Herbicide-resistant cotton (Gossypium hirsutum) plants: an alternative way of manual weed removal.

BACKGROUND: Cotton yield has been badly affected by different insects and weed competition. In Past Application of multiple chemicals is required to manage insects and weed control was achieved by different conventional means, such as hand weeding, crop rotation and polyculture, because no synthetic chemicals were available. The control methods shifted towards high input and target-oriented methods after the discovery of synthetic herbicide in the 1930s. To utilise the transgenic approach, cotton plants expressing the codon-optimised CEMB GTGene were produced in the present study. RESULTS: Local cotton variety CEMB-02 containing Cry1Ac and Cry2A in single cassette was transformed by synthetic codon-optimised 5-enolpyruvylshikimate-3-phosphate synthase gene cloned into pCAMBIA 1301 vector under 35S promoter with Agrobacterium tumifaciens. Putative transgenic plants were screened in MS medium containing 120 µmol/L glyphosate. Integration and expression of the gene were evaluated by PCR from genomic DNA and ELISA from protein. A 1.4-kb PCR product for Glyphosate and 167-bp product for Cry2A were obtained by amplification through gene specific primers. Expression level of Glyphosate and Bt proteins in two transgenic lines were recorded to be 0.362, 0.325 µg/g leaf and 0.390, 0.300 µg/g leaf respectively. FISH analysis of transgenic lines demonstrates the presence of one and two copy no. of Cp4 EPSPS transgene respectively. Efficacy of the transgene Cp4 EPSPS was further evaluated by Glyphosate spray (41 %) assay at 1900 ml/acre and insect bioassay which shows 100 %mortality of insect feeding on transgenic lines as compared to control. CONCLUSION: The present study shows that the transgenic lines produced in this study were resistant not only to insects but also equally good against 1900 ml/acre field spray concentration of glyphosate.

Molecular and Biochemical Characterization of Cotton Epicuticular Wax in Defense Against Cotton Leaf Curl Disease.

BACKGROUND: Gossypium arboreumis resistant to Cotton leaf curl Burewala virus and its cognate Cotton leaf curl Multan beta satellite (CLCuBuV and CLCuMB). However, the G. arboreum wax deficient mutant (GaWM3) is susceptible to CLCuV. Therefore, epicuticular wax was characterized both quantitatively and qualitatively for its role as physical barrier against whitefly mediated viral transmission and co-related with the titer of each viral component (DNA-A, alphasatellite and betasatellite) in plants. OBJECTIVES: The hypothesis was the CLCuV titer in cotton is dependent on the amount of wax laid down on plant surface and the wax composition. RESULTS: Analysis of the presence of viral genes, namely alphasatellite, betasatellite and DNA-A, via real-time PCR in cotton species indicated that these genes are detectable in G. hirsutum, G. harknessii and GaWM3, whereas no particle was detected in G. arboreum. Quantitative wax analysis revealed that G. arboreum contained 183 µg.cm-2 as compared to GaWM3 with only 95 µg.cm-2. G. hirsutum and G. harknessii had 130 µg.cm-2 and 146 µg.cm-2, respectively. The GCMS results depicted that Lanceol, cis was 45% in G. harknessii. Heptadecanoic acid was dominant in G. arboreum with 25.6%. GaWM3 had 18% 1,2,-Benenedicarboxylic acid. G. hirsutum contained 25% diisooctyl ester. The whitefly feeding assay with Nile Blue dye showed no color in whiteflies gut fed on G. arboreum. In contrast, color was observed in the rest of whiteflies. CONCLUSIONS: From results, it was concluded that reduced quantity as well as absence of (1) 3-trifluoroacetoxytetradecane, (2) 2-piperidinone,n-|4-bromo-n-butyl|, (3) 4-heptafluorobutyroxypentadecane, (4) Silane, trichlorodocosyl-, (5) 6- Octadecenoic acid, methyl ester, and (6) Heptadecanoicacid,16-methyl-,methyl ester in wax could make plants susceptible to CLCuV, infested by whiteflies.

Functional characterization of a bidirectional plant promoter from cotton leaf curl Burewala virus using an Agrobacterium-mediated transient assay.

The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional gene promoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant.

Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton

Background: Transgenic plants inhabiting single Bt gene are prone to develop insect resistance and this resistance has been reported in case of some important yield-devastating insect larvae of commercial crops, such as cotton and rice. Therefore, it has become essential to adapt new strategies to overcome the problem of insect resistance and these new strategies should be sophisticated enough to target such resistant larvae in broad spectrum. Among these, plants may be transformed with Bt gene tagged with some fusion-protein gene that possesses lectin-binding capability to boost the binding sites for crystal protein gene within insect mid-gut in order to overcome any chances of insect tolerance against Bt toxin. Enhanced chloroplast-targeted Bt gene expression can also help in the reduction of insect resistance. Results: In the present investigation, a combined effect of both these strategies was successfully used in cotton (G. hirsutum). For this purpose, plant expression vector pKian-1 was created, after a series of cloning steps, carrying Cry1Ac gene ligated with chloroplast transit peptide towards N-terminal and Ricin B-Chain towards C-terminal, generating TP-Cry1Ac-RB construct. Conclusions: Efficacy of pKian-1 plasmid vector was confirmed by in-planta Agrobacterium-mediated leaf GUS assay in tobacco. Cotton (G. hirsutum) local variety MNH-786 was transformed with pKian-1 and the stable integration of TP-Cry1Ac-RB construct in putative transgenic plants was confirmed by PCR; while fusion-protein expression in cytosol as well as chloroplast was substantiated by Western blot analysis. Whereas, confocal microscopy of leaf-sections of transgenic plants exposed that hybrid-Bt protein was expressing inside chloroplasts.