enhancing cotton germplasm, improving resistance to cotton ... · icarda’s pakistan-us cotton...

ICARDA’s Pakistan-US Cotton Productivity Enhancement Program (2010-15), ID-1198

Enhancing Cotton Germplasm, Improving Resistance to Cotton Leaf CurlVirus Disease (CLCVD) and Supporting Cotton Best Management Practices

(BMPs) for Small Farmers

(ICARDA Project No. 1198)

ARS Agreement No.58-6402-0-178F

Annual Progress Report (Oct 2014-Sept, 2015)

Dr. Abdul Majid, Country/Project ManagerMuhammad Arshad, Consultant/Cotton Expert

International Center for Agricultural Research in the DryAreas (ICARDA) – Pakistan Office, Islamabad

Annual Progress Report (October, 2014 – September, 2015)


The Main objective of the project is to assist the cotton research and development system ofPakistan to minimize the adverse effects of chronic and lethal Cotton Leaf curl Virus Disease(CLCuD), its scientific studies and the ultimate development of genetically-resistant varieties usingboth conventional and non-conventional techniques through highly coordinated approaches atnational and international levels.

Approaches include ( 1) supporting cotton best management practices (BMP) for small farmers(short term);( 2) enhancing Pakistan cotton germplasm to broaden its genetic base;( 3) developmentand utilization of sources of resistance to CLCuD; and (4) capacity building of Pakistani researchers,institutes and farmers for advanced cotton research and development.

Cooperators include i) ARS, USDA,( ii) University of Arizona, iii) Texas A & M College Station,Texas, iv) Cotton Incorporated, USA, v) ICARDA-HQ, vi) Borlaug Institute and thirteen (13)Pakistani cooperators (ID-1198-1 to 13).

Annual (October 2014 - September 2015) Progress: Proposed and achieved milestones asdeliberated and collected during the fourth quarter review and planning meeting of all thirteencooperators/PIs held at Institute of Agricultural Sciences, University of the Punjab, Lahore on 9 th

October, 2015 are stated as below.

Objective-1-2: Cotton Germplasm Enhancement of Pakistan: Screening and transfer ofsource of resistance to commercial varieties: I: To increase seed and send to Pakistan to screen against CLCuVD resistance. 2: To select increase and transfer to Pakistan U.S. germplasm from the USDA cotton germplasmcollection. Partners/Cooperators: 1) ARS, Stoneville, 2) ARS, College Station, Texas 3) DREC, MS 4) National Cotton Council

(NCC), Memphis, TS. 5) CCRI, Multan. 6) CCRI, Sakrand.7) CRI, Faisalabad including CRS,Multan and CRS, Vehari. 8) NIBGE, Faisalabad.9) Consultant, ICARDA. 10) Govt. Focal, Cotton Commissioner, Ministry of Textile Industry, Govt. of Pakistan. 11) Director Research HQ, PCCC, Karachi.

1-2.1: Progress of CCRI, Multan (ID-1198-2), Germplasm-I ; PI: Mr. Sajid Masood ShahSr.#

Milestones Proposed Milestones Achieved

1.Confirmation of CLCuDresistance of ratoon USgermplasm of 2013

During 2013, 500 accessions comprising of three setsi.e. C, D and E of species, G. hirsutum L, G. arboreum L andG. herbaceum L were given to CCRI Multan. The resistanceof these accessions was reconfirmed against CLCuV. In Set-C out of 200 accessions, 30 were foundresistant. Flower induction was started only in 01 accessionand this accession is being utilized in breeding program. In Set-D out of 200 accessions we found 34accessions are resistant against CLCuV and only 4accessions produced normal flowering.

In case of Set-E out of 100 accessions 99 belong toG. arboreum L and G. herbaceum L and showedresistance against CLCuV while one accession whichbelongs to G. hirsutum L was found susceptible toCLCuV.

2. Ratooning of 2014 cottongermplasm for thereconfirmation of CLCuV

The plant crop of 2014 comprising of four sets i.e. K,L, M, and N (1050 accession) were ratooned in the third weekof February 2015 to check the CLCuV infestation in ratoon



resistance. crop. The CLCuV data were recorded at 30, 60, 90 and 120DAS.

In Set -K out 200 accessions 07 were found resistantand only one accession produced flowers that is being usedin the breeding program. In Set-L out of 200 accessions 01 accession showedresistant against CLCuV and unluckily this accession did notsprout after pruning. In case of Set-M out of 50 accessions 49 belong to G.arboreum L and showed resistance against CLCuV while oneaccession which belong to G. hirsutum L was foundsusceptible toward CLCuV. In case of Set-N out of 600 accessions 57 accessionswere resistant to CLCuV, None of these accessions producedflowers.

3 Characterization ofMorphological data of2014 USDA accessions.

Data about the Morphological Characteristics of 1050USDA Cotton accessions were recorded according to thestandard protocol of CCRI Multan. Characters are Species,Plant Height (cm), No. of monopodial and sympodialbranches, First sympodial node No., Earliness, Pollen color,Petal spot, Stigma exertion, Leaf shape, Leaf hairiness, Leafcolor, Boll shape, Boll Size, Boll no. Plant-1, Nectariless,Gossypol Presence and any special Character etc.

4 Picking of selfed seed ofMac 07 & DNWC-1324

Selfing were practiced in 120 and 110 plants of Mac07 and DNWC-1324 respectively to purify these accessionsand to get true to type seed. After maturation of bolls pickingwere practiced during the month of November 2014. The seedof these accessions were planted in field on May 19 th 2015 fornext year selfing.

5 Formation of temporaryfield tunnels

Temporary field tunnels for the protection of selfedbolls of resistant/tolerant material (Mac 07 and DNWC-1324)have been installed.

6 Evolution of CLCuVresistant cottongermplasm (Raising of F2

generation)

26 F1 hybrids weighing (50 to 2400g) were delintedwith H2SO4 @ 4litre 40kgs-1. The delinted seed were dried andthe seed were sown on May 19th 2015 in field to raise F2

population.7 Picking and ginning of

crossed bolls of USDA cotton germplasm.

The crossed bolls (crossed during 2015) were pickedand ginned on 10 saw ginning machine and the seed weight ofcotton seed ranged between 0.5g to 58.0g.

8 Ginning of resistant/tolerant of F2 populations

Ginning of the resistant/tolerant of F2 single plants ofUSDA cotton germplasm were completed and the seed wereSun dried and sown in field (crop season 2015) in F3 singlelines.

9 Sowing of CLCuVresistant/tolerant (G.hirsuum L) F1 hybrids ingreenhouse.

07 F1 hybrids which were obtained as a result ofcrossing between USDA cotton germplasm and local highyielding (CLCuV susceptible cultivars) were sown ingreenhouse during the month of November 2014. During thelast week of April 2015 matured bolls were picked, ginned andwere sown in the field as F2 population.



10 Ginning of USDA cotton germplasm (2014).

All the available seed cotton of plant crop 2014 wasginned on 10 saw ginning machine. The seed weight dataranged between 0.65 - 630.5g in set-K, 1.95 - 195.0g in set-L,32.5 - 910.0g in set-M and 1.3 to 354.9g in set-N.

11 Lint% age & Fiber data

Compilation of USA

cotton germplasm (plantcrop 2014).

The Lint %age and fiber traits' of 2014-15 USDA

Germplasm were compiled.

Set-K :- Lint %age (21.1-39.7%), staple length (23.3-

32.0mm), Micronaire value (2.6-4.99µg/Inch) and fibre

strength (22.4-35.0 tppsi)

Set- L:- Lint %age (14.0-39.2%), staple length (23.58-



Set-M :- Lint %age (16.0-37.6%), staple length (17.2-



Set-N :- Lint %age (10.7-46.7%), staple length (17.9-



12 Distribution of USA

cotton germplasm

(2015)

The seed of the three sets (i.e. Set-P, Q and R)received from USA through Directorate of Research PCCCwere distributed to CCRI, Multan, CCRI, Sakrand, CRI,Faisalabad, CRS, Multan, CRS, Vehari and NIBGE,Faisalabad for sowing purpose.

13 Screening of USA

cotton germplasm

(2015) at CCRI, Multan

The seed of Set-P, Q and R received during the yearunder report were sown at CCRI Multan on May 19 th, 2015 forscreening against CLCuV. The CLCuV data were recorded at30, 60, 90 and 120 DAS. However, none of the accessionswere found resistant to CLCuV on 120 DAS.

15 Data of seed

germination

The data for seed germination in all sets of USDA

cotton Germplasm 2015-16 were recorded in

percentage as detailed below:

Set P = 69-100 % Germination,

Set Q = 80-100 % Germination,



Set R = 88-100 % Germination.

16 Sowing of USDA

resistant /tolerant Bulk

The seed of all the USDA genotypes showingresistance/tolerance against CLCuV were sown for thepurpose of seed multiplication

17 Sowing of Selfed seed

of Mac 07 & DNWC-

1324 lines.

The selfed seed of the single plants of Mac 07 andDNWC 1324 were sown in plant to progeny for further selfingto create genetic purity.

18 Sowing of material for

multiplication

The seed of CIM-496, CIM-506 and 2472 provided byconsultant of the project were sown for the purpose of seedmultiplication

19 Selection of plants in

USDA based F2 & F

3

Single plants showing CLCuV resistance andequipped with other desirable traits were selected and taggedin F2 & F3 generation produced by hybridization of localcotton cultivars and USDA Germplasm.

20 Seed preservation The preservation of the seed at the Cold RoomChamber of CCRI Multan is in progress with three timesrecording of temperature data daily.

