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Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton

View Article: PubMed Central

ABSTRACT

Cotton fiber is multigenic trait controlled by number of genes. Previous studies suggest that one of these genes may be responsible for switching cotton fiber growth on and off to influence the fiber quality produced from a cotton seed. In the present study, the Gossypium hirsutum GhEXPA8 fiber expansin gene was introduced into local cotton variety NIAB 846 by using an Agrobacterium-mediated gene transformation. The neomycin phosphotransferase (NPTII) gene was used as a selection marker for screening of putative transgenic cotton plants. Integration and expression of the fiber expansin gene in cotton plants was confirmed with molecular techniques including Southern blot analyses, real-time PCR. Cellulose assay was used for measurement of cellulose contents of transgenic cotton fiber. The data collected from 3 years of field performance of the transgenic cotton plants expressing GhEXPA8 showed that significant improvement has been made in fiber lengths and micronaire values as compared to control G. hirsutum variety NIAB 846 cotton plants. Statistical techniques were also used for analysis of fiber and agronomic characteristics. The results of this study support improvement of cotton fiber through genetic modification.

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Complete protocol of agrobacterium mediated transformation in cotton plant embryos. (A) germinated cotton seeds, (B) isolation and injuring of cotton embryos, (C) Agrobacterium treated embryos on MS medium, (D) Agrobacterium treated embryos on MS medium after 3 days, (E) implantation of cotton embryos in test tube containing MS medium with kanamycin, (F) shoot growing on MS medium containing kanamycin selection, (G) root development on MS rooting medium, (H) transgenic cotton plants shifted to soil for acclimatization.
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Figure 1: Complete protocol of agrobacterium mediated transformation in cotton plant embryos. (A) germinated cotton seeds, (B) isolation and injuring of cotton embryos, (C) Agrobacterium treated embryos on MS medium, (D) Agrobacterium treated embryos on MS medium after 3 days, (E) implantation of cotton embryos in test tube containing MS medium with kanamycin, (F) shoot growing on MS medium containing kanamycin selection, (G) root development on MS rooting medium, (H) transgenic cotton plants shifted to soil for acclimatization.

Mentions: Agrobacterium-mediated transformation was used to incorporate the GhEXPA8 gene into NIAB 846. A total of 8500 embryos were transformed with the GhEXPA8 gene and selected on MS medium containing 50 mg/ml kanamycin (Figure 1). There were 106 putative transgenic plants, an efficiency of 1.24% of 8500 transformed embryos on the base of transformation procedure and on the base of confirmed transgenic cotton plants is 0.07%, obtained and transferred to MS medium with kanamycin selection. The putative transgenic plants were screened through selection medium as shown in (Figures 1D,E). On the base of PCR analysis of transgenic cotton plants at T0 and T1 generations, these transgenic cotton plants were subjected to molecular analysis such as PCR, southern blot analysis, Real Time PCR, cellulose assay, fiber characteristics and yield parameters at T2 generation in order to protect chimeric presence of gene in few part of cotton plants. There was only one cause behind this protocol in order to screen the transgenic cotton plants from chimera or low expression of gene.


Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton
Complete protocol of agrobacterium mediated transformation in cotton plant embryos. (A) germinated cotton seeds, (B) isolation and injuring of cotton embryos, (C) Agrobacterium treated embryos on MS medium, (D) Agrobacterium treated embryos on MS medium after 3 days, (E) implantation of cotton embryos in test tube containing MS medium with kanamycin, (F) shoot growing on MS medium containing kanamycin selection, (G) root development on MS rooting medium, (H) transgenic cotton plants shifted to soil for acclimatization.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4628126&req=5

Figure 1: Complete protocol of agrobacterium mediated transformation in cotton plant embryos. (A) germinated cotton seeds, (B) isolation and injuring of cotton embryos, (C) Agrobacterium treated embryos on MS medium, (D) Agrobacterium treated embryos on MS medium after 3 days, (E) implantation of cotton embryos in test tube containing MS medium with kanamycin, (F) shoot growing on MS medium containing kanamycin selection, (G) root development on MS rooting medium, (H) transgenic cotton plants shifted to soil for acclimatization.
Mentions: Agrobacterium-mediated transformation was used to incorporate the GhEXPA8 gene into NIAB 846. A total of 8500 embryos were transformed with the GhEXPA8 gene and selected on MS medium containing 50 mg/ml kanamycin (Figure 1). There were 106 putative transgenic plants, an efficiency of 1.24% of 8500 transformed embryos on the base of transformation procedure and on the base of confirmed transgenic cotton plants is 0.07%, obtained and transferred to MS medium with kanamycin selection. The putative transgenic plants were screened through selection medium as shown in (Figures 1D,E). On the base of PCR analysis of transgenic cotton plants at T0 and T1 generations, these transgenic cotton plants were subjected to molecular analysis such as PCR, southern blot analysis, Real Time PCR, cellulose assay, fiber characteristics and yield parameters at T2 generation in order to protect chimeric presence of gene in few part of cotton plants. There was only one cause behind this protocol in order to screen the transgenic cotton plants from chimera or low expression of gene.

View Article: PubMed Central

ABSTRACT

Cotton fiber is multigenic trait controlled by number of genes. Previous studies suggest that one of these genes may be responsible for switching cotton fiber growth on and off to influence the fiber quality produced from a cotton seed. In the present study, the Gossypium hirsutum GhEXPA8 fiber expansin gene was introduced into local cotton variety NIAB 846 by using an Agrobacterium-mediated gene transformation. The neomycin phosphotransferase (NPTII) gene was used as a selection marker for screening of putative transgenic cotton plants. Integration and expression of the fiber expansin gene in cotton plants was confirmed with molecular techniques including Southern blot analyses, real-time PCR. Cellulose assay was used for measurement of cellulose contents of transgenic cotton fiber. The data collected from 3 years of field performance of the transgenic cotton plants expressing GhEXPA8 showed that significant improvement has been made in fiber lengths and micronaire values as compared to control G. hirsutum variety NIAB 846 cotton plants. Statistical techniques were also used for analysis of fiber and agronomic characteristics. The results of this study support improvement of cotton fiber through genetic modification.

No MeSH data available.


Related in: MedlinePlus