Increasing the Efficiency of Production of Chickpea Supported by The McKnight Foundation, USA Project Coordinators Vidya S. Gupta (NCL, India) F.J.Muehlbauer (WSU, USA) Progress Report (March 2004 to February 2005) Investigators Contributing to the Report INDIA USA National Chemical Laboratory Washington State University V.S. Gupta (Co-ordinator) F.J. Muehlbauer(Co-ordinator) V.V. Deshpande V. Franceschi M.N. Sainani A.P. Giri UK N.Y. Kadoo University of Durham J. Gatehouse Mahatma Phule Krishi Vidyapeeth Dr. B.M. Jamadagni AUSTRALIA Assam Agricultural University CSIRO Division of Plant Industry B. Sarmah T.J. Higgins M. K. Modi
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Chickpea: Year 3: Increasing the efficiency of chickpea production
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Increasing the Efficiency of Production of Chickpea
Supported by
The McKnight Foundation, USA
Project Coordinators
Vidya S. Gupta (NCL, India) F.J.Muehlbauer (WSU, USA)
Progress Report (March 2004 to February 2005)
Investigators Contributing to the Report
INDIA USA
National Chemical Laboratory Washington State University V.S. Gupta (Co-ordinator) F.J. Muehlbauer(Co-ordinator) V.V. Deshpande V. Franceschi M.N. Sainani A.P. Giri UK N.Y. Kadoo University of Durham J. Gatehouse Mahatma Phule Krishi Vidyapeeth Dr. B.M. Jamadagni AUSTRALIA Assam Agricultural University CSIRO Division of Plant Industry B. Sarmah T.J. Higgins M. K. Modi
TABLE OF CONTENTS
Page No.
Executive Summary 3
Research Objectives 5 Research Progress Report 7
Training Report 36 Publications and Ph.D.s from the Project 38
Work Plan for fourth Year 40 Budget Details 44
Executive Summary
EXECUTIVE SUMMARY
The goal of this McKnight Foundation sponsored research and training programme is to improve the efficiency of production of chickpea. Significant progress has been made towards this objective and the specific highlights are given below:
• Among the three genotypes developed under this programme, one genotype, Phule G-9425-5, has been accepted for release as variety for irrigated and late sown conditions.
• Expression analysis of WRKY and 14-3-3 TDFs indicated early responsiveness nature of these genes in chickpea wilt resistance variety when infected with fungal pathogen.
• Stable transformation of a large (20 to 100kb) insert into chickpea through improved protocol of Agrobacterium mediated transformation.
• QTL1 contributing to >35% of the variation for blight resistance was characterized by identifying and sequencing BAC clones in this region.
• Four constructs pBINAR/ptWCI-2, pBINAR/ptWCI-5, pVicGal-Shp/ptWCI-2 and pVicGal-Shp/ptWCI-5 involving CaMV 35S promoter and pea vicilin promoter and winged bean chymotrypsin inhibitor genes were developed.
• These four constructs were used for chickpea (Vijay) transformation and 19 putative transformants have been obtained.
• Recombinant chickpea HGPI was analysed against various proteinases, namely trypsin, chymotrypsin, subtilisin and HGP as well as by feeding it to Helicoverpa armigera larvae.
• A synthetic lectin gene effective against Helicoverpa was designed by fusing toxin and snowdrop lectin. Expression construct using this fusion was developed and expressed in Pichia pastoris system.
• A trypsin-like enzyme from H. armigera expressed in E. coli could be refolded from solubilised denatured protein to give soluble proenzyme, which could be activated by treatment with bovine trypsin.
• Three To lines were established in ICCV 89314 and Vijay, each using α-AI gene construct.
• Apart from these scientific achievements, training of students and research
associates was also completed to build up capacity.
Research Objectives
RESEARCH OBJECTIVES
1. Development of improved chickpea varieties through conventional breeding 2. Participatory evaluation and utilization of generated materials by chickpea
growing farmers 3. Development of Fusarium wilt resistant chickpea varieties 4. Development of chickpea genotypes resistant to insect attack
Research Progress Report
RESEARCH PROGRESS REPORT Objective 1. Development of improved high yielding, irrigation and fertilizer
responsive varieties of chickpea through conventional breeding
The immediate needs of farmers for chickpea improvement as identified by MPKV, Rahuri include (i) Development of high yielding, irrigation and fertilizer responsive varieties suitable for rainfed cultivation and for late sown conditions and (ii) Development of plants for efficient and economic protection schedule against major diseases and insect-pests. Activities: a. Development of improved varieties through diallele crossing and SSD
method MPKV, Rahuri, India I. Cultivar Development Programme
Eight divergent chickpea genotypes given in Table 1 were intermated in a
half diallel fashion in Jan. 1994. Progress of cultivar development by pedigree method and by single seed descent method are summarized in flow chart 1 & 2, respectively.
Flow chart-1 Progress of cultivar development by pedigree method
1993 1994 8 x 8 Diallel cross 1994 1995
1995 1996
1996 1997
1997 1998
1998 1999
1998 1999 2000
Station 2000 2001
2001 2002
2002-2003
2003-2004
2004-2005
28 F1 hybrids
Selection of 186 Plants in F2
Selection of 140 progenies from F3
Selection of 346 single plants from F4
Testing of most promising 19 genotypes in RVT under Rainfed, Irrigated and Late sown condition.
Selection of 60 progenies from F5
trial of 60 genotypes
Testing of PG-9414-7 ,PG-9425-5, PG-9425-9 in SMVT under Rainfed, Irrigated and Late sown conditions.
Testing of PG-9414-7, PG-9425-5, PG-9425-9 in SMVT (21 locations), AICRP
Testing of PG-9414-7, PG-9425-5, PG-9425-9 on Farmers Field
Testing of PG-9414-7, PG-9425-5, PG-9425-9 in SMVT (21 locations), AICRP AVT-1 (Bold) CZ (9 Loc.), SZ (3 locations).
Testing of PG-9414-7, PG-9425-5, PG-9425-9, on Farmers Fields
(52 locations).
PG-9425-9 (AVT-2 late , NWPZ) ( 9 locations )
Testing of PG-9425-5 & Vijay on farmers field (90 locations )
Prerelease of PG-9425-5
Flow chart-2 Progress of cultivar development by single seed descent method 1993-1994 8 x 8 Diallel cross
28 F1 hybrids
F2 population of each hybrid plant
F3 Single Seed Descent
F4 Single Seed Descent
Station trial [3 sets] of 18 promising genotypes in each set.
F5 Single Seed Descent
F6 Single Seed Descent
Testing of 18 promising genotypes in Regional Vrietal Trial under rainfed, irrigated and late sown conditions.
Testing of 18 promising genotypes in Regional Vrietal Trial under rainfed, irrigated and late sown conditions.
Testing of Phule G 9426-2, Phule G 9409-1 and Phule G 9421-1 in Regional Varietal Trial (6 locations) under rainfed, irrigated and late sown
Testing of Phule G 9426-2, Phule G 9409-1 and Phule G 9421-1 in Co-ordinated Trials (15 locations).
Testing of PG-9426-2 , PG-9409-1, PG-9421-1 in SMVT (21 locations)
Phule G 9425-5 Pre-released for general cultivation The genotype Phule G -9425-5 is developed at Pulses Improvement Project , Mahatma Phule Krishi Vidyapeeth, Rahuri under the McKnight Foundation Collaborative Crop Research programme , which has been initiated in year 1994. Under this, eight divergent genotypes of chickpea were intermated in a half diallel fashion in year 1994. The genotype Phule G- 9425-5 has been evolved from a cross of Phule G- 91028 x Bheema. It was evaluated in station trial at Rahuri in 1999 to 2000, Regional Varietal Trial in 2000-01, State Multilocation Varietal Trial in 2001-02 to 2003-04 and in All India Co-ordinated Varietal Trial during 2002-03. It has, surpassed the check varieties Vijay and Vishal in all these trials. The per cent increase in yield of Phule G- 9425-5 over Vijay and Vishal was 14.44 and 17.81 % respectively (Table 2). It has also shown good acceptance and higher yield i.e. 14.97% over Vijay in Farmers Participatory Programme conducted under McKinght Foundation Collaborative Crop Research Programme during 2003-04.The salient features of Phule G- 9425-5 are given below. 1. Average yield 19.00 q/ha which is higher by 14.44% than Vijay and 17.81% than
Vishal. 2. Potential yield : 35.00 q/ha. 3. Resistant to Fusarium wilt (7.40 % incidence). 4. Attractive yellow bold seeds (24.0g /100 seeds). 5. Suitable for optimum sowing, irrigated and late sown conditions. 6. Its performance under rainfed conditions is equivalent to that of Vijay.
Release proposal of the variety is attached here with
Details of chickpea variety Phule G-9425-5
1 Name of the crop and species : Chickpea (Cicer arietinum L.) a) Name of the variety under
which tested. b) Proposed name of the variety
: Phule G – 9425-5 --
2 a) Parentage with details of pedigree b) Breeding Method c) Breeding objectives
: Phule G- 91028 x Bheema Pedigree method To develop high yielding, medium bold seeds, wilt resistant chickpea variety.
3 State the varieties which are most closely resemble the proposed variety in general characteristics.
: None
4 a) Whether recommended by Seminar /Conference / Workshop / State Seed Sub. Committee.