21 Supply of cotton

germplasm

729 cotton accessions were supplied to 41 scientistsof different Research Stations/Institutions/Universities to utilizein their research programs.

22 Capacity Building I- Rehabilitation

ofGreenhouse.

II-Foreign

Trainings/

Workshops.

Installation of Wet pad, water circulation, fixing ofceramic tiles, painting/distempering of walls, white wash,fixing of wheals in table along with their painting has beencompleted.

Earthen pots of various sizes have been purchased.

Repair of doors (aluminum angles) and glass work are alsocomplete

Dr. Dill Baugh Muhammad, Head Agronomy Sectionattended Annual Meeting of American Society of Agronomy,Crop Science and Soil Science from November 2-5, 2014 atLong Beach, Cal, USA.

Mrs. Shabana Wazir, S.O. Entomology Section attendedBeltwide Cotton Conferences 05-07 Jan 2015 at SanAntonio, TX, USA

Hafiz Abdul Haq and Miss Iram Waqar S.Os of Plant



III- Local training Breeding and Genetics Section have completed 3 monthstraining at NIBGE Faisalabad.

23 Up gradation of seed testing laboratory at CCRI Multan

The main works i.e. civil, wooden and electrical worksfor the up gradation of Seed Testing Laboratory at CCRIMultan have been completed.

1-2.2: progress of CCRI, Sakrand (Project ID-1198-3, Germplasm-II) Mr. Mushtaque Ali Leghari.Sr.# Milestones Proposed Milestones Achieved

1 Evaluation of 2014 US Germplasm for botanical description at CCRI, Sakrand.

During the year 2014 the seed of two sets (Set-M and Set-N) was received on 30-05-2014 at CCRI-Sakrand forsowing. Set-M comprised of 50 accessions belonging to G.arboreum L. While Set-N consisted of 600 accessionsbelonging to G. hirsutum L. were planted on 03-06-2014and 04-06-2014 at CCRI, Sakrand respectively. The seedof only one accession USG14_2249 was missing.

Agronomic data were recorded in all accessions of set Mand N during the reporting period.

In Set-M, the plant height ranged from (120-270 cm),number of monopodial branches per plant ranged from (1-7), number of sympodial branches/plant ranged from (24-48), average boll weight ranged from (0.8g-2.7g), numberof bolls/plant ranged from (0-50) and seed cottonweight/plot ranged from (0-2500g), lint percentage rangedfrom (21.26 to 35.5%) and seed index ranged from (4.1-6.5g).

In Set-N, the plant height ranged from (35-310cm), numberof monopodial branches per plant ranged from (0-9),number of sympodial branches/plant ranged from (3-52),average boll weight ranged from (1.21-5.20g), number ofbolls/plant ranged from (5-70), seed cotton weight/plotranged from (0-1600g), lint percentage ranged from (21.6-43.2%) and seed index ranged from (7.0-12.8g) wererecorded.

In Set-M accessions belonging to G.arboreum, the staplelength ranged from 16.7 to 27.8 mm, uniformity indexranged from 70.6 to 85.9%, mocronaire value ranged from4.4 to 8.1µg/inch and fibre strength ranged from 23.3 to30.9 g/tex. In Set-N accessions belonging to G.hirsutum,the staple length ranged from 21.3 to 33.9 mm, uniformityindex ranged from 78.9 to 89.4%, mocronaire value rangedfrom 2.4 to 6.6 µg/inch and fibre strength ranged from 21.7to 36.8 g/tex.



2 Screening of 2014 US Germplasm against CLCuV .

In Set-M out of 50 accessions, 49 (G. arboreum) wereresistant. One accession USG14_1884 belonging to G.hirsutum was susceptible at 120 DAS.

In Set-N, out of 600 accessions of G. hirsutum 108 wereresistant, 94 highly tolerant, 288 tolerant, 96 susceptibleand 13 were highly susceptible at 120 DAS.

3 Reconfirmation of resistance/tolerance of identified CLCuV resistant US Germplasm 2012, 2013 and 2014.

The ratoon crop of 1386 accessions (2012), 500accessions (2013) and 650 accessions (2014) has beenmaintained at CCRI-Sakrand to reconfirm theresistance/tolerance of identified CLCuD resistantaccessions during 2014-15. No symptoms of CLCuVdisease have been observed so far in Mac-7 (Set-C) andthe CLCuV resistant shifted single plants and ratoon crop of26 accessions of Set-E.

In US Germplasm 2013, 80 out of 400 accessions wereidentified as CLCuD resistant during 2013-14 were alsomaintained to reconfirm their resistance during the year2014-15. At the end of September, 2015 only 67accessions were resistant.

Out of 108 identified CLCuV resistant accessions from 650USDA Germplasm 2014, only 75 accessions were resistantat the end of September, 2015.

4 Selection of CLCuV resistant single plants from F2 generation for planting.

77 single plants were selected from F2 on the basis ofCLCuV resistance/tolerance along with the other desirableeconomic characteristics to plant in plant to progeny row inF3 generation during next year.

5 Development of CLCuV resistant Germplam.

The local promising G. hirsutum cultivars were crossedwith resistant/highly tolerant USDA accessions and crossedbolls were picked.

6 Planting of F1 hybrids toget the F2 seed forplanting in the fieldduring 2015.

29 F1s were planted in green house on November 18,2014. The seeds of 16 out of 29 cross combinations weregerminated and growing well in green house conditionsand picked all the plants in the month of April 2015. Thesewere planted as F2 generation in field conditions duringMay, 2015.

7 To preserve the seed of US Germplasm for future use.

Fresh seed of US Germplasm imported during 2011 to2015 have been preserved as medium term (workingcollection) in cold room for future use.



8 Screening of 2015 US Cotton Germplasm against CLCuV at CCRI, Sakrand.

During the year 2015 the seed of two sets (Set-P andSet-R) was received for planting at CCRI, Sakrand. InSet-P out of 100, 21 accessions were free from virus56 were highly tolerant and 23 were tolerant at 120DAS. In Set-R out of 51, 18 accessions were virusfree, 26 highly tolerant and 07 were tolerant up to 120DAS.

8 Botanical description of 2015 US Germplasm at CCRI, Sakrand.

The data regarding morphology and agronomic charactersviz., days to open 1st flower, flower colour, petal spots, pollen colour, leaf shape, leaf size, leaf colour, boll shape, boll size, plant height (cm), number of monopodial branches/plant and number of sympodial branches/plant were recorded in all accessions of both sets during the reporting period.

9 Screen of F2 hybrids against CLCuV.

18 F2 hybrids were sown in the field for screening againstCLCuV. 240 virus resistant plants with desirable economiccharacters were selected to plant in plant to progeny rowduring next year for further screening against CLCuV.

10 Development new CLCuV resistant germplasm.

The local G. hirsutum cultivars are being crossed with theresistant/ highly tolerant USDA accessions of USG 2014and 2015 for generating new CLCuV breeding material.Bolls’ setting is in progress.

5 Capacity building:- Research system(RI/RS)

--- Researchers

One Roller ginning machine has been purchased.

Contract of Seed Cold Storage with backup generatorand 02 temporary greenhouses (Tunnels) was cancelledbecause contractors were failed to complete installationwork up to March, 2015.

Mr. Muhammad Zaheer Ahsan, Scientific officer ofPlant Breeding Section has completed three monthstraining on “Molecular Breeding of Cotton” in PGMBlab, National Institute for Biology and GeneticEngineering (NIBGE), Faisalabad under Pak-US GeneMapping Project from Jan 19 to April 19, 2015.

Mr. Abdul Razaque Channa, Scientific officer of PlantBreeding Section participated in two weeks Workshopon "Genomic Assisted Breeding in CottonImprovement in PGMB lab, National Institute forBiology and Genetic Engineering (NIBGE), Faisalabadunder Pak-US Gene Mapping Project from March 2-



13, 2015.

1-2.3; Progress of CRI, Faisalabad (Germplasm-III: ID-1198-4, PI: Mr. Khalid Mahmood).Sr.#


Cotton Research Institute, Faisalabad1 Screening and study

(characterization) of USDA germplasm imported during 2014

1250 accessions in five sets i.e.; Set-J, K, L, M and N(200+200+200+50+600) received from USDA were sown inthe field on first and second week of June 2014. Dataregarding of CLCuD incidence 30, 60, 90, 120 DAS and atmaturity was recorded. 49 accessions from set-M and 73 fromSet-N were found to be resistant against CLCuD. Quality traits(Lint percentage, Staple Length and Fibre Finess) of theseaccessions were also recorded. Lint percentage ranged from33-41 %, staple length 25-28mm and fibre finess 3-5 ug/inch.

2 Maintaining of 2013 USDAgermplasm as ratoon cropfor reconfirmation of CLCuD.

63 accessions from previous year 1140 USDA accessionswere maintained as ratoon crop. Data regarding CLCuDincidence 30, 60, 90, 120 DAS and at maturity were recorded.No accessions showed symptoms of CLCuD.

3 Reconfirmation of resistant USDA accessions (2012-13) in replicated trials

Five resistant accessions from previous year USDAaccessions were sown in the field on 09-06-2014. Data on 30,60, 90, 120 DAS and at maturity were recorded. Allaccessions were highly tolerant to CLCuD. Fibre Quality traits(Lint Percentage, Staple Length and Fibre Finess) of theseaccessions were also recorded. Lint Percentage ranged from35-42 %, staple length 27-28 mm and fibre finess 3.9-4.9ug/inch.

