Chickpea: Year 3: Increasing the efficiency of chickpea production

65
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

Transcript of Chickpea: Year 3: Increasing the efficiency of chickpea production

Page 1: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 2: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 3: Chickpea: Year 3: Increasing the efficiency of chickpea production

Executive Summary

Page 4: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 5: Chickpea: Year 3: Increasing the efficiency of chickpea production

Research Objectives

Page 6: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 7: Chickpea: Year 3: Increasing the efficiency of chickpea production

Research Progress Report

Page 8: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 9: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 10: Chickpea: Year 3: Increasing the efficiency of chickpea production

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)

Testing PG-9426-2, PG-9409-1 AVT-1 (R ) 6 locations CZ in Co-ordinated progarmme

1995 1996

1996 1997

1997 1998

1998 1999

1999 2000

2000 2001

2001 2002

2002 2003

2003-2004

2004-2005

Page 11: Chickpea: Year 3: Increasing the efficiency of chickpea production

A significant achievement

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.

Page 12: Chickpea: Year 3: Increasing the efficiency of chickpea production

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)

Page 13: Chickpea: Year 3: Increasing the efficiency of chickpea production

conditions.

9 a) Agency responsible for maintaining breeder seed

: Principal Scientist Pulses Improvement Project MPKV, Rahuri, Dist – Ahmednagar

b) Quantity of breeder seed in stock

: 3.00qtl.

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

of Cow dung + Vermicompost + Cow urine + Rhizobium + Trichoderma + PSB

T1 : Control T2 : Seed treatment T3 : Seed treatment + Vermiwash spray @ 50 ml /lit T4 : Seed treatment + Cow urine spray @ 50 ml /lit T5 : Seed treatment + NAA spray 20ppm @ 20 ml /lit T6 : Seed treatment + Sea weed extract spray @ 2

ml /lit

[Spraying thrice: - Pre – flowering, flowering and pod filling stage]

Page 14: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 15: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 16: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 17: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 18: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 19: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 20: Chickpea: Year 3: Increasing the efficiency of chickpea production

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).

Page 21: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 22: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 23: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 24: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 25: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 26: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 27: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 28: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 29: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 30: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 31: Chickpea: Year 3: Increasing the efficiency of chickpea production

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).

Page 32: Chickpea: Year 3: Increasing the efficiency of chickpea production

• 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.

Page 33: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 34: Chickpea: Year 3: Increasing the efficiency of chickpea production

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)

Page 35: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 36: Chickpea: Year 3: Increasing the efficiency of chickpea production

Training Report

Page 37: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 38: Chickpea: Year 3: Increasing the efficiency of chickpea production

Publications

Page 39: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 40: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Page 41: Chickpea: Year 3: Increasing the efficiency of chickpea production

Table 2 : Grain yield (kg/ha) performance of chickpea variety Phule G -9425- 5 in various trials

Yield (kg/ha)

% increase of Phule G-9425-5

over

Year

Trial

No.of trials/

locations

Phule G-9425-5

Vijay (ch)

Vishal (ch)

Vijay (ch)

Vishal (ch)

Irrigated 1999-00 Station Trial 1 3132 -- 1667 2000-01 Regional Varietal

Trial 2 1859 1508 1739

2001-02 State Multilocation Varietal trial

7 2407 1835 1950

2002-03 State Multilocation Varietal trial

8 2406 1954 2160

2003-04 State Multilocation Varietal trial

9 2224 -- 1896

2002-03 Initial Varietal Trial (AICRP Trial)

10 1931 1813 (SAKI-9516)

--

Mean 37 2224 1853 1968 20.02 13.01 Rainfed 2000-01 Regional Varietal

Trial 1 903 852 958

2001-02 State Multilocation Varietal trial

10 1375 1319 1097

2002-03 State Multilication Varietal trial

10 1195 1185 1041

2003-04 State Multilication vareital trial

7 1855 1910 1751

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.

Page 42: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Improvement Project , M.P.K.V, Rahuri Sr.No

Genotype Milling quality Cooking period (Min.)

Protein%

Clean dhal (%)

Broken dhal (%)

Churi + Gota (%)

Husk + whole seeds

1 Phule G 9425-5

67.4 8.6 5.2 18.8 22 23.4

2 Vijay (ch) 60.4 15.3 6.8 17.5 20 24.8 3 Vishal (ch) 78.7 6.0 4.3 11.0 24 28.5 Phule G-5(ch) 73.1 7.7 2.8 16.9 23 25.6 Phule G-

12(ch) 59.1 12.8 4.1 24.9 22 26.9

Table 5 a : Reaction to Fusarium wilt in wilt sick plot at M.P.K.V., Rahuri : 2000-01 to 2003-04. Sr.No Genotypes Per cent wilt 2000-01 2001-

02 2002-03 2003-04 Mean

1 Phule G- 9425-5 4.00 8.21 7.47 9.92 7.40 2 Vijay 8.62 7.85 58.55 62.07 34.27 3 Vishal 0.00 7.29 5.71 6.15 4.79 4 Phule G -5 87.71 -- -- 89-06 88-39 5 JG-62(ch) 100.00 100.00 100.00 100.00 100.00

Page 43: Chickpea: Year 3: Increasing the efficiency of chickpea production

Table 5 b : Reaction to Helicoverpa armigera at MPKV, Rahuri (2000-01 to 2003-04).

% pod damage Sr. No.

Genotype 2001-02 2002-03 2003-

04 Mean

PSR % PS

1 Phule G- 9425-5 6.88 21.51 29.15 19.18 5 12.61 2 Vijay 14.22 18.58 33.05 21.95 3 Vishal 11.45 21.14 35.07 22.55

Pest susceptibility rating : Up to 5 : Less susceptible than check.