: Recommended for prerelease in Research Review Committee of MPKV, Rahuri held in April 2004.
b) Specific area of its adaptation Maharashtra
5 Recommended ecology : Suitable for irrigated and late sown conditions of Maharashtra
6 Description of variety a) Plant height b) Distinguishing morphological characters c) Maturity (range in number of days) seedling / transplantation to flowering, seed to seed d) Maturity group (early, medium and late where ever such classification exists. e) Reaction to major diseases under field & controlled conditions (Reaction to physiological strains / races/ bio-types to be indicated where ever possible ) f) Reaction to major pest (Under field conditions including storage) g) Agronomic features (e.g resistance to lodging , shattering, fertilizer responsiveness, suitability to early or late sown conditions, seed rate etc. h) Quality of produce of grain / forage/ fibre including nutritive value where relevant i) Reaction to stress
: : : : : : :
33-43 cms. Semi erect, small leaves, medium bold seed size (24.0 g/ 100 seed) 100-105 days Medium maturity. Resistant to Fusarium wilt (Table 5a) Table 5b Suitable for irrigated and late sown conditions of Maharashtra Medium seed size (Table 4) Table 6 and 7
7 Description of the parents of the hybrid
: Not applicable
8 a) Yield data in regional / interregional district trials year wise (level of fertilizer application, density of plant population and superiority over local control / standard variety to be indicated
: Table 1
b) Yield data in Regional Demonstration from large – scale demonstration
: Table 10
c) Average yield under normal : 19.18 q/ha (Table 2a)
conditions.
9 a) Agency responsible for maintaining breeder seed
10 Information on the acceptability of the variety by farmers/ Consumers / Industry.
: It has been accepted by farmers due to its good yield performance, wilt resistance, attractive yellow bold seeds.
11. Specific recommendations if any for seed production
: Recommended field and laboratory seed standards are applicable.
Effect of foliar sprays of nutrients and growth substances on rainfed chickpea variety Phule G-9425-5. Background information: a) Objective : 1. To study the effect of foliar sprays of nutrients and growth substances on rainfed chickpea. 2. To investigate the most effective package and foliar sprays for yield enhancement in rainfed chickpea. b) Previous results : This is the first year of the experiment. Location : Pulses Improvement Project, Mahatma Phule Krishi
Vidyapeeth, Rahuri. Year of start : Rabi 2004-2005 Design : RBD with 4 replications Plot Size : Gross: 3.60 x 3.00 m, Net: 2.70 x 2.80 m Spacing : 45 x 10 cm Variety : Phule G 9425-5 Treatments : Seed treatment = Soaking of seed for two hours in the slurry
[Spraying thrice: - Pre – flowering, flowering and pod filling stage]
Fertilizer dose : 12.5 kg N + 25 kg P2O5 /ha at the time of sowing. Date of sowing : 05.10.2004 Date of Harvest : 22.01.2005 Date of Irrigation : Rainfed hence no irrigation Results : The grain yield data are given in Table b: Field evaluation of Recombinant Inbred Lines of Chickpea for various yield related parameters and against various races of Fusarium oxysporum Three parents viz; JG-62(highly susceptible to wilt), ICC-4958 (late wilter) and Vijay ( wilt resistant) were mutually intermated. The Recombinant Inbred Lines obtained by this crossing programme have reached F11 generation in the year 2004-05. On the basis of reaction to wilt recorded in previous years, the lines were catagorized into different groups in each of the cross. Each group included eight RIL’s having resemblance for reaction to Fusarium wilt. The field testing was done in augumented design by using corresponding parent i.e JG-62, ICC-4958 and Vijay as checks. The evaluation of RIL’s was done for agronomic characters in normal plot and for disease resistance in wilt sick plot at Pulses Improvement Project , MPKV., Rahuri. The observations on wilting due to Fusarium on each RIL’s were recorded at every fifth day up to maturity. The values of disease intensity of each successive day of evaluation were used for estimating the Area under Disease Progress Curve (AUDPC). The formula given by Wilcoxson et al. (1975)* was used for estimating AUDPC value for each RIL’s and the three parents. The formula is as below
k AUDPC = Σ [ (S i + S i-1)/2] x D i-1
Where, S i = disease intensity at ith day of evaluation
K = number of successive evaluation D = interval between i and i-1 evaluation of disease For statistical analysis the values of AUDPC were subjected to square root transformation. --------------------------------------------------------------------------------------------------------------- * Wilcoxcoson, R.D., B. Skomand and A.A. Atif. 1975. Evaluation of wheat cultivars for the ability to retard development of stem rust. Ann. Appl. Biol., 80 : 275-287
There was a wide variation for growth and yield characters as well as disease reactions among the RIL’s in each of the cross. The data of individual RIL’s are given in Table 15. The average of eight RIL’s in each group for the respective character is given in Table 12.
CROSS I From Table 11 it is revealed that the maturity period in cross I ranged from 104 to 112 days. In respect of plant height nine groups had shorter height than Vijay whereas, almost all the group means were lower than JG-62. The plant spread of JG-62 and Vijay was 20 cm and 19 cm respectively. Group No. 15, 16 and 18 had greater values of plant spread than the two checks. For pods/plant, group No. 9, 15, 17, 18, 24, and 25 were more promising than the two checks. For seeds/pod there was no variation and for 100 seed weight all the group means were lower than Vijay. For yield/plant group No. 9, 17 and 18 were found to be of greater magnitude than better parent Vijay. JG-62 had highest AUDPC value for race-1 (5812) as well as race-4 (6142) whereas, it was lowest for Vijay i.e 303 and 204. It is to note that, none of the group value of AUDPC were smaller than that for Vijay. In general, the group means for AUDPC were less than that for JG-62. CROSS II In cross-II, there were 16 groups (Group No. 26-41). The maturity duration in this cross ranged from 106 to 113 days whereas, height ranged from 39 to 44 cm and plant spread in the range of 18 to 25 cm. It is to note that, the group means of these four characters did not deviate much than those for the parents viz; JG-62 and ICC-4958. For pods/plant, group No. 28,29 30, 33, 34, 36, 38 and 40 were superior over the better parent i.e. ICC-4958. None of the group of inbred line had the test weight equivalent to better parent i.e. ICC-4958. For yield/plant however, group No. 27, 28, 34, and 41 were superior over the better parent. The AUDPC values for both the races were highest in JG-62. This check was more susceptible to race-1 than race-4. In ICC-4958, the AUDPC value for race-1 was 903 while that for race-4 was 477. Almost all the group means in this cross indicated susceptibility to both the races but it was to less extent than the susceptible check JG-62. CROSS III The cross-III was developed from Vijay x ICC-4958. In this cross total 14 groups were incorporated (Group No. 42 to 55). The group means for maturity period were in between the values for Vijay (97) and ICC-4958 (119) days. The plant height was also intermediate between two parents. None of group mean surpassed in comparison with the parent for plant spread and number of secondary branches. The group number 52 had more pods/plant and higher test weight than both the parents. For yield/plant group No. 45, 52 and 55 were superior to both of the parents. The AUDPC values for race-1 and race-4 in Vijay were minimum. None of the group means were lower than the values of AUDPC for either of the two parents. II. Reaction of individual RIL’s to Fusarium wilt Reaction to Race I A perusal of the reaction of individual RIL to Fusarium wilt (Table 15) indicated that in a cross of JG-62 x Vijay, not even a single line had higher
resistance than ICC-4958 in this cross. Further, 195 lines were found to be less susceptible than JG-62. It is to note that during the year 2003-04, three lines were resistant whereas, 8 were more resistant than ICC-4958 and 186 lines were less susceptible than JG-62. Thus, there was a slight shift in respect of number of RIL’s in respect of reaction to Fusarium wilt during the two years. In a cross of JG-62 x ICC-4958, not even a single line was more resistant than either Vijay or ICC-4958. One hundred nineteen lines were less susceptible than JG-62 whereas, 3 lines were more susceptible than JG-62. Here, in this cross the tendency to remain less susceptibility than JG-62 was consistent during the two years of study whereas, the number of lines higher susceptibility than JG-62was declined sharply. In a cross of ICC-4958 x Vijay, one line had greater resistance than Vijay whereas, 11 lines had more resistance than ICC-4958 and 96 lines had less susceptibility than JG-62. Importantly, not even a single line was more susceptible than JG-62 during 2004-05. It must be noted that during the previous year there were 5 lines with greater susceptibility than JG-62. In brief, it could be stated that the distribution of RIL’s in different groups of resistance is relatively stable during both the years. Reaction to Race-4 Among the three parents Vijay had maximum resistance (AUDPC - 204) where as ICC-4958 had AUDPC value 477. JG-62 was highly susceptible (AUDPC - 6142). The perusal of data indicated that, in a cross JG-62 x Vijay, there was only one RIL having greater resistance than Vijay and one RIL have more susceptibility than JG-62. The number of RIL’s having less susceptibility than JG-62 was 195. In second cross also two lines each were more resistant than Vijay and ICC-4958 and 2 lines had more susceptibility than JG-62. Thus, there was a kind of transgressive segregation for wilt resistance in these two crosses. In third cross, there were eleven RIL’s with greater resistance than Vijay and ICC-4958. Remaining 79 lines were less susceptible than JG-62.
III. Agronomically superior RIL’s The three parents viz. Vijay, ICC-4958 and JG-62 had yield performance of 9.7,11.7 and 7.5 gm/plant respectively. In a cross of Vijay x JG-62 among 197 RIL’s 30 exceeded the yield performance over Vijay. Where as 50 exceeded over JG-62 and 14 were better than ICC- 4958. In second cross i.e JG-62 x ICC -4958 out of 122 RIL’s 55 had higher yield than Vijay, 76 were better than JG-62 and 40 RIL’s had higher yield than ICC-4958 . In a cross of ICC-4958 x Vijay 36 RIL’s showed higher yield than Vijay , 61shown better yield than JG-62 and 17 were superior to ICC-4958.
In nut shell there was a kind of transgresive segregation indicating a scope for obtaining superior types over better parent. But there was a larger tendency to have more number of RIL’s closer to the performance of JG-62.
