Five different pulse crops viz., ricebean, Urdbean and Rajmash in Kharif; pea and lentil in rabi were evaluated in various trials for identification suitable genotypes. New crosses were
made and segregating generations advanced for developing new genotypes.
Ricebean
Evaluation of fodder ricebean
An IVT was conducted with 6 genotypes of fodder ricebean received through AICRP. Based on the green plant weight and green leaf weight 3 promising genotypes were identified. Genotype RB1 was the best performer. Green plant weigh (GPWT) in this genotype was 226 g/plant and green leaf weight (GLWT) was 41.3 g/plant. Other two promising genotypes were RB4 (196.5 g – GPWT
& 42 g GLWT) and RB 7 (155g GPWT and 40.9g GLWT). Among these genotypes green leaf weight/
plant was not significantly different. There was no significant difference in days to 50% flowering, which ranged between 100 – 102 days.
Germplasm evaluation
Sixty-nine core germplasm of ricebean were evaluated in augmented design (5.25 m2 each) with RCRB 1-6 and RBS – 16 as checks. Four genotypes, as collated in Table 1 were superior to checks in their yielding ability. But, the best yielding genotype BKSB 221 was late compared to all others.
Table 1. Promising genotypes identified from ricebean germplasm collection
Genotype DF 50% 5m2 Yield
BKSB – 221 124 771.3
BKSB – 199 98 727.7
BKSB – 165 80 712.7
BKSB-= - 233 96 803.8
RCRB 1-6 © 89 518.9
RBS 16 85 678.53
CV 11%
Advancement of segregating generations Out of 68 F6 lines of photo-insensitive ricebean crosses selected from the spring for advancement and grown in kharif, 4 lines were selected for advancement. Out of 468 F4 lines of photo- insensitive ricebean crosses, 108 lines were advanced to next generation. Eight interspecific
crosses of urdbean and ricebean were made and F1 seeds obtained.
Rajmash
Sixty-three lines were evaluated in augmented design with varieties Uday and Chitra as checks.
Six genotypes were identified as promising as collated in Table 2.
Table 2. Performance of rajmash genotypes at Umiam
Genotype Duration Yield
(days) (g/4.m2)
Carmel 87 104.70
ECAB 0524 83 73.55
ECAB 0527 87 64.22
Mexico 87 61.04
ECAB 0621 83 59.27
Mac 12-2 87 57.88
Uday 87 33.00
Chitra 83 51.60
CV 12.8%
DS – 13.8.07
Urdbean
Thirty-nine genotypes were evaluated in RBD with 3 local checks Pahelodal 4, MZ-1 & MZ-2.
Promising genotypes identified from the trial were KU-507 (0.42 t/ha), KU 504 (0.42 t/ha), Shekhar (.40 t/ha) KU 549 (.41 t/ha) and KU 551 (.40 t/ha) compared to checks Pahelodal 4 (.37 t/ha), MZ-1 (.21 t/ha) and MZ-2 (.30 t/ha). All these genotypes matured between 74-80 days.
Pea
Two coordinated trials, AVT-1 (Tall) and AVT- 2 (Tall) were conducted. Best genotype from AVT- 2 tall was P7-501 (2.36 t/ha-122 days) and from AVT-1 Tall was P7 – 526 (1.95 t/ha – 122 days). F2 genotype from 22 crosses involving TRCP-8, TRCP-9, DDR – 39, DDR-60, DDR – 63, DDR – 50, MNPL – 2, DDR – 64 and DMR – 50 were advanced to next generation. Two methods, single
pod descent and plant to row methods were followed.
Lentil
Seven identified genotypes were grown for the 3rd year for yield evaluation. Yield and maturity data of the genotypes are presented in Table 3. VL – 4 (2.52 t/ha) was the best genotype followed by VL – 126.
Table 3. Yield performance of selected lentil genotypes at Umiam
Genotype Duration Yield
(days) (t/ha)
PL – 639 150 1.38
VL –120 133 1.62
L – 414 138 .83
VL –126 140 1.91
L – 305 135 1.09
L – 306 150 .78
VL – 4 135 2.52
BIOTECHNOLOGY Pigeonpea
In pigeonpea, 12 days old leaves when inoculated in the callusing medium gives better callusing then the leaves that are younger or older to it. Among the genotypes, TRA-1 a local genotype was the best. The callusing percentage was more than 95%. BAP and 2,4-D were the hormones used for callusing. The matured callus was transferred to the regeneration medium containing BAP and Kinetin in different concentrations. The regenerated plants after attaining a height of about 2 cm were transferred to the root inducing medium.
