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 seedlingagar tubes and Leonard jars
3)Prepare wateragar plates
4)Select, surface sterilize, and germinate seeds
5)Plant pregerminated seeds in seedlingagar 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 seedlingagar tubes and Leonard jars
(Key step 2)
Prepare 54 seedlingagar slants in 22 x 250 mm tubes. The composition of the seedlingagar is detailed in Appendix 3 and its preparation in Appendix 7. A simple setup 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. Nitrogenfree nutrient solution for use in Leonard jars is of similar composition as that used for making seedlingagar.
Table 15.1. Strains of Rhizobium and hosts according to cross-inoculation groups
TAL No. / Rhizobial species / Host legume169 / 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 specieslegume 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) wateragar in a 500 ml flask and sterilize. Pour 25 ml of melted wateragar 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. Batches of seeds with more than 70% viability will be suitable. Select undamaged seeds for uniformity in size and color. 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 35 min. (To prepare 3% sodium hypochlorite solution, add 10 parts of commercial bleach [5.25% sodium hypochlorite] to 7.5 parts of water.)
Hard seedcoated 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 spoonshaped spatula.
Each batch of 100 seeds should be dispensed evenly in two or more (depending on seed size) wateragar plates and incubated at 2530C. (The large-seeded species, e.g., Phaseolus and Cicer may need more wateragar plates.) Invert the plates containing the smallseeded 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 wellspaced 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. The seedling radicles should be 0.51.0 cm long and straight. 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. Seedlingagar 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. Avoid disturbing the rooting medium during thinning. 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. Use a fresh pipette for each strain of rhizobia. 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. Note color and growth. 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 70C). 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 crossinoculation groups as recorded in Table 15.2 and the ineffectiveness and effectiveness of each rhizobial specieslegume 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 in each rhizobia/legume combination.
LegumeRhizobia / soybean / cowpea / bean / lentil / leucaena / chickpea / alfalfa / clover / siratro
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
Requirements
(a)Culturing strains of rhizobia
Slant cultures of rhizobia
YM broth in flasks
(b)Preparing seedlingagar tubes and Leonard jars
Seedlingagar slants
Leonard jars
(c)Preparing media for germination
Agarpowder, Petri dishes, 500 ml flasks
Balance
(d)Surfacesterilizing 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
Seedlingagar 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 (70C)
Scalpels or razor blades