Objective 2. Participatory evaluation and utilization of generated materials by chickpea growing farmers Activities: Evaluation of promising genotypes developed through the McKnight Foundation supported programme by fifty two farmers at thirteen locations from chickpea growing areas in the country. MPKV, Rahuri, India NCL, Pune, India The material has been provided to 90 locations in various agroclimatic regions. Data from each center need to be received and compiled. Compilation will be ready by June 2005 and will be submitted upon compilation.
Objective 3. Development of Fusarium wilt resistant chickpea varieties with
broad spectrum resistance suitable in various agrobiotic zones Activities: a. Development of intraspecific chickpea genetic linkage map b. Identification of molecular markers linked to Fusarium wilt resistance in
chickpea NCL, Pune, India MPKV, Rahuri, India WSU, Pullman, USA: The use of molecular markers has facilitated the breeding of crop plants, including resistance breeding. Molecular marker technology has made it possible to generate a genetic map of chickpea which can be used in marker assisted selection and positional cloning of disease resistance genes. In this study on development of Fusarium wilt resistant chickpea varieties, three parental lines were used, Vijay (resistant to Fusarium wilt), JG62 (susceptible) and ICC4958 (late-wilting). Three cyclic crosses were made in the following manner: Vijay x JG62 (Cross I), JG62 x ICC4958 (Cross II) and Vijay x ICC4958 (Cross III). The F9 RIL population of Cross I is being used mainly for the purpose of tagging Fusarium wilt race 1 resistant gene(s) while the F9 RIL population of cross III is mainly being used to develop intra-specific genetic linkage map and for mapping important agronomic traits. During last one year one hundred STMS primers were screened for parental polymorphism, among them, 22 primers were polymorphic. Out of 22 primers, 10 primers namely, TA002, TA005, TA018, TA025, TA078 ,TA96, TA110s, T146, TA37 and GA34 were used for population screening. Screening of remaining primers is underway. The data were analyzed for 25 and 62 primers ( RAPD, ISSR and STMS ) in cross I and III, respectively. Linkage analysis was performed using Mapmaker/Exp 3.0 (Lander et al. 1987). Linkage groups were established at a constant LOD score of 3.0 and a recombination value of 0.30 by two-point analysis using the “group” command. Once the markers were assigned to previously reported LGs, all the markers were then integrated into that group by applying the multipoint analysis “try” function. The most-likely order of loci within a group was determined using the multipoint “compare” command and these orders were verified using the “ripple” command. The Kosambi mapping function was used to determine cM distances between markers (Kosambi 1994). Interval mapping (Q gene) analysis for cross I showed the presence of major QTL in linkage group-2 for FOC-1. The STMS marker TA96s alone showed 38% contribution followed by TA59 (31.8%), TA37 (23.7%), TA96 (19.8%), UBC302 (11.5%) and TA110 (9.5%). This QTL explained 41% of total contribution from all
markers with maximum LOD-score of 10.13. Thus, QTL mapping confirms that LG2 carries at least one of the FOC-1 QTL.
group1
10.13
LOD3.0
ST12
U302
ST98
ST8
ST7
ST10
0.0
Interval analysis for trait Wt
U
TA 59- 31.8%
TA96-19.8% TA37-23.7%
(Interval QTL mapping analysis of FusariumF9 RIL population Vijay x JG-62.) In cross III data were analysed for botof QTLs for important agronomic traits like yiespread , pods per plant and grains per pod.15 groups, among them 30 markers formed aTA5 and UBC-284 flanked the FOC1 gene group 11 had 4 markers (UBC807300bp, UBC5, 8 and 9 comprised two markers each, and All 62 markers were analysed to idenagronomic traits. Results are interpreted in t c. Molecular breeding for developmen
genotypes Chickpea genotypes ( 21 in no. ) usRahuri for developing wilt resistant and screened with DNA markers reported ( CS 27resistance in chickpea. They will be used fseason. Similarly TA 80 linked to double poscreened in the cross Vijay X JG 62 and JG 6 d. Host-pathogen interactions: Chickp NCL, Pune, India WSU, Pullman, USA Expression studies of TDFs (WRKY and chickpea variety
TA96s – 38%
TA110-9.5% BC302-11.5%
wilt race1 resistance in the chickpea
h mapping of markers and identification ld, plant height, 100 seed weight, plant
Analysis of the 62 markers resulted in single group. At LOD 3.0 two markers at 28 and 30cM, respectively. Linkage 807600bp, UBC-891and UBC811). Group 11 markers remained unlinked.
tify Quantitative Trait Loci (QTLs) for he Table 18
t of Fusarium wilt resistant chickpea
ed in breeding programme at MPKV, agronomically superior varieties were , UBC 845 and UBC 855 ) to link to wilt or progeny screening in the next crop dded gene at 4.8 cM distance is being 2 X ICC 4958 for validation.
ea and Fusarium oxysporum system
14-3-3) in resistant and susceptible
We have employed cDNA-AFLP technique to find the differences between the root cDNAs of resistant (WR315) and susceptible (JG62) chickpea varieties after challenging by FOC-R1. We identified 3 defense related TDFs- WRKY, NBS-LRR, 14-3-3 protein. Expression studies of these TDFs were carried out using RNA dot blot and Northern hybridization approaches. Northern hybridization
Total RNA was extracted from chickpea root tissues using the TriZol (Invitrogen) method according to manufacturer’s instructions. Total RNA was extracted from root samples collected at 1 DAI (Days After Infection), 2DAI, 4DAI, 8DAI and 12DAI intervals. Total RNA (25 µg) was separated on 1.2% denaturing formaldehyde agarose gels and blotted onto Hybond-N membrane (Amersham Biosciences) according to standard techniques (Sambrook et al. 1989). DNA probes were prepared from selected cDNAs clones isolated from cDNA-AFLP from a cDNA library made using Fusarium oxysporum f.sp ciceri-induced roots harvested at 1, 2, 4, 8, 12, 16 and 20 days after infection. Northern blot hybridizations were accomplished using P32 radio-labeled DNA probes generated by PCR labeling and hybridized in Express-Hyb buffer (Clontech) at 65oC. RNA blots were washed under high stringency (0.1X SSC, 0.1% SDS at 50oC) and exposed to x-ray sheets (Konica) to generate the autoradiographs.
RNA dot blots
RNA arrays were fabricated by applying small volumes of total RNA from JG62 control, JG62 Infected, Vijay control and Vijay infected from 1, 2, 4, 8 and 12 DAI as spots on the Hybond N+ membrane (Amersham Biosciences). Each spot corresponded to 2 µg total RNA. Following spot application, RNA was covalently attached to the filters by UV cross-linking (UV Stratalinker), 70,000 µJ/cm2. Hybridizations were accomplished using P32 radio-labeled cDNA probes generated by PCR labeling and hybridized in hybridization buffer (5 x SSC; 50 % Formamide; 5 x Denhardt's-solution; 1 % SDS; 100g/ml heat-denatured sheared Salmon sperm DNA) at 50oC. RNA blots were washed under high stringency (0.1X SSC, 0.1% SDS at 50oC) and exposed to x-ray sheets (Konica). Alternatively, the same filters were repeatedly hybridized, stripped and re-hybridized with 3 different labeled cDNA clones (WRKY, 14-3-3 and NBS-LRR) ( Fig. 1 )
Northern hybridization as well as RNA dot blots showed that the 14-3-3 and
WRKY clone hybridized with the transcripts in resistant infected (Vijay) chickpea variety at 2 DAI only. This indicates the early responsive nature of both the genes. It is reported that, 14-3-3 proteins form a part of the defense reactions by regulating the proton pump (H+-ATPase) to initiate the hypersensitive response (Roberts et al., 2002). In potato a gene encoding 14-3-3 protein is strongly induced in the resistant cultivar than in the susceptible cultivar after 72 hours post infection with Phytophthora infestans (Barbara, et al 2004). Based on recent reports, it is becoming evident that WRKY transcription factors are implicated in the rapid responses of plants to wounding, to pathogens or to inducers of disease resistance (Cormack et al, 2002). Arabidopsis WRKY70 was identified recently, as a common regulatory component of SA- and jasmonic acid (JA)-dependent defense signaling, mediating cross-talk between these antagonistic pathways (Li et al. 2004).
Thus our expression studies indicated presence and differential early expression of WRKY and 14-3-3 in resistant chickpea variety and their role in transcriptional regulation, and also upregulation during biotic and abiotic stress.
Fabrication of cDNA arrays
Arrays were fabricated by applying small volumes of purified PCR products through slot blot apparatus on the Hybond N+ membrane. 2.5-5.0 µg DNA was used per spot. All the clones from cDNA-AFLP, cDNA-RAPD and extended TDFs were spotted on Hyond N+ membrane. Four identical filters were prepared serially, which will be hybridized separately with labeled cRNA made from each of the source RNAs; JG62 control, JG62 infected, Vijay control, Vijay infected. Hybridization of these DNA arrays is underway.