The fully grown plants were transferred to the green house for acclimatization.
Chickpea tissue culture and transformation Induction of multiple shoots from embryos
Embryos were excised from surface sterilized- pre-soaked seeds, sliced into two halves (Fig. 1a)
and grown on MS salts + B5 vitamins + BAP, various concentrations of glutamine and silver nitrate.
After 2 weeks of culture in presence of light, embryos with developing shoots (Fig. 1b) were transferred for shoot elongation on medium containing MS salts +B5 vitamins + BAP glutamine and 1/10th concentration of silver nitrate used in the previous step and cultured in the light.
Subculture to the same medium was done at 15 days interval. After 4 weeks in the elongation medium, shoots were transferred to root induction medium containing half strength MS salts and vitamins and IBA. Shoots were grown on a nutrient pad kept soaked in the medium. After about 45 days, plants with well formed roots were first transferred to MS ½ medium without hormone for 15 days and then to a mixture of autoclaved soil and cocopit. pH of all media were adjusted to 5.8 before autoclaving, for semisolid media 0.8% agar
was used and 3% sucrose was used as carbon source.
Response of embryos to different combinations of shoot induction medium is given in Table 4. As is evident from the table the medium combination
‘SMD’ was the best and produced on an average 5 shoots/ embryo slice.
Table 4. Shoot induction from embryo slices in different medium (cv. C235)*
Medium No. of embryo No. of shoots/ % slice slices/exp. slices responding
SM A 25 3-4 82.1
SM B 25 2-4 65.6
SM C 25 0-01 28.3
SM D 25 4-7 89.6
SM E 25 2-3 66.6
SM F 25 0-3 23.3
SM G 25 2-4 42.3
SM I 25 0 0
* Average of 4 sets.
Fig. 1. a. Sliced embryos. b. Multiple shoots formed from sliced embryo.
For establishment in soil, it was necessary to cover the plants with plastic cups with periodical removal of cover. The combination of cocopit and soil was better than cocopit alone or vermiculite alone. However, survival in soil after 45 days was less than 1%.
Multiple shoot induction from epicotyl segments Epicotyl from 7-days-old seedlings were cut into 1-2 cm segments and grown on medium containing MS salts + B5 vitamins + BAP + kinetin + glutamine + silver nitrate. After 3 weeks of shoot induction in this medium epicotyls with multiple shoots were transferred to the same medium but with 1/10 silver nitrate. After another 2 weeks roots were induced from elongated shoots in medium containing MS salts (1/2 strength) + B5 vitamins + IBA glutamine. Rooted shoots were transferred to soil as described before.
Callus induction and plant regeneration from epicotyl
Epicotyl explants prepared as described above were cut into 3-4 mm pieces : (i) horizontal (ii) vertical or (iii) horizontal and then vertical. Calli were induced from these pieces on medium containing MS salts + B5 vitamins + 2,4-D + kinetin glutamine and silver nitrate.
After 45 days of induction calli with small embryoids were transferred to maturation medium with the same composition as above but with IBA and BAP as hormone supplement. Another set was maintained in the medium of same composition but with 1/10th concentration of silver nitrate. Calli were allowed to mature for 20 days after which they were transferred to regeneration medium which contained the same salts, vitamins glutamine and BAP as hormone supplement but without silver nitrate. Rooted plants were transferred to soil as described in section 1.
Fig. 2. a. Shoots developing from epicotyls segment. b. Developed shoots rooted on rooting medium
Fig. 3 a. Nodular embryogenic calli formed in presence of glutamine and silver nitrate. b. Loose non- embryogenic calli in the control plates
Among the media compositions tested CIMD was best for callus induction using horizontally cut epicotyls (Table 5) and subsequent plant regeneration. It was necessary to maintain calli under diffused light as long as silver nitrate was used. It was also necessary to reduce silver nitrate concentration to 1/10th in the maturation medium.
Table 5. Callus induction from epicotyl in different medium (cv. C235)*
Medium No. of % of explant Regeneration explant/ producing %
exp. calli
CIM A 36 86.11 13.88 (5)
CIM B 31 74.19 Nil
CIM C 30 83.33 Nil
CIM D 34 67.65 52.94 (18)
CIM E 29 86.2 6.90 (2)
CIM F 21 52.38 Nil
CIM G 24 50.00 12.50 (3)
CIM H 35 91.42 8.57 (3)
CIM I 24 87.50 Nil
* Average of 3 sets
Among three different types of explants, horizontally cut explants were better than the other two (Table 6).
