Retrospective Theses and Dissertations
1970
Bacteria-free soybean plants
Jerry Fred Strissel
Iowa State University
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71-7333
STRISSEL, Jerry Fred, 1942BACTERIA-FREE SOYBEAN PLANTS.
Iowa State University, Ph.D., 1970
Agriculture, plant pathology
University Microfilms, Inc., Ann Arbor, Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED
BACTERIA-FREE SOYBEAN PLANTS
by
Jerry Fred Strlssel
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Major Subject: Plant Pathology
Approved:
Signature was redacted for privacy.
In Charge of Major Work
Signature was redacted for privacy.
Head of^^jorD^partment
Signature was redacted for privacy.
Dean of/'Qi-aduate Co^ege
Iowa State University
Of Science and Technology
Ames, Iowa
1970
11
TABLE OF CONTENTS
Page
INTRODUCTION
1
LITERATURE REVIEW
3
Gnotoblology
3
Aseptic Culture of Bacteria-free Plant Roots
3
Aseptic Culture of Bacteria-free Plants
5
Methods of Obtaining Bacteria-free Plants
6
Sterilization of Isolators
7
Assay Procedures
8
Interactions Between Plants and Bacteria
9
Peroxidase Activity In Plants
MATERIALS AND METHODS
Bacterial Population Assays
10
13
13
Seed assays
13
Sodium hypochlorite as a seed sterilant
13
Development of bacteria-free plants
l4
Assay procedure for plants in gnotoblotlc
Isolators
15
Excision of the Embryo Axis
l6
Growth of the Embryo Axis
19
Glbberelllc acid (GA)
19
Streptomycin
22
Use of Embryo Axis for Physiological Studies
23
Preparation of enzyme extract
23
Peroxidase activity
24
ill
Page
Acrylamlde gel electrophoresis
24
Transaminase activity
27
Gnotobiotic Isolators
28
Description of isolators
28
Sterilization of isolators, nutrient solu­
tion, assay material, and equipment
28
Plant material
31
RESULTS
39
Bacterial Population Assays
39
Untreated seed
39
Effect of NaOCl on bacterial survival
39
Treated seed
42
Growth of Excised Soybean Embryo Axis
43
Nutrient studies
43
Lateral root formation
46
Peroxidase activity
49
Electrophoresis of peroxidase extracts
54
Transaminase activity
62
Development of Bacteria-free Plants under
Gnotobiotic Conditions
65
Assays
65
Growth characteristics (isolator l)
66
Isolator 2
67
DISCUSSION
69
Iv
Page
SUMMARY
74
LITERATURE CITED
76
ACKNOWLEDGMENTS
85
1
INTRODUCTION
The concept of research dealing with the development of
bacteria-free plants or animals is primarily based on the
premise that a macroorganism can be physically separated from
all microorganisms.
The origin of this concept is not defi­
nite, but it probably began with the concepts of pure food and
water and the realization that microbes might be involved in
the health of man.
Research involving development of bacteria-
free plants or animals is also an extension of the pure culture
concept which is based on the fact that an organism must be
isolated from the natural complex state in which it normally
exists if the demands of the experimental approach are to be
satisfied.
The first experiment dealing with bacteria-free plants
was done in I885 by Duclaux (19) who reported that peas would
not grow under absolutely bacteria-free conditions.
This
report eventually launched the first decade of research
dealing with development of bacteria-free plants and animals
in that it raised the question, "Is life possible without
bacteria?"
Since 1885, there have been numerous reports of a rela­
tionship of bacteria with plants. Some investigators (5, 8)
demonstrated that plants grew better under bacteria-free
conditions; others (9, 54, 79) reported that plant growth
was unaffected by microorganisms.
Still other investigators
2
(13, 28, 78) demonstrated that plant growth was Improved when
microorganisms were present.
High bacterial populations have been found intimately
associated with the growth and development of soybean plants
(21).
Several strains were recently Isolated from soybeans
that are able to perform biochemical processes similar to
those found in plants.
In order to determine whether the
microflora associated with soybeans had a beneficial, neutral,
or detrimental effect on plant growth, it was decided that a
method should be devised by which bacteria-free soybean plants
could be obtained and grown under gnotoblotlc conditions.
was the goal of this research project.
This
3
LITERATURE REVIEW
Gnotobioiogy
Previous investigators concerned with relations between
plants and microorganisms found it necessary to develop a
technique for growing plants under gnotobiotic conditions.
The term gnotobioiogy has been defined as that part of biology
involving studies in which only known biota are present (56,
67).
This has involved either bacteria-free or specifically
inoculated bacteria-free plants.
It has also included con­
taminated plants only when all microbial species are known to
the investigator (53).
The design of isolators to grow plants under gnotobiotic
or aseptic conditions were influenced by the intent of the
investigator.
There are two main types of aseptic plant
culture. In one type the entire plant is grown under aseptic
conditions while in the other type only the root system Is
maintained microbe-free.
Aseptic Culture of Bacteria-free Plant Roots
Apparatuses for the production of plant-cultures with a
microbe-free root system have generally consisted of altered
bottles or flasks.
Szember (79) used an Inverted milk bottle
with the bottom removed as a culture vessel.
The soil in the
vessel was first covered with sand coated with paraffin and
then with silver-coated (catadyne) sand. Bacteria-tight
4
glass filters were used for adding water and nutrient solu­
tion. This was a marked improvement over a similar
apparatus designed by Gerretsen (28).
Bloom (7) used bottles in which the area around the
base of the plant was covered with a mixture of paraffin
and vaseline to seal out contamination. The top of the
bottle was fitted with a soft rubber stopper which could be
pierced with a longer hypodermic needle to withdraw fluids
from the root area to check for root exudates.
Blanchard and Diller (6) designed glass jars in which
cotton gauze was laid over the hole of an inverted lid.
An
aluminum cylinder was placed on top of this and screwed
into place on the jar.
The cylinder was half-filled with
vermiculite and a gauze-wrapped cotton plug was inserted at
the top until the seeds had germinated.
A similar apparatus
in which test tubes were held in place by basalt pebbles and
cotton was used by Swaby (78).
A more refined apparatus for the aseptic culture of
plant roots for studies on root respiration and the bio­
chemical nature of root exudates was developed by Stotzky
e_t a^. (76).
The apparatus consisted of a growth tube into
which a surface-sterilized seed or seedling is planted, a
root chamber containing a solid growth medium, an irrigation
chamber, and an irrigation solution reservoir.
5
Aseptic Culture of Bacteria-free Plants
The cultivation of whole plants under aseptic conditions
was usually planned with a view of discontinuing the cultiva­
tion long before maturity.
With this objective, culture
vessels such as Erlenmeyer flasks (27, 37, 75, 88), Warburg
flasks (4l), test tubes or glass tubes (5, 11, 17, 24, 45, 48,
59, 63, 80), glass cylinders (42, 79), bottles with plastic or
aluminum screw-type caps (43, 44, 84), and a bell glass
apparatus (73) were used for the culture of plants from
embryos, callus tissue, or seeds.
Various types of cabinets have been used as enclosures
for the growth of plants (38).
Estey and Smith (22) designed
one of the first cabinets that was gas sterilizable, and used
it to grow plants from seed to maturity in a sterile environ­
ment.
The cabinets, 25 x 33 x 36 inches, were constructed of
cedar wood and glass.
A front panel had armholes
bordered
by flanged aluminum rings to which were attached neoprene or
rubber gloves.
Air and water were drawn into the cabinets
through a series of filters.