4 Raising of 12 advance lines for confirmation of CLCuD.

Twelve advance lines were sown in the field on 10-05-2014Data on CLCuD incidence 30, 60, 90, 120 DAS and atmaturity were recorded. Ten accessions found to be highlytolerant to CLCuD so far. Fibre Quality traits (Lint Percentage,Staple Length and Fibre Finess) of these accessions werealso recorded. Lint Percentage ranged from 34-41 %, staplelength 27-28 mm and fibre finess 3.7-3.4 ug/inch.

5 Raising of F2 generation (2014-15) of 20 greenhouse F1 progenies in the field

F2 from 20 greenhouse F1 progenies was raised in the field on02-05-2014. Data on CLCuD 30, 60, 90, 120 DAS wererecorded. Single plants resistant/highly tolerant to ClCuV andbetter yield were selected to plant in F3 generation for furtherscreening.

6 Raising of 34 F3 progenies(2014-15) in the field

34 F3 progenies were sown in the field on 17-04-2014. Data onCLCuD 30, 60, 90, 120 DAS and at maturity were recorded.One progeny were resistant to CLCuD while fourteen werehighly tolerant. Fibre Quality traits (Lint Percentage, StapleLength and Fibre Finess) of these lines were also recorded.Lint Percentage ranged from 29-44 %, staple length 27-28 mmand fibre finess 3.7-5.1 ug/inch.

7 Screening and study 272 accessions received from USDA were sown in the field on



(characterization) of USDA germplasm imported during 2015

29th May 2015. Data regarding CLCuD 30, 60 and 90 DASwere recorded. Only one accession showed resistance toCLCuD so far.

8 Reconfirmation of resistant USDA accessions (2012-14) in replicated trials

Five resistant accessions from previous year 708 and 1140USDA accessions were sown in the field on 10-06-2015. Dataregarding CLCuD was recorded 30, 60 and 90 DAS. At 90DAS two accessions were highly tolerant to CLCuD.

9 Raising of 10 advance lines for confirmation of CLCuD.

Ten advance lines were sown in the field on 10-06-2015 Dataregarding CLCuD was recorded 30, 60 and 90 DAS. Allaccessions were highly tolerant to CLCuD.

10 Raising of F2 generation (2015-16) of 22 greenhouse F1 progenies in the field

F2 from 22 greenhouse F1 progenies was raised in the field.Data regarding CLCuD incidence 30, 60 and 90 DAS wererecorded. All progenies were highly tolerant to CLCuD exceptone which was tolerant so far.

11 Raising of 42 F3 progenies(2015-16) in the field

42 F3 progenies were sown in the field and data regardingCLCuD incidence 30, 60 and 90 DAS were recorded. Eightfamilies were tolerant to CLCuD so far.

12 Raising of 30 F4 (2015-16)in the field

30 F4 progenies were sown in the field and data regardingCLCuD incidence 30, 60 and 90 DAS were recorded. Tenfamilies were highly tolerant to CLCuD so far.

Cotton Research Station, Multan15 a. Maintinance of 28

CLCuD resistant accessions as ratoon cropand their utilization in crossing program. b. Crossing of CLCuD resistant/highly tolerant USDA material with local CLCuD tolerant G. hirsutum genotypes.

a. All 28 accessions were resistant to CLCuV. There was not any natural boll setting in these accessions due to flower shedding. All these accessions bloomed in November to March(Photoperiod sensitive). Crosses were attempted with local promising strains in tunnel.b. Total number of crosses = 31Pollinations attempted = 2400Total number of number of bolls set = 373Boll setting percentage = 15.5 %

16 Recording morphological data of F1, F2, F3, F4, PYT-5, PYT-6 and screening of germplasm

F1: Total 19 crosses were planted, out of which 07 crosses (151001, 151002 and 151015) showed tolerance against CLCuV, whereas all the other crosses showed susceptibility for this disease. CLCuV infestation ranged from 26 to 89 %. F2: 21 families have been planted. Six families (152002, 152003, 152005, 152006, 152010 and 152011) were highly tolerant against CLCuV, Maximum bolls were recorded on family No. 152006. F3: Out of 47 families, only 3 families showed tolerance against CLCuV. FH-Lalazar (check) showed 60 % CLCuD infestation. Two families 153011 and 153042 had maximum number of bolls per plant. CLCuV infestation ranged from 27 to 81 %.



F4: Out of 29 families of F4, two families 154027 and 154029 showed the lowest CLCuV incidence, while FH-Lalazar (check) showed 90 % infestation. Maximum green and open bolls were observed in family No. 154010 and 154019. Preliminary Yield Trial (PYT)-5: All the eight genotypes showed symptoms of CLCuD which ranged from 62-78%. FH-Lalazar (check) had 58% incidence of the CLCuD . Genotype USG-223 showed maximum number of bolls per plant. PYT-6: All the eight genotypes were highly susceptible to CLCuD. The value of CLCuD ranged from 44-83%. FH-Lalazar (check) had 67% incidence of the CLCuD . USG-259 showed maximum number of bolls per plant. Screening of Germplasm: It includes two sets (Set P & Set R) of USG-14 comprising 151 accessions. In Set-P, none of the accessions was tolerant to CLCuD, while in Set-R, 4 accessions (USG14-2831, USG14-2832, USG14-2833 and USG14-2850) were highly tolerant to CLCuD. CLCuD ranged from 63-100% in set P and 2-100 % in set R. Check variety FH-Lalazar had 82.5% incidence of the disease. Maximum squaring and flowering has been observed on USG14-2850.

17 Transplanting of the genetic material from green house tofield.

l 22 genotypes have been transplanted from greenhouse to the field. Out of which, six genotypes were tolerant and three were highly tolerant against CLCuD. Maximum bolls were recorded on 154001 and 154002.

18 Testing of introgressed material through grafting in green house.

22 introgressed families and MAC-07 were tested through grafting in the green house during 2014-15, and only 3 accessions from USDA introgressed material and MAC-07 showed no symptoms of CLCuD.

19 Backcrossing Pentaploid of (G. longicaly

x G. hirsutum) x G. hirsutum

(G. hirsutum x G. stocksii) X G. hirsutum

Empty bolls have been set for cross (G. longicalyx × G. hirsutum) × G. hirsutum having no seed inside while 103 bolls have been set for cross (G. hirsutum × G. stocksii) × G. hirsutum. Majority of seeds obtained from cross (G. hirsutum ×G. stocksii) × G. hirsutum were immature.

Cotton Research Station Vehari20 Picking of seed cotton and

maintaining 678 accessions of USDA Set-D as ratoon crop for reconfirmation of CLCuV data.

1. Picking of seed cotton from the resistant and flowering entries was done separately in paper bags.

2. Ginning of all the entries separately was done to obtain seed for sowing during the next year

3. In February, sticks of all the entries were removed for sprouting.

4. All entries sprouted but none of the resistant entries



showed symptoms of the disease during current year.

21 Monitoring of flower induction and seed production in sprouts (Set-D).

1. Entries like USG13-1066, USG13-1067, USG13-1087 andUSG13-1023, USG13-1111 and USG13-1140 (set-D) germplasm flowered and produced more bolls as compared to last year due to intensive care against Pink Bollworm.

22 Ratooning of resistant entries in Set-N (600 G. hirsutum) for reconfirmation of CLCuV infestation

1. Sticks of Resistant and flowering entries in Set-N were removed six inches above ground for ratooning.

2. All the entries sprouted.

3. Data regarding CLCuD infestation was recorded after 30, 60, 90,120 days and at maturity.

4. Last year’s five resistant entries USG-14-2281, USG-14-2270, USG-14-2463, USG-14-2476 and USG-14-2480 are still resistant against the disease 150 DAS but none ofthese entries has not produced flowers so far.

23 Growing of Graft screenedresistant Mac-07 plants forre-screening and crossingin the field.

1. Last year a number of Mac-07 plants were graftinoculated.

2. Leaf samples from resistant plants were sent to NIBGE toconfirm their resistance by qPCR

3. Seed harvested from these plants was sown in the field.

4. Out of 35 plants 5 plants have been found susceptible toCLCuV.

5. Resistant and flowering plants are being crossed withlocal varieties for the development of CLCuV resistantvarieties.

24 Selection of desirablesingle plants from F2s ofintra-specific crosses andharvesting of seed cottonfrom selected plants andanalysis of fibre qualitytraits

1. Desirable plants on the basis of morphological data and with respect to yield and CLCuV resistance from the crosses (F2s and DCs) were selected.

2. Seed was harvested from desirable plants to grow next generations.

3. Fibre analysis was conducted.

25 Sowing of new F1Population in theGreenhouse from theseed harvested from

1. Seed harvested from fresh crosses (tolerant local G.hirsutum lines X US G. hirsutum) was sown in the greenhouse.



crosses. 2. Two entries USG13-1112 and USG1-1066 were used incrosses with local germplasm

26 Growing of F1s, F2s, F3s and F4s in the field

1. Desirable plants were selected from the last year crosses(F1s, F2s and DCs) and fibre quality parameters were analyzed.

2. Two F1s, 18 F2s, 68 F3s and 158 F4 progenies were sown in the field during current year.

3. Data regarding CLCuV infestation and selection of desirable plants is in progress.

27 Crossing of resistant andflowering genotypes fromrationed Set-N with localdesirable germplasm inthe field

1. During last year five entries in set-N USG-14-2281, USG-14-2270, USG-14-2463, USG-14-2476 and USG-14-2480 showed resistance against CLCuD but did not flower.