Table 6. Callusing percentage from three different types of epicotyl explants
Cut type No. of Callusing Regeneration
explants/ % %
exp.
Vertical 70 64.28 35.66
Horizontal 67 80.60 54.8
Horizontal + 48 77.08 22.98
Vertical
Agrobacterium-mediated transformation of embryos
Explants were prepared by cutting embryos into two halves. Embryo pieces were plated on the shoot induction medium SMD (without silver nitrate) with 100µM acetosyringone and 5µl aliquot of bacterial suspension (OD600 – 0.06) was added to each of the embryo slice. Embryo slices were then incubated in diffused light for 4 days. After cocultivation embryos were washed for 2 hours (3 changes) in 500 mg/l cefotaxime with constant agitation. After blotting, the embryo pieces were cultured in SMD medium with 250 mg/l cefotaxime. Shoot and root inductions were done as described in section 1 in presence of 30-mg/l hygromycin as selective agent. Rooted shoots were transferred to soil in plastic cups.
Bacterial strain LBA4404 containing the plasmid pCAMBAC (CryIAc) was used.
Transformation frequency, as calculated from the shoots rooted on 30-mg/l hygromycin, was 0.7 – 3.2%.
Fig 4. Plant regeneration from epicotyls-derived calli.
Agrobacterium-mediated transformation of epicotyl segments
Epicotyl explants were prepared from 7 day old seedlings. These explants were infected (LBA4404, pCAMBAc) by soaking them in OD600 – 0.6 bacterial suspensions for 45 minutes. Before soaking, the explants were pricked at several places using a sterile needle. Infected explants were blotted dry and co-cultivated for 3 days on shoot induction medium without silver nitrate but with 2% mannitol. After the co-cultivation period, explants were washed thoroughly, blotted dry and grown on shoot induction medium with silver nitrate, 250 mg/l cefotaxime for 7 days and another 3 weeks on the same medium but with 30 mg/l hygromycin under diffused light. Surviving shoots were rooted in root induction medium containing 30mg/l hygromycin. Transformation frequency ranged from 0.5 – 1.5%.
INSECTS AND PESTS
Pigeon pea
Competitive suppression of pod boring weevil damage
Field experiments were conducted to assess yield losses due to different pod borer species in pigeon pea. During pod formation stage (March- April), three different pod borers viz. Apion clavipes, Melanagromiza obtusa and Etiella
zinckinella were recorded with dominance index significantly equal, whereas during September- October, the pod borer complex was dominated by Apion clavipes with negligible population of other two species. In the first season, the larval population peaks observed were in the last week of March, first week of April and second week of April for A.clavipes, M.obtusa and E.zinckinella, respectively. During the larval counts, three kinds of pod damage were observed viz. pods with A.clavipes and M.obtusa, pods with only M.obtusa and pods with all the three larvae. The yield loss was observed 32 % due to this pest complex.
During the second season, A.clavipes was the predominat pest while the presence of other two species was negligible which could be due to the unfavourable weather conditions and yield losses estimated was 86 %. In the first season, yield loss was significantly lower owing to the presence of M.obtusa and E.zinckinella , A.clavipes which could not multiply unlike in the second season.
A.clavipes life cycle takes longer than the other two species, especially M.obtusa and the pod consumption per larva was more than one in the case of A.clavipes whereas in M.obtusa it was only one. A.clavipes mortality was observed more whenever it was found in combination with other two species. The experiment was also conducted in the glasshouse condition and the results were similar to field conditions. Thus there exists a clear evidence of interspecific competition among the species fighting for the same niche.
Fig 5 a. Transformed plant from embryo slice growing on hygromycin. b. Transformed plant from epicotyl growing on hygromycin.
Field pea
Insect pests of gram and pea
Pod borer Helicoverpa armigera, cut worm Agrotis ipsilon and leaf miner and semilooper were observed as the the major lepidopteran pests of gram and pea, respectively. On an average pod borer causes 20-40% damage to gram annually.
Among various botanicals and conventional insecticides evaluated for their efficacy on pea aphids, imidacloprid @ 0.5 ml/l was highly effective in controlling the aphids to the extent of 95%, followed by dimethoate, achook, nimbicidine, karanjin, neem oil and anonin over control.