There was no arrangement for
the transfer of material in and out of the chamber.
In 1957, Trexler and Reynolds (82, 83) developed a
flexible film apparatus for the rearing and growth of bacteriafree animals which could also be used for growing plants.
The
dimensions of the plastic isolator could be altered easily and
either one or two operators could be accommodated.
The
iso-
6
labors were made of transparent vinyl -llm (12 to 20 mils
thick) and were sealed with heat, or by solvent cements.
Shoulder-length rubber gloves were attached to the plastic
walls by means of a clamp so arranged that only rubber and
plastic were exposed to the interior of the isolator.
A
double-door lock was also built in so that materials could
be transferred in and out of the chamber without contamina­
tion.
Lindsey ($3, 54) has used this type of chamber to grow
beans, tomatoes, corn, and peanuts under gnotobiotic conditions.
Methods of Obtaining Bacteria-free Plants
To obtain bacteria-free plant material, a large number
of different chemical preparations have been used as steri­
lizing agents.
They have been used with varying time periods,
varying concentrations and in combination with various pretreatments of the seed.
The severity of treatment has ranged
from washing seed in sterile distilled water (13) to treatment
with peracetic acid (54). A common sterilizing agent that has
been used by several workers is mercuric chloride (37, 4l, 78,
93, 94).
Kylin (47) used mercuric chloride to pre-treat wheat
seed followed by sterilizing them overnight in hydroxylamine.
/
Stotzky (76) found that Ca(001)2 was the most effective
surface-sterilant, but it did not remove deep-seated contami­
nants present in some seeds.
Benzalkonium chloride removed
most of these microorganisms and also increased germination,
but the seedlings were stunted and abnormal.
Blanchard (6),
7
Wilson (92), Bowen and Rovira (8), Rovira and Bowen (68), and
Yamashita and Goto (94) also used Ca(001)3 solutions.
Miflin (59), using excised barley embryos, first soaked
dry seed in sodium hypochlorite solution for 1 hour, then
dissected the embryos and allowed them to dry out for at least
3 weeks.
When required, the embryos were first sterilized by
exposure to mercuric chloride followed by treatment with
hydrogen peroxide.
Streptomycin was used by Ark (2) to disinfect cucumber
seed infected with Pseudomonas lachrymans and tomato seed
carrying Oorynebacterium michiganense.
Streptomycin, however,
produced a variety of effects in the plant:
germination, shoot
growth, root growth, and chlorophyll formation were reduced
(10, 15, 18, 63); internal development of chloroplasts and
mitochondria was also inhibited (46). Root growth inhibition
produced by streptomycin was reversed by addition of calcium
and manganese ions.
Antibacterial activity of streptomycin
was not reversed by these divalent ions which suggested that
inhibition of root growth and inhibition of bacteria were
caused by separate mechanisms (32).
Sterilization of Isolators
Peracetic acid has been used by Lindsey (54), and Trexler
and Reynolds (Bl, 83), to sterilize the plastic isolator
chambers used for growth of bacteria-free plants and animals
under gnotobiotic conditions. These workers reported that
8
peracetlc acid possessed germicidal activity against a broad
spectrum of gram positive and gram negative bacteria.
When
used in a 2% aqueous solution with the addition of 0.1#
sodium alkylarylsulfonate, resistant spores (Bacillus
stearothermophllus) dried on an etched glass bead carrier
were inactivated within 30 seconds in the liquid phase and
within 10 minutes in the gas phase (33).
Peracetlc acid was
considered a temporary bactericide-fungicide, breaking down
gradually after application to a surface to yield acetic
acid, water, and oxygen.
No toxic product remained as a
residue.
Assay Procedures
One of the most Important aspects of research in
gnotobiology has been the adequacy of the assay procedure.
A routine procedure for the detection of bacteria and fungi
was suggested by Wagner (87).
Samples were collected on
swabs and Inoculated (within isolator) into tubes of
thloglycollate medium and a general purpose medium (tryptic
soy broth).
Samples were removed from the isolator and
inoculated on the surface of plates of tryptic soy agar and
potato dextrose agar.
There was never an occasion when
stained smears showed the presence of large numbers of
organisms which were not subsequently Isolated.
Contami­
nants that were detected included molds, yeasts, strepto­
cocci, micrococci, Clostridia, conforms, pseudomonads and
9
aerobic sporeformers.
A similar test used at the Lobund
Institute (University of Notre Dame) for routine examination
of rat, mouse, and chicken colonies proved to be successful
in detecting 99^ of all contamination encountered (l4).
For contamination tests on bacteria-free plants,
Lindsey ($4) checked the sterility of the seeds during
germination and thereafter, the sterility of the isolators
and plants was confirmed every 2 weeks. Samples of plant
tissue, rooting medium, and debris from the isolator floor
were placed in fluid thioglycollate, Czapek-Dox broth,
nutrient broth, and brain heart infusion broth. The culture
media containing the samples were held for 2 weeks and
examined microscopically, then discarded.
The majority of other investigators tested the sterility
of their isolators and plants with only 1 or 2 types of media.
These included nutrient agar (13, 68, 73), potato-glucose agar
(63), a nutrient medium:
glucose, yeast extract, and casein
hydrolysate (6), gli.cose/peptone medium (50, 91), and a yeastagar-dextrose medium (5).
Interactions Between Plants and Bacteria
High populations of Bacillus subtilis were found by Dunleavy et. ^.l. (21) in the roots, stems, and leaves of soybean
plants.
Soybean cultivars very sensitive to high concentra­
tions of phosphorus supported high bacterial populations
prior to the appearance of toxicity symptoms in the host
10
plants; hosphorus tolerant soybean cultivars supporting
much lower bacterial populations developed toxic symptoms.
These results Indicated that a close relationship between
these bacteria and the soybean plant is operative.
Rovira and Bowen (68) found in short-term uptake
studies that non-sterile clover and tomato plants took up
45 and 85^ respectively more phosphate than did sterile
plants; translocation of absorbed phosphate to the tops was
4.4 times greater in non-sterile tomato plants.
Differences
in incorporation of phosphate between sterile and non-sterile
plants have indicated the difficulties of correctly inter­
preting metabolic patterns in plants unless precautions
are taken to exclude all microorganisms.
Many physiological
studies have relied on washing plant material to reduce the
microbial population to negligible proportions.
Studies
with 7-day-old barley roots however have shown that bacteria
counts on non-sterile roots before and after washing in
rapidly running tap water were 125 x 10^ and 2 x 10^,
respectively, per root system (68).
Peroxidase Activity in Plants
Peroxidase activity has been closely associated with
the metabolic processes which control the growth and
development of plants.
Its physiological role is somewhat
obscure, but it has been indicated that it may inactivate
11
auxin (55, 65), convert hydroxyphenylpropanes such as
conlferyl alcohol to lignin-llke materials (72), detoxify
HgOg, and oxidize phenol (29). The development of peroxi­
dase activity in plants was considered to be a part of the
normal aging process as a result of increased indole acetic
acid oxidase activity (25, 26).
The level of peroxidase activity may be regulated by
several materials such as gibberellin (34), auxin, cytokinin,
ethylene (26), nitrogen fertilizer (71), and AMO-I618 (3^).
Peroxidase activity generally increases when plant tissue is
infected (23, 40, 64, 69).
Stahmann (74) found a relative
increase in peroxidase activity was higher in resistant
varieties than in susceptible varieties when both were
inoculated with the same pathogenic bacterial .'.solate,
Umaerus (85) observed that peroxidase activity was positively
correlated with field resistance in SPlanum tuberosum,
but not in S^. demissum .