References
Barbara Ros, Fritz Thummler and Gerhard Wenzel., 2004, Analysis of differentially expressed genes in a susceptible and moderately resistant potato cultivar upon Phytophthora infestans infection, Molecular Plant Pathology; 5 (3): 191–201. Cormack R S , Eulgem T, Rushton P J , Kochner P, Hahlbrock K , Somssich I E , 2002, Leucine zipper-containing WRKY proteins widen the spectrum of immediate early elicitor-induced WRKY transcription factors in parsley. Biochimica et Biophysica Acta 1576 : 92– 100. Li J, Brader G, Palva E T, 2004, The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylatemediated signals in plant defense. Plant Cell; 16:319-331. Roberts, M R., Salinas, J and Collinge, D B. (2002) 14-3-3 proteins and the response to abiotic and biotic stress. Plant Mol. Biol.; 50: 1031-1039. Chickpea- Ascochyta rabiei system WSU, Pullman, USA: (P.N.Rajesh from India: Visiting scholar at WSU) I. Agrobacterium mediated transformation of a large genomic insert in chickpea
Agrobacterium tumefaciens-mediated transformation is the most preferred of
all available transformation methods for grain legume species. Our objective was to establish stable transformation of large genomic inserts using a suitable Agrobacterium tumefaciens (A. t) strain that will have long-term application in functional analyses of disease resistance genes in chickpea. Here we report stability analysis of seven large inserts ranging in size from 20 to 100 kb and transformation of the BAC clone, 15(O)o9 which is 46kb in size.
It is recommended that the integrity of large genomic fragments in
Agrobacterium be verified prior to plant transformation. In this study, the stability of large genomic DNA inserts in A.t. was assessed using seven fragments ranging in
size from 20 to 100 kb obtained from a chickpea Bacterial Artificial Chromosome (BAC) library constructed using the pCLDO4541 (V41) binary vector. Six inserts were associated with ascochyta blight resistance and one was associated with fusarium wilt race 3 resistance in chickpea. These BAC clones were transformed into A.t. strains Agl0 and Agl1 using triparental mating or electroporation. Stability of the clones in A.t. was assessed by transforming the BAC clones back into E. coli - ElectroMAXTM DH10BTM strain and evaluated using Pulsed Field Gel Electrophoresis. All fragments up to 100kb in size were stably transformed into Agl0 by triparental mating and recovered intact. Clone identity was confirmed by fingerprinting. All the large inserts were degraded in Agl1. Our results show that genomic fragments up to 100kb transferred by triparental mating were stably maintained in A.t. strain Agl0. However, despite the presence of intact plasmids, evidence of deletions from individual colonies was also observed in Agl0 which emphasizes the need to verify the presence and integrity of the plasmid being transferred.
15(O)o9 was shown to have 8 genes by blast analysis and 36 predicted
genes by genscan analysis. Since this particular large insert was linked to ascochyta blight resistance and also gene rich, we transformed 15(O)o9 into a chickpea recombinant inbred line 34 for complementation analysis. The explants were screened on selection media for 23 weeks. To identify the presence of the inserts in the potential transformants by PCR, we used the markers spanning this large insert as well as primers designed from the binary construct. Our investigation was positive for all markers in the To transformants. Work is underway to determine the copy number and the inheritance of this particular fragment in subsequent generations.
II. Genome characterization of QTL1 of ascochyta blight resistance in chickpea
Ascochyta blight caused by Ascochyta rabiei (Pass.) Lab. is a major limitation
to chickpea production in the US. Genetic studies indicated that blight resistance is governed by two QTLs: QTL1 and QTL2. One of the QTLs, QTL1, is estimated to account for 35% of the variation for blight resistance and is flanked by two RAPD markers (UBC 733b and UBC 181a) and contains a DNA amplification fingerprinting marker (OPS06-1) mapped between the flanking markers. To characterize the QTL1 genomic region, we used the "super-pooling” method to screen a chickpea BAC library and now have identified two BAC clones with OPS06-1. The sequences are available at URL link: http://www.genome.ou.edu/plants_totals.html . Blast and genscan analysis identified the presence of multiple genes in two BAC clones 15(O)o9 and 4m10 but did not detect NBS-LRR type genes. Concurrently, we developed a SCAR marker for UBC733b. This SCAR marker enabled chromosome walking towards OPS06-1 and identification of an additional BAC clone.
To characterize this genomic region further, we mapped the ends of BAC
clones 4m10, 15(O)o9 identified using OPS06-1 and 20(R)l12 identified using the SCAR marker. All BAC end primers except 20(R)l12-Left amplified monomorphic bands during parental analysis. In order to genetically map these markers, we developed CAPS and dCAPS markers for each BAC end primer. One Single Nucleotide Polymorphism (SNP) was observed for every 52 nucleotides in this genomic region. Our results narrow the previously reported genetic region of QTL1 from 12cM to 4.6cM using sequence based markers. Qgene analysis indicated that flanking markers have higher R2 (56%) and LOD value (19.98) than the previously reported marker (35% and 13.40) at QTL1. Comparison of sequences of 4m10 and 15(O)o9 with Medicago truncatula genome sequences identified several contigs with partial homology. Work is in progress to fine map the genomic region using additional BAC end probes and using synteny with Medicago truncatula. Identifying additional BAC clones using the BAC ends and subsequently sequencing them will reveal the gene content of this major QTL and will help to develop direct markers for marker-assisted breeding. Functional genome analysis of two ascochyta blight responsive genes in chickpea
In our earlier analysis to identify blight responsive genes, we compared the expression profile in pooled RNAs of FLIP84-92C, a resistant cultivar, and C. reticulatum (PI 489777), a susceptible wild species accession challenged with a virulent isolate of A. rabiei (Ar20) with the control plants using Differential Display Reverse Transcription (DDRT) technique. We had identified two differentially expressed DDRT products that showed 87% and 88% homology with serine hydroxy methyl transferase (SHMT) and aldolase of pea counterparts, respectively. Further, we studied the behavior of these genes at specific time intervals in C. reticulatum accession PI 599072, Spanish White, a cultivar, that are susceptible to pathotype I and II of ascochyta and FLIP84-92C, a germplasm accession that is resistant to both pathotypes, after challenging all three lines with Ar19 (low virulent Pathotype I) and Ar628 (highly virulent Pathotype II) using quantitative PCR analysis. Our results confirmed the differential expression of these two genes. The expression patterns of
aldolase and SHMT were different between wild and cultivated susceptible chickpea plants upon infection.
Significant accomplishments • CAPS and dCAPS marker development in chickpea • Development of an improved transformation protocol with the decreased time
from lab to greenhouse
Objective 4. Development of chickpea genotypes resistant to insect (H. armigera) attack
Among the biotic stresses, insect pests are the major problems which hamper the productivity of chickpea. Increased scientific and public concerns over the widespread use of chemical insecticides have steered research which is environmentally friendly and involves sustainable methods for pest control. Biotechnological tools such as transgenic expression of antifeedent proteins are being increasingly investigated. Plant-derived defense genes have enormous potential for sustainable pest management. It is, therefore, planned to incorporate suitable defense protein genes such as proteinase inhibitors (PIs), lectins, amylase inhibitors and Bt in chickpea for enhancing resistance to Helicoverpa armigera and storage pests and thereby increase the production of chickpea. The transgenic H. armigera resistant chickpea cultivars would substantially boost the chickpea production in India to make it a more profitable crop for the farmers. Such a technology would be an ideal example for other important crops like pigeonpea and cotton that are severely damaged by H. armigera. Activities: a. Exploitation of novel Proteinase Inhibitors in conferring resistance to
insect attack :
(I) Transfer of Winged bean protease inhibitor gene into chickpea NCL, Pune, India WSU, Pullman, USA AAU, Jorhat, India MPKV, Rahuri, India
Agrobacterium tumefacience mediated transformation of chickpea, var. Vijay for devoloping resistance against pod borer: Helicoverpa armigera. Personnel: Gauri Bhat/Dr. Vincent Francheschi
Agrobacterium mediated transformation of the following four constructs
pBINAR/ PtWCI-2; pBINAR/ PtWCI-5; pVicGal-ShP/ PtWCI-2; pVicGal-ShP/ PtWCI-5 is described below. pBINAR recipients involving CaMV 35S promoter for constitutive plant gene expression and pVicGal-ShP involving pea vicillin promoter for seed specific plant gene expression.
These four constructs were transferred into Agrobacterium tumefaciens strain,
Agl-0 by triparental mating. Vijay, a cultivar developed at MPKV, Rahuri, India, was transformed with all of these constructs. Approximately 400 explants (T0) were transformed in three different sets of experiments. Explants were the immature embryonic axis sliced longitudinally and were grown on regeneration medium (RS) in presence of kanamycin before inducing the roots. After four cycles of screening on RS1, RS2 and RS3 (two weeks each), they were transferred to rooting medium. The
rooted explants were transferred onto soil less mixture and grown in controlled conditions in a growth chamber for two weeks before transferring to the greenhouse to grow under normal day-night conditions.
Explants were screened for the putative transformants at an early stage of
development using gene specific primers, promoter specific primers and NPT-II specific primers. For example, a total of 19 explants grown in vitro were found to be PCR positives in transformation event involving PtWCI-5 gene under CaMV 35S promoter. Of 19, finally, nine plants survived in ex vitro conditions. Similar PCR screening was performed for other three transformation events and found several explants positive. In a nutshell, we have identified positive putative transformants for all four independent transformation experiments. Southern hybridization is in progress to determine the copy number. Work is underway to analyze the expression of these genes in the putative transformants using RT-PCR. The effectiveness of transgenically expressed PIs will be analyzed using standardized enzyme inhibitor assays and direct feeding assays that will be carried out in T1 or T2 generation of transgenic plants.
(II) Chickpea Helicoverpa gut protease inhibitor (CHGPI) NCL, Pune, India.