More than 6 : More susceptible than check

%PD in check entry - % PD in test entry % Pest susceptibility = ------------------------------------------------------------- x 100

%PD in check entry Pest susceptibility rating (PSR) % pest susceptibility (PS)

1 100 2 75 to 100 3 50 to 75 4 25 to 50 5 10 to 25 6 -10 to -10 7 -25 to -10 8 -50 to -25 9 -50 or less (PD = Pod damage)

Page 44: Chickpea: Year 3: Increasing the efficiency of chickpea production

Table 6: Grain yield (kg/ha) and drought characters of different chickpea genotypes (2003-04)

I0 : Moisture stress I1 : Irrigated condition

Yield (kg/ha)

Membrane injury index

Harvest index

Sr. No

Genotype

I0 I1

% reduction in yield

DTE %

DSI %

I0 I1 I0 I1

1 Phule G-9425-5 1215

1555

21.86 78.1 0.90 0.15 0.10 38.3 37.0

2 Phule G-9426-2 1291

1687

23.47 76.5 0.96 0.17 0.14 38.5 36.9

3 RSG-888 757 896 15.51 84.5 0.64 0.21 0.14 39.2 38.6 4 IPC-9767 102

8 1354

24.08 75.9 0.99 0.20 0.14 37.8 38.1

5 Phule G-96006 1236

1458

15.23 84.8 0.62 0.19 0.13 37.2 38.2

6 C-235 646 979 34.01 66.0 1.39 0.21 0.14 40.1 38.7 7 Phule G-5 736 108

3 32.04 67.9 1.31 0.19 0.16 36.6 36.3

8 RSG-143-1 757 1155

32.70 67.3 1.34 0.15 0.15 34.0 38.9

Table 7 : The grain yield of rainfed chickpea as influenced by seed and different foliar sprays

Treatments Mean grain yield ( kg /ha)

T1 Control 1458 T2 Seed treatment 1527 T3 Seed treatment + Vermiwash spray @ 50

ml /lit 1733

T4 Seed treatment + Cow urine spray @ 50 ml /lit

1547

T5 Seed treatment + NAA 20ppm @ 20 ml /lit 1316 T6 Seed treatment + sea weed extract @ 2 ml

/lit 1843

SE+ : 70.2 CD at 5% : 211 CV % : 8.94

Page 45: Chickpea: Year 3: Increasing the efficiency of chickpea production

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

Page 46: Chickpea: Year 3: Increasing the efficiency of chickpea production

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 --

Page 47: Chickpea: Year 3: Increasing the efficiency of chickpea production

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.

Days to maturity

Height (cm)

Spread (cm)

Secondary branches / Plant

Pods / Plant

Seed/ Pod

100 Seed wt.(g)

Yield / Plant

AUDPC value (Race-1)

AUDPC value (Race-4)

Cross I (JG-62 x Vijay) Group 1 113 31 18 8 27 1 13 5 1738 2286Group 2 109 35 17 8 27 2 16 6 2013 3201Group 3 105 34 17 9 32 1 13 6 2554 3488Group 4 109 30 14 6 17 1 14 4 2388 2764Group 5 107 35 18 9 28 1 16 4 2939 3363Group6 107 36 22 10 39 1 14 6 2920 4066Group7 109 37 18 10 30 1 12 4 2586 3884Group 8 109 36 19 9 44 1 14 8 3229 3642Group 9 111 39 21 8 59 1 19 11 3304 3513Group 10 112 35 16 7 28 1 17 5 3430 3681Group 11 110 32 16 7 16 1 13 2 3353 2233Group 12 107 36 17 8 34 2 13 4 4186 2911Group 13 107 30 15 8 22 1 13 2 3808 3811Group 14 107 32 16 8 21 1 14 3 4364 3130Group 15 107 35 24 12 42 1 14 7 4687 3095Group 16 110 34 22 16 28 1 14 4 4320 2890Group 17 97 31 18 10 42 1 13 10 4613 2909Group 18 105 38 22 10 51 1 17 11 4733 3491Group 19 110 33 18 7 21 1 16 3 4839 3101Group 20 109 33 15 7 19 1 14 3 4750 1909Group 21 109 36 19 8 31 1 15 6 5208 3765Group 22 111 35 15 6 19 1 13 4 4578 3203Group 23 114 38 19 8 32 1 14 3 4977 3048Group 24 112 38 22 9 45 1 17 10 5326 3133Group 25 105 38 22 11 53 1 14 10 5660 1783JG-62 109 40 20 12 30 1 14 8 5812 6142Vijay 97 34 19 9 32 1 19 10 303 204

Page 48: Chickpea: Year 3: Increasing the efficiency of chickpea production

Cross II (JG-62 x ICC-4958) Group 26 113 40 21 9 42 1 22 9 2242 2494Group 27 111 44 23 10 39 1 23 8 2649 2766Group 28 114 40 25 11 53 1 23 13 3538 2892Group 29 111 41 27 11 47 1 20 12 3607 2752Group 30 113 43 21 11 52 1 23 9 3356 2363Group 31 112 39 18 8 34 1 18 10 3987 2603Group 32 113 41 19 10 40 1 18 8 4047 2563Group 33 106 38 20 10 48 1 22 12 4397 1813Group 34 109 42 21 9 60 1 25 14 4490 2966Group 35 115 42 22 12 53 1 23 13 4534 2116Group 36 107 38 22 12 49 1 21 9 5027 1787Group 37 0 0 0 0 0 0 0 0 5138 3238Group 38 113 39 23 11 58 1 17 8 4974 1695Group 39 112 43 19 9 43 1 22 11 5579 2406Group 40 113 40 19 11 45 1 22 8 5298 2967Group 41 111 44 20 9 39 1 22 17 5306 3750JG-62 109 40 20 12 30 1 14 8 5812 6142ICC-4958 119 43 22 11 30 1 28 12 903 477Group 42 111 36 17 10 36 1 22 8 1999 1461Group 43 113 36 19 10 36 1 20 8 1083 541Group 44 116 38 18 9 34 1 25 6 1361 1642Group 45 115 41 19 10 44 1 25 11 1463 871Group 46 116 39 20 11 39 1 24 8 2033 839Group 47 117 42 20 10 36 1 21 8 1844 1059Group 48 115 43 18 10 36 1 25 8 1903 1369Group 49 116 40 17 9 37 1 21 6 1662 1350Group 50 116 35 16 8 34 1 27 7 2196 1972Group 51 117 36 17 10 34 1 21 7 2035 916Group 52 113 39 18 10 45 1 29 12 2359 2280Group 53 116 43 19 10 35 1 25 11 2444 1866Group 54 117 41 16 9 34 2 27 8 2510 1871Group 55 113 41 17 9 41 1 26 12 2387 1136Vijay 97 34 19 9 32 1 19 10 303 204ICC-4958 119 43 22 11 30 1 28 12 903 477

Page 49: Chickpea: Year 3: Increasing the efficiency of chickpea production

Table 12 : Grouping of Chickpea RIL’s according to AUDPC values Fusarium oxysporum ciceri, race-4

No. of RIL’s Sr. No.