In many plants, peroxidase activity was resolved into a
number of isoenzymes electrophoretlcally separable on starch
gel or polyacrylamide gel (36, 57, 77). Buttery and Buzzell
(12) used disc electrophoresis to separate cultivars of soy­
beans into two main groups on the basis of high or low peroxi­
dase activity in the seed coat.
Seed coat extracts from the
high level group exhibited more than 78 times the peroxidase
activity of seed coat extracts of the low level group.
A
12
major band of isoperoxidase was common to both groups.
Two
minor bands of isoperoxidase were also identified in all
high-level cultivars and in two of the five low-level
varieties tested.
13
MATERIALS AND METHODS
Bacterial Population Assays
Seed assays
The bacterial populations commonly associated with the
seed coat, cotyledons, and the embryo axis of 1968 Ford
soybean seed were assayed.
The seeds were soaked in sterile
distilled water for 6 to 10 hours and the seed coat,
cotyledons, and embryo axis were then separated with a
sterile scalpel, placed in sterile distilled water in test
tubes, crushed with a sterile glass rod, diluted, and plated
on trypticase soy agar (TSA). The plates were incubated at
30 C and examined after 40 hours.
Sodium hypochlorite as a seed sterilant
A bacterial isolate was obtained from seeds used in
the previously described seed assay.
Effectiveness of
sodium hypochlorite (NaOCl) as a seed sterilant was
determined by growing the culture in mineral media (70) in
500 ml side-arm flasks.
The flasks were placed in a
shaker incubator at 30 C and growth was measured by optical
density in a spectrophotomer at 600 m^.
When the culture
was in the log phase of growth, NaOCl was added to the
flasks at concentrations of 0.26, 1.31, and 2.63^.
was not added to control flasks.
replicated twice.
Each treatment was
NaOCl
14
Development of bacteria-free plants
An attempt was made to excise and grow the embryo axis
in the absence of the seed coat and cotyledons. The embryo
axis was excised after the seed was treated with 0.53 or 2.63#
NaOCl for 0.25, 1, 3, 5, 10, or 30 minutes. The excised
embryo axis was surface sterilized with 0.53 or 2.63^' NaOCl
for 10, 15, 30, or 60 seconds and grown for 8-l6 days in
agar.
The entire plant was then crushed in sterile distilled
water, diluted (l/lOO dilution series-sterile distilled water),
and assayed on TSA.
Plates were incubated at 30 C for 72 hours
and were examined for bacterial or fungus growth.
Each treat­
ment was replicated 5 to 16 times with 1 plant/flask/rep.
Bacterial populations of seed coats and cotyledons were
determined by soaking Ford seed in 2.63# NaOCl for 1, 5, 10,
and 30 minutes. The seeds were rinsed 3 times in sterile dis­
tilled water, allowed to imbibe sterile distilled water for 6
hours, after which the seed coat, cotyledons, and embryo
axis were separated immediately, placed in sterile distilled
water, crushed, and assayed on TSA plates.
Each treatment
was replicated 10 times with 1 plant/flask/resp.
Bacterial populations were also determined in plants
from Ford seed that was germinated and grown for 8 days in
agar after the following treatments; (a) no treatment with
NaOCl (control), (b) seed soaked in 2.63# NaOCl for 30 minutes,
(c) seed soaked in 2.63# NaOCl for 30 minutes, seed coat re­
15
moved and discarded, intact cotyledons and embryo axis dipped
in sterile distilled water or in 2.63^ NaOCl, and (d) seed
soaked in 2.63# NaOCl for 30 minutes, embryo axis excised
(seed coat and cotyledons discarded) and surface sterilized
in 2.63# NaOCl.
After 8 days, the entire plant was crushed
in sterile distilled water and assayed on TSA.
Each treatment
consisted of 20 replications with 1 plant/flask/rep.
Assay procedure for plants in gnotobiotic isolators
Samples assayed from plants grown from seed or from the
excised embryo axis consisted of trifoliolate leaflets,
unifoliolate leaves, cotyledons, stems, entire germinated
seedlings, and entire dead plants, including the root system.
In isolator 1, the plants grown from seed were assayed every
7-10 days for 89 days after germination. At first, entire
germinated seedlings were assayed until 5 plants/pot re­
mained.
After this, a trifoliolate leaflet was sampled.
If a plant died, the entire plant was assayed.
The plants
grown from the excised embryo axis were not large enough to
sample until 39 days after plants were placed in the isolator.
After 39 days, a trifoliolate leaflet was sampled from each
plant every 7-10 days.
All plant samples were placed in 50
ml Erlenmeyer flasks containing 20 ml of trypticase soy
broth (TSB).
Control flasks for checks on contamination in
the isolators consisted of TSB in flasks that were left open
and placed in each corner of the isolators.
The plant samples
16
were kept in the isolator for J-20 days to check for any
change in turbidity due to bacterial growth. The plant
samples were then removed, crushed with a sterile glass rod,
and 0.3 to 0.5 ml was plated on TSA, nutrient agar (NA),
potato dextrose agar (PDA), heart infusion agar (HIA), and
on horse serum agar (HSA) (6l). The plates were incubated
at 30 C for 7-10 days and then examined.
The plates of plant
samples assayed at 89 days after germination were also incu­
bated under anaerobic conditions in Torbal, Model AJ-2, stain­
less steel anaerobic containers (obtained from The Torsion
Balance Co., Clifton, N.J.).
Before discarding, all plant
samples were examined for bacteria under a phase microscope.
In isolator 2, the plants were assayed only at the end
of 81 days, at which time the isolator program was terminated.
The entire plant, supporting agar medium, reserve nutrient
solution, and control flasks were assayed on the same media
and in the same manner as described for isolator 1.
Excision of the Embryo Axis
All plants were excised under a plastic microvoid hood
(obtained from Air Control, Inc., Norriston, Pa.) previously
wiped down with a-3 (Airkem Inc., Carlstadt, N.J.) (Figure l).
The seed, either treated (NaOCl) or untreated (directly from
seedlot source), was soaked in sterile distilled water for 6
to 10 hours.
The embryo axis was excised by making an
incision at the hilum and along one side of the radicle with
Figure 1.
Plastic mlcrovold transfer hood used for aseptic
excision of the embryo axis from soybean seed
Figure 2.
Excision of embryo axis from Imbibed soybean seed
with a sterile scalpel and tweezers
19
a sharp, sterile scalpel (Figure 2). The cotyledons were
then separated. The embryo axis was removed from the
remaining cotyledon with a careful incision near the seed
leaves (Figure 3); the axis Immediately was placed in
sterile distilled water or dipped In NaOCl, and then placed
In 125, 250, or 500 ml Erlenmeyer flasks (Figure 4). The
flasks contained Howell's nutrient solution (39), White's
amino acld-vltamin solution (89),
sucrose, ferrous
ethylenedlamlnetetraacetate (Fe - 1.12 ppm), and 0.4# Bactoagar (which gave a solution dense enough to keep the embryos
floating).
All flasks were Incubated in the dark at 30 C.
From this point on, embryos excised from untreated seed will
be referred to as normal, and embryos from treated seed will
be referred to as bacteria-free.
Growth of the Embryo Axis
Glbberelllc acid (GA)
Howell's nutrient solution and a soybean callus tissue
medium described by Miller (60) were used as the basal medium
for studying the effects of glbberelllc acid.
