I= REGENERATION RESPONSE OF GOSSYPIUM HIRSUTUM, L
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Title of Thesis
REGENERATION RESPONSE OF GOSSYPIUM HIRSUTUM, L

Author(s)
Rozina M.Ali
Institute/University/Department Details
National centre of Excellence in Molecular Biology University of the Punjab, Lahore
Session
2003
Subject
Molecular Biology
Number of Pages
144
Keywords (Extracted from title, table of contents and abstract of thesis)
GOSSYPIUM HIRSUTUM, Somatic embryogenesis, Agrobacterium mediated transformation, Biolistic medicated transformation, Paraticle bombardment, pest management, Herbicide, Embryo germination

Abstract
Plant genetic engineering offers unique opportunities to a) overcome compatibility barriers, exploit the expression potential of genes from unrelated genetic resource materials and also avoid the problems associated with transfer of large blocks of DNA between the donor and the recipient plants. The first successful plant genetic transformation was achieved only two decades ago and during the intervening period. significant progress has been made. As consequence hundreds of plant species have been genetically transformed. with a variety of different genes possessing novel traits. Most notable of these novels genetic traits is insect resistance, herbicide tolerance and nutritional improvement.

Two main methodologies employed to achieve stable expression of an unrelated novel gene in any crop plant are predominantly though not exclusively to yield stable transformation. Both methodologies, namely biolistic transformation and Agrobacterium mediated transformation, employ an appropriate tissue ex-plants as the starting material, which after the introduction of exogenous DNA, are allowed to regenerate into fertile plants. Evidently, therefore, regeneration response of a target crop is of critical importance. Succes3ful regeneration of any tissue ex-plant into a fertile plant depends upon the genetic make-up of the target crop, the nature of the ex-plant chosen, in vitro growth medium used, growth conditions including light, temperature etc. Tissue explants d certain crops such as cotton, secrete toxic metabolites such as phenolic compounds into the medium, which must be removed to allow sustained, in vitro development into a fertile plant.

Previous studies in CEMB laboratories had demonstrated that cotton and chickpea are recalcitrant crops and different cultivars of cotton exhibit extremely varied regeneration response. Whereas Upland cotton, Coker has proved to be the most successful in regeneration response while in Pakistan (Gossypium hirsutum) is one of the most difficult crops to regenerate.

In a background of the foregoing, different ex-plants of various Pakistani cotton cultivars including elite varieties were studied for their ability to regenerate into fertile plants. The effects of different growth hormones in the growth media, temperature of incubation, intensity of incident light and nature of the ex-plant were investigated to establish parameters from that would be used in the research project on transformation of elite varieties of Pakistani cotton with exogenous genes to breed novel traits.

As part of these studies eighteen local cultivars, were studied among them CIM 482, FH 901, CIM 443, CIM 448, BH 36 and FH 900 were selected on the basis of germination response somatic embryogenesis and multiple shoot formation was observed only in variety CIM 482. During these studies, two phenomenan namely. recurrent somatic embryogenesis and twin embryo production were observed for the first time in cotton. When germinating embryos were transferred to MSO medium. growth and production of new somatic embryo from old somatic embryos (Recurrent) was observed Twins and multiple embryos were also formed.

In another part of these studies different ex-plants of various local varieties were tested for their ability to form multiple shoots. Induction of multiple shoots was achieved in CIM 482 and CIM 443 with meristem and cotyledonary nodes. Among the entire tested media, maximum shoot multiplication response 6.'72 and 4.92 per meristum and cotyledon ex-plants was observed in MS medium where SAP 1.0 mg /L and NAA 0.05 mg /L were used.

Conditions for biolistic transformation were optimized, 46.7% GUS expression was observed. Selection of transforl1ants was found optimal at 100ug/ml of the Kanamycin for cotton ex-plants. Using optimal selection and biolistic transformation conditions, Pakistani cotton cultivars CIM 482, was genetically transformed with Cry/Ac under wound inducible promoter (MPI) and constitutive promoter (CaMV 35S) and the comparative strengths of the two promoters were studied. The results of these studies showed the maximum transformation efficiency 4.0 % was observed in case of bombarded embryos in horizontal orientations while a transformation efficiency 3.2% was observed where the embryos were bombarded in vertical orientation.

Moreover studies on Biosafety aspect of transgenic crops was also carried out. Because transgenic cotton plants were not available at large numbers therefore these studies were done on transgenic rice transformed in CEMB.