Genotypes More resistant than Vijay

More resistant than ICC-4958

Less Susceptible than JG-62

More susceptible than JG-62

JG-62 x Vijay 1 0 195 1 1 AUDPC 0 0 562-5250 7750 JG-62 x ICC-4958 2 2 116 2 2 AUDPC 0 281 562-5550 2750 - 9000 ICC- 4958 x Vijay 11 18 79 0 3 AUDPC 37 - 202 229 - 476 501 - 4070 --

Genotype Reaction to wilt AUDPC Value Vijay : Resistant : 204 ICC-4958 : Late wilter : 477 JG-62 : Highly Susceptible : 6142 Table 13 : Grouping of Chickpea RIL’s according to AUDPC values for Fusarium oxysporum ciceri, race-1

JG-62 x Vijay Year

JG-62 x ICC-4958 Year

ICC- 4958 x Vijay Year

Sr. No

2003-04 2004-05 2003-04

2004-05 2003-04 2004-05

No. 3 0 1 0 1 1 1 More resistant than Vijay

AUDPC 498 -

529

---- 426 ---- 412 274 - 303

No. 8 1 3 0 4 11 2 More resistant than Icc-4958

AUDPC 529 -

841

710 - 903

614 - 802

---- 656- 841

354 - 884

No. 186 195 120 119 98 96 3 Less Susceptible than JG-62

AUDPC 854 -

5523

935 - 5802

688 - 5571

1114 - 5782

863 - 5419

910 - 3761

No. ---- 1 ---- 3 5 0 4 More susceptible than JG-62

AUDPC ---- 5802 - 8420

---- 5871 - 7727

5884 - 7874

----

AUDPC Value Genotype Reaction to wilt

2003-04 2004-05 Vijay Resistant 529 303 ICC-4958 Late wilter 841 903 JG-62 Highly Susceptible 5625 5812

Page 50: Chickpea: Year 3: Increasing the efficiency of chickpea production

Table 14 : Categories of RIL’s according to yield per plant

Number of RIL Sr.No

Cross Higher than Vijay Higher than JG-62 Higher than ICC- 4958

1 JG-62 x Vijay

38,125,47,96,154,130, 132,122,185,120,78, 164,177,99,195,61,117, 16,66,191,12,35,110, 179,184,123,133,196, 37,76

38,19,2,45,94,125,46,10147,17,96,154,131,132,122,185,120,78,164,22,82,44,10,72,177,116,66,191,12,35,188,146,139,145,110,179,184,123,67,283,133,196,37,197,76

38,154,120,78,164,177,99,195,61,66,191,12, 110,76

2 JG-62 x ICC- 4958

36,93,48,78,33,50,118,11,6,69,15,115,10,74, 62,108,87,30,2,103,91,54,12,113,72,3,9,37,66,22,101,112,97,109,95,65,105,5,42,111,64,1, 79,1122,40,116,76,29, 85,28,63,67,56,45,44, 57

36,16,93,18,48,94,78,33,50,46,20,118,98,11,6,69,15,115,10,74,53,62,108,87,30,2,103,31,7,91,54,107,12,26,113,72,3,9,37,66,49,22,101,112,97,109,14,95,65,105,5,42,111,64,1,79,122,40,116,77,76,29,85,28,58,73,63,80,67,35,56,120,45,25,44,57

48,50,118,11,69,15, 115,10,62,87,30,2,31, 91,54,12,113,72,9,37, 66,22,101,112,97,109, 95,105,5,42,64,1,122, 40,116,76,85,67,45,44

3 ICC- 4958 x Vijay

67,94,52,38,58,10,16, 28,71,47,27,1,74,102,3,41,37,85,17,24,50,72, 39,69,70,9,100,101,61,15,78,81,56,65,2,23

49,31,89,67,94,106,59,9557,84,42,68,52,38,58,10,16,28,80,21,22,71,62,98,47,27,1,7,74,102,3,41,37,85,53,17,24,25,93,108,50,48,72,3966,43,30,69,70,9,14,100,101,6,61,15,78,81,56,65,2,23

67,94,52,58,10,71,27, 32,37,50,72,43,70,101,61,2,23

Page 51: Chickpea: Year 3: Increasing the efficiency of chickpea production

Table 15

Evaluation of chickpea RIL's for agronomic characters and reaction to Fusarium wilt at Pulses Improvement Project MPKV, Rahuri during rabi 2004-05

Cross & Group no.

RIL Days to maturity

Height (cm)

Spread (cm)

Secondary branches / Plant

Pods / Plant

Seed/ Pod

100 Seed wt.(g)

Yield / Plant

AUDPC value (Race-1)

AUDPC value (Race-4)

Cross I (JG-62 x Vijay) 15 0 0 0 0 0 0 0 0 1439 208329 112 23 14 4 9 1 21 1 3196 172585 0 0 0 0 0 0 0 0 1145 1969

106 119 25 14 5 19 1 0 3 711 491741 114 37 20 10 40 1 14 7 936 2464

100 113 34 20 9 34 1 15 7 2199 135026 116 33 17 9 32 1 12 5 1729 2063

1

43 103 34 22 8 29 1 15 5 2551 1714 Total 677 187 107 46 163 7 77 27 13905 18285 Mean 113 31 18 8 27 1 13 5 1738 2286

87 103 35 15 6 36 5 13 4 1414 4650157 109 35 17 7 17 1 15 3 3040 1312103 109 38 15 6 16 1 24 3 2136 4050

86 102 35 15 6 18 1 15 5 2342 5250134 115 37 20 12 36 1 16 6 1303 3750107 113 37 19 11 43 1 13 7 2435 563

3 111 30 14 7 23 1 14 7 2194 2500

2

38 113 35 24 9 26 1 16 13 1239 3536 Total 875 282 139 65 215 12 126 48 16102 25610 Mean 109 35 17 8 27 2 16 6 2013 3201

166 102 34 18 6 24 1 12 2 3391 405020 98 33 19 9 26 1 12 4 4106 439319 115 38 15 11 42 1 14 8 1888 3000

2 106 37 16 10 5 1 13 9 2943 300088 103 31 16 7 30 1 12 5 1996 196945 103 31 18 7 30 1 12 5 2883 285016 107 33 16 9 51 1 13 8 1258 4250

3

94 102 32 17 10 48 1 14 10 1965 4393 Total 836 269 135 69 256 8 102 51 20430 27904 Mean 105 34 17 9 32 1 13 6 2554 3488

