EXERCISE 15
TO TEST FOR GENETIC COMPATIBILITY BETWEEN RHIZOBIA AND LEGUMES
Specificity and promiscuity in the symbioses are studied in
cross-inoculation experiments.
The specific requirements of
certain legumes for particular rhizobia are demonstrated.
Key steps/objectives:
1)
Culture strains of rhizobia
2)
Prepare seedling-agar tubes and Leonard jars
3)
Prepare water-agar plates
4)
Select, surface sterilize, and germinate seeds
5)
Plant pregerminated seeds in seedling-agar tubes and Leonard
jars
6)
Thin seedlings in Leonard jars
7)
Inoculate seedlings in Leonard jars and tubes
8)
Make periodic observations of nodulation
9)
Harvest after 5 weeks
10)
Evaluate results
(a)
Culturing strains of rhizobia
(Key step 1)
Culture each of the Rhizobium spp. and Bradyrhizobium spp. listed
in Table 15.1 in 100 ml of YM broth in 250 ml Erlenmeyer flasks.
(b)
Preparing seedling-agar tubes and Leonard jars
(Key step 2)
Prepare 54 seedling-agar slants in 22 x 250 mm tubes.
The
composition of the seedling-agar is detailed in Appendix 3 and
its preparation in Appendix 7.
A simple set-up for dispensing
the melted agar into the tubes is illustrated in Appendix 7
(Figure A.9).
Set up 108 Leonard jars as explained in Appendix 11.
Nitrogen-free nutrient solution for use in Leonard jars is of
similar composition as that used for making seedling-agar.
Table 15.1.
Strains of Rhizobium and hosts according to cross-
inoculation groups
TAL No.
Rhizobial species
Host legume
169
Bradyrhizobium sp.
Macroptilium atropurpureum
(siratro)
169
B. sp.
Vigna unguiculata (cowpea)
182
R.l. bv. phaseoli
Phaseolus vulgaris (bean)
379
B. japonicum
Glycine max (soybean)
380
R. meliloti
Medicago spp. (alfalfa,
sweet clover)
382
R.l. bv. trifolii
Trifolium spp. (clover)
1145
R. sp. (Leucaena)
Leucaena sp.
620
R. sp. (Cicer arietinum)
C. arietinum (chickpea)
634
R.l. bv. viceae
Lens culinaris (lentil)
Each treatment (rhizobial species-legume host combination and
controls) in this exercise will be done in duplicate.
Refer to
Table 15.2 for the treatments and the various combinations to
test genetic compatibility between rhizobia and legumes.
(c)
Preparing germination plates
(Key step 3)
Make 300 ml of 0.75% (w/v) water-agar in a 500 ml flask and
sterilize.
Pour 25 ml of melted water-agar into 12 or more Petri
dishes and allow to cool.
Surface sterilized seeds will be
pregerminated in these plates.
(d)
Surface sterilizing seeds
(Key step 4)
Check percentage germination of each legume species in advance of
experiment.
be suitable.
color.
Batches of seeds with more than 70% viability will
Select undamaged seeds for uniformity in size and
Surface sterilize enough seeds (at least 200 of each
species) to give at least 100 germinated seeds.
Surface sterilize the seeds (Appendix 10) by immersion in a 3%
sodium hypochlorite solution for 3-5 min. (To prepare 3% sodium
hypochlorite solution, add 10 parts of commercial bleach [5.25%
sodium hypochlorite] to 7.5 parts of water.)
Hard seed-coated species (e.g., leucaena, siratro) are scarified
and sterilized simultaneously by immersion for 10 min in
concentrated sulfuric acid.
Drain off all excess acid prior to
rinsing with sterile water.
(If acid is used, the first rinse
should be done quickly to prevent loss of viability of the seeds
caused by the heat generated when water is added to the acid.)
Rinse seeds with six to eight changes of sterile water after
surface sterilization.
Allow the seeds to imbibe water by
soaking for 1 h and then rinse twice.
Transfer the seeds
aseptically to agar plates with a spoon-shaped spatula.
Each batch of 100 seeds should be dispensed evenly in two or more
(depending on seed size) water-agar plates and incubated at
25-30C.
(The large-seeded species, e.g., Phaseolus and Cicer
may need more water-agar plates.)
Invert the plates containing
the small-seeded species to provide straight radicles that are
much easier to handle in later steps of the exercise.
(e)
Planting and inoculating
(Key steps 5, 6, and 7)
Soybean, cowpea, bean, chickpea, lentil, and leucaena seeds will
be planted in Leonard jars.
Make three well-spaced holes in the
rooting medium to a depth that will accommodate the pregerminated
seeds 1 cm below the surface.
Pick up well germinated seeds with
sterile forceps and place one seed in each hole with the radicle
entering first.