2. During current season Set-N was ratooned for flowerinduction and to make crosses of resistant and floweringgenotypes with local desirable germplasm in the field

3. Up till now none of the retooned resistant entries in Set-Nhas started flowering.

28 Screening of new USgermplasm 2015

1. During current year 100 accessions in Set-P, 32 accessions in Set-Q and 51 accessions in Set R were provided.

2. These accessions have been planted according to the given protocol.

3. All the accessions germinated and have produced reasonable number of plants

4. Data regarding Plant population and CLCuD infestation data after 30, 60, 90 and 120 DAS have been collected in all the three sets.

5. Six entries in Set-Q and one in Set-R are free of symptoms 120 DAS

29 Monitoring of floweringand crossing with localgenotypes

1. All the six entries in Set-Q and one in Set-R are producing flowers.

2. Crosses between desirable local genotypes and US resistant accessions are under progress.

30 Capacity Building Mr. Khalid Mahmud, Director, CRI, Faisalabad, Dr. Saghir



Ahmed, Cotton Botanist, CRS, Multan and Dr. Saeed Ahmed Assistant Cotton Botanist, Vehari attended Beltwide Cotton Conference held at San Antonio, TX, USA from January 5-7, 2015.

31 Next Year Plan of CRI,Faisalabad.

Recording of CLCuD data 120 DAS and at maturity and their quality parameters of 272 newly received accessions

Recording of CLCuD data 120 DAS and at maturity and their quality parameters of five resistant accessions

Recording of CLCuD data 120 DAS and at maturity and their quality parameters of 10 advance lines and breeding material in F2, F3, F4, F5 and F6 generation..

32 Next Year Plan on CRS, Multan.

Recording morphological data of USDA Germplasm.

Crossing of CLCuD resistant/highly tolerant USDA

material with local CLCuD tolerant G. hirsutum genotypes to

generate 30 crosses.

Backcrossing of G. longicaly × G. hirsutum and G.

hirsutum × G. stocksii with G. hirsutum.

Testing of resistant genetic material through grafting.

Fibre testing of all the genetic material.

Multiplication of promising lines in green house.

Cytological studies of interspecific genetic material.

Field evaluation of F1, F2, F3, F4, F5 and selected

accessions from 155 USDA accessions.

Testing of promising lines in replicated trials

33 Next Year Plan of CRS, Vehari.

Grafting and Ratooning of Set-D& N (G. hirsutum)

Collection of CLCuV infestation data after 150 daysand at maturity from the graft inoculated plants of Mac-07 and rationed Set-D & N.

Monitoring of flower induction and seed production in



ratooned sprouts of Set-D & N.

B. Hybridization of USA X Local Genotypes

Collection of data regarding CLCuV infestation of F2s,F3s and F4s after 150 days and at maturity.

Selection of desirable plants on the basis of CLCuDresistance, yield components and fibre quality traitsfrom F2, F3 and F4 in the field.

Ginning to harvest seed from desirable plants fromdifferent generations of crosses to grow advancedgenerations in the field.

Analysis of fibre quality traits of selected plants.

Attempting of new crosses of resistant and floweringgenotypes with local desirable germplasm in the field.

1-2.4: Progress at NIBGE (ID-1198-5), Germplasm-IV; PI: Dr. Mahboob-U-Rehman.Sr.#


1 Participation in USDA germplasm trail 2015

Like previous years, NIBGE also participated in US

germplasm screening trials. A total of 272 accessions were

planted this year on May 29, 2015 in the fields of CRI,

Faisalabad. NIBGE 2 Collection of disease

response data of US 2015 germplasm

The disease response data of each accession was collected

by rating the disease symptoms on designed scale after

regular intervals (30, 60, 90 and 120 DAS). However, none of

the accessions were found resistant. 3 Maintenance of

interspecific F2s and collection of disease data of interspecific F2:3

progenies

In the previous growing season, three interspecific F2 mapping

populations were planted. During current season, F3 progenies

of these individual F2 plants were planted in the field and

collected 60, 90 and 120 DAS disease response data. F2

populations were also maintained by ratooning to acquire

more seed.4 Planting of single plants

selected from 2014 USDA germplasm and their disease rating

Individual plant progenies of all population from Set-K and

Set-L from 2014 USDA germplasm were planted at CRI,

Faisalabad fields. Scored the individual plants of these

progenies on rating scale from 30 DAS to 120 DAS.

5 Maintenance of ratoon (set K and Set-L)

USDA germplasm (set-K and L) planted in 2014 was

maintained as ratoon to monitor its disease response and also



to acquire more seed from these populations.

6 Breeding for CLCuD resistance using USDA accessions

Tremendous development in breeding for development of

CLCuD resistance material has been made utilizing USDA

germplasm. MAC-7 and its sister accession have been used

extensively in crosses with local highly productive lines and

advanced to F3 and F4 generation. Additionally, for interspecific

population, F5 and F6 generations have been achieved. New

crosses have been attempted to pyramid different sources of

resistance. 7 Planting of NIBGE

germplasm and its disease response data collection.

This year ~1800 genotypes have been planted in the field of

NIAB/NIBGE to evaluate their disease response. Disease

response was monitored at 60, 90 and 120 DAS.

8 Raising of resistant accessions.

Some important resistant accessions (local and USDA) were

planted to test the stability of their resistance.

9 Construction of greenhouse

The greenhouse construction work of new green house facility

was completed during this year and it is fully functional now.

10 Plan for the Next Quarter Harvesting and picking of USDA germplasm trials

Monitoring the disease response of resistant

accessions identified so far

Planting of new germplasm and maintenance of Set-K

and Set-L populations to obtain seed

Collection of CLCuV data of advance population.

New crosses of resistant and highly productive local

lines.

Continued maintenance and protection of interspecific

Maintenance of resistant USDA accession as a ratoon

of germplasm 2012.

Genetics of tolerance/resistance to CLCuVD

Data compilation and statistical analysisObjective-3: To develop and map DNA markers to track transfer of the CLCuVDresistance trait into cotton lines.Partners: 1) ARS, Stoneville 2) University of Arizona 3) Texas A&M University 4) NIBGE,Faisalabad (Gene- mapping component ID-1198-5).Progress of NIBGE, Faisalabad (ID-1198-6; Gene-Mapping project; PI: Dr. Mehboob-u-Rahman

Sr.#




1 Establishment of ratoon of crop.

Ratoon population of the following crosses was established

1. G. hirsutum 2472-3 / G. barbadense 66 F2 2014

Total Population at first sampling = 610

Total No. of dead plants = 6

Total No. of ratoon plants = 604

2. G. hirsutum Mac 7 / G. barbadense-PIMA S-7 F2 2014




3. G. hirsutum 2472-3 X G.barbadense Pima S-7 F2 2014




2 Primer survey 1.Parental survey of 9 parents

The following parental genotypes were surveyed

G. hirsutum 2472-3, 2. G. barbadense 66, 3. G hirsutum Mac-7, 4. G. barbadense 66 PIMA- S7, 5. G. hirsutum IR-3701, 6.G. hirsutum 3661, 7. G. hirsutum 3300, 8. G. hirsutum 2381,9. G. hirsutum IR-6). In total 221 SSRs were surveyed 19were found polymorphic. The following SSRs were surveyedTable 1.

Table 1. SSRs syrveyed nine parents



2. G. barbadense-PIMA S-7/. G. hirsutum Mac 7 F2 2014

Survey with the following SSRs was carried out on this

population. In total 429 were surveyed out of which 18 have

been surveyed in the F2 during this year. The list of primers

surveyed is shown in Table 2.

Table 2. SSRs surveyed on PIMA S-7/ MAC-7 F2

3 Identification of QTLs

We were able to identify three QTLs after the SSR survey of

the PIMA S-7/ MAC-7 F2 population whicha re as follows in

Table 3.

Table 3. Identified QTLs from PIMA S-7/ MAC-7 F2



4 Introgression studies

Introgression study of G.arboreum into G. hirsutum

Leaf samples of both parents (G. arboreum and MNH-886)

with four progenies were collected from CCRI, Multan. DNA of

these samples was extracted by CTAB method. A total of 155

SSRs at random were surveyed on both parents. In total, 18

SSR were found polymorphic and surveyed on progenies for

introgression detection. The findings are shown in the Table 4.

Table 4. Introgression studies results



5 Human Resource development

Three months training programme

Second batch of scientists started their three months training

on molecular cotton breeding, Mr. Hafiz Muhammad Imran.

Scientific Officer, CCRI Multan and Dr. Fazl-I-dayim Shehzad.

Scientific Officer, CCRI Multan is participating. They are being

trained in various molecular techniques like DNA Extraction,

PCR, Gel electrophoresis, and currently engaged in

population survey independently.

Two weeks training workshop on Genomic Assisted Breeding

in cotton improvement from 2nd March to 13th March was

organized. About 25 young scientists attended the workshop.

6

Plan for the next year.

1. Continued development Fine mapping of F2 and F2:3

populations.

2. Microsatellite analysis on new F2 population of 2014 using

polymorphic SSRs

3. Primers will be checked for possible linkage with the

CLCuD in order to develop linkage map

4. Preparation of final manuscriptObjective-4: Biodiversity analysis of virus and its vector: To identify, characterize andmonitor the Cotton Leaf Curl Virus (CLCuVD) and whitefly vector.



Partners: 1) ARS, Stoneville, MS 2) University of Arizona 3) NIBGE-Project ID-1198-6(Biodiversity analysis/virology component). 4) Institute of Agricultural Sciences (IAGS),University of Punjab, Lahore Project ID- 1198-7(Biodiversity analysis/virology component).4-1: Progress of Institute of Agricultural Sciences (IAGS), University of Punjab, Lahore(ID-1198-9, Virology- I; PI: Dr. Saleem Haider).Sr.#


1 Extraction of total DNA, detection of begomoviruses through specific primers. Full length genome cloning and sequence analysis

A total of 220 full length begomovirus sequences were annotated and analyzed and the important findings aregiven below.

