**8. Novel trends and perspectives**

Introgression breeding procedures have been deployed to introgress important traits from *G. arboreum* L. like, resistance to CLCuD, tolerance to drought etc. into the cultivated *G. hirsu‐ tum* L. cotton varieties. In these experiments, chromosome of *G. arboreum* L. was doubled by applying colchicine followed by hybridization with the allotetraploid *G. hirsutum* L. under natural conditions. Exogenous treatment of hormones 50 mg/L gibberellic acid and 100 mg/L naphthalene acetic acid were applied for overcoming the problem of boll shedding. It has been demonstrated that the percentage of pollen viability in F1s was 1.90% in 2(*G*. *arboreum*) x *G. hirsutum* versus 2.38% in *G. hirsutum* x 2(*G*. *arboreum*). Further confirmations were made through cytological studies; found that all F1s were sterile. All the F1 plants exhibited resistance to the CLCuD after exposing through grafting of the infected buds, indicating the chances of success for transferring resistance into the cultivated tetraploids [36]. Currently the progenies/ advance generations are being screened against the disease at CCRI Multan (Project PI Mr. Zahid Mehmood, Pr Scientist) and CRS Multan (Project PI Dr. Saghir Ahmed, Botanist). Similarly, emphasis was also made for improving quality parameters of the local cultivated species var FH-1000 by crossing it with the *G. barbadense* L. The introgressions of the DNA fragments from *G. barbadense* L. were monitored through SSR markers [37]. The resultant hybrids or progenies had two types of leaves, narrow versus broad lobed types. It was reported that bolls of the narrow lobed types did not mature due to bad opening. However, the broad lobed types were found comparatively high yielder but poor in lint quality. Efforts on various

In early seventies, activities related to the collection as well as conservation of germplasm have been started in Pakistan. In this regard, cotton germplasm have been collected from various countries largely of upland cotton. Recently, more than 3000 accessions have been imported from US under the Pak-US cotton productivity enhancement project. Before this, cotton material (accessions of *G. hirsutum*) was imported from different countries including USA, Uzbekistan, and France, etc. Most of the accessions are available with CCRI Multan, and maintained descriptions of each accession both hard as well as soft copy which can be obtained upon the request. However, no website is available showing the description of the cotton

Efforts for conservation of genetic material are also under way at PGRI, Islamabad. Passport data of around 75% of the accessions of different crops has been entered in the form of dBase files. Users can get information in the form of computer print outs on request. However, local scientists on a limited scale can get direct on-line access to the files. Consultation of data books that are maintained by the institute can be done at any time by local scientists. Linkage of documentation section with all PGRI laboratories is made sure by availability of a local network that is not in working condition yet. However, currently there are no options available for networking with other gene banks for the data exchange on crop basis as well as regional basis. The process of data base and information system establishment is on the move. PGRI

fronts are going on for evolving useful germplasm or varieties.

has plans for its connection to other gene banks in future.

**7. Databases**

156 World Cotton Germplasm Resources

germplasm in Pakistan.

Exotic Bt cotton strains were first cultivated in Sindh in 2000. This introduced material showed high susceptibility to sucking insect pests and CLCuD. Breeding to introgress the *Bt* gene (*Cry1Ac*) by backcross hybridization was initiated in 2000 by various public and private sector organizations of Pakistan but resulting lines could not be tested in the field due to the sus‐ pension of biosafety rules in Pakistan. The National Institute for Biotechnology & Genetic engineering (NIBGE) initiated development of Bt cotton and field testing under the "voluntary code of conduct" issued by the Ministry of Environment. A huge quantity of data for the safe release of Bt cotton in the field was generated using rabbit as an experimental animal, and also the impact of Bt cotton residues on weed and soil microbial populations. It was demonstrated that the cultivation of Bt cotton is safe to wild as well as domesticated animals, and its impact, if any, will be low or negligible. This approach for characterizing risk is consistent with the accepted risk assessment procedures and shared similarities with the previous assessments over a wide range of situations (Zaman & Co-workers, unpublished).

The area under Bt cotton cultivation has been increased dramatically. Around 40,000 kg of seed of the Bt cotton strains IR-FH-901 (later approved as IR-NIBGE-901), IR-NIBGE-2 (later approved as IR-NIBGE-1524 in 2010), IR-CIM-448 (later approved as IR-NIBGE-3701) and IR-CIM-443, was provided to farmers and was grown on over 3,238 ha (hectares) in 2005-2006 [38]. IR-NIBGE-1524 was approved for general cultivation in 2010 and 2011 for Punjab and Sindh (Table 4). This variety was drought tolerant, with an open canopy and bears small bolls. It was planted on a large area (more than 5%) in 2007 and retained ~2% of the area, particularly in the drought prone, each year until 2012 in Punjab. In Sindh, it was planted on ~10% of the area in 2012 (Director General Agriculture Sindh). The NIBGE Bt cotton strains were used exten‐ sively in breeding programs as a source for developing Bt cotton varieties by various research organizations, and established the foundation of Bt cotton cultivation in Pakistan.

