I= Genetic Transformation of Cotton with Galanthus Nivalis Agglutinin (GNA) Gene
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Title of Thesis
Genetic Transformation of Cotton with Galanthus Nivalis Agglutinin (GNA) Gene

Syed Sarfraz Hussain
Institute/University/Department Details
University of the Punjab/National Centre of Excellence in Molecular Biology
Molecular Biology
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
cell tissue, organ culture, bacillus thuringiensis, seed sterilization, embryo germination, regeneration system, somatic embryogenesis, agrobacterium, transgenic plants

Genetic engineering is a breeding strategy that attempts to avoid the problems associated with the transfer of large blocks of genetic material between two parents. The current state of technology allows only a very limited number of foreign genes (from any life source) at a time to be introduced into a plant. However, single gene traits cause the least disruption of the existing plant genome and are much easier to develop in subsequent breeding efforts. A well-established regeneration system is desirable for cotton improvement. In this perspective, different explants were used to judge their regenerability and during these attempts, two phenomena i-e, recurrent somatic embryogenesis and twin embryo production were described for the first time in cotton. Similarly, conditions were established to enhance the regenerabilty of immature embryos of cotton. In-ovule embryo culture was used for the first time as a method of cotton transformation and a marker gene was also used to transform cotton embryo. This tissue culture protocol developed generated large amounts of embryogenic calli and copious numbers of somatic embryos in the existing cotton germplasm. Although the number of regenerated plants was low, the protocol has the potential to be used for the regeneration of other elite commercial cultivars. Similarly, a minimal amount of genetic change was associated with this protocol as the somatic embryos and regenerated plants were mostly genetically uniform. In vitro and In planta shoot tip grafting of Gossypium hirsutum was found more effective than direct soil rooting method since it increased the survival rates by >40%. Shoot grafting was particularly helpful in recovering non-rooting putative transgenic shoots from culture and it has provided biotechnologists an edge over hormonal or other In vitro root induction procedures.

Agrobacterium provides one of the main vehicles for introducing DNA into the plants but the transformation frequencies were very low. In order to enhance transformation rates, improvements have been made in the existing transformation procedures. Recently, sonication has been used to enhance Agrobacterium mediated transformation of many different plant species. Many parameters were optimized for the enhancement of GUS transient expression in cotton using mature embryos and zygotic ovules as explants. The beauty of this method is that the cavitation caused by sonication results in thousands of micro-wounds on and below the surface of the plant tissue. This permits Agro bacterium to travel deeper and more completely throughout the tissue, thus increasing the probability of infecting plant cells. Development of Bt transgenic plants to protect crop losses due to insect attack is a technically feasible and otherwise viable strategy. However, there are concerns that insects could evolve genetic resistance to transgenic plants harboring single insecticidal gene. One of the strategies to delay insect resistance build up is the use of multiple genes (gene pyramiding). Two different insecticidal gene cry1Ac and gna were selected on the basis of their different modes of action against different insects. These genes were first transferred to a binary vector pBinGNAC and then transformed in cotton using sonication assisted Agrobacterium mediated transformation (SAAT) method. Molecular analysis of transgenic plants generated through this procedure confirmed stable integration and expression of the introduced genes. This is the first report for the control of major insects using a non-Bt gene gna and the stable integration of two highly significant insecticidal genes as a result of single step transformation for pyramiding resistance against insect pests of cotton. This present approach will delay or perhaps in combination with integrated pest management practices prevent evolution of insect populations resistant to single insecticidal gene.

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2196.89 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
112.11 KB
2 1 Introduction 4
58.85 KB
3 2 Review Literatur 4
601.2 KB
  2.1 Taxonomy 8
  2.2 Domestication 10
  2.3 Cell Tissue & Organ Culture of Cotton 11
  2.4 Expression and Segregation of Transgenes 43
  2.5 Lectins: Plant Defense Proteins 49
  2.6 Bacillus thuringiensis: The History 64
4 3 Materials& Methods 83
378.26 KB
  3.1 Selection of Varieties 83
  3.2 Delinting of Seeds 83
  3.3 Seed sterilization 83
  3.4 Preparation of Culture Media 84
  3.5 Preparation of Explant 84
  3.6 Cell Suspension Culture & Maintenance 87
  3.7 Embryo Germination 87
  3.8 Somatic Embryogenesis from Immature Zygotic Embryos 91
  3.9 In-Ovule embryo Culture 91
  3.10 In vitro & In planta Shoot Tip Grafting 92
  3.11 Establishment of Regenerated Plants in Soil 92
  3.12 Construction of Vector 94
  3.13 Optimization of Conditions for Sonication of Mature Embryos & Zygotic Ovules 105
  3.14 Transformation of CIM-446 with GUS Reporter Gene 108
  3.15 Agrobacterium Mediated Transformation 109
  3.16 Preparation for Transformation Studies 110
  3.17 Transformation of Gossypium hirsutum cv. CIM-446 with pBinGNAc 112
  3.18 Molecular Analysis of Transgenic Plants 114
  3.19 Western Blot analysis 115
  3.20 Genomic DNA Isolation 116
  3.21 Polymerase Chain Reaction (PCR) 117
  3.22 Southern Blot Analysis 118
  3.23 Insect Bioassay 120
5 4 Results 121
891.63 KB
  4.1 Cotton plant Formation from Mature Embryos 121
  4.2 Regeneration Response from Shoot Apices 121
  4.3 Callus Initiation 123
  4.4 Immature Zygotic Embryo 127
  4.5 In-ovule Embryo Culture 130
  4.6 In vitro & In planta Shoot Tip Grafting 134
  4.7 Establishment of Regenerated & Grafted Plants in Soil 139
  4.8 Construction of Vector 143
  4.9 Comparison of BMT & SMT Transformation Methods 151
  4.10 Transformation of Cotton Variety CIM-446 with pBinGNAc 151
  4.11 Molecular Analysis of transgenic Plants 155
6 5 Discussion 174
131.64 KB
7 6 References 190
253.39 KB
8 7 Appendices 214
40.06 KB