Cotton leaf curl Burewala Virus (CLCuBuV) was the most dominant virus infecting cotton in all three sampling sets. Aside from CLCuBuV, OELCuVand SLCV were also detected from a few cotton samples. First infection of cotton by OELCuV and SLCV; also detected in US Germplasm samples. SLCV is a New World virus introduced into Middle East ~ 8 years ago; now apparently spread to Pakistan ~8% divergent from type virus from US-Mexico; not known in SW-US cotton (host shift, asymptomatic, or mixed infection w/ Cotton leaf crumple virus).

Aside from cotton, CLCuBuV is occasionally found infecting other malvaceous hosts including okra and Chinese rose but in this study we reported the first infection of CLCuBuV into Luffa cylindrica family cucurbitaceae .

Tomato leaf curl new delhi Virus (ToLCNDV) is wide spread in subcontinent. It was detected in all of the species of plants sampled in this study including cotton and emerging as serious threat to crop production in the region.

Chickpea chlorotic dwarf virus (CpCDV) is a dicot infecting mastrevirus. Our results indicated the first report of CpCDV C- strain infecting cotton, first reported CpCDV infection of okra, tomato, and cucumber.

Asiatic cotton (G. arboreum) that is native to Pakistan and India never showed the disease symptoms and thought to be immune to geminiviruses.Few samples were collected from asymptomatic G. arboreum to confirm the presence of any geminivirus. Presence of three isolates of L strain of CpCDV was confirmed in asymptomatic G. arboreum

Parthenium is an invasive weed in Pakistan and has hampered crop production because of allelopathic effects. ToLCKV was detected in invasive weed



Parthenium, reflecting an emergent situation.

Nine species of begomoviruses have been reported to

cause CLCuD in the region, in association with Cotton

leaf curl Multan betasatellite. The recent studies

revealed that Cotton leaf curl Burewala virus

(CLCuBuV) is the dominant virus infecting cotton in

Pakistan and India. Many of the CLCuD associated

virus species identified in Pakistan were subsequently

identified in India. However, Cotton leaf curl Rajasthan

virus (CLCuRV) has been identified extensively in

cotton in India but, except for being identified in a

collection of exotic (not cultivated) cotton species in

Multan and tomato in Faisalabad, it was not identified

in Pakistan. In this study we report a distinct strain of

CLCuRV infecting cotton in Sindh province of

Pakistan. The virus was identified along with

Shahdadpur strain of CLCuMB (CLCuMBsha) and

Tomato leaf curl alphasatellite (ToLCA). This is the first

report of CLCuRV invading cultivated cotton in

Pakistan.

Some differential distribution of helper monopartite, beta/alphasatellites, and bipartite begomos in cotton showing different symptom ratings (grades 0-4)

Cotton leaf curl Multan betasatellite (CLCuMB) was detected from Cotton (92), Chilli (1), Squash (4), Okra (2), Black nightshade (3) and Cucumber (4) samples. All the three strains of CLCuMB (CLCuMB_Bur , CLCuMB_Sha & CLCuMB_Mul) were detected but CLCuMB_Bur was the dominant strain. OLCuB, BYVB, ChLCB and KLCuB were also found to infect cotton.

1680 Alphasatellite Sequences were annotated and analyzed comprising of 576 from Sentinel Plots, 839 from farmer’s fields and 265 from US Germplasm trials. Most of the alphasatellite sequences (~70%) were CLCuMA and GDarSLA. Rest includes OLCuA, ToLCA, HoYVA, AcYVMA, AYVIA, GDavSLA, AYVPKA, AYVSGA, GMusSLA, HoLCA, SiYVCA, BYVA, VeYVA.



2 Extraction of DNA from white flies, PCR amplification of Cytochrome Oxidase-I gene. Sequencing and analysis for haplotyping.

Phylogenetic analyses of whitefly sequences revealed that Asia II 1 clade with 544 sequences is the dominant haplotype in Pakistan.

3 Trainings/workshops/symposiums

Four researchers (Dr. Muhammad Zia-Ur-Rehman, Dr.Usman Hameed, Mr. Muhammad shafiq and Miss Ayesha Bibi) attended Borlaug fellowship meeting at NUST Islamabad.

Prof. Dr. Muhammad Saleem Haider and Dr. Muhammad Zia-Ur-Rehman attended Plant and Animal Genome conference, San diego, USA(January,2015).

Dr. Muhammad Zia-Ur-Rehman visited Dr. Brown’sLab for 6 months for training on analyses of Next Gensequence data.

4.2: Progress at NIBGE, Faisalabad ( ID-1198-7, Virology-II; PI: Dr. Rob W. BriddonSr.#


1 Sample collection and storage

Some 200 plant samples and ~1000 whitefly samples were collected from the diverse locations of Pakistan. Samples were collected from interior Sindh and KPK province of Pakistan.

2 Cloning and sequencing of 150 full length clones

A further 135 clones (representing virus, beta- and alphasatellites) have been obtained this year and sequencingis being completed. Our results have shown that most prevalent virus in the field is CLCuKoV-Bur while CLCuMA and CLCuMB are the most abundant satellites. As part of a project to monitor for possible genetic changes in the CLCuD virus complex in Pakistan, some recent cotton samples have been shown to harbor CLCuKoV-Bur isolates with more complete TrAP encoding genes, with the potential to encode a 127 amino acid protein. This finding suggests that, in the absence of the resistance, CLCuKoV-Bur can rapidly revert toa form with an intact TrAP gene; with the viruses identified here being the first step towards a complete TrAP gene. Prior to this CLCuKoV-Bur variants with an intact TrAP (134 amino acids) was reported from India and Pakistan.

3 Quantification of viralload in infected cotton samples and comparison of the relative levels of resistance

The qPCR method for detection/quantification of all three components was used to examine the 2014 National Coordinated Varietal Trials and the Mac7 plants grown in the field during the last year. Mac-7 plants from different locationswere collected and subjected to qPCR assay. Four control plants (highly susceptible) were also used as a control in these analyses. Our results have shown that begomovirus level in Mac-7 plants from all the locations were below the detection limit. However in two of the ten samples small



amounts of alphasatellite was detected in them. No betasatellite was quantified in these samples. However, in control susceptible plants all components were present in high quantities. Overall, these results indicated that Mac-7 is highly resistant but do support virus replication in very small amounts.

4 Cloning and sequencing of whitefly COI

Total number of 550 Sequences of COI-3' of Bemisia tabaci was collected from Genebank/CSIRO in order to assign name to the specimens showed their correspondence with six of the 34 putative species (D Barro et al 2012) of B. tabaci. Further Analysis of the 290 COI-3 sequences of the B. tabaci from out data showed the presence of six putative species of B. tabaci: Asia 1, Asia II 1, Asia II 5, Asia II 7, Asia II 8 and MEAM 1. In our study Asia II 8 was found for the first time in Pakistan and was collected near the border of Pakistan and neighboring country India. Asia II 8 lineage was phylogenetically closer to Asia II 11 reported from China but fall in Asia group. Pair wise distances among these sequences range from 0.0% to 19.4%.Maximum distance among COI-3' sequences of Asia II 1 is (3.3%), Asia 1 (0.2%) and MEAM1 (1.1%), Asia II 7 (0.1%) following the distance limit (3.5%) for B. tabaci species differentiation, these were assigned the same species. Maximum sequence divergence was found in Asia II 1 specimens (3.3%), Following the species demarcation limit 3.5% may be in future this species split into two different species. Due to less three sequences of Asia II 7 and Asia II 8 their pair wise distances was not measured but in comparison with Asia II 8 and Asia II 7 reported from India, our species Asia II 8 showed 1.6%, Asia II 7 showed 2.6% and Asia II 5 showed 1.8% divergence. The Asia II 7 showed maximum divergence with specimens reported from India, may be a different haplotype. Pairwise distance analysis among all the sequences obtained in our study showed minimum distance between Asia II 1 and Asia II 7 is 9.2% and placed these two biotypes as closely related. The minimum distance among Asia II 1 and Asia II 5 is 10.5%, Asia II 1 and Asia II 8 is 12.7%and among Asia II 5 and Asia II 7 is 10.4%, Asia II 5 and Asia II 8 is 12.6%, Asia II 7 and Asia II 8 is 12.6%. The phylogenetictree of B. tabaci showed a close relationship among four species of Asia II group (Asia II 1, Asia II 5, Asia II 7, Asia II 8).The maximum divergence between Asia II 1 and MEAM 1 is 16.9% than Asia I which is 15.8% making Asia I phylogentically more closer to Asia II 1 than MEAM 1 with strong bootstrap value. The divergence among the Asia II 8 and MEAM 1 is 15.8% and among Asia I and MEAM1 is 15.9%

5 Determine the diversity of whitefly endosymbionts and GroEL

A total of 120 16S clones sequenced. The diversityendosymbionts in B. tabaci samples for which COIsequences are available were also examined usingdiagnostic PCR without sequencing. These results haveshown that Arsenophonous is the most prevalent.



6 Pathogenicity of virusclones in Nicotiana benthamiana and cotton.

The cotton plants infected with clones arebeing used to investigate the old resistance bygrafting.