In post-Bt era, preference for cultivating compact to semi-compact varieties has been given for sowing in normal season. Earlier, semi-compact to compact type cotton varieties like CIM-448, CIM-497, NIAB-111 and BH-160 were released for general cultivation but could not capture significant area. There were two major reasons. Firstly, it is difficult to control insect pests especially bollworms in compact shaped plant versus open type plant. Secondly, compact shaped plant does not compensate for low population density compared to the open shaped plant. Before Bt cotton cultivation, major area >10% covered by open type varieties, viz. B-557, NIAB-78, MNH-93, S-12, CIM-240, NIAB-Karishma, CIM-473 and CIM-496 etc. Bt cotton varieties offered inbuilt resistance to *Heliothus*, spotted and marginally to pink bollworm. Thus one of the disadvantages of cultivating compact shaped varieties has been addressed. First Bt cotton variety, IR-NIBGE-3701—semi-compact shaped variety, tested for yield in National Coordinated Bt Trials (NCBT) in 2009, out yielded all candidate lines and standard cotton variety CIM-496. IR-NIBGE-3701 formed the basis for cultivation of compact shaped variety among the farming community. Later on, CIM-886 dominantly a compact shaped variety covered a significant area in 2012.


**Name of variety**

15 Sitara-009

16 A-One

by bollworms.

**9. Conclusions**

**Center of release**

13 FH-114 CRI, Faisalabad 2012

14 IR-NIBGE-3 NIBGE, Faisalabad 2012

Agri Farm Research Center,

Multan

Weal Ag Corporation, Multan

**Table 4.** List of approved Bt-cotton varieties

**Year of release**

**Pedigree/parentage**

variety

of FH-1000

x MNH-786}

\$=Approved for Punjab and Sindh provinces while rest of the varieties are only approved for Punjab; £=Approved for

**Source:** Minutes of 42nd meeting of Punjab Seed Council at Lahore dated Feb 16, 2012, approval documents of cotton

Now the emphasis is on releasing varieties with a high boll count and a low shedding rate. It has dramatically been changed after the introduction of Bt cotton as it offers inbuilt resistance to the cotton plant; otherwise this trend has not been observed in varieties released before the Bt-era. Also, spring cultivation is gaining popularity in the Punjab province. Around 5-10% of the area is sown early (Feb-March) because of the Bt varieties are not prone to early infestation

In Pakistan the provision of high quality seed has been a major issue that emerged after the first epidemic of CLCuD. The informal seed sector (growers/breeders/private seed companies) profited by selling unapproved seed of advanced resistant lines resulting in the release of unstable cotton lines in early 1990s and onward. This situation was further exaggerated after

2012

Sindh province only; ¥=Ginning out turn percentage; **€**=Thousand pounds per square inch

varieties and personal communication with breeders of the cotton varieties.

Non-*Bt* early maturing cotton lines FH-925 with Australian *Bt*

Bollgard-1(*Cry1Ac*)

Developed through selection from IR-NIBGE-2381);Bt version

{(CIM-496 x Sitara-008)

**GOT %¥**

2012 {FVH-53 x Exotic *Bt*} 38.01 29.91 4.56 96.6

**Staple length mm**

**Fineness**

39.64 28.12 4.85 95.5

38.68 28.3 4.96 97.6

39.8 25.7 4.87 97.6

**µg/inch**

Cotton Germplasm of Pakistan http://dx.doi.org/10.5772/58620

**Strength**

**tppsi€**

159


\$=Approved for Punjab and Sindh provinces while rest of the varieties are only approved for Punjab; £=Approved for Sindh province only; ¥=Ginning out turn percentage; **€**=Thousand pounds per square inch

**Source:** Minutes of 42nd meeting of Punjab Seed Council at Lahore dated Feb 16, 2012, approval documents of cotton varieties and personal communication with breeders of the cotton varieties.

**Table 4.** List of approved Bt-cotton varieties

Now the emphasis is on releasing varieties with a high boll count and a low shedding rate. It has dramatically been changed after the introduction of Bt cotton as it offers inbuilt resistance to the cotton plant; otherwise this trend has not been observed in varieties released before the Bt-era. Also, spring cultivation is gaining popularity in the Punjab province. Around 5-10% of the area is sown early (Feb-March) because of the Bt varieties are not prone to early infestation by bollworms.

