Genetic Diversity and Expression Analysis of Cry1Ac Gene in Transgenic Cotton (Gossypium hirsutum L.)

Abstract

Upland cotton (Gossypium hirsutum L.) is also known as a “white gold” due to their high fibre producing quality in the world. Diverse genotypes of cotton have been used to improve the yield related traits. In the current study, two years data of morphological and fibre quality traits of 71 cotton genotypes were recorded under field conditions. Highly significant diverse responses were recorded in morphological and fibre traits. The results of agro-morphological traits illustrated that seed cotton yield showed positive correlation with plant height (0.14), No. of bolls (0.17), internode distance (0.05) and boll weight (0.26) while yield negatively correlated with DFI (-0.15). Fibre strength was positive correlated with uniformity ratio (0.33**) and length (0.25*) whereas negative correlated with fineness (-0.36**), elongation (-0.25*) and GOT (-0.18). Based on principal component analysis, the first component contributed 32.4% diversity in the morphological traits while 31.5% in fibre traits. Morphological traits of 71 cotton genotypes were distributed into various clusters. In the second part of this study, molecular diversity through 63 SSR markers in 71 cotton genotypes were determined. Most of the SSRs showed polymorphic bands having overall PIC values range 0.03 - 0.78. Genetic similarity index in all genotypes based on 63 SSR markers were ranged from 0.3 to 0.8. Through UPGMA method, dendrogram of 71 cotton genotypes was constructed in which they were distributed into five groups. Group B alone has contributed 46.48% of diversity responses in all genotypes. In 71 genotypes Mon-531 event (Cry1Ac gene) was confirmed through PCR and immunostrip assays. At 80 DAS, validation of relative fold expression of Cry1Ac gene transcript analysis in all cotton genotypes were performed in cotton parts and the overall relative expression level ranged (0.45% to 4.81%). In the next part of this study, spatio-temporal expression of Cry1Ac gene was checked in field grown 71 cotton genotypes for two years at 80, 120 and 150 DAS in leaf, boll and square tissues. Results illustrated that diverse responses of Bt protein contents were recorded in all genotypes. Optimum Cry1Ac protein content was recorded in leaf tissue followed by boll and square tissues while 80 DAS showed high Bt protein followed by 120 and 150 DAS respectively. In the last part of this study, top ten cotton genotypes in terms of Bt protein contents at 80 DAS were further subjected to Hoagland hydroponic culture to evaluate physiological, biochemical and expression analysis of Cry1Ac gene under salt and drought stresses for three and six days. Three treatments i.e. 100, 150 and 200 mM of salt and 10%, 15% and 20% PEG were used for drought stress. Under both abiotic stresses, physiological responses i.e. chlorophyll contents, stomata conductance and net photosynthetic rate in ten genotypes were reduced as compared to control. More reduction was noted after 6 days and high concentration of salt and drought stresses as compared to three days of stress. Diverse biochemical responses (SOD, POD, CAT, MDA, hydrogen peroxide and proline) were achieved under salt and drought stress treatments. While elemental analysis under salt stress revealed that sodium ions were increased while calcium, potassium and magnesium ions were reduced. Similarly Cry1Ac gene expression under salt and drought stresses were more declined after 6 days of stress than 3rd day. Based on abiotic stress treatments results, PR-129 and NAR-34 genotypes showed salt tolerant responses while NB-61 genotype exhibited drought tolerant response. These genotypes can be further used under salinity and drought field areas in the country