that fresh weight was used. Then
two blocks of experiments on PM medium.were run with three samples of
tissue of each cultivar instead of two (see section on cultivar by
NaZSO3 factorial experiments). Two samples were stained with TTC, and
the third sample was used to determine growth rate. The samples were
about 100 mg fresh weight and were grown for 13 days. Growth rate was

the ratio of the final to the initial weight.

24

Preliminary experiments. A number of preliminary experiments were
performed on callus to determine proper experimental conditions for
treatment with Na2803. Firstly, the effects of ionic strength and pH
on callus cultures were determined by adding 5 cc of a solution of
either 100 mM, 50 mM, 12.5 mM, 6.25 mM or 3.125 mM NaCl at a pH of
either 5.2-5.7 or 2.9-3.0. The cultivars used were 990 and 997.
Treatment was for 3 hours, and the calli were stained with 3 cc of TTC
and scored the next day. Secondly, duration of exposure to Na2803 solu-
tions was determined by treating calli of 990, 993, and 997 with either
distilled water or 1000 ppm (ca. 8 mM) Na2803 for times of l, 6, 24, or
49 hours. TTC was added immediately after removal of the treatment
solutions, and the calli were scored 24 hours later. Thirdly, the
potency of Na2803 solutions was evaluated zero, 1, 4, 24, and 48 hours
after preparation. Five cc of either distilled water or 1500 ppm Na2803
solutions were added at appropriate times to calli of 990, 993, and 997.
Treatment was for two hours. Fourthly, the effect of pH on Na2503
damage was determined by adding 3 to 5 cc of solutions of 500 ppm Na2303
dissolved in liquid PM medium.at a pH of 7.0, 6.5, 6.0, 5.5, 5.0, 4.5,
4.0, 3.5, 3.0, 2.5, and 2.0 to cultures of 990, 993, and 997. Titration
of the Na2803 solution to pH 2.0 caused evolution of some $02 gas.
Treatment was for 24 hours, 2.5 cc of TTC were added, and calli were
scored the following day.

Cultivar by Na §Qfl concentration factorial experiments. From the
J

2
results of the preliminary experiments the following experimental pro-

cedure was used to treat callus cultures of 1003, 1006, 1012, 1015,

25

and 1016. Due to oxidation of sulfite to sulfate and subsequent loss

of potency and the escape of SO2 from.water solutions of Na2803,
especially at high temperatures, it was necessary to prepare fresh solu-
tions by filter sterilization. The solutions were prepared as follows:
a 10 mM stock solution at pH 5.5 was prepared by dissolving 10 milli—
moles (1.26 g) of Na2803 in 500 cc of distilled water; and the solution
was simultaneously titrated and brought up to volume by adding 500 cc of
water to which 0.7 cc of concentrated HCl was added. Solutions of 7.5
mM, 5.0 mM, and 2.5 mM Na SO were made by dilution with distilled water.

2 3
At pH 5.5 the predominant form of ion was H80

3.

The experimental design wasaisplit-plot with Na2803 concentrations
as the main plot treatment split by cultivars. The treatments of the
calli were carried out as follows: 1) two samples of calli (about 100
mg) of each of the five cultivars were placed together (in random posi-
tions by cultivar) on petri dishes of either PM or MSNP medium, 2) three
to seven days later each dish of calli was treated with 10 cc of either
a control solution of 20mM NaCl at pH of about 4.2 or a treatment solu-
tion of 2.5 mM, 5.0 mM or 7.5 mM NaZSOB’ 3) solutions were removed 3
hours later using sterile, disposable pipettes, 4) 10 cc of filter-
sterilized water were used to rinse the cultures, 5) 24 hours after
initiation of the exPeriment, the dishes were treated with 5 cc of
filter-sterilized 0.5% solution of TTC, and 6) dishes were assigned
random numbers and visually rated from 0 to 10 on the basis of color

development, 10 being the most viable, 24 hours after application of

the TTC. The rating values of the two pieces of each cultivar were

 

26

averaged. The data were normalized and analyzed as ratings of treat-
ments divided by controls multiplied by 10 for convenience. This normal-
ized rating was actually percent of control divided by 10. Since large
amounts of callus and much manipulation of materials were necessary to
run an experiment, they were usually carried out two blocks at a time.
Blocking was by callus source and by day of experiment. There were 6
blocks on MSNP medium and 7 on PM medium. Data were analyzed using AOV
for a split-plot design.

NaCl stress experiment. Calli of 1003, 1006, 1012, 1015, and 1016
were treated with 0, 3, 6, or 9% NaCl solutions for 24 hours. The TTC
staining solution was added after removal of the NaCl solutions and
rinsing with sterile water. Three replications were run, and the AOV
was performed for split-plot design.with NaCl concentrations as the main
plot split by cultivars. Data were normalized as percent control
divided by 10.

