INTRODUCTION• More than 10 million hectares of Bt cotton were

cultivated in India in 2012. With a total of 11.6 millionhectares of cotton, this equates to an adoption of 93 %,an increase of 5 % compared to 2011.

• More than seven million of the eight million farmersopted for Bt cotton in 2012. These usually smallfarmers cultivate cotton on an average agricultural areaof 1.5 ha.

• Prior to the introduction of Bt cotton in India,approximately 5.9 g of pesticides was required for theproduction of 1 kg of cotton.

• In 2010, less than 0.9 g of pesticides was used for theproduction of 1 kg of Bt cotton, which equates to analmost seven-fold reduction in the use of pesticides.

• Indian Bt cotton farmers spend 31 % to 52 %

less on insecticides and achieve a 34 % to 42

% higher cotton yield per ha than farmers who

cultivate traditional cotton.

• Although the total production cost price of Bt

cotton is 15 % higher than that of non-Bt

cotton, the income of Bt cotton farmers is 53

% to 71 % higher.

• The Bt technology is at least 19 % responsible

for the increase in yield per ha of the Indian

cotton cultivation in the past 10years.

• Partly due to Bt cotton, India has developed intothe second biggest producer of cotton since 2006,after China, but ahead of the United States.

• Today, India accounts for 21 % of the globalcotton production.

• India changed from a net importer of cotton fibersto an exporter: in 2009 – 2010, more than 8million bales of 170 kg were exported, accountingfor US$ 2.3 billion in revenue.

• Cotton is the second most important field crop inIndian for the production of table oil: 13.7 % ofthe 7.88 million tons of table oil produced in Indiais obtained from cottonseed.

• Cottonseed has also become the main ingredient(33 %) in processed animal fodder, with soy,rapeseed and groundnuts lagging far behind.

THE CHRONOLOGY OF BT COTTON DEVELOPMENT BY

MAHYCO IS AS FOLLOWS:• 1994 - Formation of IBSC and application for transgenic Bt cottonseed

import.

• 1995 - Permit from DBT received to import 100 g. Bt Cotton seed ofCoker 312 from Monsanto, USA.

• 1996 - Imported seed and Green House trial initiated.

• 1996 - Limited field trial (1 Location) to assess pollen escape.

• 1996 - Back crossing (Ongoing) breeding for transfer of Bt gene into

elite parental lines in green house.

• 1997/98 - Limited field trials (5locations) to assess pollen escape.

• 1998 - Toxicological (Ruminant goat model) and Allergenicity (BNR

model) studies.

• 1998/99 - Multi-centric research trials (15 +25 locations) to assess efficacy

of Bt gene in Indian elite germplasm.

• 1999 - Multi-centric research trials (11 locations) to assess efficacy of

Bt gene in Indian elite germplasm.

• 2000/01 - (a) Large-scale trials (100ha) to assess efficacy of Bt gene in

Indian elite germplasm and the performance of Bt hybrids.

• (b) Hybrid seed production (150 ha)

• (c) Various biosafety studies.

• (d) ICAR trials at 6 locations.

2001 - 02- (a) Large-scale trials (100ha) toassess efficacy Bt gene in Indian elite germplasmand the performance of Bt hybrids,

• (b) Hybrid seed production (300 ha)

• (c) Biosafety studies.

• (d) ICAR Trials at 11 locations.

