An efficient and highly reproducible approach for the selection of upland transgenic cotton produced by pollen tube pathway method

Pollen tube pathway mediated transformation is most favorable genotype-independent approach and has been widely used for the production of transgenic crops all over the world. We used this technique to transform Zheda B, an upland cotton, by two different vectors i.e. pCAMBIA1301 with hpt gene for hygromycin resistance and pCAMBIA 2301 having nptII gene for kanamycin resistance. A preliminary experiment was conducted to find out suitable concentration of kanamycin (100-600 mg/L) and hygromycin (25-150 mg/L) for the screening of transgenic plants. Results revealed that both antibiotics caused a significant reduction in seed germination, seedling survival, plant height, root length, fresh and dry weight of root and shoot as compared to their respective controls. Furthermore, 500 mg/L kanamycin and 75 mg/L hygromycin were established as effective dozes to eliminate 8085% of non-transgenic seedlings during seedling stage. The outcomes of the preliminary experiment were applied to select transgenic plants from T1 population, which were later confirmed by PCR and GUS staining. The results were identification of positive transgenic plants with successful integration of GUS gene into recipient cotton genome. Comparing both antibiotics, upland cotton was found to be more sensitive to hygromycin application as compared to kanamycin. Therefore, designing gene constructs for pollen tube pathway mediated transformation; hptII gene seems to be more appropriate choice than nptII gene for selection of transgenic plants. The protocols are robust and can be utilized for other crop species as well.