NaCl, Nazgggdouble stress experiments. A number of double stress
experiments were carried out on 1006 callus cultures to determine the
effect of environmentally induced variability of viability on $02 re-
sponse. In the first attempt, callus was subjected to a stress of 5%
NaCl solution with a control of sterile water. After 24 hours, the
solution or water was removed, the callus was rinsed, and subjected to
either a second stress of 5 mM NaZSO3 or a control of 20 mM NaCl.
Treatment time was for 24 hours. The TTC stain was added after another
24 hours. In the second experiment, the first stress was either 2, 3,

4, or 5% NaCl and the second stress was 3.5 mM NaZSO3' In the third

27

experiment, the first stress was 2 or 4% NaCl and the second stress was

5 mM Na2803.

4% NaCl and the second stress was 2.5 or 5.0 mM NaZSOB'

ADV's were performed when possible, and correlation coefficients were

And in the fourth experiment, the first stress was 2 or

Appropriate

determined for ratings of controls (NaCl treatment) !§,normalized

ratings of treatments (NaZSO3 treatment).

RESULTS

Whole Plant Experiments

Exposing whole plants to 5 ppm SO2 for 16 hours resulted in damage
consisting of wilting, chlorosis, and necrosis. Cultivars 1003, 1006,
and 1016 were most resistant, while 1012 was less resistant, and 1015
was susceptible (Table 2). This ranking was similar to that of Feder
st 21. (15) where 1003, 1006, and 1016 were resistant and 1012 and 1015
were susceptible. The ADV is given in the Appendix as are the AOV's

of the results of the other experiments.

Whole Leaf Experiments

Injury caused by exposing whole leaves to 10 mM NaZSO3 solutions
consisted of water-soaking with loss of turgor, yellowing, and necrotic
lesions (Table 3). Variation among the four blocks was substantial
(block variance % error variance was 25.2), but no differences were

observed among the cultivars tested.

Leaf Disc Experiments

The damage to leaf discs exposed to Na2803 solutions consisted of
bleaching of the tissue to a tan color (Tables 4 and 5). Since the
values given are absorbances by chlorophyll, the larger the value is,
the less damage occurred. There were significant differences among the
cultivars and among the Na2803 concentrations, but there was no inter-

action between these two variables. The effect of Na2803 concentration

28

29

Table 2. Mean R values of undetached petunia leaves after 16 hours

 

 

exposure to 5 ppm SO2 gas.
Cultivar 1006 1003 1016 1012 1015
Mean R value 19.2 21.6 23.3 28.2 57.7

 

(L'S'D°.OS " 13.27)

 

Table 3. Mean percent damage to whole detached leaves after 24 hours
exposure to 10 mM Na2803 of initial pH of 5.0 to 5.5.

 

 

Cultivar 1003 1015 1006 1012 1016
Mean percent damage 51.6 55.6 61.6 63.1 65.6

 

(LcSoDo.05 g 2505)

 

Table 4. Normalized absorbances of chlorophyll extracts of leaf discs
of 5 petunia cultivars averaged over NaZSO concentrations
after 3 hours exposure to 2.5, 5.0 or 7.5 EM Na $0 at initial

pH of 5.0 to 5.5. 2 3

 

 

 

 

Cultivar 1006 1003 1012 1015 1016

Mean normalized 41.47 34.32 32.18 32.10 20.20
absorbance ---
(665 nm)

(LoSoDo.05 - 7.66)

 

Table 5. The effect of Na SO concentration on mean normalized absorb-
ances of chloropEle extracts of petunia leaf discs, all
cultivars,after 3 hours exposure to solutions at initial pH
of 5.0 to 5.5.

 

 

Na2803 concentration 2.5 mM 5.0 mM 7.5 mM

Mean normalized absorbance (665 nm) 54.21 23.88 18.07
(LoSoDo.05 . 5093)

 

30

was straightforward with treatments of 5.0 and 7.5 mM causing signifi-
cantly greater damage to the leaf discs than the treatment of 2.5 mM.
However, the differences among the cultivars as leaf discs were not the
same as observed with whole plants. Cultivar 1006 was still rated

as resistant, but 1003, 1012, and 1015 were intermediate. The line
1016, which.was resistant as a whole plant, was very susceptible to
injury when tested in the leaf disc assay.

Notably, plants of cultivar 1016 appeared to be a lighter green
than the others. This was confirmed by a significantly lower chloro—
phyll content in the leaf discs of the controls. Consequently, there
was a significant correlation between the chlorophyll content of the
controls and the sum of the normalized chlorophyll contents of the
treatments. However, there were no significant differences among the
mean chlorophyll contents of the controls of the other cultivars. The
correlation was not significant if 1016 was excluded. Therefore, the

significant differences for NaZSO damage between 1006 and 1012 and

3
1006 and 1015 could not be contributed to differences in the initial

chlorophyll content of the leaf discs.

Epidermal Strips

No extensive experiments were carried out using epidermal strips
because a suitable staining procedure to demonstrate viability could not
be devised. Freshly prepared epidermal strips did not stain with TTC
even after 24 hours of exposure. Freshly prepared epidermal strips took
up the phenosafranin stain indicating that the cells in these tissues

were no longer viable. Strips exposed to Neutral Red stain revealed

31

viable and non—viable regions. However, when strips were exposed to
control and 7.5 mM Na2803 treatment solutions (exposed with leaf discs),
no differences between control and Na2803 treated strips could be

determined. Therefore, all staining procedures were deemed unreliable.