• April 2002 Commercial cultivation in Six States(Andhra Pradesh, Gujarat, Karnataka, MadhyaPradesh, Maharashtra, Tamil Nadu)

No. of hybrids/varieties per event approved for cultivation in

India till august 2009

A list of the Bt cotton events approved

for cultivation in India

Availability of Cottonseed for Processing in India

(2001-2002 to 2003-2004 and 2005-2006 to 2011-2012)

Items2001-

02

2002-

03

2003-

04

2005-

06

2006-

07

2007-

08

2008-

09

2009-

10

2010-

11

2011-

12

Cotton Production

(lakh bales)158 140 167.5 241 280 310 305 305.00 339.00 347.00

Cottonseed Production (@333kg/bale) (lakh tonnes)

52.61 46.62 55.77 80.25 93.24 103.23 101.57 101.57 112.89 115.51

Retained for Sowing &

Direct Consumption

(lakh tonnes)

11.3 10 11 13 13 13 5 5.00 5.00 5.00

Marketable Surplus

(lakh tonnes)41.31 36.62 44.77 67.25 80.24 90.23 96.57 96.57 107.89 110.51

Production of Washed

Cottonseed Oil (lakh

tonnes)

4.13 3.66 4.77 7.4 8.83 9.92 11.59 11.59 12.94 13.26

TYPES OF HYBRIDS

In Cotton hybrid seed production is done either by

conventional hand emasculation and pollination or by

nonconventional (male sterility based).

Conventional Hybrids

Majority of the hybrids released so far are conventionalones. Development of such hybrids involve three stepsviz.

(i) Identification and growing of male and femaleparents,

(ii) emasculation of female parent and

(iii) pollination of female parent with identified maleparent.

Cotton is an often cross pollinated crop.

The average outcrossing is 6%.

The pollen is heavy and sticky and hence cross

pollination occurs only by insects i.e. honey

bees and bumble bees.

In diploid cottons conventional method is

highly uneconomical since boll setting is low

due to small flower size and brittle pedicel.

MALE-STERILITY BASED HYBRIDS

The development of hybrids using malesterility eliminates the process of emasculationsince the anthers are sterile in female parentwithout pollen.

The cost of hybrid seed production can be reduced.

pollination has to be done manually.

In cotton, mainly two types of male sterilitysuch as genetic male sterility and cytoplasmicgenetic male sterility are used for seedproduction.

ISOLATION DISTANCE

Maintenance of genetic purity for certifiedseed production of conventional or malesterility based hybrids depends upon the use ofsafe isolation distance.

The cotton hybrid seed production plot shouldhave 30 m isolation distance on all sides fromother fields.

The isolation distance between parents shouldbe minimum 5 m.

The other field standards to be met forcertification

Planting ratio : varies (4:1 or 5:1)

Seed rate : female parent 3.75kg per ha

: male parent 2.5kg per ha

Spacing : row to row 150cm

: plant to plant 100cm (female)

: 50cm (male)

RAISING OF PARENTAL LINES

An inbred line to be eligible for certification shall

be from a source such that its identity may be

assured and approved by the certification agency.

Hybrid seed to be certified should be the progeny

of two approved inbred lines one of which shall

be male sterile.

An inbred line shall be a relatively true breeding

strain resulting from self pollination with

selection.

The foundation class seed shall consist of anapproved male sterile line to be used as femaleparent and an approved inbred line to be used as amale parent for the purpose of producing hybridseed.

A male sterile line shall be a strain carryingcytoplasmic genetic male sterility which sheds noviable pollen and is maintained by the normalsister strain which is used as a pollinator.

The certified class seed shall be the hybrid seed tobe planted for any use except seed production.

SOWING

Breeder/ foundation seeds of selected hybrids.Female and male parents are planted in the samefield in separate plots with 5 m isolation betweenparents and 30 m from other cotton crop.

The sowing dates of parental lines are so adjustedin such a way that there is synchronization offlowering in female and male parent and therewill be continuous supply of pollen till thecrossing season is over.

Staggered planting of male is generally donedepending on the date of flowering in male andfemale.

STEPS IN HYBRID SEED

PRODUCTION (Conventional)

EMASCULATION OF FEMALE PARENT

This is a highly skilled, laborious andcumbersome process.

Different emasculation methods have beendevised which can be adopted depending uponthe flower types.

Flower buds which are likely to open thenext-day are chosen for emasculation forwhich the best time is after 1 P. M.