(Proper orientation of the radicle during
planting is important to ensure proper emergence of the shoot and
establishment of the seedling.)
After placement of the seed,
inoculate (1 ml per seed) with the rhizobial culture and cover
the hole with the rooting medium.
If vermiculite is used as the
rooting medium, autoclaving will cause swelling and loosening of
the vermiculite.
This leads to poor anchorage of the root.
Therefore, gentle compacting of the vermiculite will be required
before planting/sowing of the seeds.
Firmness of the rooting
medium can be restored by pressing it down with the bottom
(sterilized by flaming) of a 125 ml Erlenmeyer flask.
After planting and inoculation are completed, add sterile gravel
over the surface of the rooting medium.
Set up 18 jars for each
species.
Siratro, clover, and alfalfa will be cultured on agar slants in
tubes.
Select and plant one seedling on the agar surface.
Observe the usual aseptic precautions, taking care to sterilize
the hands with 70% alcohol, flame sterilizing the inoculating
loop and mouth of the tube etc. when transferring the seedling.
Using an inoculating loop, pick up the pregerminated seedlings
and transfer them into the tubes.
be 0.5-1.0 cm long and straight.
The seedling radicles should
After planting, tubes should be
kept in a slant position for the radicles to adhere to the agar
surface for at least 2 h.
Dispense 1 ml of culture over the
roots of the seedlings in the agar slants.
Use a fresh pipette
for each new rhizobial species or strain.
Aluminum foil wrapped
around the lower part of the tubes will shield the roots from
light and heat.
Seedling-agar tubes need to be placed in
suitable wooden racks and kept in a growth chamber (environmental
growth chamber or in a temperature controlled greenhouse) for
proper seedling development.
Thin plants in the Leonard jars to two uniform plants per jar
after 5 days.
Excise the shoot of the unwanted plant aseptically
using scissors.
thinning.
Avoid disturbing the rooting medium during
To facilitate proper inoculation, carefully clear
(with a sterile glass rod) the rooting medium around the root of
the plant, to a depth of 1 cm.
Dispense drops of rhizobial
culture (totaling 1 ml) into the cleared area around the root.
Dispense 1 ml of rhizobia culture over the roots of the seedlings
in the agar slants.
rhizobia.
Use a fresh pipette for each strain of
Place the inoculated jars on the benches in the green
house.
(f)
Observing periodically and harvesting
(Key steps 8 and 9)
Examine the plants over a period of 5 weeks.
growth.
Note color and
Replenish tubes and Leonard jars with sterile water as
required.
At the end of the fifth week, excise the tops and
determine their dry weight (dry for 48 h at 70C).
Remove roots
from the jars and tubes and wash them free of rooting medium.
Where nodules are present, describe nodule shape, size,
pigmentation, and distribution.
(g)
Evaluating the experiment
(Key step 10)
Note cross-inoculation groups as recorded in Table 15.2 and the
ineffectiveness and effectiveness of each rhizobial
species-legume combination.
Effectiveness will be apparent from
the green coloration of the plant and abundant nodules that are
red/pink when sliced open.
Table 15.2.
For recording presence (+) or absence (-) of nodules
B. japonicum
TAL 379
Bradyrhizobium
sp.
TAL 169
R. l. bv.
phaseoli
TAL 182
R.l. bv. viceae
TAL 634
Rhizobium sp.
(leucaena) TAL
1145
Rhizobium sp.
(chickpea) TAL
620
R. meliloti
TAL 380
R.l. bv.
trifolii
TAL 382
Uninoculated
siratro
clover
alfalfa
chickpea
leucaena
lentil
bean
cowpea
Legume
Rhizobia
soybean
in each rhizobia/legume combination.
Requirements
(a)
Culturing strains of rhizobia
Slant cultures of rhizobia
YM broth in flasks
(b)
Preparing seedling-agar tubes and Leonard jars
Seedling-agar slants
Leonard jars
(c)
Preparing media for germination
Agar-powder, Petri dishes, 500 ml flasks
Balance
(d)
Surface-sterilizing seeds
Seeds of cowpea, bean, soybean, alfalfa, clover, leucaena,
siratro, chickpea, and lentils
3% sodium hypochlorite solution or other sterilants
(Appendix 10)
Concentrated sulfuric acid
Sterile water
Sterile flasks or beakers
Incubator
(e)
Planting and inoculating
Pregerminated seeds of the various species
Leonard jars
Seedling-agar slants, wooden racks, growth chamber
Aluminum foil
Alcohol, forceps
Sterile pipettes (1 ml), cultures of rhizobia
(f)
Observing periodically and harvesting
Sterile water
Scissors, paper bags or envelopes
Drying oven (70C)
Scalpels or razor blades