Tissue Culture Experiments

Generalpgrowth of callus. Unorganized callus cultures of 1003,
1006, 1012, 1015, and 1016 were established on both MSNP and PM media.
The callus growth of some lines was better on.MSNP than on PM and XEEET
‘gggga (see Table 6). However, growth was sufficient to allow subcultur-
ing of all cultures on the same day, so that callus of all the cultivars
for a given experiment were identical with respect to age and the number
of subcultures. Aside from callus growth rate differences among the
cultivars, there were coloration and friability differences. Most
obvious was the constant brownish color of the callus of line 1016 on
MSNP‘medium.
Table 6. Callus growth rates of 5 petunia cultivars on two media as

determined by the ratio of the fresh weight after 12 days to
the initial fresh weight.

 

 

Cultivar 1003 1006 1012 1015 1016
Growth rate on MSNP 3.30 4.50 1.26 2.06 2.80
Growth rate on PM 1.46 4.30 2.77 2.41 1.55

 

Correlation of growth and visual rating of TTC stainipg, Correla-
tion coefficients were calculated for growth rate with visual rating of

TTC staining. Correlation using each raw datum value was highly

32

significant (r = 0.638**). Correlations of raw data summed over blocks,
over cultivars, and over cultivars and blocks were highly significant

(r . 0.784**, 0.890**, and 0.994**, respectively). The correlation of
each value normalized as percent of control was significant (r = 0.438* ).
Therefore, TTC staining was a quick, convenient and reliable means of
operationally defining viability.

Effects of ionic strengph andng. The effects of treatment of
calli with solutions of ionic strength to 100 mM and pH to 3.0 are shown
in Table 7. There were no effects of ionic strength and pH in the range
that the callus cultures were subjected to in the later experiments with
Na 803, where ionic strength was less than 20 mM and the pH did not drop

2
below 4.0.

Table 7. Effect of ionic strength and pH on TTC viability ratings of
petunia callus cultures after 3 hours exposure.

 

 

 

NaCl conc. (mm) 3.125 6.25 12.5 25 w so 100

Cultivar 990 997 990 997 990 997 990 997 H9909 997 990 997
pH 5.5 5 9 4 9 l 4 9 8%: 3 7
pH 3.0 4 8 4 10 i 3 s 4 8 ‘4 4 8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Duration of exposure to NaS The results of exposure of callus

2— —3

cultures to 1000 ppm Na2803 for various durations are shown in Table 8.

Much of the damage occurred after an hour of exposure; hence, duration
of exposure for subsequent experiments was 3 hours to allow for a more
thorough treatment. A longer exposure was not chosen because the

potency of NaZSO solutions decreased rapidly (see the next section).

3

33

Table 8. Mean TTC viability ratings of petunia callus cultures after
1, 6, 24 and 49 hour exposure to 1000 ppm of Na $0 at

 

 

 

 

initial pH of 5.5. 2 3
Cultures 1 hr. 6 hrs. 24 hrs. 49 hrs.
Control 990 5.5 8.5 8.0 9.0
993 8.5 8.5 8.5 9.5
997 8.5 9.0 9.0 *
Treatment 990 5.0 4.5 4.5 0.0
993 1.5 1.5 0.0 0.0
997 0.5 0.0 0.0 0.0
*
Contaminated

Potency of NaZSQp solutions. Callus cultures were exposed to
either a water control or 1500 ppm NaZSO3 treatment 0, 1, 4, 24, and 48
hours after preparation of the solutions. Damage to callus decreased

with time after preparation; therefore, the solutions had to be freshly
prepared before use (Table 9).
Table 9. Mean TTC viability ratings of petunia callus after 2 hours

exposure to 1500 ppm NaZSO starting 1, 4, 24, and 48 hours
after preparation of the salution.

 

 

Time after preparation: 0 hr. 1 hr. 4 hrs. 24 hrs. 48 hrs.

 

Control 990 5.5 7.5 9.5 6.0 --
993 7.0 9.5 9.5 10.0 --
997 7.0 10.0 10.0 10.0 --

Treatment 990 1.5 1.5 6.0 5.5 8.0
993 0.0 0.0 0.0 4.0 9.0
997 0.5 0.0 0.0 4.5 9.0

 

 

 

34

Effect of pH on Nazgga damage. Callus cultures were exposed to
500 ppm NaZSO3 solutions at different pH's (Table 10). The effect of pH
was dramatic (Figure l, the values are the means over cultivars). Since

Na2303 solutions become more acidic due to oxidation of H803 to H802

and the ionization of H804 to H+ and SOZZ, solutions of Na2803 must be

prepared at a high initial pH so that the solutions do not approach the
critical pH of about 3.5 during the course of an experiment. The in-
crease in damage closely follows the increase in the theoretical concen-

tration of H2303 molecules in solution (pka=l.8). This, perhaps, indi-

cates more uptake of the uncharged H2803 molecules in comparison to the

charged HSO3 ions.