DOAK METHOD OR THUMBNAIL METHOD:

In Tetraploid cottons 40 to 50% or more seedsetting is obtained.

The method involves removal of corolla alongwith anther sheath by giving shallow cut at thebase of the bud with thumb nail and removingcorolla and anther column in one jerk twistingaction (Doak, 1934).

Care should be taken to ensure that the whitecover membrane of the ovary is not damaged orremoved during this operation as this affects theboll setting.

It should also be verified that no anther sacremains at the base of ovary at the time ofemasculation. This will cause selfing and causegenetic impurity by increased number of seed ofthe female parent.

Emasculated flower buds are generally coveredwith tissue paper bag (9cmx7cm) so as to preventcontamination from foreign pollen.

If no open flowers are left over in the field offemale parent, bagging may be avoided butmarking of emasculated flower helps inidentification during

PINCHING OFF OF TOP OF COROLLA /SURAT METHOD

The top portion of flower bud is pinched offusing thumb and first finger nails of hand sothat the tip of stigma get slightly exposed andthe bud is covered with mud.

As the buds mature the stigma tip extendsufficiently for enabling pollination.

In genotypes where stigma tip exertion iscomparatively low, instead of cutting the topportion of corolla, the entire corolla is removedand lint dipped in mud is applied on unopenedanther sacs. The pollen is applied next morning(Mehta et al, 1983).

STRAW TUBE/COPPER STRAW METHOD

The top of the corolla of bud is pinched offand a piece of straw tube is used by inserting iton the style so as to separate all anthers fromanther column and leaving the tube in the same1 position till pollen is applied next morning(Hays et al, 1953).

However during the process of emasculationrelatively more time is required for insertingthe straw tube exactly on the style so thatanthers would get dislodged.

PETAL REMOVAL AND BRUSHING OFF ANTHERS:

Only flower petal is removed by thumb nailmethod and the anthers brushed off by lightlytouching and moving the thumb and first fingerdown and up along the staminal column.

A light tapping of the flower pedicel will dislodgeany anthers sticking in the bracts.

This method is useful in herbaceum cotton budswhere anthers are of granule type, get removedvery easily and drop to the ground

PROBLEMS ENCOUNTERED IN EMASCULATION

CROSSING OF THE PARENTS

The emasculated buds are covered with red coloured tissue bags foreasy identification.

The emasculated buds are pollinated the next day between 8 and 11 ambecause stigma receptivity is maximum during this period.

When crossing is done during the months Oct and – Nov end, the maleflowers need to be spread on a cloth and put under sun for few hours foreffective pollen dusting.

The crossed flowers are again covered with a white colored tissue bagto distinguish them from emasculated bud awaiting cross pollination.

A thread is tied to the pedicel for identification of crossed bolls at thetime of picking.

If proper isolation is given, and early morning

removal of open flowers in female parents is

assured bagging before and after is not

required.

For good setting and boll development, it is

necessary that crossing programme should be

confined to about 10- 14 weeks after the

commencement of flowering.

Nowadays tieing of thread to pedicel after

crossing is also avoided by removing bracts, it

is better if only one bract is removed and two

bracts are intact.

SEED STANDARDS FOR CERTITIED HYBRID COTTON SEED

USE OF CYTOPLASMIC GENETIC MALE STERILITY

The sterility of G. arboreum and G. anomalumis not stable and is influenced byenvironmental factors.

The CMS line of G. harkensii cytoplasm ishighly stable and fertility.

Restoration is also possible which is beingwidely used for hybrid seed production.

The A, B and R lines are maintained by asgiven above and are planted in separate blocks.

DEMERITS OF CGMS SYSTEM

Availability of limited number of restorers is a

major drawback in this system of male

sterility.

Transfer of undesirable characters along with

sterile cytoplasm is also a major concern in

development of CGMS lines.

USE OF GENETIC MALE STERILITY GMS lines are generally simple in inheritance and

controlled by one or two pairs of nuclear recessive or dominant genes.