7 High throughput sequencing of virus samples

A further 96 samples have been sent to Mississippi forNGS. The results obtained with the previous samples arebeing used in publications

8 Brief plan for the next year(2015-16)

Finish the virus/satellite sequencing, completethe analyses and publish Continue the comparison of the relative levelsof resistance in cotton using qPCR. Continue the production of constructs for theinfectivity of selected begomoviruses, betasatellitesand alphasatellites. Comparison of the pathogenicity of virusspecies/strains in the model host Nicotianabenthamiana and cotton and assess of the effects byqPCR. Analyse the next generation sequencingresults. Support the other parts of the project whererequired such as providing qPCR results forpublication of the Mack7 resistance. Repeat the analysis of virus/satellite load inB. tabaci across a cotton growing season

4.3: Progress of ASAB, NUST, Islamabad, (ID-1198-13;PI; Dr. Muhammad Tahir):Sr.#


1. Procurement of the Equipment/software’s

Procurement of consumables and two equipments;refrigerated centrifuge and incubator shaker which were inpipeline, were made. The procurement of one of theequipments, Universal oven, was not preceded due totechnical issues in payments. The payment of the Universaloven will be made in next month.

2. Procurement of lab reagents/supplies

Purchase of reagents and other project relatedchemicals was completed. Few items related to cell cultureand hybridomas are underway. The items are delayed, sincecells lines were not available in Pakistan and the procurementof cells lines from USA is underway.

3. Plant sample The development and maintenance of healthy controlplants under glass house conditions are in progress. Alongwith. whiteflies are rearing in the Insectary which would beused to test the transgenes



4. Development of T/A and pET expression constructsfor development of antibodies

CP constructs didn’t express initially so optimization toreduction free energy of secondary structures by silentmutation, and expression of CP gene of CLCBuV wasachieved. The protein was expressed in the form ofinclusion body, the optimization and refolding of theprotein was successfully achieved. Ni chromatographybased purification was not successful, hence the protocolwas optimized for direct elution of the protein from SDSPAGE gel and we are successful in getting 80% purifiedprotein using this meth

5. Isolation and cloning ofCyt C gene from cotton and N. benthamiana plants

We are successful in replacement of βC1 gene withCyt c. we designed two constructs; one with the wild type Cytc and other with codon optimized Cyt c.

Development of modified cotton leaf curl Multan betasatellite (mCLCuMB)

The partial tandem repeat constructs of both, wildtype and optimized Cyt c, have been developed

Development of transgenic N.benthamiana

In progress. Transgenes are T2 (tissue culture) stage

Mice Inhabitation Analysis and expression optimization is in progress

Human Resource Development

Dr. Muhammad Tahir attended Borlaug fellowship meeting at NUST Islamabad.

Dr. Muhammad Tahir visited Dr. Brown’s Lab for 6 months for training on analyses of Next Gen sequence data.(3 months under Borlaug fellowship and 3 months supported by the Cotton Project.

Dr. Muhammad Tahir attended Plant and Animal Genome conference, San Diego, USA (January, 2015).

Workshop on Omics of Begomoviruses and its Impact on National Economy was organized from March 25-27, 2015 for the training of young scientists. About 30 scientists from different institutes and universities attended the Workshop.

Brief plan for the next year(2015-16)

1) Protein purification

2) Mice immunization

3) Development of hybridomas

4) Development of transgenic N. benthamiana plants and itsverification.

Objective -5: Transformation for the development of virus resistant transgenic cotton:Direct transformation of promising cotton cultivars and Indirect transformation using



Cocker cotton and tomato/tobacco plants. Partners: 1) ARS, Stoneville 2) Danforth Centre 3) NIBGE, Faisalabad (Transformationcomponent, 4) CEMB, Lahore (Direct-Transformation component).5.1: Progress of NIBGE, Faisalabad, (ID-1198-8; Indirect Transformation: PI: Dr. Shahid Mansoor). Sr.#


1. Transformation of cotton(Coker 312) with genecassettes containing RNAi Amplicon 1 (DNA-A based)

RNAi amplicon cassettes targeting DNA-A, 102 events of calli(DNA-A based) were achieved and embryogenesis wasachieved in 5events. T0 seed of three events (1, 2 and 3)were collected andT1 (135plants)of two events (1(133 plants),2( 2 plants) was planted for progeny collection. T1 seed ofevent 1 was collected and screened for kanamycineresistance. T2 (41 plants) of event1 was planted for progenycollection. Experiments are underway. Genomic southernresults verify single gene integration pattern in event 1 and 2.

2. Cotton Coker-312transformation with RNAi Amplicon 2 (betasatellite based)

RNAi amplicon targeting betasatellite, 153 calli events wereachieved. Embryogenesis is achieved in 5 events. Plants of 4events (55 plants) are in soil pots in containment. Event 1, 2(15 plants each) 3 (10 Plants) & 4 (15 Plants) are incontainment while embryogenesis is achieved in Event 5.PCR confirmation has been done for all events currently incontainment (Event 1, 2, 3, 4). Flowering has been started inthree events (Event 1,2 and 3) and boll setting expected withinnext couple of weeks. Sub culturing of rest of the calli eventsare in process. Gene integrating detection through southernhas been done for two events (Event 1, 2). Results are theevidence for single integration in Event 2 and doubleintegration in Event 1.

3. Transformation of miRNA targeting whitefly

Artificial miRNAs cassette (targeting white flies genes) wastransformed into cotton coker 312. Out of 740 explants, 52kanamycin resistant calli were obtained and embryogenesiswas achieved in two events. One plant (sterile) from one eventis in earthen pot and two plants of 2nd event are in containmentand 10 plantlets of 2ndevent are also in jars in growth.

4 Transformation of miRNA targeting virus

In case of virus targeting miRNA construct 1013 explants wereinoculated. 102kanamycin resistant calli were obtained andembryogenesis was achieved in 7 events. Plants of fiveevents (ca 21 plants) are in containment for progenycollection.T0 seeds of event 4(12 plants) were collected and inparallel T1 is being planted (7 plants).Single copy geneintegration was confirmed through southern hybridization inevent 4. Plantlets of 6th event are in jars and embryos arebeing generated from 7th event.

5 Transformation of RNAi construct targeting whitefly genes

RNAi constructs (G1 & G2) targeting whitefly genes weretransformed into coker 312 (recently added). A total of 3100explants of both construct were inoculated in 8 independentbatches. Kanamycin resistant calli of G1 (108) while 117 of G2



were obtained and proliferating on emryogenic medium.

6 Infectivity assay of seeds obtained from CEMB

Four lines of (MC1-1-2, MC1-3-2, VC1-1-4, VC1-12-1)generation harboring miRNA virus constructs were obtainedfrom CEMB. 20 seeds of each plant were grown in pots andchallenged with virulifurous whitefly and placed in containedenvironment. After three months of sowing all lines showedleaf curl symptoms. However, one line (MC1-1-2) showed verymild symptoms in 7 plants and rest of the plants remainedasymptomatic. In other three lines all plants showed mild tosevere leaf curl symptoms. Further analyses based on PCR,qPCR are underway

7 Analyses of Coker 312 plants harboring miRNA whitefly construct

Leaf disc bioassay was carried out in perforated bottle placedon petri dish filled with 1% agar, 100–110 freshly hatched adultwhiteflies were collected in bottle. Three transgenic lines wereanalyzed, and three leaves per plant were tested in thebioassay. Fresh leaves of transgenic and control plants wereplaced on the agar base plat and whitefly carrying bottle wereplaced on petri plat and tightly sealed with parafilm .Bioassaywas continued for 6 days. All experiments have shown thatmortality rate of whitefly was higher in transgenic plants ascompared to control plants. The results are given in tablebelow

Leaf Disc Bioassay of white fly

Experiment # 1 Date 2/09/15

Plant Name Mortality rateDay 2

Mortality rateDay 4

Mortality rateDay 6

Control 18 25 29

Plant 1 27 40 63

Plant 2 29 51 72

Plant 3 32 53 75

Experiment # 2

Plant Name Mortality rateDay 2

Mortality rateDay 4

Mortality rateDay 6

Control 13 25 35

Plant 1 20 42 51

Plant 2 30 53 67

Plant 3 21 47 60

Experiment # 3

Plant Name Mortality rate Mortality rate Mortality rate



Day 2 Day 4 Day 6

Control 15 22 30

Plant 1 27 41 65

Plant 2 30 50 68

5.2:- Progress of CEMB, University of the Punjab, Lahore, (ID-1998-10); Direct Transformation. P I: Dr. Tayyab Hussnain. Sr.#


1 Determination of copyno of Construct 2through FISH.

FISH and Karyotyping of construct 2 has been achieved onecopy no. of transgene at chromosome no. 6 was obtained

2 Southern blot analysis

of C1 and 2. Achieved in Construct 1 and results coincides with FISH

Results. In Process in Construct 2.

3 Quantification of Virus titerin constructs 2 and 4transgenic plants.

Quantification of virus titer in transgenic plants of construct 2 has been achieved while for construct 4 are underprocess.

4 Transformation ofconstruct 8 and 9.

Achieved nine experiments of each of Construct 8 and 9 were done. Total 9 transgenic plants of construct 8 out of which 7 are PCR Positive and 4 of construct 9 which are PCR Positive are in tunnel inT0 generation.

5 Selfing of at least 10flower each plant of T1generation of C3, C4,C5,C6 and C7.

Achieved: Selfing of C 3, C 4, C 5, C 6 and C 7 plants in T1generation has been done.

6 Grafting of Resistant planton symptomatic plants ofC3, C4, C5, C6 and C7constructs for evaluationthrough virus pressure.