97 113 32 15 7 22 1 15 3 1772 243839 114 32 15 7 20 1 15 3 2362 3000

142 109 31 17 6 15 1 16 3 2181 90089 103 29 13 5 14 1 15 2 3035 2550

152 116 33 13 5 20 1 10 3 2841 28934 109 29 15 5 17 1 12 2 1972 1500

125 103 24 12 5 14 1 15 16 2862 3583

4

104 103 28 15 4 15 1 14 2 2077 5250 Total 870 239 116 46 138 8 112 34 19102 22114 Mean 109 30 14 6 17 1 14 4 2388 2764

Page 52: Chickpea: Year 3: Increasing the efficiency of chickpea production

161 102 28 12 8 25 1 15 3 1318 405031 109 30 13 7 15 1 13 2 2718 375069 102 30 12 6 20 1 15 3 4285 525046 102 34 22 12 54 1 14 9 2013 262554 116 38 18 8 22 1 27 5 2997 225058 110 42 22 12 38 1 12 3 2479 3250

174 113 39 20 9 30 1 12 4 3392 2625

5

138 101 38 21 7 21 1 16 4 4309 3107 Total 855 280 141 68 225 8 124 34 23511 2690

7 Mean 107 35 18 9 28 1 16 4 2939 3363

101 110 40 19 10 47 1 12 9 2401 3750105 110 36 18 9 33 1 14 6 2660 525093 106 32 21 8 35 1 13 5 3077 375098 106 36 15 8 29 1 13 4 3269 525074 112 37 20 10 27 1 16 7 2757 412547 99 34 36 16 76 1 14 10 4216 450048 102 37 22 9 31 1 15 3 3359 1688

6

112 109 35 24 10 33 1 14 6 1623 4219 Total 854 286 176 79 312 8 111 51 2336

4 32531

Mean 107 36 22 10 39 1 14 6 2920 406628 113 40 22 11 27 1 14 5 2643 353691 109 38 19 11 40 1 12 4 2875 4500

108 106 37 16 9 23 1 11 3 1229 482114 100 33 13 6 15 1 10 2 3070 375071 108 37 19 12 38 1 11 6 2783 2625

189 108 34 26 9 36 1 10 4 2756 435050 109 36 18 12 38 1 13 5 2454 2667

7

79 116 41 9 8 23 1 12 5 2878 4821 Total 869 295 141 77 240 8 93 34 2068

9 31070

Mean 109 37 18 10 30 1 12 4 2586 3884167 114 36 21 10 31 1 16 5 4227 3750129 109 33 19 7 26 1 11 3 2782 4500190 113 36 19 8 35 1 15 6 3407 2344147 111 38 17 6 37 1 15 6 2779 157517 102 33 20 11 43 1 16 9 4411 525096 106 37 18 10 64 1 16 11 2568 5250

154 106 36 20 12 69 1 10 13 2228 2344

8

131 110 39 20 8 47 1 16 10 3434 4125 Total 871 289 155 71 352 8 115 62 2583

6 29138

Mean 109 36 19 9 44 1 14 8 3229 3642

Page 53: Chickpea: Year 3: Increasing the efficiency of chickpea production

102 114 38 19 8 31 1 21 7 2925 3750132 106 35 24 7 66 1 18 10 3214 2250122 106 43 22 11 102 1 18 17 2602 3750185 108 40 20 10 65 1 18 11 3614 2625120 113 40 21 6 59 1 18 13 3281 337578 113 40 14 9 49 1 22 15 3465 2357

164 115 40 28 10 73 1 18 13 3606 5250

9

159 114 40 18 7 27 1 16 5 3727 4750 Total 889 315 166 67 472 8 149 92 26435 28107 Mean 111 39 21 8 59 1 19 11 3304 3513

160 113 36 17 7 31 1 16 7 5149 225022 112 36 17 8 39 1 17 8 3440 412582 113 39 21 7 39 1 16 9 2779 439395 113 32 13 6 22 1 16 5 3406 4393

111 113 37 15 7 33 1 16 5 3779 439332 103 31 16 7 24 1 18 5 2911 225042 114 37 14 7 20 1 18 4 3646 3250

10

90 115 35 14 7 16 1 15 0 2327 4393 Total 896 283 127 56 224 8 132 43 27438 29446 Mean 112 35 16 7 28 1 17 5 3430 3681

55 108 32 13 5 13 1 14 2 2809 583186 116 35 17 8 13 1 16 2 2659 1875169 0 0 0 0 0 0 0 0 4744 4250

1 109 31 21 9 28 1 12 5 2814 33757 108 25 14 5 15 1 0 2 3054 1406

137 101 32 15 7 16 1 16 2 3866 337533 116 32 18 8 18 1 14 2 3270 1125

11

140 109 35 13 5 12 1 20 2 3610 1875 Total 767 222 111 47 115 7 92 15 26827 17865 Mean 110 32 16 7 16 1 13 2 3353 2233

80 106 38 13 8 27 1 18 5 3463 18448 101 36 11 5 32 1 0 2 4678 1875

135 102 32 15 7 20 5 18 4 4649 158318 113 35 16 8 37 1 16 4 4014 4219

158 113 39 27 14 54 1 14 7 3956 4393144 109 34 16 7 34 1 14 3 4384 3250109 0 0 0 0 0 0 0 0 4219 3375

12

62 0 0 0 0 0 0 0 0 4124 2750 Total 644 214 99 50 205 10 80 24 33486 23289 Mean 107 36 17 8 34 2 13 4 4186 2911

Page 54: Chickpea: Year 3: Increasing the efficiency of chickpea production

92 109 33 17 7 32 1 13 3 3226 375060 109 26 14 5 16 1 10 2 3879 3107

119 103 28 17 7 25 1 11 1 4610 396481 103 34 18 8 28 1 12 3 4563 900

153 109 32 15 6 21 1 17 4 3336 300021 110 27 14 6 17 1 14 2 3611 4050

156 102 32 15 18 29 1 16 2 4407 3964

13

57 108 26 8 4 11 1 14 1 2831 7750 Total 853 239 117 61 178 8 107 18 30464 30485 Mean 107 30 15 8 22 1 13 2 3808 3811

118 114 32 14 7 12 1 16 2 3319 4650170 108 25 17 6 14 1 14 2 4820 3964

5 102 28 14 5 12 1 10 1 4898 3000127 102 28 13 5 12 1 11 1 5188 3536171 110 37 19 15 21 1 21 5 2387 325023 109 35 13 6 28 1 12 3 5389 25 110 34 15 8 26 1 16 4 4300 2438