The lines controlled by recessive genes are used to produce hybrid seed.

Maintenance of genetically double recessive male sterility is done by crossing male sterile plants by pollen obtained from corresponding heterozygous male fertile plants.

The major drawback in this is that the cross population segregates into male sterile and male fertile plants in the ratio of 1:1.

One way to overcome this is to sow two or

three seeds per dibble and fertile plants

thinned out at the flowering stage to ensure

that enough male sterile plants are left.

A total of 17 GMS lines have been identified.

ms14and ms5ms6 are being utilized for hybrid

seed production in India and China.

Maintenance of male sterile population in

GMS system of hybrid seed production is

difficult as compared to cytoplasmic genetic

male sterile system and also less quantity of

seed is produced.

Low ginning in male sterile line which is not

inherited in F1 hybrids is also advantageous in

obtaining higher proportion of hybrid seed.

LIMITATIONS OF HYBRID SEED PRODUCTION

High cost of hybrid seed (conventional and male sterilehybrids).

Difficulty in seed production (diploid hybrids) due tovery poor seed setting (about 25%).

The availability of pure hybrid seeds in time is also amajor limitation due to the requirement of Grow-outtest for genetic purity which does not fit in the periodbetween seed production and planting in the nextseason.

The high cost of conventional hybrid seeds is due torequirement of emasculation and pollination which canbe reduced to some extent through use of male sterility.

The yield of male sterility based hybrids is 10-15% lower than conventional hybrids sincerestoration of sterility is again a problem.

In Hybrid seed production pollen transferredby wind, and requires insects especiallyhoneybees.

In addition to above there is also the problemof parrot beak boll formation due to lowdusting of pollen when same flower is used topollinate more than 4 female flowers.

HONEY BEES AS POLLINATORS INHYBRID

SEED PRODUCTION These pollinating agents when used in male

sterile hybrid seed production enable us to

significantly reduce the cost of hybrid.

It is known that 50 pollen grains per stigma are

enough to fertilize all the ovules (McGregor,

1976), in insect pollinated cultivated crop plants.

On an average 10 bees per 100 cotton flowers are

reported to be sufficient to practically coat all

stigma with pollen.

The yield of seed cotton in A line also depends on

the bee population and distribution of bee

colonies.

It was found that 13-15 bee colonies are

adequate for economical seed production.

The reasons attributed to this are shift in visit

of honey bees from cotton to other more

rewarding crops in the area, higher gossypol

content in cotton which may affect the

foraging behaviour and the heavy pesticidal

schedule used for the control of pests.

Few basic studies show that sucrose content in theflower nectar especially in CMS flowers affectedsignificantly the honey bee population density andmight be a key factor for higher hybrid seed productionin CMS lines (Mofett et al, 1976; Wang et al, yet to bepublished).

Among various sugar components of the cotton nectar,only sucrose showed highly positive and significantcorrelation with visiting honey bee frequency.

Thus breeding lines with higher sucrose content isneeded for increasing bee visits which in turn canincrease the boll setting per plant and decrease thenumber of aborted seed, resulting in higher yield ofhybrid seed.

FACTORS AFFECTING YIELD AND

QUALITY OF HYBRID SEED

1. The planting ratio of male and female parents.

2. No: of male flowers used in pollinating one female flower.Using one male flower to pollinate more than three femaleflowers is found to decrease the hybrid seed yield and quality.

3. Staggering sowing pattern of parents. If staggering is notfollowed as required seed yield will decrease due to failure insynchronization.

4. Stage and period of crossing : Seeds produced after crossingduring the second fortnight (15 days) of flower initiation hasthe highest germination compared to later produced hybridseeds.

5. Vigour of female parental seed.

6. Pollen production ability of male parent.

7. Position of boll set on the plant.

Problems during emasculation:

Bract damage during emasculation, damage to

style and stigma cause low seed set.