Grafting of transgenic plants of constructs C 3, C 4, C 5, C 6 and C 7 plants is under process.

7 Raising of T2 generationof each of construct3,4,5,6 and 7.

Achieved; Four events of Construct 3, Three events of Construct 4 while four events of Construct 5 and one event of Construct 7 has been confirmed through PCR in T2 generation. While confirmation of other events is in process.

8 Molecular Confirmation ofT3 generation ofConstruct 1 & 2.

Achieved Two events each of Construct 1 and 2 in cotton varieties VH-289 and MNH-786 has been confirmed through PCR in T3 generation. While confirmation of other events is in process

9 Handing over of material Transgenic material of construct 1 and 2 has been



to NIBGE

handed over to NIBGE.

10 Capacity building of scientists:

Institutional capacity building (up gradations and procurements):

Dr. Tayyab Husnain & Dr. Idrees Ahmad Nasir attended Plant and Animal Genome Conference 2015 held in USA from 10-14January 2015

Aneela Yasmeen attended Workshop on Omics of Begomoviruses and its impact on National Economy held in ASAB-NUST 25-27 March 2015

Internship Training of five Student from Different Universities of Pakistan has been done

Ms. Maryam and Ms Sheza from Kinard College Mr. Muhammad Zahid from University of Agriculture Ms Misbah from IAGS& Ms Saira Mahmood of BZU Multan Mr. Aftab Ahmad has completed his M .Phil in this Project.

11 Next year (2015-16) plans:

Generation advancement of transgenic plants of construct 1-9 will be done.

Selection of best events in advance generation will be done Molecular Confirmation of transgenic plants in advance

generation will be done. Determination of location of Transgene in construct 3 and 4

through FISH will be done. Research findings will be published in reputable journals

Br Summary of Transformation experiments during Quarter July to September:SrNo

Construct

Varieties

used

Total number of events at stage;

(Ten) (Three)1. VH2892. MNH7863. CIM496

Laboratory / Shoots in Test Tubes

Greenhouse/ plants in soil pots under acclimatization

Containment trials/ T0

Containment / T1

Containment / T2

Containment / T3

Results/Remarks

1 C1 VH-289

MNH-786

-

-

-

-

-

-

-

-

2 Events10 Plants

2 Events10 Plants

2 Events10 Plants

2 Events10 Plants

Molecular Analysis: Generation advancement of construct 1 from T2 to T3 was achieved by confirmation through PCR with gene specific primers which showed desired bands of 217 bp in all four events and twenty plants

2 C2 VH-289

MNH-786

-

-

-

-

-

-

-

-

2 Events10 Plants

2Events10 Plants

2 Events10 Plants

2Events

Molecular Analysis: Generation advancement of construct 2 from T2 to T3 was achieved by confirmation through PCR with gene specific primers which



10 Plants

showed desire bands of 430bp in all 4 events and 20 plants

3 C3 VH-289CIM-496FH942

0

0

0

0

0 (1 event)8 plants

0(1 event)8 plants

04 events

(20plants)

0

NA

NA

Generation advancement of construct 3 from T1 to T2 was achieved by PCR with gene specific primers which showed desire bands of 221 bp in all plants of T1 and T2 generation

4 C4 VH-289CIM-496FH942

000

000

0 (3events)

27plants

0(7

events)68 plants

0(3 events)15 plants

0

NA

NA

Generation advancement of construct 3 from T1 to T2 was achieved by PCR with gene specific primers which showed desire bands 500 bp in all plants of T1 and T2 generation.

5 C5 VH-289

CIM-496

0

0

0

0

0 (8events)

67 plants

(1events) 9

plants

0(4 events)20 plants

NA Generation advancement of construct 5 from T1 to T2 was achieved by PCR with gene specific primers which showed desire bands of 228bp in all plants of T1 and T2 generation.

6 C6 VH-289CIM-496

0shoots

0 06 plants

0(1 event)

NA NA Generation advancement from T0 to T1 was done by PCR with gene specific primers which showed desire bands in 1 plant.

7 C7 VH-289CIM-496

0shoots

0shoots

0 0 00

0(1 event) 10 plants

NA Generation advancement of Construct 7 Plants from T1 to T2 was done by PCR with gene specific primers which showed desire bands in ten plants.

8 C8 VH-289CIM-496

0 0 0405

0 0 Generation advancement of construct 8 plants from T0 to T1 was achieved by confirmation through PCR

9 C9 VH-289CIM-496

0 009 05

0 0 Generation advancement of construct 9 plants from T0 to T1 was achieved by confirmation through PCR

10

C0 VH-289CIM-496

00

00

080

(3 events) 29 plants

NAN.A

Objective-6: Cotton agronomy research: Researching agronomic crop practices to improvecotton production by reducing CLCuVD at farmers’ fields in Pakistan (Proejct-ID-1198-11), P I: Dr. Abdul Majid.Partners: CCRI, Multan, CRI, Faisalabad and ARS, Bahawalpur).Sr.#


1 Cotton as Relay Cropping The objective of the study was to enhance the cotton

productivity and CLCuD management without scarifying the



wheat crop. At CCRI Multan the cotton sowing in standing

wheat at 75 cm (T2) and 150 cm (T3) was completed on 25-

04-2015. While cotton on fallow land early (T1) and after

wheat (T4) was sown on 18-04-2015 and 04-06-2015,

respectively. The standard cultural practices were carried out

as per need of crop and data recorded on various growths and

development. The results revealed that cotton sown as a sole

crop produced highest plant height (110.2 cm) and while

maximum bolls (205 m-2) were produced in cotton sown in

standing wheat (75 cm apart row) on account of highest plant

population (83000 ha-1). The lowest plant height and bolls per

meter square were recorded in cotton sown after wheat. The

cotton sown as sole crop observed the lowest CLCuD

incidence (45.0%) and the highest figures for CLCuD (100 %)

was recorded in cotton sown after wheat harvesting

ARS, Bahawalpur:

At ARS Bahawalpur the wheat sown as sole crop on ridges

produced the highest grains yield (3890 kg ha-1). Wheat

planted on ridge and alternate furrow closed for cotton

planting on both sides of furrow (Row x Row = 75cm) (T1) and

wheat planted on ridges and one furrow closed after every two

furrows and cotton planting on both sides of furrow (Paired

row of cotton after every 150cm) (T2) produced 2825 and

2510 kg ha-1 grains yield which was 19.7 and 8.8% less over

sole crop. The treatments were comprised of T1= wheat

planted on ridge and alternate furrow closed for planting

cotton (Row x Row = 75cm and PXP=30cm), T2= wheat

planted on ridges and every 3rd furrow closed for planting

cotton (Row x Row = 150cm and PXP =15cm), T3= Wheat

planted on flat in 60cm spaced strips (90cm) and cotton

planting on furrows after each strip (double row), T4= cotton

after wheat and T5= Early cotton (on beds). The results

indicated that highest (42083 ha-1) and lowest (38958 ha-1)

plant population was produced in crop sown after wheat



harvesting and wheat planted on ridge and alternate furrow

closed for planting cotton (Row x Row = 75cm and

PXP=30cm, respectively. However, the highest plant height

(152 cm) was produced in T3 and maximum number of bolls

per plant (74) was produced in T5. Agronomy Research

Station (Bahawalpur) Punjab conducted one farmers’ field day

on demonstration site of cotton as relay cropping during the

month of August, 2015. Total 150 farmers participated in

farmer field day.

Objective-7: To reduce infection levels of CLCuVD at the field level through improved cropmanagement practices by small farmers in Pakistan (Proejct-ID-1198-1), PI: Mr.Roshanzada Khattak.

Partners: NARC, Integrated Virus Management component.

Sr.#


1 Curriculum Review Workshop & Refresher training

A Refresher Training Course on “Facilitation skills for FFSFacilitators” was organized at Agriculture Training Institute(ATI) Sakrand Sindh from 18-19 Feb. 2015 where 22 FFSFacilitators from 4 cotton growing districts of Sindh (Khairpur,Sanghar, Nawabshah and Umerkot attended the training. Themain objectives were to refresh facilitation skills and FFSphilosophy among FFS Facilitators, to assess existing skills offacilitators as well as to bring facilitators at the same levelbefore FFS establishment. 2nd Refresher Training Course wasorganized in Multan Punjab from 31 March – 1st April 2015where 23 FFS Facilitators from Punjab province belong toBahawalpur, Dera Ghazi Khan (DG Khan), Khanewal, Multan,Vehari attended the training. Curriculum Review Workshop on “Management of CottonLeaf Curl Virus (CLCV) Disease through Integrated PestManagement (IPM) Techniques by adopting Farmer FieldSchool (FFS) approach” was organized at ATI Sakrand on 20th

Feb. 2015 while on 2nd April 2015 in Multan for Sindh andPunjab provinces respectively where various resourcepersons/experts of Cotton gave their input regarding cottoncrop best management practices. The FFS facilitatorsdiscussed current cotton crop problems with the experts. FFScurriculum was developed in the light of previous years’ FFSexperiences, experiments and management practices toreduce the incidence of CLCuV in the field conditions. Keyissues and major interventions were identified in each andevery phase of cotton crop which were incorporated in thecurriculum. On the basis of the proposed curriculum, 25sessions of FFS were scheduled for the current cotton cropseason with emphasis to replicate and refine successful



management practices in the field.