Glbberelllc
acid was added to the media at concentrations of 0.3 and 10
ppm.
Control flasks had no GA added.
Glbberelllc acid was
sterilized with a Seitz filter (0.01 micron porosity) prior
to adding It to the medium, or GA was added to the medium
which was then autoclaved (15 mln at 121 C). Embryos were
excised from Ford seed that had been treated with 2.63# NaOCl
Figure 3.
Removal of embryo axis from remaining soybean
cotyledon
Figure 4.
Excised embryo axis grown on nutrient enriched
agar following surface sterilization with NaOCl
21
^^,3"A
22
for 30 minutes.
Each treatment was replicated 4 times with
4 embryos/250 ml flask (50 ml media)/rep.
The flasks were
incubated at 30 C In the dark until the epicotyl began to
elongate.
The flasks then were placed in diffuse light.
Each plant was checked dally for (a) initiation of lateral
roots (b) initiation of more than 5 lateral roots/plants, (c)
epicotyl elongation, and (d) first notable expansion of the
unifollolate leaves.
Streptomycin
In order to determine whether streptomycin could be used
to prevent bacterial contamination without Injurylng these
plants, streptomycin sulphate was added to culture flasks of
bacteria-free, excised Ford soybean embryos.
Streptomycin
solution was filtered in a Seltz filter (0.01 micron porosity)
prior to adding it to the medium at concentrations of 10 and
50 ppm.
Control flasks contained no streptomycin.
An addi­
tional treatment consisted of excised embryos from untreated
seed incubated with no streptomycin added. The plants were
grown for 8 days, and presence or absence of lateral roots
and necrotic lesions were recorded.
and assayed on TSA.
All plants were crushed
Each treatment was replicated 20 times
with 1 plant/250 ml flask (50 ml media)/rep.
Streptomycin,
at concentrations of 10 and 50 ppm, was also used when plants
were prepared for the gnotoblotic Isolators.
The number of
lateral roots/plant were recorded for 256 embryos after 8 days
23
of growth.
Use of Embryo Axis for Physiological Studies
Preparation of enzyme extract
To determine whether the bacterial population associated
with soybean seed had an effect on the enzyme activity of
developing plants, an enzyme extract was prepared from
bacteria-free plants (excised embryo axis) and from normal
plants (excised embryo axis) of Ford, Corsoy, and Harley
soybean cultlvars.
The plants were grown for 8 days in 125
ml Erlenmeyer flasks containing nutrient agar medium already
described. Each treatment was replicated 4 times with 6
plants/flask and 4 flasks/rep.
After 8 days of incubation
in the dark (30 C), the plants were washed in distilled water,
blotted dry, weighed, and crushed in a mortar with a pestle,
in O.IM sodium phosphate buffer, pH 7.1, (2 ml/g fr wt).
The
extracts were centrlfuged at 12,100 g for 20 minutes in a
Sorval RC2-B centrifuge.
The supernatants (enzyme extracts)
were collected and stored.
All crus! Ing and centrlfuging
procedures were carried out at 4 C.
Enzyme extracts from whole seed were prepared as follows:
(a) seed was soaked for 6 hours in sodium phosphate buffer,
pH 7.1, at 4 C, and crushed in a mortar with a pestle; the
extract was strained through 4 layers of cheesecloth, and was
centrlfuged as described above, or, alternatively, (b) seed
was crushed first in a mortar, then was soaked in sodium
24
phosphate buffer, pH 7.1, for 2 hours at 4 C, and then was
centrlfuged.
Peroxidase activity
Assay of peroxidase activity, based on the oxidation of
p-phenylenedlamlne, was made spectrophotometrlcally at 485 mu.
The reaction medium consisted of 1.0 ml O.IM sodlum-cltrate
phosphate buffer, pH 4.1; 1 ml IM p-phenylenedlamlne; 1.0 ml
extract (diluted 1:1000); 0.4 ml of 0.1# H2O2, and enough
distilled water to bring the total volume to 6 ml.
Change in
absorbance was recorded at 20 second Intervals.
Acrylamlde gel electrophoresis
Electrophoresis of the peroxidase extracts was carried
out on acrylamlde gel.
A separation and spacer gel was formed
according to a procedure described by Davis (16), except that
Cyanogum 4l (obtained from Fisher Scientific Company) (35, 66)
was substituted for acrylamlde and N,N-methyleneblsacrylamide.
A special apparatus was devised to carry out the electro­
phoresis in a conventional plastic chamber made by Buchler
Instruments, Inc.
The apparatus consisted of the following:
a glass tube 76 mm long with an internal diameter of 7 mm; a
plastic tube 11.2 cm long with an internal diameter of 10 mm;
2 plastic plugs; and 2 platinum-copper alloy wires 8 cm long
(Figure 5).
After the separation and spacer gels were formed
in the glass tube, a 6 mm diameter Whatman No. 2 filter paper
Figure 5. Disc-gel electrophoresis apparatus consisting of
a plastic tube (112 mm long; Inside diameter -10
mml, a glass tube (76 mm long; Inside diameter -7
mm), 2 platinum-copper alloy wires (80 mm long),
and 2 plastic plugs with a 1 mm hole drilled In
the top
Figure 6. Buchler migration chamber containing disc-gel
electrophoresis tubes
26
27
disc with 10 ni of enzyme extract was placed directly on top
of the spacer gel.
The glass tube was then placed inside
the plastic tube.
The ends of the tube were stoppered with
the plastic plugs.
A 1 mm hole drilled through the top of
the plug and the plastic tube allowed the addition of Trisglycine buffer (pH 8.1) and bromophenol blue with a #25 gauge
hypodermic needle.
After the tube was filled, the apparatus
was placed in the electrophoresis migrating chamber (Figure
6). The chamber reservoirs were filled with Trls-glyclne
buffer (pH 8.1), and the wires were placed in the ends of the
tubes to complete the circuit.
A constant current of 2.5 ma
per tube was applied; 8 tubes were run at a time.
Approxi­
mately 2-1/2 hours were required to move the migrating front 55
to 65 mm.
Following electrophoresis, the gels were removed
from the glass tubes and immersed in 0.02M gualacol for 20
minutes and then in 0.3# HgOg
15 minutes. The number of
isopercxidase bands formed and the distance they moved were
recorded Immediately.
Transaminase activity
Transaminase activity was determined on the same enzyme
extract used in the peroxidase test.
An aspartate-alpha-
ketoglutarate system (58) was used in which the resultant
oxalacetate was measured by the formation of a highly colored
compound with a dlazonlum salt (3, 4).
A standard curve of
oxalacetlc acid (OAA) (O-5OO nmoles) was plotted to determine
28
umoles OAA formed per minute per mg of protein.
The protein
concentration of the enzyme extracts was determined by a
biuret method as described by Gornall et al. (31). A standard
curve was also plotted for protein standards (0-2.5 mg BSA
protein/ml).
Gnotobiotic Isolators
Description of isolators
Flexible film isolators described by Trexler and Reynolds
(83) were used for growing plants in gnotobiotic environments.
These isolators were 6l cm x 97 cm x 97 cm (Figure 7).
They
were obtained from Standard Safety Equipment Co., G-F Supply
Division, Palatine, 111.
The isolators were fastened to a
plywood base (1.2 cm x 79 cm x 134 cm) which was placed on a
cart with a flat top.
second shelf.
An air blower was placed below on a
Light was supplied by 8 overhead Sylvania
P40T12/CW cool-white, preheat, 40 W fluorescent bulbs.