The developmental and spatial expression of Bt genes was studied and it was found out, the toxin titer declined substantially with increasing age of the plant. The cry protein was detected 15 days after harvesting of plants and none of Bt proteins were found in rice straw.

The plants containing two cry genes were used for these studies. Both gene(s) showed expression in all the parts of plants studied like roots, stem, panicle, straw husk and seed. It was also determined that transgenic rice has no allelopathic effect on wheat. A laboratory study was also done on spider survival after feeding on Rice-leaf folder and yellow stem borer. These insects fed on transgenic rice has no effect on predators. The effect on non-target insects of rice like Hemiptera, Homoptera and Orthoptera was also studied. Damage was compared with target and non-target insects. This indirectly proves that transgenic rice has no effect on non-target insects. In other experiments, it was also determined that proteins were secreted from transgenic plants to liquid and solid media as well as in the soil. The secreted protein is taken up by non-tr8nsgenic plants.

These studies describe the optirnization of tissue culture conditions like media composition effect of phytohormones, effect of ex-plant and effect of genotype on the regeneration of local varieties. Subsequently somatic embryogenesis and multiple shoot formation was observed in local cotton variety CIM 482 and complete plant formation was observed in American Upland cotton variety. This variety CIM 482 was used for transformation with vector containing MPI, promoters and plant developed showed the expression of cry gene as detected by ELISA and bioassay of transgenic plants. The integration of gene was determined by PCR amplification of cry gene in the genome of cotton. Biosafety studies were done that concern about gene flow, effect on non-target insects and allelopathy. These studies showed that cotton is recalcitrant plant however, conditions can be developed where mature embryos can be used to transform the cotton plant and transgenic plant has no or insignificant effect on environment, in the experiments designed for this purpose.

Download Full Thesis
2455.04 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
125.7 KB
2 1 Introduction 1
45.47 KB
3 2 Literature survey 5
227.35 KB
  2.1 Tissue and cell suspension culture 7
  2.2 Somatic embryogenesis and regeneration 9
  2.3 Agrobacterium mediated transformation 9
  2.4 Biolistic medicated transformation 9
  2.5 Paraticle bombardment and agrobacterium mediated transformation( a combined approach) 12
  2.6 Role BT. Intergrated pest management system 12
  2.7 Herbicide resistant crops possible becoming wed s in following years 20
  2.8 Risk assessment of GM crops 22
4 3 Materials and methods 23
146.62 KB
  3.1 Source of different varieties 23
  3.2 Treatment with seeds 23
  3.3 Tissue culture media 23
  3.4 lant explant mature embryos 24
5 4 Results 35
1563.72 KB
  Germination response 35
  4.2 Plant development 35
  4.3 Regeneration response from meristem (shoot nod ) 46
  4.4 Regeneration from residual meristem( cot. Nod ) 46
  4.5 Callus initiation 46
  4.6 Somatic embryogenesis and somatic embryo initiation 47
  4.7 Response of medium sued for osmotic regulation for embryogenic callus culture 47
  4.8 Effect of basal media on regeneration and plant recovery 54
  4.9 Somatic embryo initiation and development in variety CIM 482
  4.10 Recurrent somatic embryogenesis , twin and multiple embryo production 58
  4.11 Embryo germination 58
  4.12 Morphology of callus 58
  4.13 Establishment of regenerated plants in soil 64
  4.14 Multiple shoots formation 67
  4.15 Cotton tissue culture for the transformation 67
  4.16 Optimization of kanamycin selection condition for cotton embryos (CIM 482)
  4.17 Biolistic transformation with construe pMPAc containing constitutive promoter(MPI) & PBinAc containing constitutive promoter( CaMV35S) optimized condition 73
  4.18 Molecular analysis of putative transgenic plants 73
  4.19 Bioafety studies of transgenic plants 83
6 5 Discussion 96
330.08 KB
  5.1 Multiple shoots 99
  5.2 Selection condition improvement of cotton 100
  5.3 Genetic transformation of cotton by optimized condition of biolist bombardment 100
  5.4 Molecular analysis of the transgenic plants 100
  5.5 Protein based expression studies of wound inducible promoter (MPI) & constitutive promoter( 35S) 101
  5.6 Bioassay based expression studies of insecticidal genes 101
  5.7 Reference 103
  5.8 Appendices