Table 10. The effect of pH on mean TTC viability ratings of petunia
callus after 24 hours exposure to 500 ppm.NaZSO3.

 

 

 

PH: 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
cultivar 990 8.0 700 700 800 700 900 700 700 800 500 300
993 10.0 10.0 8.0 10.0 8.0 8.0 9.0 10.0 8.0 4.0 0.0
997 10.0 9.0 8.0 8.0 5.0 9.0 8.0 7.0 4.0 1.0 0.0

 

Cultivar by Na SOg_concentration factorial experiments. The

effects of various Na2803 concentrations on callus cultures of various

cultivars are shown in Table 11. Since the ADV's showed no interaction

between Na SO concentrations and cultivars, the means are averages

2 3
over blocks and cultivars. The higher the mean, the less damage
occurred. The effects of each concentration within a given medium were

significantly different from each other (by L.S.D.), but the effect of

Viability Rating

 

35

 

O O O O
8.0 -b 9
.0
O O
6.0 .t
4.0 --
o
2.0 ‘~
0
I L 4 .1 l n 1 1 l __l
—r I i l l
7.0 6.0 5.0 4.0 3.0 2.0
pH
Figure l. The effect of pH on mean TTC viability ratings of

petunia callus after 24 hr exposure to 500 ppm

N82803°

36

the same concentration between the two media were not significantly

different from each other (by the t-test).

Table 11. The effect of Na SO3 concentrations on mean TTC viability
ratings of petunia callus, all cultivars, after 3 hours

exposure to solutions at initial pH of 5.0 to 5.5.

 

 

Na2803 concentration 2.5 mM 5.0 mM 7.5 mM
Mean normalized viability MSNPl 8.34 4.84 1.92
Mean normalized viability PM2 7.92 3.32 0.5
1(LSD =128)

. . ..05 .

2
(L.S.D. .05 - 1.26)

 

Table 12 shows the comparison among the cultivars within a medium.
Again, since there was no interaction, cultivar values are averages over
blocks and Na2803 concentrations. Duncan's multiple range test (34) was
used for mean separation. Only 1006 cultured on.MSNP was significantly
different from the others while there were no significant differences
among the cultivars on PM. The analyses of pairs of blocks that were
run together are shown in Table 13. The results are not consistent with
each other. This was probably due to some environmentally induced
variation of critical physiology as reflected by variation of the
viability of the controls.

Correlation coefficients were determined for the ratings of the
controls with the sum of the normalized rating of the three treatments.
If all blocks and all cultivars were analyzed individually, then the

correlations were highly significant for MSNP (r = 0.560**) and PM

37

 

 

 

 

 

 

 

Table 12. Mean TTC viability ratings of the callus of 5 petunia culti-
vars after 3 hours exposure to 2.5, 5.0 or 7.5 mM Na 80 at
initial pH of 5.0 to 5.5. AOV showed no significant di fer-
ences among the cultivars on PM medium.

Cultivar 1006 1016 1012 1003 1015

Mean normalized viability MSNPl 7.29 4.93 4.67 4.66 3.57

Cultivar 1003 1016 1015 1006 1012

Mean normalized viability PM2 5.38 3.60 3.60 3.55 3.41

1(L.S.D. = 1.40)

.05
2
(L.S.D. .05 = 1.62)

Table 13. Mean TTC viability ratings of the callus of 5 petunia culti-
vars after 3 hours exposure to 2.5, 5.0, and 7.5 mM Na2803.
Analysis by pairs of blocks treated simultaneously.

Cultivars 1006 1003 1015 1012 1016

Means, MSNP, blocks III & IV 7.05 6.20 3.92 3.75 3.30

Cultivars 1006 1016 1003 1012 1015

Means, MSNP blocks V & VI 5.17 5.13 2.72 2.25 1.00

Cultivars 1003 1015 1006 1016 1012

Means, PM, blocks II & III 6.76 4.15 3.92 2.08 0.53

Cultivars 1003 1015 1006 1016 1012

Means, PM, blocks IV & V 5.75 3.71 3.03 2.80 2.77

Cultivars 1016 1012 1006 1003 1015

Means, PM, blocks VI 6 VII 5.62 5.02 4.83 4.43 3:32

 

 

 

 

 

 

 

 

 

38

(r = 0.440**) analyzed separately, and very highly significant if MSNP
and PM were analyzed together (r - 0.467***). Correlation coefficients
were determined for means over blocks of controls with means of sums of
normalized ratings for each cultivar. The correlation was significant
on MSNP (r = 0.906*) where there was a concurrent significant difference
among the means of the cultivars (Table 12). However, the correlation
was not significant on PM (r = 0.265 us) where there was not a concur-
rent significant difference among the means of the cultivars. Correla-
tion coefficients between controls and corresponding sums of the
normalized ratings within cultivars were also determined (Table 14).
Generally, these were not significant.