Light
intensity at plant height ranged from 300 ft-c at the center
to 180 ft-c at the corners of the isolators.
Air temperature
in the isolators was 25 t 5 C with the lights on.
When the
air conditioning units in the building were not functioning,
the air temperature increased to 30 C.
Sterilization of Isolators, nutrient solution, assay material,
and equipment
Isolators were sterilized by spraying the interior with
5O-IOO ml of peracetic acid solution (PAA, 2^ peracetic acid
Figure 7.
Flexible film isolators used in growing plants
under gnotoblotlc conditions
30
31
and 0.1^ sodium alkylarylsulfonate in distilled water).
The
isolators were unused for 12 hours after spraying, before the
blower was turned on.
Wet cheesecloth was used to wipe up
the excess peracetic acid.
hood for 5 days.
The isolators were vented to a
Non-autoelavable material was placed in the
isolator and was sterilized with PAA.
Autoclavable material and equipment were placed in
autoclavable nylon bags (obtained from Vail Medical Products,
Los Angeles, California) (Figure 8) and were autoclaved twice
for 15 minutes at 121 C. Square-pak flasks which automatically
seal after autoclaving were used in supplying the isolators
with nutrient solution (Figure 9).
Autoclavable material to be transferred into the isolators
was placed in a stainless steel cylinder which was covered with
mylar film and was autoclaved twice for 50 minutes at 121 C
(Figure 10).
Following autoclaving, the cylinder was attached
to the isolator by means of a vinyl transfer sleeve.
The
transfer sleeve was sprayed with PAA (Figure 11) and was not
used for 30 minutes.
The double-door locks were then removed
and the mylar film was cut with a scalpel.
A set of 46 cm
tongs was used to reach into the cylinder to transfer the
material inside the chamber.
Plant material
Seeds used in isolator 1 were sterilized with NaOCl for
30 minutes before they were transferred to the isolator.
Figure 8.
Autoclavable nylon bags used In transferring
material into gnotoblotic chambers
33
Figure 9. Square-pak flasks used In transferring sterile
nutrient solution Into gnotoblotlc chambers
35
I. i-jA L
f
Figure 10.
Stainless steel sterilizing cylinders used for
transferring autoclavable material into plastic
isolators (cylinder on right covered with mylar
film)
Figure 11.
Sterilization of transfer sleeve with peracetic
acid
37
38
Inside the isolator, a total of 50 Ford soybean seeds were
germinated in white sand in 8 cm and 10 cm clay pots.
Howell's nutrient solution was added as needed.
Plants from the excised embryo axis were also used in
isolator 1 (40 plants), and exclusively in isolator 2 (48
plants).
These plants were excised from seed that was
treated with NaOCl for 30 minutes. The plants were grown
In the dark in 25O and 500 ml Erlenmeyer flasks for one week
before they were transferred to the isolators.
Howell's
nutrient solution was added to the flasks once a week after
the agar began to dry.
39
RESULTS
Bacterial Population Assays
Untreated seed
High populations of bacteria forming smooth white
colonies (rods - typical of Bacillus previously Isolated by
Dunleavy (21)) and smooth yellow colonies (rods - typical of
Xanthomonas previously Isolated by Dunleavy (unpublished
data)) were regularly isolated from untreated Ford soybean
seed.
Mean populations of 5.3 x 10^, 9.4 x 10^, and 8.6 x
10^ bacteria/ml were assayed from the seed coat, cotyledons,
and the embryo axis, respectively.
In-25^ of the cases, how­
ever, no bacteria were detected in the embryo axis.
Effect of NaOCl on bacterial survival
Growth of a bacterial isolate, from the smooth white
colonies obtained from untreated Ford seed, was completely
inhibited with 3 different concentrations of NaOCl (Figure
12).
A concentration of NaOCl as low as 0.53^ completely
inhibited growth of the bacteria, and thus, was equally as
effective as 1.31 or 2.63# NaOCl.
Growth of the bacteria
in the control flasks continued at a high rate.
The higher
the concentration of NaOCl used, the greater was the bleaching
effect on the growth medium.
medium was practically clear.
At a concentration of 2.63^ the
Trypticase soy broth, as a
growth medium for the bacterial Isolate, was not used as
Figure 12.
NaOCl inhibition of the growth of a bacterial
isolate normally associated with Ford soybean
seed
41
0.20
0.18
"CONTROL (NO NaOCl)
0.14
O 0.12
§
^ 0.10
0.06
_0._26% NaQCl^^
0.04
r."3Î% NaOCl
0.02
2.63% ,NaOCl
0.00
TIME (HOURS)
42
addition of 2.63^ NaOCl changed the TSB to a heavy white
suspension, the absorbance of which was too great to read
on the spectrophotomer scale.
Treated seed
Bacteria-free soybean plants were obtained when the
embryo axis was excised from Ford and Blackhawk soybean seed
that had been soaked in 2.63# NaOCl for 30 minutes.
Soaking
the seed in 0.53 or 2.63# NaOCl for lesser periods of time
(0.25, 1, 3, 5, or 10 minutes) did not remove all bacterial
contaminants.
Soybean plants grown from the excised embryo
axis of normal seed supported a high bacterial population
after 8 days of growth.
Ford plants had a mean population of
5.6 X 10^ bacterla/ml while Blackhawk plants had a mean popula­
tion of 6.0 X 10^ bacterla/ml.
A large percentage of the
Blackhawk plants (60#), from normal seed, were bacteria-free,
whereas only 15# of the Ford plants from normal seed were
bacteria-free.
Growth of the plants, under gnotoblotic conditions, from
the excised embryo axis could be maintained for 3 weeks in
the flasks without transfer.
When the plants were assayed
each week for a period of 3 weeks, again, no bacteria were
found.
When Ford soybean seed was treated with 2.63# NaOCl for
30 minutes and were assayed directly, no bacteria were found.
If the treated seed was germinated on agar, bacteria were
43
detected In 10$ of the plants after 10 days of plant growth.
No bacteria were detected in seedlings in which the seed
coat had been removed after the seed was treated with NaOCl,
The cotyledons and intact embryo axis had been dipped in
2.63$ NaOCl and placed in the flask without rinsing after
the seed coat was removed.
Germinated seedlings from normal
seed, with seed coats intact, had a mean bacterial population
of 1.5 X 10^ bacteria/ml after 10 days of growth.
Growth of Excised Soybean Embryo Axis
Nutrient studies
Radicle elongation of the soybean embryo axis was noted
within 24 hours after excision.
Lateral root formation
developed in an average of 9 days and development of plants
with more than 5 lateral roots/plant required at least 12
days.
When the medium was supplemented with either 0.3 or
10 ppm gibberellic acid, initiation of lateral root formation
occurred earlier, with a high percentage of the plants having
lateral roots, as early as 4 days (Figure 13A). Development
of a large percentage of plants with greater than 5 lateral
roots/plant required only 6 days (Figure 13B).
Initial growth
of the epicotyl required 10 days in media without gibberellic
acid, and only 5 days with 10 ppm gibberellic acid added.
Expansion of the unifoliolate leaves occurred 6 days earlier
when 10 ppm gibberellic acid was added to the medium.
Gibberellic acid, at a concentration of 0.3 ppm, had no
Figure 13.