14

176 102 39 26 12 42 1 13 5 4613 2850 Total 857 258 130 64 168 8 113 23 34914 25038 Mean 107 32 16 8 21 1 14 3 4364 3130

44 115 31 25 12 45 1 15 9 4183 2250150 108 34 23 10 46 1 14 7 4009 225010 113 41 29 18 71 1 17 8 4723 964

136 109 36 22 12 33 1 11 6 5142 3964163 101 34 22 10 37 1 12 6 5156 4393165 102 39 25 10 34 1 14 7 4738 5250115 0 0 0 0 0 0 0 0 5771 3750

15

49 103 31 21 10 30 1 15 5 3776 1938 Total 751 246 166 81 295 7 98 47 37499 24759 Mean 107 35 24 12 42 1 14 7 4687 3095

52 0 0 0 0 0 0 0 0 4581 3250193 0 0 0 0 0 0 0 0 5279 1636155 0 0 0 0 0 0 0 0 4234 405059 109 31 28 25 10 1 13 1 4262 3375

16

192 108 34 16 7 17 1 12 2 4501 96472 113 38 23 17 56 1 18 10 3474 4875

151 0 0 0 0 0 0 0 0 3883 2250

11 0 0 0 0 0 0 0 0 4345 2719 Total 330 103 66 49 84 3 43 12 34558 23119 Mean 110 34 22 16 28 1 14 4 4320 2890

1350

Page 55: Chickpea: Year 3: Increasing the efficiency of chickpea production

77 0 0 0 0 0 0 0 0 4933 1667177 116 47 29 17 90 1 14 18 5190 135073 113 39 19 11 23 1 16 9 4796 412599 112 44 20 10 51 1 15 13 4309 1406

195 112 41 19 11 53 1 16 14 3220 202561 114 35 21 11 43 1 16 13 4791 3375

117 113 8 19 10 51 1 12 12 4957 4821

17

187 98 38 20 9 27 1 17 3 4711 4500 Total 778 251 147 79 339 7 106 81 36906 23269 Mean 97 31 18 10 42 1 13 10 4613 2909

53 100 39 19 9 27 1 15 4 5226 5250116 102 35 19 10 61 1 15 10 4295 384466 103 41 23 13 73 1 15 20 4624 1312

191 110 38 25 10 57 1 15 15 4358 440612 115 44 29 13 72 1 18 13 4360 364335 108 38 21 9 51 1 15 10 5197 2875

188 100 37 24 13 44 1 15 10 4833 2850

18

84 98 30 18 5 20 1 26 3 4971 3750 Total 836 301 177 83 405 8 134 85 37865 27930 Mean 105 38 22 10 51 1 17 11 4733 3491

149 103 35 13 6 29 1 13 6 5472 425056 110 30 21 7 20 1 13 3 5242 2250

141 113 36 20 7 16 1 15 1 5096 450065 115 33 19 8 23 1 18 3 4940 2025

162 113 36 18 10 27 1 15 5 5085 2125114 106 41 21 7 26 1 15 3 4016 1688173 107 28 17 5 15 1 20 1 4456 4219

19

13 113 28 15 4 13 1 15 1 4410 3750 Total 880 267 143 54 168 8 124 24 38715 24806 Mean 110 33 18 7 21 1 16 3 4839 3101

124 0 0 0 0 0 0 0 0 4338 4050168 0 0 0 0 0 0 0 0 5251 112563 0 0 0 0 0 0 0 0 5233 112540 0 0 0 0 0 0 0 0 3919 21783 102 34 16 8 16 1 13 3 4660 1200

148 109 30 15 6

20

17 1 14 2 5240 155875 110 32 14 6 14 1 12 1 4800 4875

181 115 35 17 9 29 1 15 5 4558 1125 Total 436 132 62 28 75 4 54 12 37999 15274 Mean 109 33 15 7 19 1 14 3 4750 1909

Page 56: Chickpea: Year 3: Increasing the efficiency of chickpea production

34 102 32 18 10 41 1 14 7 5268 4050146 110 39 20 8 35 1 15 9 5058 2571

21

139 113 35 22 9 31 1 13 8 5003 500024 111 35 18 8 30 1 26 7 5125 4250

113 106 41 20 7 20 1 14 3 5420 46509 113 35 19 8 33 1 12 4 5061 3000

121 0 0 0 0 0 0 0 0 5028 2679130 108 39 16 7 24 1 12 2 5701 3917

Total 763 255 133 58 214 7 105 40 41664 30117 Mean 109 36 19 8 31 1 15 6 5208 3765

175 110 37 16 7 19 1 14 1 5089 267927 115 39 17 8 26 1 14 6 5522 396451 114 39 12 7 31 1 13 6 5196 3750

172 102 37 15 7 16 1 12 6 5156 20366 106 34 16 6 23 1 10 4 4176 5155

194 110 35 17 5 7 1 14 4 2087 2438128 113 28 12 5 15 1 13 0 4649 3917

22

180 115 30 15 6 19 1 14 2 4749 1688 Total 885 279 120 51 155 8 104 30 36625 25625 Mean 111 35 15 6 19 1 13 4 4578 3203

182 116 35 18 5 20 1 15 3 4808 237536 115 38 17 9 30 1 12 6 3764 2659

145 110 40 21 9 33 1 14 8 4570 2125178 110 37 17 10 48 1 13 0 5421 112530 119 35 21 7 29 1 16 3 4520 3210

126 109 38 17 7 31 1 12 3 5597 439368 118 36 18 6 14 1 12 2 5661 5250

23

70 113 41 24 12 48 1 15 2 5479 3250 Total 910 300 153 66 253 8 109 26 39819 24387 Mean 114 38 19 8 32 1 14 3 4977 3048

110 113 40 23 9 53 1 16 12 5575 3150179 110 34 24 10 48 1 16 11 5655 2588184 114 37 19 9 47 1 16 13 4910 1125123 110 37 22 9 51 1 17 10 5543 180067 114 43 20 9 42 1 16 8 5400 4650

183 113 43 23 13 44 1 18 9 5500 2000143 114 32 19 7 19 1 16 4 5139 5250

24

133 110 40 22 8 52 1 20 11 4882 4500 Total 898 306 173 74 356 8 135 79 42604 25063 Mean 112 38 22 9 45 1 17 10 5326 3133