Table 14. Correlation coefficient within cultivars of mean TTC viabil-

ity ratings of controls with the sum of the normalized TTC

viability ratings of the Na2803 treatments.

 

 

Cultivars 1003 1006 1012 1015 1016
MSNP (df - 4) 0.819** 0.317 0.312 0.638 0.604
PM (df - 5) 0.950** 0.195 0.539 0.103 0.390

MSNP & PM (df - 11) 0.738** 0.310 0.438 0.454 0.437

 

Results of NaCl experiments. The ADV of experiments of stressing
callus with various concentrations of NaCl showed no significant differ-
ences among the cultivars, although the effect of the stress was signif-
icant. Also, the appearance of the NaCl-stressed callus was different
from NaZSOB-damaged callus, the former being brown, and the latter

being bleached. Correlation coefficients were determined for individual

39

controls with the sum of the normalized ratings (r = 0.438 ns) and for
means over blocks (r = 0.878*). The value for the means over blocks
was significant at the 5% level. Also, the Spearman rank coefficient
(59) for the means over blocks was 1.00; i.e., perfect correlation.
Results of double stress experiments. Results of the first double
stress experiment are given in Table 15. Each value is an average of
the ratings of four pieces of callus. Although the NaCl treated callus
was damaged disproportionately more by Na2S03 than the callus not
treated with NaCl, this difference was not significant. This was be-
cause the treatment with Na2S03 on the callus not treated with NaCl was
not significantly different from zero. Also, it was felt that the NaCl
stress was too damaging. Consequently, the second experiment was con-
ducted. The correlation coefficient between the rating of the controls
and the normalized rating of the treatment was 0.439 and was not signif-
icant. However, AOV showed that the treatment of 3.5 mM Na2S03 was
also not significant. Therefore, a third experiment was conducted.
Not only was there bacterial contamination, but the concentration of
5 mM Na2SO3 was too high since most of the ratings were close to 0%
viability. The fourth experiment yielded some useful information. The
raw data are shown in Table 16. The bacterial contamination of the 4%
NaCl, 2.5 mM Na2803 complicated any analysis. However, it is obvious
from the raw data that the stressed callus was not damaged by Na2803
disproportionately more than the unstressed callus. The correlation
coefficient using 0mM Na SO as controls and the sum of the normalized

2 3
ratings of 2.5 mM and 5.0 mM for the treatments of the 02 and 2% NaCl

40

 

 

 

 

Table 15. Mean TTC viability ratings of callus of cultivar 1006 after
24 hours exposure to 5% NaCl and/or 2 hOurs exposure to
5 mM Na SO .
2 3
NaCl Na2803 Block I Block II Block III
- - 8000 2000 9075
- + 0.50 0.00 1.25
+ — 1050 1000 1050
+ + 0.00 0.00 0.00
Table 16. TTC viability ratings of callus of cultivar 1006 after 21

hours exposure to NaCl solutions and 3 hours exposure to

Na2303 solutions.

 

 

 

Rep. I II III IV V

0% NaCl 0.0 mM Na2S03

2.5 mM Na2303

5.0 mM Na2S03
2% NaCl 0.0 mM Na2803

2.5 mM Na2803 9

5.0 mM Na2803
4% NaCl 0.0 mM Na2S03 3 4 4 3 3

2.5 mM NaZSO3 Contaminated

5.0 mM Na2503 3 4 5 3 7

 

41

groups was 0.363, which was not significant. The correlation coeffi-
cient between 0mM and 5.0 mM normalized to the 0mM value of the 0, 2,
and 4% NaCl groups was 0.632 which was significant. The 4% NaCl
stressed callus being damaged disproportionately less than the others.
From the results of these four experiments it is obvious that although
the double stress experiment was logical, it was difficult to obtain
consistent results. Also, it is apparent that variation in the
susceptibility to damage by Nazso3 was not strictly related to environ-
mentally induced variation within a given cultivar.

Dose Response of Leaf Discs and Callus Cultures

to Na2§(_)__3

Leaf discs and callus on MSNP and PM treated with NaZSO3 were
evaluated by dose response curves. The response of material of two
levels of biological organization were in close agreement with one

another (Figure 2).

Summary of Experiments
The ranking for $02 or Nazso3 injury of the various cultivars at
the different levels of biological organization are given in Table 17.

The ranking was different for each level of organization.

42

 

 

 

100--
90 -r-
0 Leaf Discs
+- MSNP Callus
80-3b
0 PM Callus
'3
u 70-_
u
a
o
c:
m 50 a_
o
E, O
3 50+
u
32
1.
,fi 40-w- C)
>.
u
'H
:1 30-w-
‘8
... i-
>»
20-m- C) .
lO-v- 4'
C)
J 1 J
l T I
0 2.5 5.0 7.5
mMNa2803

Figure 2. The effect of Na 80 concentration on the mean normal-
ized absorbance of chlorophyll extracts of petunia
leaf discs and on the mean normalized TTC viability
ratings of petunia callus cultures. Exposures were
for 3 hr.

43

Table 17. Rank of 5 petunia cultivars to S0 or Na SO susceptibility
at different levels of biological organizat on.