Growth response of excised soybean embryos to
glbberelllc acid measured by (A) time to
Initiate lateral root formation, (B) time required
for more than 5 lateral roots/plant to develop,
(C) time to initiate eplcotyl elongation, and (D)
time required to Initiate expansion of the
unifoliolate leaves
PERCENTAGE OF PLANTS
WITH EPICOTYL ELONGATION
(Tl 00 o
M
O
O O o
o
H3
~r ~r n—r -T~
H
o
PERCENTAGE OF PLANTS WITH
LATERAL ROOT INITIATION
M
O
to
00
o
o
o
o
O
-|—1—I—r T"
O
D^*
ifi.
g
w
n
%
H
W
M
»
m
%
In
H
Crt
H
i
H
§
if\
4^
vn
PERCENTAGE OF PLANTS WITH
PERCENTAGE OF PLANTS WITH
EXPANSION OF THE UNIFOLIOLATES
MORE THAN 5 LATERAL ROOTS
"3
S _o(
g vo > o
D
>»o •w
to S oï"
to •c^ <T* 00
o
o
O o
-r T" -r T"
M
<T> 00
O
-r T" T" "f
o
o
to
o
H
§
M
°D
D —P
s
m 3
ol
M
5d
W
%
H
tn
M
§
e.
if?
a
o
M K^
O
§
o
M
%
t-*
o
M
w
W
o
o
T
to
46
effect on the time needed to Initiate epicotyl elongation
(Figure 13c) or expansion of the unifollolates (Figure 13D).
Even after 12 days of growth, only a small percentage of the
plants, in media with 0.3 ppm GA or no GA, had unlfollolate
expansion.
The stimulatory effect of glbberelllc acid was
unaltered when it was either autoclaved or filter sterilized
prior to adding it to media.
Also, it was noted that the
soybean callus tissue medium was as satisfactory as Howell's
nutrient solution.
Lateral root formation
Bacteria-free soybean plants from the excised embryo
axis were healthy and rapidly developed white lateral roots.
Plants from seeds that were not previously treated with
sodium hypochlorite supported a high bacterial population
that limited the elongation of the radicle, inhibited lateral
root formation, and generally produced a brown to black dis­
coloration of the radicle (Figure 14).
Lateral root formation of bacteria-free embryos was
inhibited when streptomycin was added to the growth medium.
None of the plants formed lateral roots with 50 ppm strepto­
mycin present.
Only 10^ of the plants had lateral roots with
10 ppm streptomycin present while 90^ of the control plants
(no streptomycin) formed lateral roots.
Excised embryos
from normal seed had a bacterial population of 7 x 10^
bacteria/ml with 90^ of the plants having a lesion on the
Figure l4.
Bacterial Inhibition of radicle elongation and
lateral root formation
48
49
radicle.
None of the normal plants had lateral root forma­
tion.
Streptomycin inhibition of lateral root formation on
bacteria-free plants was also observed when plants were
prepared for the gnotobiotlc Isolators.
In media with no
streptomycin present, a mean number of 13 lateral roots/
plant developed within 8 days after excision of the embryo
axis.
A mean number of 5 lateral roots/plant was observed
with 10 ppm streptomycin, and no lateral roots developed with
50 ppm streptomycin even after 3 weeks of plant growth (Figure
15).
Peroxidase activity
The bacterial population associated with normal 8-dayold soybean plants (excised embryo axis) had a direct effect
on the peroxidase activity of Corsoy, Ford, and Harley
cultivars (Figure I6).
More activity was noted in Corsoy
normal plants than in bacteria-free Corsoy plants.
Bacteria-
free Ford plants, on the other hand, had a moderate Increase
in peroxidase activity over normal Ford plants, and bacteriafree Harley plants had over twice the peroxidase activity as
found in normal Harley plants supporting a high bacterial
population.
Mean bacterial populations of 1.7 x 10^, 2.7 x 10^, and
6.5 X 10^ bacteria/ml were assayed from the flasks of normal
plants of Corsoy, Ford, and Harley cultivars, respectively,
Figure 15.
Streptomycin Inhibition of lateral root formation
of bacteria-free soybeans in culture flasks (left
to right: control (no streptomycin), 10 ppm
streptomycin, 50 ppm streptomycin)
51
Figure 16.
Peroxidase activity of extracts from excised
bacteria-free embryos and normal excised
embryos of Ford, Corsoy, and Harley
53
BACTERIA-FREE
160 0
80
160 0
TIME (SECONDS)
53
FORD
CORSOY
BACTERIA-FREE
NORMAL
/
HARLEY
K- •
.20
.10
160
TIME (SECONDS)
54
prior to preparation of the enzyme extract. Ford and Harley
plants that were excised from seed treated with NaOCl were
bacteria-free.
Corsoy plants excised from NaOCl treated
seed had bacteria In 4 of the l6 flasks, but they were of
small enough numbers that they had little or no effect on
the peroxidase activity.
No lateral roots formed on the normal plants of Corsoy
and Ford cultlvars, and on only 31^ of the Harley plants.
All of the bacteria-free Ford and Harley plants had lateral
root formation, and 88^ of the Corsoy plants had lateral
roots.
Brown lesions on the radicles were present on 100, 63,
and 46$ of the normal Corsoy, Harley, and Ford plants,
respectively.
None of the bacteria-free Ford and Harley
roots had lesions.
Bacteria-free Corsoy plants In the flasks
which had contamination had started to produce lesions.
This
was found on 20$ of the total number of plants used in the
assay.
Electrophoresis of peroxidase extracts
Disc-gel electrophoresis of the same enzyme extracts
used In the peroxidase activity test also revealed that the
bacterial population associated with soybean plants can
directly affect the number and Intensity of peroxidase bands
formed (Figure 17). The number of bands and the distance
moved were recorded immediately, as the bands started to fade
Figure 17.
Peroxidase isoenzyme patterns following discgel electrophoresis of soybean sap extracts
prepared from (left to right) (a) bacteria-free
Corsoy, (b) normal Corsoy. (c) bacteria-free
Ford, (d) normal Ford, (e) bacteria-free Barley,
and (f) normal Harley plants
56
57
within 30 minutes after the reaction with gualacol and hydro­
gen peroxide.
The bands that were least Intense faded the
fastest, and did not show up well, and sometimes not at all.
In the photograph. Therefore, a dlagramatlc representation
(Figure 18) has also been Included to represent location of
the minor bands In addition to the major bands.
Isoperoxldase bands of normal plants were of less
Intensity In all 3 cultlvars tested (Figures 17 and i8).
Bacteria-free Corsoy plants had the largest number of bands,
with 4 distinct bands formed.
Corsoy normal plants had the
same number of bands and with the same Rp value, but all were
of less Intensity. The number 2(Rp .7) and 3 (Rp .6) bands
of the normal Corsoy plants were only faintly visible.
Bacteria-free Ford plants had 3 distinct bands present, while
only 2 were present for the normal Ford plants.
Bacteria-
free Harley plants also had 3 bands present while only 1 band
was present in the normal plants or those having a large
bacterial population.
A comparison was made between the embryo peroxidase
extracts used In the previous test with enzyme extracts
prepared from crushed normal seed of the same cultlvars.
The
extracts from Corsoy and Ford seed did not have as many bands
as the 8-day-old plants.
Corsoy seed extract had 1 major
band. In comparison to 4 for the bacteria-free plants (Figure
19). The leading, or major band of the seed extract also had
Figure l8.
Diagramatic representation of isoperoxidase
bands of bacteria-free (b-f) and normal (n)
plants of Corsoy, Ford, and Harley
59
CORSOY
(b-f)
0—
.1
.2
.3 —
.4 —
.5 —
.6 —
.7 —
.8
.9
1.0 —
Rp
ANODE
H
(n)
FORD
(b-f)
(n)
HARLEY
(b-f)
(n)
Figure 19.