2 Planning meetings in project districts

FFS planning meetings were conducted with FFS Facilitatorsof Sindh and Punjab provinces separately where all pre FFSactivities and plans were finalized. Seven Farmer basedorganizations working in the project districts viz. KissanWelfare Organization (KWA), Bahawalpur, Kissan DostAssociation (KDA), Farmer Integrated DevelopmentAssociation (FIDA) Vehari, Sindh Agriculture DevelopmentAssociation (SADA), Umerkot, Farmer Organization DuthroMinor, Sanghar and Sustainable Agriculture Organization(SAO) Khairpur, Sakhi Development Organization,Nawabshah were responsible for quality assurance monitoringof FFS process on regular basis.

3 Establishment of FFS and PLGs

Overall 101 Farmer Field Schools (FFS) and 135 ParticipatoryLearning Groups (PLGs) were established in nine projectdistricts involving 44 FFS Facilitators where 10 FFS and 24PLGs in district Bahawalpur, 15 FFS and 13 PLGs in DGKhan, 10 FFS and 25 PLGs in district Khanewal and Multaneach, 6 FFS and 12 PLGs in district Vehari, 9 FFS and 5PLGs in district Khairpur, 15 FFS & 5 PLGs in districtNawabshah, 14 FFS and 14 PLGs in Sanghar and 12 FFSand 12 PLGs in district Umerkot. Each FFS Facilitator wasalso provided with the printed FFS Data Book for proper andtimely record the data.

Overall 5473 cotton farmers/growers underwent trainingthrough FFS and PLGs during the current year cotton season.The regular and FFS registered farmers are 3003 while 2462farmers were facilitated in groups time to time and as per need.Overall the majority (34%) of FFS Facilitators were having SSClevel of education while age-wise the majority (36%) werebetween 41-50 years. Among FFS Farmers of Sindh, majority(26.69%) was illiterate and 37.75% were between 21-30 yearsof age while among farmers of Punjab province majority(28.03%) were having middle level education with age range31-40 years.

The curriculum covered in FFS/PLGs included identificationof problems of the cotton production technology, Cotton Agro-Ecosystem Analysis, land preparation, variety & seedselection, Seed treatment, Germination Test, Sowing onRidges, Observing seed growth, rational use of water,fertilizers and pesticides, preventive measures for pesticides,optimum plant population, weed management, CLCuVdisease, incidence and severity scale, symptomatology &management, beneficial and harmful insects and theiridentification, Economic threshold level of insect pests, role of



white fly in CLCuV disease, white fly life cycle andmanagement, management strategies for CLCuV andmanagement of arising and economically important insectpests through various available alternatives. Among major FFS issues and constraints, the prevalence ofvarious insect pests especially white flies, Dusky Bugs, MealyBugs, Jassids and Thrips etc. non-availability of certified seed,water shortage, high summer temperature and less interest offarmers in FFS sessions especially during Ramadan, cottonpicking and flooding rains. Delay in release of funds was alsoone of the issue which affected many FFS activities.

4 Monitoring and Technical assistance:

Seven Farmer based organizations working in the projectdistricts viz. Kissan Welfare Organization (KWA), Bahawalpur,Kissan Dost Association (KDA), Farmer IntegratedDevelopment Association (FIDA) Vehari, Sindh AgricultureDevelopment Association (SADA), Umerkot, FarmerOrganization Duthro Minor, Sanghar and SustainableAgriculture Organization (SAO) Khairpur, Sakhi DevelopmentOrganization, Nawabshah were responsible for qualityassurance monitoring of FFS process on regular basis.

5 FFS data collection & compilation:

A variety of experiments were initiated by FFS farmers andfacilitators especially regarding management of whitefly andgreen aphid, management of CLCuV using urea andmicronutrients, management of various sucking pests usingvarious herb/plants/planting materials/ biopesticides,management of crop health, vigor and flower shedding. Likeprevious years, promising results have been achieved by thefarmers and facilitators especially regarding the managementof whitefly and CLCuV disease.

Field data revealed that CLCuV attack was more severe inareas with uncertified crop varieties, unawareness amongfarmers and multi-crop fields. High infestation of thrips,whiteflies, dusky bugs, jassids, and pink bollworm was foundin majority of cotton fields of all areas. Overall it was observedthat the incidence of the diseases was less as compared tolast year which could be due to climatic changes as well ascotton varieties cultivated. Early and mid-sowing gave bettercrop situation especially regarding CLCuV prevalence. Lessdisease incidence was observed in late April (early sowing)and May (mid sowing) planting especially within two monthsperiod as compared to June sowing.

4. Farmer Field Day A Farmer Field day was organized by Sindh AgriculturalDevelopment Association, (SADA) Umerkot during the monthof September 2015 where FFS farmers, non-FFS farmers,Facilitators, District Agriculture Extension personnel andmembers of SADA attended and participated in the saidactivity. Representatives of FFS farmers presented their



progress and shared their learning through FFS process. Theyalso discussed the management practices for CLCuV besidebest management practices of cotton crop.

5 Technical guidelines for management of CLCuVD for farmers in English/Urdu:

A manual on “Cotton Best Management Practices” wascompiled earlier in English and printed while the manual hasbeen translated in Urdu and Sindhi languages for facilitatinglocal cotton growers and farmers with special reference toeconomically important CLCuV disease and its managementin the field in order to reduce the crop losses. The manualshave been finalized and are under the process of printing.

6 Additional Work In order to aware the FFS village children about cotton cropecology, environment, pesticides & their hazards, diseases andpests Children Ecological Clubs (CEC) were conducted inFFS villages of eight project districts (Bahawalpur, DG Khan,Khanewal, Multan, Vehari, Khairpur, Sanghar and Umerkot).Overall, 40 CEC were organized in eight districts duringsummer vacations where 1419 school children attended andparticipated in the said sessions. The children age range was4-15 years while educational level was primary to middle class.

7 Next year plans: As per proposal

Objective-8: To facilitate training opportunities and exchanges for Pakistani researchers.

Partners: 1) ARS, Stoneville, 2) Borlaug Institute, 3) Texas A&M College Station, 4) University ofArizona, 5)Cotton Incorporated, 6)U S Embassy, Islamabad. 7) ICARDA-HQ, Lebanon 8)ICARDA–Pakistan, 9) Concerned ministry/Department

Sr.#


1. Borlaug Fellowship 2014-15

Four scientists completed their training in USA under Borlaugfellowship during the year under report.

2 International Conferences and Meeting/workshops

Dr. Dill Baugh Muhammad, Focal person Agronomycomponent attended Annual Meeting of Agronomy, CropScience Society of America & Soil Science from November 2-5, 2014 at Long Beach, Cal, USA.

6 scientists of different collaborating institutions attended theBeltwide Cotton Conference from 05-07 Jan 2015 at SanAntonio, TX, USA.

5 Scientists from different Collaborating Institutes participatedin International Plant and Animal Genomic Meeting fromJanauary10-14, 2015 at San Diego, CA, USA. A projectmeeting was also held there chaired by Dr. Brian Scheffler.Project collaborators from IAGS, CEMB, NUST and Universityof Arizona attended the meeting.



Hafiz Abdul Haq and Miss Iram Waqar S.Os of Plant Breedingand Genetics Section have completed 3 months training atNIBGE Faisalabad.

Local Trainings/ workshops

Workshop on Omics of Begomoviruses and its Impact onNational Economy was organized at ASAB, National Universityof Science and Technology from March 25-27, 2015 for thetraining of young scientists. About 30 scientists from differentinstitutes and universities attended the Workshop.

Two weeks training workshop on Genomic Assisted Breedingin cotton improvement from 2nd March to 13th March, 2015 wasorganizes by NIBGE, Faisalabad. About 30 young scientistsparticipated in this workshop.

Three months training on “Molecular Breeding of Cotton” wasorganized in PGMB lab, National Institute for Biology andGenetic Engineering (NIBGE), Faisalabad under Pak-USGene Mapping Project from Jan 19 to April 19, 2015.

9- Other major achievements/activities.

1 Annual Review and Planning Meeting.

Annual Review and Planning Meeting was convened fromDecember 8-11, 2014 at Seminar Hall of PGRI, Islamabad. Themeeting was chaired by Dr. Brian Scheffler and co-chaired byDr. Jodi Scheffler. The meeting was attended by all the PIs andFocal persons. Achievement and next year work plan of all thecomponents of the project was discussed in detail.

2 Two days’ workshop of theproject.

Two days’ workshop was convened from July 18-19, 2015 atAuditorium of National agricultural Research Center,Islamabad. Inaugural Session was chaired By Mr. SakanderHayat Bosan, Federal Minister, for Ministry of Food Securityand Research, Govt of Pakistan. The workshop was attendedby no. of farmers, politicians, and scientists.

Collaborators of the project from USA, Dr. Brian Scheffler, Dr.Jodi Scheffler and Professor Dr. Jodith Brown also came toattend the workshop.

Workshop was followed by the meetings with PIs and Focalpersons of all the components of the project to review theproject activities.

3 Quarterly project reviewand planning meeting(July-September 2015).

Forth quarterly review and planning meeting was convened atIAGS, University of the Punjab, Lahore on October 9, 2015 tocollect progress reports of various project components anddeliberate on various issues.



4 Other meetings of theproject.

Meetings with the individual PIs/Focal persons at variouslocations in the field and in the lab. To discuss the projectresearch activities.

5. Financial Progress of theproject.

Annual financial report (October, 2014-September, 2015) shallbe submitted by the Account office.

Selected Plants of Highly CLCuD Tolerant F4 Line 154029



Farmer Field Day on Relay Cropping of cotton at Bahawalpur 14 August 2015



Glimpses of Farmer Field Schools


enhancing cotton germplasm, improving resistance to cotton ... · icarda’s pakistan-us cotton...

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