Page 57: Chickpea: Year 3: Increasing the efficiency of chickpea production

25 196 109 39 24 12 57 1 14 10 5662 2025 37 113 38 24 15 53 1 13 10 3735 2750 197 102 40 22 10 45 1 12 9 4678 2425 64 102 33 19 10 31 1 16 5 8420 1714 76 100 38 21 10 81 1 16 15 5803 0 Total 526 188 110 57 267 5 71 50 28298 8914 Mean 105 38 22 11 53 1 14 10 5660 1783 JG-62

109 40 20 12 30 1 14 8 5812 6142

Vijay 97 34 19 9 32 1 19 10 303 204 Cross II (JG-62 x ICC-4958)

26 36 113 44 19 9 49 1 19 10 1920 2786 16 113 44 25 10 48 1 20 9 1115 2625 93 112 28 24 11 49 1 25 10 2139 3750 17 116 43 17 8 37 1 16 6 1754 750 34 113 38 19 10 23 1 18 7 1366 2000 61 115 38 20 8 41 1 16 6 3755 3469 18 113 45 26 9 38 1 26 8 2740 2750 48 110 42 17 7 51 1 32 13 3147 1821 Total 905 323 167 72 336 8 172 70 17937 19951 Mean 113 40 21 9 42 1 22 9 2242 2494

27 92 115 47 38 12 56 1 21 0 2718 5550 94 112 45 21 8 29 1 19 8 3089 2250 78 111 44 23 9 42 1 16 11 2045 281 33 113 45 24 11 45 1 32 10 2254 1714 119 112 40 20 10 25 1 36 7 3733 2750 50 102 44 19 9 36 1 28 12 2296 1688 46 107 42 23 10 41 1 16 9 2922 3643 20 113 43 20 12 40 1 19 8 2134 4250 Total 885 351 187 80 315 8 187 65 21191 22126 Mean 111 44 23 10 39 1 23 8 2649 2766

28 27 115 42 18 5 15 1 23 3 3271 2000 118 116 40 21 11 37 1 28 13 2386 3536 98 113 38 21 10 55 1 28 9 3227 3536 11 114 39 25 13 45 1 15 17 3964 1875 68 112 46 41 19 104 1 15 7 1521 2906 6 116 36 20 9 30 1 25 12 2865 3750 69 114 42 34 15 94 1 25 30 3340 3750 15 111 41 18 10 45 1 21 13 7728 1781 Total 911 323 199 92 425 8 181 104 28302 23134 Mean 114 40 25 11 53 1 23 13 3538 2892

Page 58: Chickpea: Year 3: Increasing the efficiency of chickpea production

115 113 46 34 14 69 1 30 23 4445 287510 115 43 23 9 44 1 25 12 2724 310723 102 35 26 14 52 1 10 6 3632 375074 110 41 29 11 41 1 25 11 3088 178153 108 42 22 11 31 1 24 9 3568 225055 113 38 18 10 38 1 15 7 3771 178162 113 44 34 13 70 1 11 15 4180 1975

29

108 110 37 26 9 34 1 17 10 3449 4500 Total 884 327 213 90 378 8 156 93 28856 22020 Mean 111 41 27 11 47 1 20 12 3607 2752

87 111 40 20 14 56 1 16 12 3250 262530 112 40 21 12 53 1 18 18 4207 39642 113 45 26 12 58 1 30 23 3051 211

114 113 38 17 9 48 1 18 3 3035 375051 112 42 18 11 49 1 31 6 2843 191790 114 49 17 10 46 1 26 0 2146 93821 113 45 24 10 53 1 24 2 3325 3250

30

84 0 0 0 0 0 0 0 0 4993 2250 Total 788 298 144 78 362 7 163 64 26850 18905 Mean 113 43 21 11 52 1 23 9 3356 2363

103 113 40 18 10 37 1 21 11 3910 353631 110 39 16 8 28 1 17 16 3891 21567 112 37 17 7 35 1 20 8 4299 2813

91 113 41 15 7 29 1 25 16 3786 225054 113 42 23 11 54 1 2 14 4357 178141 113 40 24 9 45 1 21 5 3743 345060 108 35 14 8 20 1 15 0 4236 2400

31

107 113 41 15 7 21 1 22 8 3671 2438 Total 895 316 143 68 270 8 143 79 31893 20823 Mean 112 39 18 8 34 1 18 10 3987 2603

12 113 47 23 9 40 1 18 12 3233 1688117 116 36 17 12 32 1 14 4 4221 375096 115 48 16 10 41 1 20 9 4031 96426 112 39 22 12 49 1 12 8 5116 345083 113 40 14 7 30 1 26 4 3438 281275 113 41 25 12 33 1 22 6 3455 3375

121 112 38 18 8 33 1 13 5 4321 1917

32

113 113 42 20 11 60 1 20 12 4560 2550 Total 907 331 155 82 318 8 145 62 32376 20506 Mean 113 41 19 10 40 1 18 8 4047 2563

Page 59: Chickpea: Year 3: Increasing the efficiency of chickpea production

72 108 35 18 13 47 1 16 13 3750 13933 110 42 23 9 50 1 20 10 4361 37509 102 35 16 8 58 1 18 12 4911 0

37 104 39 28 14 59 1 23 15 4458 112566 108 36 16 9 44 1 24 12 4421 150049 102 40 22 9 50 1 19 9 5073 405022 109 38 20 9 46 1 24 16 3016 2125

33

8 107 36 16 7 31 1 34 6 5184 563 Total 850 301 159 78 386 8 178 93 35175 14506 Mean 106 38 20 10 48 1 22 12 4397 1813

101 109 40 24 10 69 1 18 15 5321 6750112 114 47 28 9 69 1 27 20 5214 139397 107 45 21 12 75 1 24 18 4506 1313

109 112 41 20 11 70 1 31 16 4795 166788 109 41 17 6 38 1 18 7 4204 160714 102 39 16 6 28 1 30 9 3822 437595 110 47 26 10 70 1 23 13 3424 3375

34

65 108 38 17 8 59 1 29 11 4637 3250 Total 871 338 169 70 478 8 200 109 35924 23729 Mean 109 42 21 9 60 1 25 14 4490 2966

105 113 42 23 12 58 1 20 13 4038 48215 122 46 28 15 73 1 20 18 4658 2438

86 113 37 17 10 34 1 21 6 2777 112542 114 42 19 12 56 1 21 14 5004 1875

111 113 43 21 12 31 1 32 11 4372 112564 117 40 25 11 55 1 23 12 5061 16671 116 37 25 14 54 1 14 16 5439 1625