MANAGEMENT TECHNIQUES AND

HYBRID SEED PRODUCTION

It was observed that the reduction in pollination

period from 60 to 30 days followed by the

removal of fruiting forms other than pollinated

flowers and developing cross bolls, at specific

intervals significantly increased the boll setting

percentage maintaining the yield level similar to

the 60 day pollination period.

Reducing the pollination period during rainy

season would make available the hybrid seed by

December providing sufficient time to conduct

grow-out test which in turn will enable the timely

availability of hybrid seed in the market.

PLANT TRIMMING AND PRUNING

Pruning of sympodia and removal of basalmonopodia along with aged leaves allows us tomanage a second off-season crop (summer) and athird (second rainy season) crop.

The trimmed crop responds well to irrigation andnutrient inputs and is different from a ratoon cropin that the incidence of pests and diseases is less.Also enable a further reduction of pollinationperiod from 30 to17 and 13 days respectively inthe subsequent two crops.

This management technique could be adoptedwherever the irrigation facility is available toincrease the per unit time productivity of hybridseed cotton.

Mixed planting of parental lines:

Mixed planting fashion of parental lines has

also been found to increase hybrid seed

production instead of the normal spaced row

planting method.

This helps in reducing the distance between

MS and restorer lines in the seed production

field, thus increasing the pollen transfer from

fertile to sterile flowers.

USE OF GAMETOCIDE INDUCED MALE

STERILITY

This method of inducing male sterility is fast andinexpensive compared to backcross method and there isno necessity to use maintainer lines.

The effective chemicals used in various crop plants areSodium Methyl Arsenate, Zinc Methyl Arsenate,Naphthalene Acetic Acid (NAA), Gibberellins, Ethrel,Maleic Hydroxide and FW 450.

Highest pollen sterility has been found to be caused by1.5% FW-450 applied twice i. e before bud initiationand during bud initiation. (P. Singh and Sanjeev Singh– 1999).

PROPERTIES OF AN IDEAL GAMETOCIDE

It should be selective in inducing male

sterility with affecting ovule fertility.

It should give consistent/reproducible results.

It should be economical with simple

application method.

It should be safe with minimum side effects

on plant growth.

THERMOSENSITIVE GENETIC MALE STERILITY In cotton GMS lines of G. arboreum were observed

showing sensitivity to different temperature regimesespecially under lower minimum temperatures (<180C)where there is a sterility breakdown.

Histological studies showed that male sterility in cottonis post meiotic and that microspores are not releasedafter the tetrad stage due to non degradation of callosewall surrounding the tetrad.

Later the microspores inside become shriveled andaborted.

Callase enzyme mediates this degradation process ofcallose wall releasing microspore from tetrad which isactive under reduced temperature and inactive at highertemperature expressing the fertility and sterilityrespectively.

Merits of the system

• (1) It helps to overcome the problems of crossing for maintenance of parental lines

• (2) It helps to overcome the problem of linkage drag due to transfer of sterile cytoplasm

• (3) Prevents the problem of low restoration since any fertile line could be used as a pollen parent.

Demerits of the system

• (1) Any sudden change in the environmental factors will influence the sterility of EGMS leading to more contamination.

• (2) The multiplication of EGMS lines and hybrid seed production will be restricted by space and season.

APOMIXIS

Apomixis helps to fix the hybrid vigour once a

desirable combination has been selected and

made. The hybrid seed could be multiplied and

maintained just like a straight variety.

Bt cotton seeds being too expensive

development of apomictic Bt hybrids will

prove economical since the seeds need not be

purchased every season.

In cotton, development of apomictic lines is in

progress and possibilities of evolving

apomictic Bt hybrids is being explored.

BT COTTON/ TRANSGENIC COTTON

In Bt cotton toxin producing gene has beenutilized from the soil bacterium Bacillusthuringiensis.

The Bt gene is very effective against theAmerican Boll worm (Helicoverpa armigera) themajor pest of cotton.

