Pakistan Research Repository Home

Title of Thesis

Tahira Fatima
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)
tissue culture, rice (oryza sativa l.), insect, pests, basmati, food crop, lepidopteran, bt gene, callus formation

Rice is one the world’s most important food crop. It is the second major cash crop in Pakistan. Pakistani Basmati rice holds a great commercial value for its distinguished long grain and aroma. Two lepidopteran insects pests of rice are leaf folder and yellow stem borer cause massive losses of yield. Generation of insect resistant transgenic plants with Bt gene is one of the solutions to combat this problem. The aim of present study was to transform elite variety Basmati rice with insect resistant Bt gene. For this purpose the tissue culture for efficient regeneration and optimization of conditions for stable integration and expression of foreign genes by using both Agrobacterium and biolistic method were studied.

In regeneration experiments, six indica and three japonica varieties were used. The response of callus formation on different callus inducing media was observed. The highest frequency of callus formation (97%) was observed in N6 medium supplemented with praline (300mg/L) and casein enzymatic hydrolysate (500mg/L). When embryogenic calli of different varieties was checked on regeneration medium, Super Basmati, Basmati 370and JP 5 showed 88%, 87% and 97% efficiency respectively. Regeneration of Basmati 370 and Super Basmati was also achieved from cell suspension obtained from scutellum-derived calli.

Eight varieties including Bsmati 370 and Super Basmati were assessed for their regeneration capability using meristematic leaf base tissue. The response of meristem egeneratiuon was better for Basmati 370, Super Basmati and DR 60 than other varieties. The conditions for cryopreservation of these cell suspension culture were optimized. The post-thaw recovery of viable cells after different time periods was studied. The frequency of survival was improved up to 90% by using hydroponic solution to devlelop vigorous rooting system. The regenerated plants produced 100% viable seeds on maturity.

Considering the better tissue culture performance and economic importance of the variety Basmati 370 it was selected for transformation. Conditions of biolistic bombardment were optimized by using GUS reporter gene. The best results 75% explants positive for GUS were achieved by using osmoticum (0.4M sorbitol) treatment. The scutellum derived calli were used as target tissue. The transformation efficiencies were found to be 3.5%2.43% in case of biolistic co-transformation and Agrobacterium respectively. Biolistic transformation with genes on single replicon was better (3.9%) as compared to gene residing onseparate replicons.

The integration and expression of introduced genes was studied by PCR analysis, Southern hybridization, GUC assay Western blot analysis and insect feeding assay. The transgenic plants from T0, T1 and T2 generation were subjected to bioassay at whole plant level. A mortality of 100% was observed of second instar larvae of leaf-folder.The stable integration of cry1Ac and cry2A originating from separate replicons proved a useful strategy for gene transformation to delay the resistant against Bt gene. The inheritance study for successful transmission of foreign gene into next generations was done. The progeny plants of T1, T2, T3, and T4 generation were subjected to different molecular and biological analysis. The plants with Mendelian and non-Mendelian inheritance pattern were observed and T2 and T3 homozygous lines were identified for field trial.

Download Full Thesis
1628.47 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
108.95 KB
2 1 Introduction 1
58.39 KB
3 2 Literature Review 7
196.78 KB
  2.1 In Vitro Tissue Culture Of Rice 7
  2.2 Transformation Of Rice 13
  2.3 Bt As A Biological Control Agent For Insect Pests Of Rice 23
  2.4 Selection, Molecular And Biological Analysis Of Transgenic Rice Plants 25
4 3 Materials And Methods 30
212 KB
  3.1 In Vitro Tissue Culture Of Rice 30
  3.2 Transformation Of Basmati Rice 36
  3.3 Inheritance Of Transgenes In Basmati 370 52
5 4 Results 55
870.33 KB
  4.1 In Vitro Tissue Culture Of Rice 55
  4.2 Transformation Of Rice 70
  4.3 Molecular And Biological Analysis Of Primary Transformed Rice Plants 81
  4.4 Inheritance Of Transgenes In Basmati 370 106
6 5 Discussion 121
139.6 KB
  5.1 In Vitro Tissue Culture Of Rice 122
  5.2 Transformation Of Rice 127
  5.3 Molecular Analysis For Integration And Expression Of Genes 130
  5.4 Inheritance Of Transgenes Genes In Basmati 370 134
7 6 References 138
214.41 KB