35

79 113 45 21 8 61 1 30 10 4926 2250 Total 921 334 179 93 421 8 181 102 36275 16926 Mean 115 42 22 12 53 1 23 13 4534 2116

122 112 41 24 13 79 1 18 16 4911 267840 102 43 21 12 57 1 18 12 4633 437581 116 39 15 6 23 1 19 4 4773 1393

116 113 41 27 12 71 1 18 13 5467 28177 102 30 18 11 45 1 19 9 5167 285039 100 31 23 11 29 1 19 4 5615 56371 106 39 27 16 41 1 33 4 4270 657

36

82 0 0 0 0 0 0 0 0 5381 1500 Total 751 265 155 82 346 7 144 63 40218 14296 Mean 107 38 22 12 49 1 21 9 5027 1787

Page 60: Chickpea: Year 3: Increasing the efficiency of chickpea production

70 0 0 0 0 0 0 0 0 4518 938104 0 0 0 0 0 0 0 0 4899 2500100 0 0 0 0 0 0 0 0 5098 9000102 0 0 0 0 0 0 0 0 5626 4393110 0 0 0 0 0 0 0 0 5154 275032 0 0 0 0 0 0 0 0 5096 162543 0 0 0 0 0 0 0 0 5472 1250

37

4 0 0 0 0 0 0 0 0 5238 3450 Total 0 0 0 0 0 0 0 0 41100 25906 Mean 0 0 0 0 0 0 0 0 5138 3238

76 111 39 30 13 88 1 15 12 4959 960106 111 36 21 8 41 1 13 2 5765 166713 110 37 29 13 55 1 14 5 5287 121919 0 0 0 0 0 0 0 0 5321 089 120 39 19 11 39 1 25 5 4394 300029 110 44 18 9 49 1 22 10 5186 225059 116 38 24 10 50 1 14 6 5554 3500

38

85 113 42 23 13 80 1 18 16 3324 964 Total 791 275 164 76 403 7 121 55 39790 13560 Mean 113 39 23 11 58 1 17 8 4974 1695

28 113 43 21 13 70 1 18 12 5691 325058 112 44 18 12 50 1 16 8 5746 121973 113 46 27 9 38 1 21 9 5902 495063 112 49 22 10 41 1 33 11 5191 93838 104 39 21 11 62 1 14 6 5386 375080 115 45 17 8 29 1 30 8 5549 64367 114 43 14 8 36 1 23 29 5522 1500

39

99 116 36 13 6 16 1 21 2 5646 3000 Total 899 345 155 75 341 8 176 84 44632 19249 Mean 112 43 19 9 43 1 22 11 5579 2406

35 114 40 19 12 66 1 26 8 4597 225056 114 43 20 11 52 1 22 10 5658 187524 116 34 19 12 33 1 14 4 5519 4821

120 112 44 21 12 53 1 22 8 4964 525052 116 42 18 7 26 1 25 6 4456 214345 111 44 17 11 48 1 27 12 5536 225025 116 32 22 11 37 1 16 8 5871 2250

40

47 108 38 19 9 43 1 22 7 5782 2893 Total 907 316 154 85 358 8 174 64 42383 23732 Mean 113 40 19 11 45 1 22 8 5298 2967

Page 61: Chickpea: Year 3: Increasing the efficiency of chickpea production

41 44 114 45 19 10 29 1 20 23 5172 4250 57 108 43 21 9 49 1 23 11 5440 3250 Total 222 88 40 18 78 2 43 34 10613 7500 Mean 111 44 20 9 39 1 22 17 5306 3750

JG-62

109 40 20 12 30 1 14 8 5812 6142

ICC-4958

119 43 22 11 30 1 28 12 903 477

Cross III (Vijay x ICC-4958)

49 110 42 19 10 52 1 23 10 3083 78731 109 36 18 13 37 1 20 9 2219 50282 110 40 17 8 32 1 27 9 2031 31044 108 34 17 8 33 1 18 6 884 192036 111 36 17 9 31 1 24 7 1129 375073 115 36 15 13 28 1 21 7 1992 40889 113 31 17 10 41 1 19 10 1661 3031

42

76 110 38 16 10 35 1 27 6 2990 979 Total 886 291 136 81 290 8 179 62 15990 11687 Mean 111 36 17 10 36 1 22 8 1999 1461

67 111 39 23 13 50 1 23 15 1852 19394 108 32 21 12 58 1 20 16 1420 784

106 110 38 23 10 42 1 20 8 274 89877 107 38 19 7 32 1 18 6 508 65464 116 38 17 9 29 1 19 5 1271 37559 116 34 15 6 17 1 21 3 931 55640 117 31 20 13 29 1 18 9 1611 107

43

8 121 33 16 10 26 1 18 6 797 763 Total 906 285 154 80 285 8 157 67 8663 4331 Mean 113 36 19 10 36 1 20 8 1083 541

26 122 37 12 6 14 1 34 3 1188 67695 120 37 18 10 22 1 28 10 1792 209457 119 38 22 10 35 1 24 8 1897 215684 116 34 20 11 46 1 17 8 879 59142 113 39 18 9 40 1 21 8 1688 282968 112 36 16 10 43 1 20 9 1163 40255 115 38 13 7 33 1 27 6 911 2163

44

51 109 41 21 10 40 1 30 0 1368 2224 Total 926 300 140 72 274 8 201 51 10886 13135 Mean 116 38 18 9 34 1 25 6 1361 1642

Page 62: Chickpea: Year 3: Increasing the efficiency of chickpea production

52 115 44 22 13 80 1 21 18 2308 92086 112 32 19 8 28 1 19 4 731 78738 113 40 18 9 40 1 20 10 2458 86390 114 40 17 9 36 1 19 6 1430 75358 116 38 19 9 42 1 30 13 865 61510 116 49 19 11 52 1 33 15 783 37516 117 44 19 13 38 1 26 11 506 170

45

28 118 41 22 9 37 1 30 11 2619 2488 Total 921 328 155 80 352 8 198 88 11700 6970 Mean 115 41 19 10 44 1 25 11 1463 871

80 113 37 20 9 43 1 25 9 1795 16621 114 38 23 13 42 1 21 9 3007 242722 116 37 18 6 28 1 34 8 2410 18371 117 47 20 11 40 1 20 12 581 28154 116 37 16 12 38 1 16 6 1683 12192 115 38 21 17 49 1 27 6 2295 287562 120 42 18 7 36 1 24 8 2613 295