Peroxidase isoenzyme patterns following discgel electrophoresis of (left to right) (a)
bacteria-free Corsoy plant, (b) normal Corsoy
plants, and (c) normal Corsoy seed extract
61
62
a different Rp value than did the main band of the plants.
The Ford seed extract had only 1 faint band (Rp 0.7) in
comparison to 3 distinct bands for the bacteria-free Ford
plants.
No differences in intensity or Rp values of peroxidase
bands were detected between 1968 and 19^9 Corsoy seed.
How­
ever, the method of preparing the enzyme extract did make a
difference.
Enzyme extract that was prepared by first crushing
the seed and then letting it soak in the cold produced 1 band
that was of moderate Intensity.
Enzyme extracts that were
prepared by letting the seed soak first and then crush it
produced a very Intense band In both the 1968 and 1969 seed
(Figure 20). The 1969 seed extract had 1 additional band that
was fairly faint. The leading band in each case had the same
Rp value.
Transaminase activity
The enzyme extract of normal and bacteria-free Ford and
Harley plants from the previous test was also used to test
for transaminase activity.
In both cultlvars, the bacteria-
free plants had more transaminase activity than did normal
plants which supported a high bacterial population.
Bacteria-
free Ford plants had an activity of 47.2 umoles OAA/mln/mg of
protein while the normal plants had an activity of 35.8 ^moles
OAA/mln/mg of protein.
Bacteria-free Harley plants had an
activity of 40.7 fimoles OAA/mln/mg of protein in comparison
Figure 20.
Peroxidase patterns of seed extracts of (left to
right) ( a) 1968 Corsoy soybean seed soaked before
crushing, (b) 19^9 Corsoy seed soaked before
crushing, and (c) 1968 Corsoy seed crushed
previous to soaking in phosphate buffer
64
65
to 27.0 umoles OAA/mln/mg of protein in normal plants.
Development of Bacteria-free Plants under
Gnotobiotic Conditions
Assays
No bacteria were detected in any of the samples assayed
from plants that were grown, either from seed or from the
excised embryo axis, for 89 days. The control flasks also
had no bacteria present.
A fungus with the cultural char­
acteristics of a heterothallic and homothallic species of
Diaporthe phaseolorum was isolated from the cotyledons, stem,
and unifoliolate assays 25 days after germination of the
treated seed grown in 10 cm clay pots.
Several of the plants
began to wilt and dry up 17 days after the fungus was first
isolated.
Only 1 plant out of 10 was alive 36 days after the
first isolation of the fungus.
One of the plants that died
had a black discoloration of the stem at and immediately
below the cotyledonary node.
The fungus spread to the plants developed from excised
embryos 40 days after the fungus was first detected in the
plants grown in clay pots.
The fungus was also detected in
two of the four control flasks at this same time.
The plants
showed no injury at the time of the last assay (89), but had
started to dry at 104 days as the fungus had started to grow
on the agar and on the outside of the lower portion of the
stems.
After 120 days, most of the plants had died due to
66
the fungal activity.
The fungus was isolated from the plants every week after
it was first assayed.
It readily produced stroma on all PDA
plates, few stroma on HSA, and none on TSA, NBA, or HIA,
When the Isolates on TSA, NBA, and HIA were transferred to
PDA, stroma was produced in all instances.
Growth characteristics (isolator l)
Growth development of bacteria-free plants from seed and
from the excised embryo axis followed a normal developmental
process.
In general, plants grown from the excised embryo
axis had smaller trifoliolate leaves and shorter internodes
than plants grown from seed.
Trifoliolate leaflet dimensions
ranged from 2 x 2 mm to 25 x 22 mm with an average dimension
of 19 X l6 mm.
In comparison, dimensions of trifoliolate
leaflets of bacteria-free plants grown from seed ranged from
5 X 5 nun to 38 X 26 mm with an average of 27 x 25 mm.
Both
groups of plants had 5-7 trifoliolates after 10.5 weeks of
growth. The trifoliolate leaves at first were of light to
moderate green color, but eventually developed marginal
chlorosis, with necrotic areas.
These symptoms were sugges­
tive of a nutrient Imbalance, and was also noted in other
experiments conducted in the greenhouse.
Some of the
trifoliolates of the plants grown in agar had interveinal
chlorosis also, with the bottom trifoliolates having a tendency
to abcise as new trifoliolates developed at the apex.
67
Plants from excised embryo axis ranged in height from
23 to 29 cm after 10.5 weeks of growth.
The internode length
was short with an average length of 3 cm.
Plants grown from
seed, on the other hand, were much taller, ranging in height
from 57 to 69 cm. The internode length was also considerably
larger, with a mean length of 10 cm.
Transplanting of the excised embryos from agar to a
flask containing nutrient solution did not prove successful.
Plants that were not covered with a wet nylon bag after being
transferred died within 2 hours.
Plants that were covered
but had the nylon bag removed after 24 hours showed signs of
wilting within 30 minutes.
the plants turgidity.
Replacing the nylon bag restored
If the bags were removed after 48 hours,
the plants did not show any wilting.
After 3-4 weeks, however,
the transplants still did not show any increase in growth and
eventually died.
Embryos that were not transplanted but in
which nutrient solution had been added, developed normally.
Isolator 2
No bacteria, bacteria protoplasts, or fungi were detected
in any of the plant assays, media assays, or from the control
flasks, after 8l days of growth.
Again, the bacteria-free plants developed in a normal
fashion, but they also had small trifoliolate leaves and short
internodes.
The average dimensions of the trifoliolate leaf­
lets were 10 x 8 mm.
These plants were also short with a
68
range In height of 6 to 30 cm. The trlfollolates had the
same marginal chlorosis and necrosis problem as was previ­
ously described for isolator 1.
69
DISCUSSION
Sodium hypochloride proved to be an inexpensive and very
effective agent for obtaining bacteria-free plants.
Sodium
hypochlorite had previously been used effectively in con­
junction with mercuric chloride and hydrogen peroxide to
obtain bacteria-free excised barley embryos from seed (59).
Lindsey (53)^ on the other hand, found that it was difficult
to obtain bacteria-free peanuts, using sodium hypochlorite.
Treating soybean seed with NaOCl eliminated all of the
bacteria in the cotyledons and seed coat so that bacteriafree plants could also be grown from seed; however, it did
not remove all the fungi in the seed coat and cotyledons.
Excision of the embryo axis and discarding the cotyledons and
seed coat eliminated all fungal contaminants.
Similar success
was obtained by Goos e_b aj. (30) who found that NaOCl did not
remove deep-seated contaminants (Botryodiplodia theobromae)
from Musa balbisiana seed, but that the fungus never developed
from excised embryos, even if a contaminated seed lot was
used.
The soybean plants obtained from excised embryos were
termed bacteria-free and fungi-free on the basis of an assay
procedure consisting of growth trials on 5 different media
and microscopic examination of tissue for bacteria.
Any
bacteria or fungi that was previously isolated from soybeans
by Dunleavy (20, 21), using similar media, were not isolated
70
from the bacteria-free plants grown In this study.
In addi­
tion, no bacterial protoplasts were Isolated on horse serum
agar which was recently used by Dunleavy (unpublished data)
to Isolate bacterial protoplasts from soybeans.
White lateral roots rapidly developed on bacteria-free
embryos, -While the bacterial populatluns associated with
normal excised embryos severely limited radicle elongation,
inhibited lateral root formation, and generally produced a
brown lesion.