In continuation with the characterization of HGPI (Helicoverpa armigera Gut Proteinase Inhibitor), which was purified from chickpea (Cicer arietinum) seeds, identified by MALDI-TOF as well as N-terminal amino acid sequencing, and finally whose gene was isolated and cloned into a yeast expression vector, the following work has been carried out: Stability of HGPI towards proteolytic degradation by HGP Approximately 5 µg of HGPI was mixed and incubated with 10 µL of a fresh HGPs preparation and incubated at 37°C for 0, 30 and 180 min. As a positive control, 5 µg of untreated HGPI was incubated for 180 min. After incubation, the mixtures were immediately separated by 12% native polyacrylamide gel electrophoresis (PAGE), following which, HGPI activity bands were visualized by the gel - X-ray contact print method. HGPI was observed to retain HGP-inhibiting activity even after three hours of incubation in presence of control or sensitized HGPs. Secondly, the activity band representing HGP inhibition by HGPI was observed to co-migrate in case of both, the HGPs treated as well as the untreated sample. This suggested that the inhibitory activity was not lost due to action of HGPs and also that the structural stability of HGPI was not affected in presence of HGPs. Hence it is considered that the native form of HGPI is stable to proteolytic degradation by HGPs (Fig. 2).
Inhibitory potential of HGPI against commercial proteinases
Inhibitory potential of expressed protein was assayed against the various proteinases, viz., trypsin, chymotrypsin, subtilisin and HGP preparation. Total HGP activity was assayed by using the semi-synthetic non-specific substrate azocasein. BApNA was used as the substrate for trypsin as well as HGPs-trypsin. SAAAPLpNA
and SAAPFpNA was used as substrates for chymotrypsin and subtilisin respectively. For all assays 0.4 U of each enzyme was used as a standard. Inhibition of trypsin, HGPs-trypsin and total HGP activity was also assayed with commercial Soybean Kunitz Trypsin Inhibitor (SKTI).
Stoichiometric inhibition of trypsin activity was observed with HGPI, comparable to that seen with SKTI. HGPI mediated inhibition of chymotrypsin or subtilisin was not recorded. HGPI exhibited 60% maximum inhibition of HGPs-trypsin, less than that exhibited by SKTI (71%). Total HGP activity was inhibited to a maximum of 69% by HGPI, significantly higher than with SKTI (41%). IC50 for standard trypsin activity was 2.5 x 10-7 M for HGPI, comparable to that of SKTI for trypsin. IC50 of HGPI for total HGP activity was <10-7 M and that of SKTI was 10-7 M. IC50 for HGP trypsin like activity were 10-7 M and <10-7 M for HGPI and SKTI respectively (Fig. 3). Feeding assays on Helicoverpa armigera larvae
Anti-metabolic effects of HGPI on growth of H. armigera larvae were investigated by insect feeding bioassays. Composition of artificial diet (for 650 mL) was: chickpea seed meal, 77.7 g; wheat germ, 5.6 g; dried yeast powder, 19.2 g; casein, 12.8 g; ascorbic acid, 4.6 g; methylparahydroxy benzoate, 1.5 g; sorbic acid, 0.8 g; streptomycin-sulphate, 0.2 g; cholesterol, 0.2 g; vitamin B complex, 1 capsule (approx. 0.2 g); formaldehyde (37%), 1 mL; multivitamin drops, 0.8 ml; vitamin E, 0.8 mL; agar-agar, 12 g. HGPI was used at a concentration of 0.5x (40.2 µg) per gram artificial diet. Two sets of 25 insects each was used; one set was allowed to feed on the artificial diet without HGPI and the other fed on HGPI containing diet. Larval weights were recorded every alternate day after 48 h of feeding.
The HGPI fed larvae showed a distinct lag in growth and weight gain. Differences between control and HGPI fed sets in the first and second instars (second day and sixth day respectively) were insignificant. At the third instar (eighth day) the average HGPI fed larval weight was almost 64% lower than the average control weight. Similarly, at the fourth instar, average inhibitor fed larval weight was 47% lower than the average control weight. By the fifth instar (eighteenth day), the average inhibitor fed larva weighed 39% lower than the control (Fig. 4).
Larval responses to HGPI incorporation in diet Characterization of midgut proteinases BApNA, SAAAPLpNA and SAAApNA were used as substrates for assaying HGPs-trypsin, HGPs-chymotrypsin and HGPs-elastase, respectively. Total HGPs activity was assayed with azocasein. HGPs corresponding to 0.4 U of each proteinase activity was then incubated with increasing amounts of HGPI (2, 4 and 6 µg) or the chemical inhibitors (0.1-1 µM PMSF and TPCK, 0.01-0.1 µM TLCK and elastatinal) for 10 min and residual activity was assayed. The entire set was carried out for both control as well as HGPI fed larval HGPs.
HGPI feeding caused an increase in the total HGPs activity by 7.35%, HGPs- trypsin activity by 7.03% and HGPs chymotrypsin activity by 0.67%. HGPs-elastase activity was not measurable. HGPI inhibited upto 65% of total activity of control
HGPs, and upto 73% for sensitized HGPs (Fig. 5A). There was no difference in inhibition of total HGPs activity by PMSF (Fig. 5B). HGPI also exhibited higher inhibition of HGPs-trypsins in sensitized HGPs (66%) as compared to control HGPs (61%) (Fig. 5C). With TLCK, higher inhibition of HGPs-trypsin was observed in sensitized HGPs (95%) as compared to control HGPs (90%) (Fig. 5D). Inhibition of HGPs-chymotrypsin was either absent or very low with HGPI in case of both control as well as sensitized HGPs. No difference was observed in inhibition of HGPs-chymotrypsins in either control or sensitized HGPs by TPCK (Fig. 5E).
Differential expression of larval midgut proteinases Oligonucleotide primers were synthesized based on available sequence information of eighteen unique H. armigera gut proteinases including five trypsins, three chymotrypsins, five aminopeptidases, three carboxypeptidases, one elastase and one cathepsin-B like proteinase. These primers were employed in a quantitative RT-PCR using dilutions of the cDNA derived from a midgut mRNA preparation. The number of cycles required for efficient PCR, have been previously standardized as to give 50% amplification of the target transcript. Three trypsin transcripts were detectable, and were up-regulated due to HGPI feeding (Fig 5A). Two, chymotrypsin transcripts were detectable, of which, one was up-regulated and the other was de novo transcripted in sensitized larvae (Fig 5B). Four aminopeptidase transcripts were detected, of which, two were de novo transcripted, and the other two were over-expressed in sensitized larvae (Fig. 5C). None of the carboxypeptidases, cathepsin or elastase transcripts were detectable in both sets (not shown).
b. Use of lectins to increase effectiveness and durability of resistance to
insect (Helicoverpa armigera) attack UD, UK, NCL, India I. Design of a synthetic lectin gene with enhanced toxicity towards H. armigera. Plant lectins in general have limited toxicity towards lepidopteran larvae. Results from the previous reporting period show that the lectin selected as a possible insecticide for H. armigera, winged bean lectin, has significant negative effects on survival and development of larvae of the target pest, but that those effects are not sufficient to result in effective protection of plants producing the lectin. We therefore investigated a method to increase the toxicity of plant lectins toward insects, by producing fusion proteins in which the lectin sequence is joined to an insecticidal protein or peptide (Fitches et al., 2002; Fitches et al., 2004). This technique has been developed jointly by Durham University and the Central Science Laboratory, DEFRA, and is subject to patent restrictions; its use in this programme was as part of an academic research investigation into developing and extending the use of fusion proteins as insecticides.