 

 

 

 

 

Least susceptible Most susceptible
Whole Plant 1006 1003 1016 1012 1015
Leaf Disc 1006 1003 1012 1015 1016
Callus culture MSNP 1006 1016 1012 1003 1015

 

Callus culture PM 1003 1016 1015 1006 1012

 

DISCUSSION

Although the results of fumigating whole plants with 80 were

2
from only two experiments, the overall trend of cultivar susceptibility
was the same as reported by Feder st 31, (15). Since variances were
not reported by Feder £3 £1. (15), significant differences in the
ratings of the cultivars could not be determined. However, there seems
to be one point of discrepancy: plants were treated by Feder gt a},

(15) with 2.5, 5.0 or 10.0 ppm 80 for one hour; whereas, in this report,

2
plants were treated with 5.0 ppm for 16 hours in order to obtain a
similar degree of injury. The difference in exposure times may have
been necessary to compensate for the difference in stomatal opening
associated with a difference in light intensity. Plants fumigated by
Feder 25 31. (15) were in chambers in the greenhouse with a minimum
illumination of 2.7 mwatts/cmz; whereas, in this experiment, plants were
under artificial lighting of 1.5 mwatts/cmz. Relative humidity,

temperature, CO concentration, etc., could also have affected stomatal

2

opening which in turn could have affected 80 injury.

2
Experiments using whole leaves were not useful in determining
variation for $02 response due to cultivar differences. Environmental
factors dominated the response as evidenced by the large block variance
in relation to the cultivar and error variances. However, the experi-

ments were useful in providing the necessary link between treatment

44

45

2503 solutions. Although leaves in

control solutions exhibited water-soaking, they did not lose turgidity

with $02 gas and treatment with Na

as did the Na2503 treated leaves. Plants fumigated with $02 showed

similar symptoms of water-soaking and wilting. Unlike the controls,
the treated leaves also exhibited chlorotic and necrotic lesions as did

fumigated plants. The use of Na2S03 solutions is further justified by

the results obtained by various authors (1,2,12,22,4l,48,56,60,67) who

also used Na2S03 or K28205 solutions instead of SO2

Experiments with leaf discs were more reliable than with whole

gas.

leaves because 1) the effects were reproducible and consistent, and

2) the results were easier to quantify. Whole leaves were rated visual-
ly and equal weight was given to water-soaked damage, chlorosis, and
necrosis; whereas, leaf discs were evaluated on an objective basis by
spectrophotometric analysis of chlorophyll content. Another significant
aspect of the leaf experiments was that the dose response of the discs
was similar to that of the callus cultures (see Figure 2). Furthermore,
these dose responses were of physiological significance as well. The
concentrations of 2.5 mM, 5.0 mM, and 7.5 mM Na2303 correspond to 160,

320, and 480 ppm 80 dissolved in water. 'According to Malhotra and

2

Hocking (37), at low concentrations of S0 gas there is about a thousand-

2
fold higher concentration in the water phase than in the gas phase at
equilibrium. Therefore, the experimental conditions for leaf discs and
callus cultures were equivalent to exposures of SO2 gas at concentra-
tions of about 0.16, 0.32, and 0.45 ppm. These concentrations are an

order of magnitude lower than that given to whole plants by Feder gt El:

(15) and herein.

46

Although the experiments with the leaf discs were physiologically
significant, the results did not correlate with the whole plant
response. Namely, 1016 was resistant as whole plants but was suscepti-
ble as leaf discs. Moreover, 1015 was susceptible as whole plants but
was intermediate as leaf discs. Therefore, the making of leaf discs
and exposing them to an aqueous Na2503 solution was sufficient pertuba-
tion of the whole plant-SO2 gas system to allow different genetically
controlled morphological and physiological factors to confer resistance
or susceptibility. By-passing of the stomata is a logical hypothesis
for the difference of response of whole plants in comparison to leaf

discs because the primary mode of entry of S0 gas into the leaf is

2
through the stomata as evidenced by the fact that most injury to a given
plant occurs when the stomata are open (26,35). Likewise, the fact
that significant damage occurs to leaf discs at a concentration that is
an order of magnitude lower suggests that the stomata are no longer
the primary point of entry. Instead the solutions seem to enter through
the cut edges of the disc and diffuse towards the center of the disc as
some discs remained green in the center while the edges were bleached.
The treatment of callus cultures with Na2S03 damaged the cells.
This damage was not due to pH or to excessive ionic strength as demon-
strated by preliminary experiments of varying pH and NaCl concentrations.
The NaCl stress experiments required approximately 10 times the ionic
strength before symptoms developed, and the symptom before staining was

browning of the cells instead of bleaching of the cells as caused by

Nay-SO3 treatment.