Other investigators (5, 8) have also found
that microorganisms can inhibit root growth; however, they
created quite artificial conditions, in that soil Inoculum,
consisting of various field and garden non-sterile soil
samples, were added to the flasks of bacteria-free plants.
Lindsey ($4) grew bacteria-free beans, tomatoes, and corn
and found that, in no case did microorganisms inhibit plant
growth, even though roots of plants grown with microorganisms
were discolored and less turgid than the white roots of
bacteria-free plants; Lindsey also used soil inoculum.
Glbberelllc acid, at concentrations of 0.3 and 10 ppm,
stimulated the initiation of the lateral root formation of
bacteria-free soybean embryos.
Initiation of eplcotyl
elongation and expansion of the unlfollolates occurred
earlier when 10 ppm GA was added to the growth medium, but
0.3 ppm GA had no effect in this respect.
Lack of stimulatory
effect of eplcotyl elongation by the low concentration of GA
71
Is similar to results of Nanda and Dhindsa (62) who observed
that high concentrations of gibberellic acid (10 and 100 ppm)
stimulated growth of normal soybean stems, but low concen­
trations (1 ppm) had no effect.
Streptomycin, when added to the culture flasks at a low
concentration (10 ppm), severely inhibited lateral root forma­
tion of bacteria-free soybean embryos.
This suggested that
excised embryos are probably more sensitive to low concentra­
tions of streptomycin than normal Intact seedlings, as Ander­
son and Nienow (l) found that streptomycin inhibition of
lateral roots of normal intact soybean seedlings occurred at
streptomycin concentrations greater than 50 ppm.
Because
streptomycin causes root inhibition of excised embryos at such
low concentrations. It would not be feasible to Incorporate the
antibiotic into the culture medium to prevent any contamina­
tion.
As an illustration, Anderson and Nienow (l) found that
soybeans grown in 50 ppm streptomycin solution retained 5-10
units of streptomycin in expressed sap; however, when they
were Inoculated with Xanthomonas phased1 var. sojense typical
symptoms of bacterial pustule formed.
Obtaining bacteria-free plants is especially important
in understanding metabolic and bacterial interrelationships
in plants.
Many plant researchers in the past have Ignored
bacteria in determining physiological events in plants ; however
recent investigators (49, 51, 52, 90, 9l) have shown that
72
bacterial changes can have a tremendous Influence on inter­
preting physiological studies.
I found that bacteria could be Isolated from all parts
of normal soybean seed, with the highest population being In
the seed coat. These bacteria, associated with the seed,
could directly Influence the number and Intensity of Isoperoxldase bands found In actively growing excised soybean
embryos.
Previous Investigators (12), without considering
bacterial Influences, differentiated cultlvars of normal
soybeans Into high and low activity groups based on peroxidase
activity of the seed coats.
If they (Buttery and Buzzell)
used dehulled seed, the differences in peroxidase activity
between the two groups were eliminated.
My results indicate
that bacteria could possibly have been a determining factor
in their case also, due to the fact that, at least in this
study, soybean seed coats supported high bacterial popula­
tions, and that these bacteria had the capability of influ­
encing peroxidase activity of the plants.
In the 3 cultlvars used in this study, the isoperoxidase
bands of normal excised embryos were of much less intensity
than those found in bacteria-free embryos.
In contrast, a
high peroxidase activity, with Isoenzyme bands of greater
intensity, has generally been found in plants infected with
a bacterial pathogen, especially in resistant plants (40, 64,
69» 86). There are several factors or materials such as
73
gibberellins, auxins, cytoklnlns, and ethylene which can
possibly influence the activity of peroxidase In plants.
Possibly one of the most Interesting lines of research that
might be investigated would be to determine the role, if any,
of the catalase activity of the bacteria associated with soy­
beans.
Rudolph and Stahmann (69) demonstrated a strong
bacterial catalase in bean plants Infected with Pseudomonas
phaseollcola and suggested that bacterial catalases may be a
factor for virulence by depressing the activity of peroxidases
Involved In defense reactions of the plant.
Growth of the bacteria-free soybean plants for 12.5
weeks was normal.
The most notable observation was that
plants from bacteria-free embryos were smaller than bacteriafree intact plants.
Probably the main reason for this was
the absence of the cotyledons during the early growth stages.
Joy and Polkes(4l) found that the growth rate of excised
barley embryos was less than that of intact seedlings kept
under comparable conditions.
There is presently no direct
evidence for the exact nature of the growth rate difference.
Addition of several materials such as amino acids, sugars,
vitamins, purine and pyrlmidine bases have not been found,
in this study or theirs, to Increase the growth rate of
excised embryos to the level of intact plants.
74
SUMMARY
Bacteria-free Ford and Blackhawk soybean plants were
obtained by excising the embryo axis from seed that was
treated with 2.630 NaOCl for 30 minutes. These embryos
rapidly developed a white lateral root system in agar when
either 0.3 ppm or 10 ppm gibberellic acid was added to the
growth medium.
Gibberellic acid, at a concentration of 10
ppm, also stimulated epicotyl elongation and expansion of
the unifoliolate leaves.
Lateral root development was partially inhibited with 10
ppm streptomycin added to the growth medium, and was com­
pletely inhibited with 50 ppm.
In medium without strepto­
mycin, 13 lateral roots/plant developed within 8 days after
excision of the embryo axis.
Only 5 lateral roots/plant
occurred in medium with 10 ppm streptomycin.
Bacterial populations associated with excised embryos
from normal seed not only inhibited lateral root formation,
but also limited radicle elongation and generally produced
a brown to black lesion on the radicle.
In addition, the
bacterial population had a direct effect on the enzyme
activity of young developing plants.
Bacteria-free Ford and
Harley cultivars of soybean plants had a higher peroxidase
and transaminase activity than normal plants which contained
bacteria.
Bacteria-free Ford, Corsoy, and Harley plant
extracts had more isoperoxidase bands that were of greater
75
intensity than extracts of normal plants, as revealed by
acrylamide gel electrophoresis.
Bacteria-free plants also
had more isoperoxidase bands than crushed seed of the same
cultivars.
Bacteria-free plants developed normally under
gnotobiotic conditions.
The plants that were developed
from excised embryos had small trifoliolate leaves, and were
short, reaching a height of 23 to 30 cm after 12.5 weeks of
growth.
Bacteria-free plants grown from seed were larger,
reaching a height of 58 to 69 cm.
a species of Diaporthe
phaseolorum was isolated from all of the cotyledons and stem
sections of plants grown from seed 25 days after germination.
In 40 days, the fungus spread to the plants grown from excised
embryos.
It was also detected in the control flasks.
No
bacteria or bacteria protoplasts, however, were detected in
any of the weekly assays during 12.5 weeks of growth.
No
fungi, bacteria, or bacteria protoplasts were detected in an
isolator used exclusively to grow plants from the excised
embryo axis.
76
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ACKNOWLEDGMENTS
The author wishes to express his sincere appreciation to
Dr. J. M. Dunleavy for his professional guidance and valuable
suggestions during the course of this research and during
the preparation of this dissertation.
The author also wishes to express his appreciation to
Dr. D. C. Norton, Dr. R. Q. Landers, Dr. L. Y. Qulnn, and Dr.
R. E. Atkins for serving on my graduate committee and for
assistance In editing this dissertation.
Further acknowledgment is also extended to Jerry Fisher
for his expert assistance in preparation of the photographs.