All scorpion species investigated produce a range of insecticidal toxins, of varying specificities towards both insects and higher animals; their activity is usually exercised through interaction with membrane proteins, leading to neurotoxic effects. These toxins have varying degrees of specificity in their action; some toxins are effective against a wide range of species, whereas others are specifically toxic to insects. A toxin from the Indian red scorpion, Mesobuthus tamulus, was selected as being of interest in that it has been reported to be specifically toxic towards lepidopteran insects. Tests on purified toxin ButaIT showed toxicity towards larvae of corn earworm (Heliothis virescens), but not towards blowfly larvae or mice (Wudayagiri et al., 2001). This toxin was therefore selected as suitable for enhancing the insecticidal activity of plant lectins. The sequence of the toxin, and a cDNA encoding it, are publicly available from the global sequence database; the protein is similar to a series of small toxins characterised as having insecticidal properties (Fig. 7). The fusion protein was designed by combining the mature toxin polypeptide N-terminally to the sequence of the mature snowdrop lectin (Galanthus nivalis agglutinin; GNA) polypeptide, with a short linker sequence, (Ala)3, joining the two (Fig. 8). The lectin was selected on the basis of previous work in Durham which has shown that this lectin is transported effectively to the haemolymph in lepidopteran larvae after oral ingestion (Fitches et al., 2001). The expression system selected was the yeast Pichia pastoris, which produces snowdrop lectin as a secreted protein in a soluble, functionally active form, and has previously been used to produce a similar fusion protein containing a spider venom toxin (Fitches et al., 2004). II. Assembly of expression constructs for synthetic lectin gene. Since access to the Indian red scorpion was not available, a synthetic coding sequence corresponding to the mature scorpion toxin (minus signal peptide) was assembled from oligonucleotides. Eight 30-mers and two 15-mers were synthesised which overlapped with each other to produce a complete double-stranded DNA sequence, with additional restriction sites, encoding the entire mature scorpion toxin polypeptide (Fig. 9). The signal peptide found in the cDNA sequence and the stop codon were not included. The oligonucleotides were mixed, heated and allowed to cool and hybridise. The resulting mixture was then subjected to PCR amplification using the 15-mer primers at either end of the double strand as primers. A 135 bp product was obtained, which was purified by agarose gel electrophoresis and cloned into the PCR cloning vector pCR2.1. The sequence was then checked to confirm the correct product had been obtained. The scorpion toxin coding sequence was then assembled into the final expression construct by restriction-ligation into a pre-existing yeast expression vector based on pGAPZα, containing the mature GNA coding sequence. The final construct contained an open reading frame encoding the yeast α–factor prepro- sequence, the complete scorpion toxin mature polypeptide (RST), a linker region (encoding Ala-Ala-Ala) and the complete GNA mature polypeptide, with C-terminal myc epitope and (His)6 tags. As a control, a separate expression construct encoding the yeast α–factor prepro- sequence and the complete scorpion toxin mature polypeptide with a
C-terminal (His)6 tag was also assembled. The constructs were checked by DNA sequencing. III. Expression of recombinant proteins in Pichia pastoris. Expression constructs for the RST-GNA fusion and RST alone were transformed into Pichia pastoris strain X-33, and transformed yeast were selected by plating on media containing zeocin, as described in the protocols supplied by Invitrogen. Selected colonies were grown in small-scale cultures (10ml) and culture supernatants were screened for the presence of recombinant proteins by dot-blotting onto nitrocellulose and probing with anti-(His)6 antibodies (Fig. 10). Clones giving the highest expression levels for the recombinant proteins were selected, and were grown up in a 2 litre laboratory scale fermenter (Rogelj et al., 2000). Recombinant proteins were purified from culture supernatant by hydrophobic interaction chromatography on a column of phenyl-Sepharose; in both cases the supernatant was made 2M in NaCl, and the column was eluted (after washing) with a linear salt gradient, 2M – 0M. Recombinant proteins eluted in water in both cases. The RST-GNA was obtained at approx. 90% purity as a major band on SDS-PAGE of approx. 18,000 mol. wt. (Fig. 11). RST alone stained very poorly after gel electrophoresis, giving a faint and disperse band at approx. 5,000 mol. wt. Approx. 1mg of RST, and 5mg of RST-GNA were purified. IV. Assays of insecticidal activity of synthetic lectin. Purified RST-GNA was assayed for toxicity towards larvae of the model lepidopteran tomato moth (Lacanobia oleracea) by injection. RST alone was used as a positive control, and either water or GNA were used as negative controls. Varying amounts of protein were injected into 5th instar larvae, in the weight range 12-14mg, and survival was monitored over a 3-day period. Both positive and negative controls gave expected results. Survival for both water- and GNA-injected larvae was >90%, and in most assays was 100%. Doses up to 10µg GNA had no effect on survival. On the other hand, injection of RST at doses in the range 0.5 – 3.0µg caused 100% mortality over 3 days; 0.1µg of RST had only a slight effect on survival (Fig. 12). The lowest dose for 100% mortality corresponds to approx. 40µg toxin / g insect, making this toxin highly effective at low dose. The RST-injected insects showed flaccid paralysis, which led to death. Injection of RST-GNA showed a similar effect to injection of RST alone, although the fusion protein was not so effective on a weight basis, with mortlaity at does of 2.5µg and 5µg per insect being approximately 55% and 75% respectively (Fig. 13). The injected insects showed the same symptoms of flaccid paralysis, but the effects were less severe, and some insect were still alive, although moribund, after 3 days. These assays demonstrate that the RST is showing the expected toxicity towards lepidopteran larvae, which is also shown by the RST-GNA fusion. The fusion is less toxic than RST alone on a weight basis, by a factor of approx. 10x, and on a mole basis, by a factor of approx. 3x. The RST-GNA fusion protein was also assayed for its toxicity towards rice brown planthopper, a homopteran pest of rice, by oral delivery via artificial diet. The
toxicity observed was the same as that produced by GNA at the same level, and thus the RST was not observed to have any toxicity towards this insect, as expected. Further material is ciurrently being produced and purified to allow the toxicity of RST-GNA towards larvae of a lepidopteran species to be assayed when fed as part of the diet (oral administration). V. Characterisation of H. armigera gut proteinases. Work was carried out in Durham prior to this project to characterise the biochemistry and molecular biology of the digestive serine endopeptidases of H. armigera (Johnston et al., 1991; Bown et al., 1997). Attempts to produce these enzymes as recombinant proteins, to allow the properties of individual enzymes rather than mixtures to be assayed, were not successful. Non-functional proteins were produced in bacterial systems, but refolding to active proteinases was not achieved. Attempts to produce functional enzymes in yeast or insect cell expression systems did not yield any products. Initial work carried out by Dr. Nana Chougule during Feb. 2005 used a construct previously assembled in Durham, based on a pET vector containing a DNA sequence encoding the pro-form of a H. armigera trypsin-like protease (HaTC16; Bown et al., 2004). This construct had been shown to produce recombinant protein in an insoluble form in E.coli strain BL21 DE3. After expression, the insoluble protein fraction was isolated from lysed E. coli cells by centrifugation, dissolved in 8M urea s a denaturant, and the recombinant protein was purified under denaturing conditions by chromatography on immobilised nickel, exploiting the presence of a C-terminal (his)6 tag added to the protein by the vector. The purification was combined with refolding by exposing the bound protein to a gradient of decreasing urea concentration, from 8M urea to 2M urea, while bound to the column. Protein was finally eluted with 0.3M imidazole. The purified H. armigera trypsin was soluble after elution, and gave a band of mol. wt. approx. 25,000 by SDS-PAGE; however, yields of soluble protein were very low (approx. 1-2 �g per 100ml culture). The purified protein had no hydrolytic activity against a synthetic substrate for trypsin, but when treated with bovine trypsin (50-100ng; approx. molar equivalence) appeared to activate, in that the resulting activity was at least 2x the activity of the bovine trypsin alone. Work on this part of the programme is being actively pursued to allow amounts of functionally active H. armigera trypsins, sufficient for assay against proteinase inhibitors, to be produced. The recombinant enzymes will be used to characterise the activities of H. armigera gut proteinases. Conclusions • Synthetic gene encoding lepidopteran-specific scorpion toxin constructed. • Recombinant scorpion toxin is insecticidal towards a model lepidopteran (tomato
moth) when injected. • Gene encoding snowdrop lectin with scorpion toxin fused N-terminally has been
constructed. • Recombinant fusion protein produced by expresssion in yeast (Pichia pastoris).
• Scorpion toxin-snowdrop lectin fusion protein is insecticidal towards model lepidopteran (tomato moth) when injected.
• Constructs for expressing digestive trypsin- and chymotrypsin-like proteinases in H. armigera as recombinant proteins have in both E. coli and yeast (Pichia pastoris) been produced; yeast constructs fail to express, whereas E. coli produces proteins as insoluble aggregates.
• Preliminary results suggest that a trypsin-like enzyme from H. armigera expressed in E. coli can be refolded from solubilised denatured protein to give soluble proenzyme, which is activated by treatment with bovine trypsin.
References Bown, D. P., Wilkinson, H. S. and Gatehouse, J. A. (1997). Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochemistry and Molecular Biology 27, 625-638. Bown, D. P., Wilkinson, H. S. and Gatehouse, J. A. (1998). Midgut carboxypeptidase from Helicoverpa armigera (Lepidoptera: Noctuidae) larvae: enzyme characterisation, cDNA cloning and expression. Insect Biochemistry and Molecular Biology 28, 739-749. Bown, D.P. and Gatehouse, J.A. (2004). Characterisation of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues. European J. Biochem. 271, 2000-2011. Fitches, E., Gatehouse, A. M. R. and Gatehouse, J. A. (1997). Effects of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on the development of tomato moth (Lacanobia oleracea) larvae in laboratory and glasshouse trials. Journal of Insect Physiology 43, 727-739. Fitches, E., Woodhouse, S. D., Edwards, J. P. & Gatehouse, J. A. (2001) In vitro and in vivo binding of snowdrop (Galanthus nivalis agglutinin; GNA) and jackbean (Canavalia ensiformis; Con A) lectins within tomato moth (Lacanobia oleracea) larvae; mechanisms of insecticidal action, Journal of Insect Physiology 47, 777-787. Fitches, E., Audsley, N., Gatehouse, J. A. & Edwards, J. P. (2002) Fusion proteins containing neuropeptides as novel insect contol agents: snowdrop lectin delivers fused allatostatin to insect haemolymph following oral ingestion, Insect Biochemistry and Molecular Biology. 32, 1653-1661; Fitches, E., Edwards, M. G., Mee, C., Grishin, E., Gatehouse, A. M. R., Edwards, J. P. & Gatehouse, J. A. (2004) Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion, Journal of Insect Physiology. 50, 61-71. Johnston, K. A., Lee, M. J., Gatehouse, J. A. and Anstee, J. H. (1991). The partial purification and characterisation of serine protease activtiy in the midgut of larval Helicoverpa armigera. Insect Biochemistry 21, 389-397.
Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering, 10, 1-6. Rogelj, B., Strukelj, B., Bosch, D. & Jongsma, M.A. (2000) Expression, purification, and characterization of equistatin in Pichia pastoris. Protein Expr. Purific. 19, 329-334. Wudayagiri, R., Inceoglu, B., Herrmann, R., Derbel, M., Choudary, P.V., & Hammock, B.D. (2001) Isolation and characterization of a novel lepidopteran-selective toxin from the venom of South Indian red scorpion, Mesobuthus tamulus. BMC Biochemistry 2, 16. c. Co-expression of Bt Gene with pI and/or lectin and/or α-ai genes in chickpea for effective control of insects d. Introduction of cotyledon specific α-amylase inhibior gene into popular Indian chickpea cultivars for tolerance against storage pest CSIRO, Australia AAU, India NCL, India
Chickpea (Cicer arietinum) is one of the most important grain legumes of India in terms of area and production. Stored grains of this legume are highly susceptible to attack by bruchid pests such as Callosobruchus spp causing extensive loss during storage. No germplasm, resistant to these pests has yet been reported. A bean α-amylase inhibitor gene (α ai) has been shown to confer resistance against these pests. Earlier, we transformed an Australian cultivar of chickpea with this gene and the transgenics were found to be resistant to C. maculatus and C. chinensis (Sarmah et al., 2004. Mol Breed. 14:73-82). Attempts are being made to incorporate this gene directly into two Indian chickpea cultivars using gene technology as well as by crossing the Australian transgenic line with adapted Indian germplasm.