47

Aside from 1006 cultured on MSNP, there were no consistent signif-
icant differences among the cultivars when placed in culture. As whole
plants, the 1003, 1006, and 1016 cultures were damaged less than 1012
and 1015. Therefore, no correlation was observed between.ig;!i££g-
cultured cells and their whole plant response to 802 (Na2803). There
was a significant correlation between the viability of the cell cultures,
as measured by TTC staining of the controls, and their response to
Na2803 treatment. Less viable cultures were damaged disproportionately
more than the more viable cultures. This same correlation existed when
the cells were subjected to NaCl stress. Therefore, the variations and
differences among blocks and cultures including 1006 on MSNP was not
only correlated with, but may also have been due to, variation in via-
bility (vigor) of the cultures. This does not necessarily imply that
the resistance of 1006 on MSNP was not genetic in nature. The differ-
ence in viability (vigor) in zitrg could have had a genetic as well as
an environmental component. However, this possible genetic component
was not the same one involved in determining the physiochemical struc-
tures directly affected by $02; for if it were, then 1006 should have
been resistant on PM as well as on MSNP.

The idea that genetic factors are partially responsible for the
resistance of 1006 on.MSNP and not totally due to environmental factors
is substantiated by three other observations. There was basically no
correlation between viability and response within a given cultivar. If
environmental factors were totally responsible for this correlation

among the cultivars, then the correlation should have existed within

48

the cultivars. Likewise, there was no correlation between viability
and response in the double stress experiment on 1006 when the viability
was purposely altered. Again, if the correlation was due to environ-
mental effects then correlation should have existed in these experi-
ments. Helgeson.g£p§l, (25) found that although a dominant gene was
expressed in culture on one medium, that gene was not expressed on
another medium. Hence, there was a genetic-environment interaction.
Therefore, it seems that the differences in response to 802 damage in
culture was not simply due to environmentally induced differences in

viability. Genetic factors affecting S0 response, perhaps by affect-

2
ing viability in gitgg, were involved as well, for there was a definite
genetic-environmental interaction: response of the cultivars on medium
PM was different from the response on.medium MSNP.

Experiments using plant material at three different levels of
organizations--whole plants, organs (leaf discs), and cells in XEEEQT'
have given different results when treated with S02 (Na2S03). As whole
plants, 1015 was significantly more susceptible to S02 injury than the
rest. As leaf discs, 1016 was significantly more susceptible to Na2803
injury than the rest and 1006 was signifiCantly more resistant than
1012, 1015, and 1016, but not 1003. Finally, as cells in culture, 1006
was significantly more resistant than the rest on medium MSNP, but not
on medium PM. All of these differences could have had a genetic basis
for 802 response at each level of organization. By artificially remov-

ing the constraints and complexities going from whole plants to cells

in culture, different genetic systems affecting the response to $02

49

(Na2803) treatment may have been unmasked; but apparently the genetic
systems of the different cultivars determining the proteins and/or struc-

tures that are physiochemically affected by $0 or its ions were not

2

exclusively compared. Such a comparison would involve removing one more
constraint; that is, testing and comparing proteins and/or structures
in a cell-free system. Unfortunately, the most physiologically import-

ant system(s) directly involved with $0 injury are not known, although

2
many systems have been implicated and, therefore, such comparisons
would be of questionable significance at this time. Ballantyne (2)
found that $02 injury was not related to mitochondrial response.
Although Phaseolus vulgaris was more susceptible to 802 injury than

 

ZEENE§Z§.33 whole plants, the responses of isolated mitochondria in_
zi££g_were not significantly different from each other. Likewise, a
selection system involving cell—free proteins or structures would be
impractical for a mutation-selection breeding scheme because plants can
not be regenerated from selected, resistant, proteins or structures as
from cells in culture.

Filner and Bressan (personal communication) have shown with
cucurbits (whole plants) that resistance and susceptibility to $02

injury among cultivars correlated with SO uptake by the plants.

2
Furthermore, leaf discs of the various cultivars floated in K28205 solu-
tions showed no significant differences in injury. They concluded that
genetic differences in cuburbits as whole plants was stomate related

and not cellularly related. Their results and the results presented

herein lead to the same conclusion: the genetic basis of resistance

50

of whole plants to $02 damage is whole plant related and not leaf, organ,
or cellularly related.

A speculative model may be presented in order to summarize the
responses and the significance of the differences of the responses at
different levels of organization. As whole plants stomata may be prim-
arily responsible for resistance or susceptibility. Their number, size,

response to $02 by closing, or a combination of these may be important.

3

structure of the intercellular matrix may lead to differential uptake.

As leaf-discs differences in active transport of H80 or physiochemical

Likewise, a hypersensitive reaction to physical injury by cells at the

3

of the intercellular matrix to a less permeable form or by the release

cut surface could form a partial barrier to HSO uptake by alteration

of cellular components that oxidize sulfite to sulfate. And general

3
excluded by cells in culture. The importance of this model is not its

physiological vigor may be necessary in order for HSO to be actively
accuracy but the idea that resistance and susceptibility could be con-
trolled by different genetic systems at different levels of organization
and that none of these genes could have any direct influence in determin-
ing the proteins and structures that are injured by exposure to $02
(Na2803).