## **9. Conclusions**

**Serial #**

1

2

**Name of variety**

158 World Cotton Germplasm Resources

IR-

IR-

3 Neelum-121

4 FH-113

5 AA-802

6 AA-703

7 MG-06

8 Sitara-008

11 Tarzen-1

12 Neelum-141

**Center of release**

NIBGE-3701\$ NIBGE, Faisalabad <sup>2010</sup>

NIBGE-1524\$ NIBGE, Faisalabad <sup>2010</sup>

Neelum Seeds Corporation

Ali Akbar Seeds, Multan

Ali Akbar Seeds, Multan

ThattaGurmani Research Center, KotAdu, Muzafarghar

Agri Farm Research Center,

Multan

9 IR-NIBGE-901£ NIBGE, Faisalabad 2011

9 MNH-886 CRS, Multan 2012

4-Brothers Seed Corporation, Multan

Neelum Seeds Corporation

CRI, AARI, Faisalabad **Year of release**

2010

**Pedigree/parentage**

Selection from IR-CIM-448

Transgenic line as a donor parent for Bt gene.NIBGE-2 as an adapted parent

[{(FH-1000 x HK-303) x LRA-5166} x Linea-100]

Transgenic line as a donor parent for Bt gene. FH-901as an adapted parent, used in

FH-207 x MNH-770 x Bollguard-1

{(CIM-496 x hk 303) x

backcrossing

10 Bt. CIM-598 CCRI, Multan 2012 CIM-446 x IR-CIM-448 41.82 29.03 4.38 94.8

Linea-100}

2012

**GOT %¥**

2010 A-92 x exotic variety 41.87 28.70 4.81 29.5

2010 FH-925 x Bollgard 38.13 28.61 5.00 24.85

2010 CIM-482 x Exotic Line 38.8 29.8 4.45 99.98

2010 CIM-443 x IR-448 38.0 29.32 4.7 28.7

2010 NIAB-III x IR-448 40.0 27.3 4.6 95.9

2012 IR-448 x C-2-2 41.05 29.0 4.9 101.5

**Staple length mm**

**Fineness**

43.23 27.52 5.43 90.2

38.55 30.15 4.73 92.5

43.26 29.49 4.77 92.8

38.86 27.06 5.38 90.8

41.01 28.21 4.95 99.5

42.6 29.15 4.96 95.0

**µg/inch**

**Strength**

**tppsi€**

In Pakistan the provision of high quality seed has been a major issue that emerged after the first epidemic of CLCuD. The informal seed sector (growers/breeders/private seed companies) profited by selling unapproved seed of advanced resistant lines resulting in the release of unstable cotton lines in early 1990s and onward. This situation was further exaggerated after the introduction of Bt cotton varieties. Thus a number of varieties, not properly bred, have been released in a very short time period, which accelerated the varietal replacement rate. All these issues hampered the process of production of certified seed.

at CRS Multan and CCRI Multan. Similarly, QTLs/genes conferring high quality traits have been transferred into *G. hirsutum* L. using DNA markers at NIBGE, Faisalabad. Another strategy for the creation of genetic variability is the deployment of various mutagens (radiation and chemical). In this regard, leading genotypes of *G. hirsutum* L. and G*. arboreum* L. have been treated with EMS to develop TILLING populations—would help in understanding the genes

Cotton Germplasm of Pakistan http://dx.doi.org/10.5772/58620 161

The introduction of new genes from distantly related species using gene cloning and trans‐ formation approaches has emerged as a revolutionary genomic tool worldwide. In common with many other major cotton growing countries, the public sector in Pakistan has made substantial investment in developing GM-cotton conferring resistance to biotic and abiotic stresses. Bt cotton containing *Cry1Ac* gene is cultivated on 82% of the area of Pakistan. The first step towards the introduction of two genes (*Cry1Ac* and *Cry2Ab*) has been taken at CEMB Lahore and NIBGE Faisalabad. The material is being tested in multiple trials. For commerci‐ alization of advanced cotton lines containing these two genes, proposals for approval from the National Biosafety Committee have been submitted. Secondly, the expressing transgenes in different genetic backgrounds should be quantified to identifying genotype (s) best suited for commercial cultivation. This practice will reduce the possibility of resistance against the target pest developing—will help in formulating IPM strategies. Thirdly, Bt toxins are lethal to insects belonging to different orders. Hence, proper characterization of Bt gene cultivars is imperative before their release into the environment. To evaluate the possible impact of transgene containing cotton, or their byproducts, it is important to establish dedicated biosafety labs, which are lacking at the moment in the country, Ethyl methanesulfonate to ensure the safe

involved in conferring various traits of interest.

release of GM crops and their products.