Another, devastating pest of chickpea is pod borer (Helicoverpa armigera)
that infests the crop from the vegetative stage to maturity. Wing bean protease inhibitor conferred protection against the pod borer in vitro. The NCL group in Pune has cloned the wing bean protease inhibitor gene in collaboration with WSU, USA. At AAU we are trying to develop transgenic line against pod borer using wing bean protease inhibitor (wbpi) gene that was reconstructed earlier in collaboration with NCL for chickpea transformation. The objectives of our programme in 2004-05 were 1. Introduce bean alpha amylase inhibitor (αai) gene into Indian cultivar of chickpea
to confer resistance against storage pest. 2. Introduce wing bean protease inhibitor (wbpi) gene into Indian cultivars of
chickpea to confer resistance against pod borer.
Achievement Putative transgenic chickpeas were obtained using cotyledon specific alpha amylase inhibitor (α-ai) gene A binary vector harboring bean α ai gene and npt II expression cassette flanked by lox sites (floxed) of Bacteriophage P1 was used for transformation. This vector was introduced into two Agrobacterium strains AGL 1 and GV 3101 and was used for chickpea transformation. Transgenics were developed using two different Indian cultivars viz ICCV 89314 and Vijay (Table 16). In the cultivar ICCV 89314 three primary transgenic (T0) lines were established and the presence of transgene was confirmed by PCR (Fig 14). Analyses of T1 progeny of one line confirmed transmission of the transgene into segregating progeny. However, progeny of this line (Chickpea-� AI-1) shall have to be tested in order to determine segregation ratio. T2 seeds were harvested from all four T1 plants shown in Fig 15 and T2 plants and are now being grown in ICRISAT, Hyderabad. ICRISAT has good facilities for multiplication of transgenic seeds. Collaboration has been established between AAU and ICRISAT. The Department of Biotechnology, New Delhi has given us permission to exchange our transgenic material with ICRISAT for further seed multiplication and insect bioassays as per the order No. Bt/17/11/99-PID. Dated 25.5.04. Further molecular analyses will be carried out using advanced generation plants. Three putative transgenics were also established using the cultivar Vijay and they were positive by PCR. Only 1-2 T1 seeds could be harvested from these lines but they were also sent to ICRISAT for multiplication. Segregation analysis will be done on the T2 progeny. Molecular analyses such as Southern (to determine copy number of transgene) and Western (to determine level of expression of the transgene) using segregating progenies will be carried out using all the transgenics developed so far in collaboration with NCL, CSIRO, and ICRISAT. New transformation experiments are also in progress to establish more primary transgenic lines so that one or two high expressing lines can be obtained for insect bioassays. Transfer of alpha amylase inhibitor (α ai) gene into adapted Indian germplasm using conventional backcross breeding:
Transgenic chickpea lines harboring the bean � amylase inhibitor gene were developed at CSIRO using the Australian chickpea cultivar Semsen by Dr Sarmah under the guidance of Dr T J Higgins. Seeds of two lines (BK39C and BK40D) were earlier brought to AAU via the import permit No. BT/BIS/17/11/99-PID/IBSAAU, dtd. 4.01.2001. Seeds of these lines were tested for resistance against the stored grain pest C. chinensis. We recorded a good level of tolerance in these two lines. Our observations were published in “Transgenic chickpea seeds expressing high levels of a bean α- amylase inhibitor” (Sarmah et al., 2004. Mol Breed.14:73-82). We multiplied these lines in our polyhouse at AAU. Only three seeds of the line BK39C germinated but they did not bear pods. On the other hand, 2 seeds of the line BK40D germinated under our condition and we harvested a few seeds. These seeds have been sent to ICRISAT, Hyderabd for further multiplication. More seeds of these lines will be obtained from CSIRO for multiplication so that large-scale, replicated bioassays can be carried out for confirmation of resistance to stored grain pests. Apart from these lines we also propose to import two more lines (1O1 and 3A1)
developed by CSIRO and which have been characterized there to show mendelian inheritance and single copy inserts. These lines will be used in a backcross breeding programme to transfer the transgene into popular Indian cultivars involving a breeder at ICRISAT. Efforts to make the cross (Vijay X 39C) were made at AAU and we obtained 8 seeds out of one hundred crosses. Unfortunately these seeds did not germinate in soil. This season fresh crosses (Vijay X 40D) are being made (Table 17) at AAU and ICRISAT and we have already obtained few seeds. Some of these seeds will be utilized to for transfer of transgene by PCR analysis. Transfer of winged bean protease inhibitor gene into chickpea:
The wing bean protease inhibitor (wbpi) gene was reconstructed in collaboration with NCL using the binary vector pBK11 harbouring nptII expression cassettes flanked by Lox (Locus for crossing over) sites of bacteriophage P1. Transformation experiments were initiated in AAU Jorhat using the chimeric gene (35S-wbpi) and 12 putative transgenic lines were established and all of them were found to be positive for both nptII and wbpi gene by PCR. However only 4 lines produced T1 seeds. These T1 seeds were sown in soil to raise T1 plants. Only one seed per line germinated to give rise to T1 plants. These plants were stressed by fungal infection we successfully grafted green shoots of these plants in the glasshouse using the non-transgenic root-stocks and were able to recover survivors. PCR analyses using these plants confirmed transmission of the transgenes. Further biochemical and molecular analyses will be carried using these plants to confirm transgene expression. Experiments are in progress to generate more T0 lines at AAU and transformation experiments are also in progress at WSU using wbpi gene.
At AAU in a separate programme, transgenics have been developed in
chickpea using a Bt-cry1Ac gene. The best WBPI line may be crossed to one best Bt line in order to have both genes (having different modes of action against pod borer) in one elite cultivar. This may enhance the level of tolerance against the pod borer since both WBPI and Cry1Ac have insecticidal properties. They have different modes of action against lepidopteran pests and could jointly provide a durable level of protection. However before we proceed we will need to evaluate the biosafety of WB PI for human consumption.
Training Report
TRAINING REPORT
Training at WSU, Pullman; USA • Dr. Rajesh PN, a research associate from NCL, Pune, India has been receiving
training from Dr. Fred. Muehlbauer, Department of Crop and Soil Sciences, WSU from September, 2002, to work on [i] Screening of BAC library for blight resistance genes and physical mapping of this region [ii] Identification of BAC clones having wilt marker [iii] Studies on comparative genomics of chickpea and Medicago, the closely related model legume crop [iv] Agrobacterium mediated transformation of large genomic insect in chickpea.
• Mrs. Manasi Telang, a graduate student at NCL, India worked with Dr. Vincent
Franceschi, School of Biological Sciences, WSU to receive training on cloning and expression of non host proteinase inhibitor genes in yeast and Arabidopsis for a period of ten months (Sept1,2003 -June30, 2004).
• Mrs. Gauri Bhat, a graduate student at NCL, India has been working with Dr.
Vincent Franceschi, school of Biological Sciences, WSU to receive training on Agrobacterium tumefacience mediated transformation of chickpea
Training at University of Durhum, UK • Dr. N. Chougule, Research Associate, NCL has been working with Prof. J. Gatehouse on lectin gene and protease genes for a period of nine months Training at NCL, Pune, India • A team of five graduate students has been working on host- pest / host-
pathogen interaction at NCL. Apart from these regular students, many M.Sc. students carry out project on chickpea at NCL towards partial fulfillment of M.Sc. degree
Publications
PUBLICATIONS 1. Srinivasan A., Giri A.P., Harsulkar A.M., Gatehouse J.A.and Gupta V.S. (2005) A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) that exerts anti- metabolic effect on pod borer (Helicoverpa armigera) larvae", Plant Molecular Biology. (Accepted) 2. Srinivasan A, Chougule NP, Giri AP, Gatehouse JA, Gupta VS (2005) Adaptive
Responses in Helicoverpa armigera towards the Cicer arietinum Kunitz Proteinase Inhibitor. (manuscript under preparation).
3. Telang M., Giri A.P., Sainani M.N.and Gupta V.S. (2004) Characterization of two midgut proteinase of Helicoverpa armigera and their interaction with proteinase inhibitor Journal of Insect Physiology (in press) 4. Barve M.P., Santra D.K., Ranjekar P.K.and Gupta V.S. (2004). Genetic diversity
analysis of a worldwide Collection of Aschochyta rabiei isolates using sequence tagged microsatellite markers. World J. Microbiology and Biotechnology 20:735-741
5. P. N. Rajesh, Kevin McPhee, Fred J. Muehlbauer (2004) Detection of polymorphism using CAPS and dCAPS markers in two chickpea genotypes (accepted in ICPN)
6. P. N. Rajesh, Kevin McPhee, Fred J. Muehlbauer. Stability of chickpea large genomic DNA inserts in Agrobacterium. (To be resubmitted to Plant cell reports in February 2005) 7. P. N. Rajesh, Weidong Chen, VS Gupta and Fred J Muehlbauer. Functional genome analysis and molecular mapping of ascochyta blight responsive genes in chickpea (To be submitted in 2005) 8. P. N. Rajesh, Majesta Siegfried, Kevin McPhee, Bruce Roe and Fred J. Muehlbauer (2004) Genome characterization of QTL1 of ascochyta blight resistance in chickpea (Cicer arietinum L.). (To be submitted in 2005)
9. Presented posters at Plant and Animal Genome XIII conference (January 2005)
i) P. N Rajesh, Majesta Siegfried, Kevin McPhee, Bruce Roe and Fred J.