In India, Bt cotton was first approved forcommercial cultivation by the CentralGovernment in March 2002.

Initially three Bt hybrids viz; MECH 12,MECH162, MECH 184 were released forcommercial cultivation.

By now 133 Bt cotton hybrids (Private) have beenreleased for cultivation in three cotton growingzones of India.

Bt gene is being incorporated in the parental lines(either male or female or both) used fordevelopment of hybrids.

Difference in expression levels of toxin in theplants as well as seeds of Bt hybrid when maleparent alone or female parent alone or both usedas Bt parent need to be studied from seed testingperspective.

With growing demand for Bt hybridsGovernment has formulated certain laws for seedquality control of such hybrids.

HYBRID GROW OUT TEST Once Seed Bill, 2004 starts operational all Bt

cotton hybrids will have to undergo geneticpurity testing by a field grow out test.

Genetic purity testing of Bt cotton hybridusing Grow out test is confronted by variousissues.

Determining the threshold level for Btexpression in the hybrid.

Significant variation for CrylAc expressionhave been reported in different Bt cottonhybrids despite having a common geneinsertion event.

The crylAc expression is found to declineprogressively over the crop growth with toxinlevels falling below the critical level of1.9mg/g after 110 days.

Identifying the ideal plant part which need tobe examined for cry protein expression. This isbecause expression of crylAc is found to behighly variable in different plant parts.

Highest level was found in leaves of seedlingsfollowed by squares, boll rind and flowers.

Submitted sample size: 25g

Working sample size: 10 seeds

For Bt transgenics Bt toxin level (minimum)

450 nanog/sq.cm or /g seed

Purity for Bt protein expression 90% (9 out of

10 seeds tested should be Bt+ve)

BT DETECTION METHODS: Three Bt detection methods, viz; (i) Bt express

(ii) Bt detection (iii) Bt quarantine are in commonuse.

Bt Express:

This is an instant check method where a stripprovided with the kit is dipped into a vialcontaining seed crushed with supplied buffer.

If two bands develop at the top it indicatespresence of Bt gene Cry1 Ac and Bt positive.

If only one band develop at the top end of thestrip it indicates that the sample is Bt negative.

It is a very easy method that can be used even byfarmers besides laboratories and industries.

FUTURE PROSPECTS

In diploid hybrids the seed setting is very poor(about 20%). Hence efforts have to be made toimprove seed setting in diploid hybrids.

In future, more emphasis has to be laid on thedevelopment of CGMS Based hybrids whichwill help in providing hybrid seeds to farmersat cheaper rate.

Research on use of two line and one line andmethod of hybrid development and seedproduction needs to be intensified.

Most of the presently available Bt hybrids are conventional. There isneed to develop CGMS based transgenic hybrids.

To have a successful seed testing programme on Bt cotton it isessential to understand the level of toxin expression in seed coat fordifferentiating F2 seeds from F1 hybrid seeds.

Difference in transgene expression in direct and reciprocal Btcrosses i.e. Bt x NBt and NBt x Bt and Bt x Bt to solve the problemof varied expression in same hybrid while testing Level of toxinexpression in seeds when more than one transgene is present.

Expression of toxin levels on different qualities of same seed lot.

Study on the effect of Bt gene on various seed vigor and storability.

Efforts are also needed to further improve the planting value ofhybrid seeds especially with respect to enhancing shelf life duringseed storage.

REFERENCESHybrid seed production in field crops- principles and

practices by N. C. SINGHAL (2013).

Seed technology by R.L. AGARWAL.

Nucleus and breeder seed production manual byNational seed project-IARI by P. K. CHOWDARY.

Cotton seed production by CICR Nagpur.

Cotton statistics at a glance by VIB.

Cotton statistics by INDIASTAT.

Egypt to Produce Bt Cotton Soon By Prof. Mohamed Abdel Hakeem Nagib. Director, CRI.

Cotton seed production by TNAU Agri portal.