46

98 119 37 18 10 34 1 22 8 1883 365 Total 930 313 156 85 311 8 189 66 16267 6712 Mean 116 39 20 11 39 1 24 8 2033 839

47 109 42 18 10 38 1 20 11 2149 65627 113 40 23 13 71 1 22 14 1662 14551 117 40 16 7 34 1 22 10 3513 23135 116 38 19 8 23 1 20 6 1362 555

88 125 44 20 8 27 1 20 7 1600 29583 122 43 19 8 35 1 24 3 1579 184

107 117 42 20 9 20 1 24 3 1179 706

47

33 119 44 21 13 43 1 15 7 1705 2308 Total 938 332 156 76 290 8 167 62 14748 8471 Mean 117 42 20 10 36 1 21 8 1844 1059

105 125 46 23 9 39 1 31 6 1362 6277 116 44 20 10 65 1 27 8 1628 717

74 115 46 21 14 29 1 30 14 2240 220632 118 43 12 8 30 1 18 4 2419 1801

102 108 44 19 15 34 1 23 11 2435 82346 111 36 16 10 34 1 17 5 1997 18513 114 41 17 8 39 1 26 11 2607 2767

48

60 112 43 17 9 20 1 27 5 536 159 Total 919 343 145 81 289 8 199 64 15224 10951 Mean 115 43 18 10 36 1 25 8 1903 1369

Page 63: Chickpea: Year 3: Increasing the efficiency of chickpea production

34 113 45 19 12 33 1 16 6 2281 171820 115 39 14 6 23 1 19 4 1443 2591

104 119 41 16 6 23 1 25 4 1402 73841 118 43 19 12 53 1 23 10 1634 392437 115 33 20 9 48 1 21 12 1936 41211 115 45 15 7 28 1 25 5 2279 22985 115 39 17 11 48 1 20 10 941 444

49

18 116 32 18 8 37 1 20 0 1383 744 Total 926 317 139 70 293 8 169 51 13299 10799 Mean 116 40 17 9 37 1 21 6 1662 1350

99 117 36 14 7 38 1 16 5 1699 2694 110 39 16 9 36 1 23 6 3179 2138

53 115 37 18 8 28 1 32 9 1902 25017 114 36 18 6 33 1 35 10 1688 219124 115 42 18 11 45 1 26 10 1735 256313 120 27 13 6 28 1 30 6 2330 243235 119 35 14 7 27 1 27 4 2847 3340

50

25 118 30 16 8 37 1 25 9 2183 2596 Total 928 282 126 62 272 8 214 60 17564 15778 Mean 116 35 16 8 34 1 27 7 2196 1972

103 122 41 17 9 30 1 25 5 1905 5275 120 31 15 8 44 1 17 7 1669 20329 123 31 11 6 16 1 18 3 2990 217887 122 36 15 9 44 1 20 6 1190 15393 110 37 17 14 34 1 25 9 2869 1831

108 116 38 16 8 28 1 22 8 2141 126650 114 35 22 13 45 1 21 13 1298 610

51

48 112 41 24 12 28 1 20 8 2217 1034 Total 939 289 136 79 269 8 168 59 16279 7327 Mean 117 36 17 10 34 1 21 7 2035 916

72 113 40 20 10 43 1 30 15 2423 240939 113 40 17 10 41 1 22 12 2453 261766 112 35 16 7 31 1 26 8 2107 288497 110 38 13 7 29 1 28 7 2531 167430 111 37 17 10 32 1 27 10 2387 190196 116 38 17 8 38 1 24 7 1953 86043 115 41 24 17 83 1 35 23 2258 4070

52

69 113 38 22 12 64 1 38 11 2763 1823 Total 903 309 147 81 360 8 230 93 18876 18238 Mean 113 39 18 10 45 1 29 12 2359 2280

Page 64: Chickpea: Year 3: Increasing the efficiency of chickpea production

70 114 41 20 11 48 1 18 12 3484 5659 112 45 21 10 42 1 28 11 2586 386

14 115 46 17 9 30 1 33 8 1627 840100 116 50 23 12 37 1 27 12 1919 258519 115 34 11 6 20 1 25 5 1436 15979 0 0 0 0 0 0 0 0 354 2708

101 125 44 20 10 35 1 20 17 2239 2281

53

45 0 0 0 0 0 0 0 0 1017 1672 Total 697 260 112 57 212 6 151 65 14661 11197 Mean 116 43 19 10 35 1 25 11 2444 1866

12 122 39 20 8 24 1 25 6 1648 28546 120 43 16 10 52 1 26 9 2093 1625

61 119 38 14 12 48 1 20 14 2485 37115 120 39 15 11 19 5 22 10 2036 243863 115 41 17 10 37 1 23 0 2477 205891 115 39 16 9 24 1 28 7 3762 260778 113 47 15 8 36 1 38 10 2906 284

54

81 115 45 16 8 37 1 36 11 2672 2730 Total 939 332 128 74 275 12 218 67 20079 14966 Mean 117 41 16 9 34 2 27 8 2510 1871

56 112 44 17 9 41 1 24 11 1867 47665 110 37 15 9 36 1 18 12 974 592

55

2 113 40 17 10 43 1 35 13 3232 1546 23 118 43 19 9 43 1 26 12 3474 1928 Total 453 163 70 38 162 4 103 48 9547 4542 Mean 113 41 17 9 41 1 26 12 2387 1136 Vijay 97 34 19 9 32 1 19 10 303 204

ICC-4958

119 43 22 11 30 1 28 12 903 477

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 +

Page 65: Chickpea: Year 3: Increasing the efficiency of chickpea production

* 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 (%)

Plant Height (cm UBC859250bp (12%), RGA6600bp (10.9%)

24

Plant spreading (cm2) UBC859250bp (5.9%), STMS9 (4.1%)

12.7

Branches/plant UBC815200bp (5.6%), UBC807300bp (6.8%)

14.7

Pods/plant STMS80 (5.2%), UBC859250bp (4.9%)

7.0

Grains/pod STMS11 (6.9%), UBC346 (5%)

8.9

Yield/plant (g) UBC859250bp (11.9%), STMS87 (8%)

18.3

100 seed weight (g) UBC859250bp (6.9%), RGA6600bp (8.4%)

19.6

Fusarium wilt UBC859250bp(12.9%), UBC465 (10.8%), UBC859230bp (10.1%)

21.4%