Muehlbauer. Genome characterization of QTL1 of ascochyta blight resistance in chickpea.
ii) P. N. Rajesh, Fred J. Muehlbauer, Kevin McPhee. Agrobacterium mediated transformation of a large genomic insert in chickpea.
Table 1 : Parents used in diallel mating Sr. No. Genotype Pedigree Special Features
1. Phule G-89219 ICCL-80074 x ICCC-30 High yielding potential, wilt resistant.
2. Vijay P-1270 x Annigeri Wilt resistant, drought tolerant, high yield potential suitable for rainfed, irrigated and late sown condition.
3. ICCV-10 P-1231 x P-1265 Wilt resistant, drought tolerant, high yield.
4. Phule G-12 GW-5/7 x Ceylon-2 Wilt resistant, suitable for rainfed as well as irrigated conditions.
5. Phule G-91028
(K-850 x BN-31) x MSDP-62
High yield potential.
6. Vishal K-850 x ICCL-80074 Attractive yellow colour, bold seeds, wilt resistant and high yield.
7. ICC-4958 GW-5/7 Drought tolerant, bold seeds.
8. Bheema Selection from RPR-30-43-1-2-2-18-1
Bold seeds.
Table 2 : Grain yield (kg/ha) performance of chickpea variety Phule G -9425- 5 in various trials
Mean 28 1414 1402 1236 0.86 14.4 Late sown 2000-01 Regional Varietal
Trial 2 2385 1929 2440
2001-02 State Multilication vareital trial
3 2157 1742 1809
2002-03 State Multilication vareital trial
3 2232 1915 1750
2003-04 State Multilication vareital trial
3 1987 1639 1644
Mean 11 2173 1795 1805 21.06 20.39 General Mean 76 1918 1676 1628 14.44 17.81
Note : 1. In 37 trials under irrigated condition the genotype Phule – 9425-5 show 20% higher yield than Vijay and 13% than Vishal 2. In 11 trials under late sown condition Phule G- 9425-5 showed 21% and 20% higher yield over Vijay and Vishal, respectively. 3. In rainfed trial it was equivalent to Vijay but 14% higher yield than Vishal.
Table 3 : Duration and morphological characters at Rahuri : 2001-02 to 2003- 04 Sr. No
Character/Variety Phule G- 9425-5
Vijay (ch) Vishal (ch)
1 Days to 50% flowering 42-46 38-43 43-48 2 Days to maturity 90-105 93-110 109-114 3 Plant height (cm) 34-43 25-30 33-38 4 Plant spread (cm) 12-16 15-20 12-16 5 Number of fruiting
branches / plant 9-11 9-12 6-10
6 Number of pods /plant 28-33 34-45 25-32 7 100- grain weight (g) 23-25 18-20 28-30
Table 4 : Quality parameters of chickpea genotypes grown at Pulses
Table 8 : Consumers preference for grain quality Character/ Variety Phule G 9425-5
Vijay Vishal
Size 3.1 2.3 3.8 Shape 3.4 1.8 3.4 Colour 3.1 2.3 3.7 Mean 3.2 2.1 3.6 Judged by 10 persons using 1 to 4 scale, where 4: Excellent, 3: Good, 2: Fair and 1: Poor Table 9: Prevailing market rates of chickpea varieties as on 27.04.2004 at Agril. produce Market Committee, Rahuri, Dist. Ahmednagar Sr. No. Variety Rate (Rs/q)
1 Phule G-9425-5 1525/- 2 Vijay 1425/- 3 Vishal 1525/- Table 10 : Yield performance (kg/ha) of Phule G-9425-5 in Farmers Participatory Programme of Mcknight Project (2003-04
Yield kg/ha Sr.
No. Name of farmers and Address Soil type
PG-9425-5
Vijay
1 Shri. R.R. Chavan Deolalipravara, A’nagar
Medium Black 2200 1751
2 Shri C.B. Taware, Deolalopravara, A’nagar
Medium Black 1540 1240
3 Shri Vilas Tarade, Deolalipravara, A’nagar
Medium Black 1880 1370
4 Shri M.K. Pawar, Khudsargaon, A,nagar Deep Black 1825 2235 5 Shri.B.G. Pawar, Khudsargaon, A,nagar Deep Black 2060 1650 6 Shri.S.D. Dethe, Khudsargaon, A,nagar Deep Black 1650 1050 7 Shri.B.G. Dethe, Khudsargaon, A,nagar Deep Black 1570 1300 8 Shri.R.L. Pawar Khudsargaon, A,nagar Deep Black 2050 1967 9 Shri. Balkrishna.J. Kolse, Ambi, A,nagar Medium Black 2470 1970 10 Shri. Bhagwat.J. Kolse, Ambi, A,nagar Medium Black 1860 1367 11 Shri.B.K Kolse, Ambi A,nagar Medium Black 2280 1960 12 Shri. Amit Gugale Kolhar Khurd, A,nagar Deep Black 2875 2330 13 Shri. B.S. Patil Kolhar Khurd, A,nagar Shallow 2000 1500 14 Shri.D.G. Patil Kolhar Khurd, A,nagar Shallow 1970 1730 15 Shri.D.T. Khose, Hingani, Shrigonda,
A,nagar Medium Black 1750 1375
16 Shri.P.E. Khise, Wadu Khurd, Pune Medium Black 1654 1530 17 Shri. H.N. Khise, Wadu Khurd, Pune Deep Black 2508 2371
18 Shri.N.D. Kand, Loni Kand, Pune Deep Black 2298 2516 19 Shri.S.R. Rokade, shivapur, Haveli, Pune Shallow 800 600 20 Shri.S.G. Haralikar, Gadhinglaj, Kolhapur Medium Black 2500 1500 21 Shri.C.I. Moldi, Gadhinglaj, Kolhapur Deep Black 2910 2050 22 Shri.S.S. Vandi, Gadhinglaj, Kolhapur Deep Black 1667 834 23 Shri.G.T. Patil Mehergaon Dhule Sandy loam 1068 1192 24 Shri.S.I. Bhamre, Mehergaon, Dhule Shallow 1120 1005 25 Shri.P.D.Shinde, Kavathi, Dhule Medium Black 1550 1750 26 Shri.G.H. Patil, Kavathi, Dhule Medium Black 1900 1400 27 Shri.S.P. Shinde, Kavathi, Dhule Medium Black 1750 1250 28 Shri. P.B. Patil, Padawat, Dhule Shallow 1200 1000 29 Shri.R.A. Patil Chirai, Nashik Clay loam 3500 3250 30 Shri.N.D. Patil Chirai Nashik Sandy loan 2020 1650 31 Shri.M.B. Patil Chirai, Nashik Sandy loan 2520 2105 32 Mrs.M.D. Jadhav Nandgaon, Nashik Medium Black 2420 1933 33 Mrs.P.S. Jadhav, Nandgaon Nashik Medium Black 1900 2050 34 Mrs.H.D. Tarade, Nandgaon, Nashik Deep Black 2299 1562 35 Shri. M. K. Ghare Kaluste, Nashik Medium Black 2150 1700 36 Shri. A.N. Ghare, Kaluste, Nashik Sandy loan 1778 1290 37 Shri. V.T. Nakat Tandali Bk, Akola Deep Black 1282 855 38 Shri.S.B. Wankhede, Alewadi, Akola Deep Black 1027 1010 39 Shri. M.S. Jadhav, Kasegaon Sangali Medium Black 850 950 40 Shri. P.D.Patil,Kasegaon Sangli Medium Black 990 810 41 Shri. P.H. thosare Mera Bk. Buldhana Medium Black 1128 1110 42 Shri. B.R. Patil, Sawargaon Buldhana Medium Black 1310 1050 43 Shri. N.S. Gupta, Chikhali, Buldhana Medium Black 1415 1190 44 Shri. P.M. Gupta, Buldhana Medium Black 1533 1067 45 Shri. S.N. Ambhore, Selgaon, Jalana Deep Black 2110 2000 46 Shri. S.P. Ambhore, Selgaon, Jalana Deep Black 2619 2750 47 Shri. A.K. Ambhore, Selgaon, Jalana Deep Black 2500 2670 48 Shri. V.P. Kapadia, Vichvad, Junagarh,
Gujarat Medium Black 2778 3272
49 Shri. K.K. Hirpara, Navania, Junagarh, Gujarat
Medium Black 1587 2222
50 Shri. S.S. Hirpara, Navania, Junagarh, Gujarat
Medium Black 1852 2315
51 Shri. G.L. Asodaria, Tadaka-Pipalia, Gujarat
Medium Black 2667 1926
Mean (kg/ha) 1905 1657 Per cent increase over Vijay 14.97 --
Table 11 : Group averages of chickpea RIL's for agronomic characters and reaction to Fusarium wilt at Pulses Improvement Project MPKV, Rahuri during rabi 2004-05
(Average of 8 RIL's in each group) Cross & Group no.
Table 16: Summary of work done at AAU using chimeric α ai gene
PCR using transgene
specific primers
T0 lines
T0 T1 + : -
Protein dot blot (T2)
+ : -
ICCV 89314 3 (1)* + 6 : 1 (1)**
In progress
Vijay 3 +
* Figure in parenthesis indicates number of lines producing T1 progeny ** Figure in parenthesis indicates number of T0 lines used for analyses of their progenies Table 17: Summary of crossing experiments done at AAU and ICRISAT using
40D line Crosses Seeds obtained With Desi cultivars
1. Vijay X 40D 2. JG 11 X 40D 3. ICCC 37 X 40D
25 25 7
With Kabuli cultivars
1. Chefe X 40D 2. KAK 2 3. JGK 1
25 16 30
Table 18 : Contribution of markers to agronomic traits
Agronomic Trait Major contributing markers Total contribution (%)