AARI Ayub Agricultural Research Institute AFLP Amplified Fragment Length Polymorphism

CEMB Centre of Excellence in Molecular Biology

ARI Agricultural Research Institute CBD Convention on Biological Diversity CCRI Central Cotton Research Institute

CLCuD Cotton Leaf Curl Disease CRI Cotton Research Institute CRS Cotton Research Station DAS Days After Sowing

GM Genetically Modified

**Abbreviations**

EMS

The low germination of most cotton varieties, particularly in the post-Bt cotton era, is another area of concern for growers, regulators and policy makers. The germination rate can be improved by avoiding the use of early opened bolls and seed cotton exposed to excessive rain. Similarly, proper control of the moisture content of seed and proper storage conditions can also ensure the good health of cotton seed.

For Bt-cotton, the mixing of various types, mixing of non-Bt seed with the Bt variety, and the expression level of Bt genes in different varieties are the major issues which need to be addressed. The marketing of earlier released cotton varieties/strains under different names in the market is another area of concern which has affected the reputation of the cotton seed industry. In this regard, FSC&RD must ensure the distinctness of each of the newly developed varieties and or advanced strains with authenticated pedigree that may be verified by the use of DNA fingerprinting.

The deterioration of a cotton variety leads to reduced seed cotton yield. One of the major causes of this is a high natural cross pollination rate, largely by honeybees, in the Bt cotton era due to a reduction in the number of insecticide applications. Most of the breeding centers are located near urban areas where farmers also grow fodder and vegetable crops which provide alter‐ native hosts for pollinators. Under such circumstances, selfing of plants is recommended on the representative plants of the variety/genotype which would help in maintaining the typical features of the variety.

Cultivation of hybrid cotton showing heterosis for seed cotton yield has remains a major challenge in Pakistan. Conventional methods of hybrid seed production (manual emasculation of floral buds), low seed setting, high cost of production resulting in high cost of seed and purity of seed are the major issues for cultivating hybrid cotton on significant area. Though limited efforts by the private sector (Mr Siddique Akbar Bukhari spent ~30 years; Guard and Four Brothers Seed Corporation Pakistan.) and public sector organizations (CCRI Multan, CRI Faisalabad, NARC Islamabad etc.) have been made, but are unable to provide seed which can cover even one percent of the total cotton growing area of Pakistan. In this regard, the development of male sterile and restorer lines, deployment of new genomic tools (such as RNAi technology), and also chemical emasculation, are the most plausible approaches for overcoming the issue surrounding the widespread adoption of hybrid seed.

Cultivated cotton has a narrow genetic base which limits future breeding progress. The selection and crossing of well adapted cotton varieties for developing new varieties are the main causes of the narrow genetic base. This problem can be partly overcome by involving genetically diverse parent genotypes in the genealogy of a new variety. For example, genes conferring resistance to abiotic stresses, particularly drought, and biotic stresses, particularly resistance to CLCuD, can be introgressed into *G. hirsutum* L. from *G. arboreum* L. and or *G. herbaceum*L. Tissue culture tools may help in overcoming the phyletic barriers. Preliminary steps have already been taken for introgressing useful genes into the cultivated cotton varieties at CRS Multan and CCRI Multan. Similarly, QTLs/genes conferring high quality traits have been transferred into *G. hirsutum* L. using DNA markers at NIBGE, Faisalabad. Another strategy for the creation of genetic variability is the deployment of various mutagens (radiation and chemical). In this regard, leading genotypes of *G. hirsutum* L. and G*. arboreum* L. have been treated with EMS to develop TILLING populations—would help in understanding the genes involved in conferring various traits of interest.

The introduction of new genes from distantly related species using gene cloning and trans‐ formation approaches has emerged as a revolutionary genomic tool worldwide. In common with many other major cotton growing countries, the public sector in Pakistan has made substantial investment in developing GM-cotton conferring resistance to biotic and abiotic stresses. Bt cotton containing *Cry1Ac* gene is cultivated on 82% of the area of Pakistan. The first step towards the introduction of two genes (*Cry1Ac* and *Cry2Ab*) has been taken at CEMB Lahore and NIBGE Faisalabad. The material is being tested in multiple trials. For commerci‐ alization of advanced cotton lines containing these two genes, proposals for approval from the National Biosafety Committee have been submitted. Secondly, the expressing transgenes in different genetic backgrounds should be quantified to identifying genotype (s) best suited for commercial cultivation. This practice will reduce the possibility of resistance against the target pest developing—will help in formulating IPM strategies. Thirdly, Bt toxins are lethal to insects belonging to different orders. Hence, proper characterization of Bt gene cultivars is imperative before their release into the environment. To evaluate the possible impact of transgene containing cotton, or their byproducts, it is important to establish dedicated biosafety labs, which are lacking at the moment in the country, Ethyl methanesulfonate to ensure the safe release of GM crops and their products.