In conclusion, this project has demonstrated two ideas of import-
ance in studying the genetics and physiology of traits of complex
systems and the use of such studies in cell culture techniques for

plant improvement. In general, tissue culture is a useful system in

studying the physiological genetics of certain traits. By removing the

51

complexities and interactions of organized tissues, organs, or whole
plants, the traits of the unconstrained cells can be studied. Likewise,
the physiological genetics of a trait may be different at each level of
organization. In particular, by removing these constraints, it has been
shown that at different levels of organization, different genetic sys-

tems may be important in determining responses of Petunia hybrida to

 

treatment with $02 (Na2803). Consequently, the genetic basis for $02

resistance apparently does not involve the genes that determine the

proteins and/or structures that are damaged by $02 or its ions in solu-

tion; but rather resistance involves genes that control indirect effects
such as SO2 uptake by stomata. Therefore, at this time the most effec-

tive method of selection of desirable phenotypes for resistance to SO2

in Petunia hybrida is to screen whole plants by fumigation with $02.

APPENDIX

RECOMMENDATIONS

To further understand the physiology and genetics of SO2 resist-
ance, the following approaches are recommended:

1) Investigate the possible correlation of the genetics of stomate
parameters and 802 damage by studying inbred parents, F1, F2, etc.,
generations of Petunia hybrida cultivars 1006 and 1015, as the response
of these two cultivars to $02 treatment as whole plants were at extremes.

2) Study whole plant responses as influenced by manipulating
stomatal function.

3) Use tissue culture of divergent species and/or genera in order

to obtain a wide genetic base of SO resistance to see if there are

2
genetic differences in proteins and structures of the cell affected by
802.

4) Compare cell-free protein and/or structures implicated in 802

damage of these divergent species and/or genera to possibly determine

which systems are most significant at the subcellular level.

52

APPENDIX

TABLES 0F AOV'S

Table A1. AOV: Whole Plants-

 

 

 

Source df SS MS F F.05
Total 9 2292

Blocks 1 198

Cv's 4 2002 501 21.78 6.39
Error 4 92 23

 

Table A2. AOV: Whole Leaves-

 

 

 

Source df SS MS F F.05
Total 19 24554

Blocks 3 20733 (6911) (25.22)

cv's 4 532 133 0.49 3.26
Error 12 3289 274

 

53

54

 

 

 

 

 

 

 

Table A3. AOV: Leaf Discs.

Source df SS MS F F.05

Total 44 16330

Blocks 2 504

Treatments 14 14065
Cv's 4 2109 527.25 8.38 2.71
Na280 2 11295 5647.50 89.80 3.34
Cv x NaZSOB 8 661 82.63 1.31 2.29

Error 28 1761 62.89

Table A4. AOV: MSNP Medium.

Source df SS MS F F.05

Mp1 total 17 935.9

Blocks 5 263.8

Na2S03 2 622.5 311.25 62.75 4.10

Error a 10 » 49.6 4.96

Sp2 total 89 1347.5

Mp total 17 935.9

Cv's 4 134.3 33.58 7.61 2.52

Cv x Na $03 8 12.9 1.61 0.37 2.10

Error b 60 264.4 4.41

 

1Mp = Mainplot
2Sp - Subplot

55

 

 

 

Table A5. AOV: PM Medium.
Source df SS MS F F.05
Mp total 20 1148
Blocks 6 98
Na2803 2 980 490.0 84.0 3.88
Error a 12 70 5.83
Sp total 104 1765
Mp total 20 1148 1
Cv's 4 57 14.25 2.05 2.501
Cv x Na SO 8 59 7.38 1.05 2.07
Error b 72 501 6.96

 

lThese F values are for 70 degrees of freedom for the error variance.

Table A6. AOV:

NaCl Stress.

 

 

Source

df

SS

 

.05
Mp total 8 371
NaCl 2 290 145 10.75 5.14
Error a 6 81 13.5
Sp total 44 623
Mp total 8 371
Cv's 4 44 11 2.53 2.78
Cv x NaCl 8 104 13 3.00 2.36
Error b 24 104 4.33

 

56

Table A7. AOV: Second Double Stress.

 

 

D.
HI

Source SS MS F F

 

.05

Mp total 9 1396

Blocks 1 58

NaCl 4 1237 309.25 12.24 5.19
Linear 1 1232 1232 48.79 7.71
Residual 3 5 1.66 ns

Error a 4 101 25.25

Sp total 19 1529

Mp total 9 1396

Na SO3 1 7 7 .301

NaCl x Na S03 4 10 2.5 .107

Error b 5 116 23.2

 

BIBLIOGRAPHY .

10.

ll.

12.

13.

BIBLIOGRAPHY

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Binding, H. (1971). Organogenese an Kallus von Petunia hybrida. g,
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Binding, H. (1974). Regeneration von haploiden und diploiden
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Binding, H., K. Binding, and J. Straub (1970). Selektion in
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Brunold, C. (1976). Sulfur dioxide as a sulfur source in duckweed
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Carlson, P. S. (1970). Induction and isolation of auxotrophic

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Cecil, R. and R. G. Wake (1962). The reactions of inter- and intra-
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