Abstract
Genetic transformation is a powerful tool to study gene function, secondary metabolism pathways, and molecular breeding in crops. Cotton (Gossypium hirsutum L.) is one of the most important economic crops in the world. Current cotton transformation methods take at least seven to culture and are labor-intensive and limited to some cultivars. In this study, we first time achieved plantlet regeneration of cotton via embryogenesis from transformed hairy roots. We inoculated the cotyledon explants of a commercial cultivar Zhongmian-24 with Agrobacterium rhizogenes strain AR1193, harboring a binary vector pBI-35S::GFP that contained the NPT II (neomycin phosphotransferase) gene and the GFP (green fluorescent protein) gene as a fluorescent marker in the T-DNA region. 82.6% explants produced adventitious roots, of which 53% showed GFP expression after transformation. 82% of transformed hairy roots produced embryonic calli, 12% of which regenerated into stable transformed cotton plants after 7 months of culture. The integration of GFP in the transformed cotton genomes were confirmed by PCR (Polymerase chain reaction) and Southern blot analysis as well as the stable expression of GFP were also detected by semi-quantitative RT-PCR analysis. The resultant transformed plantlets were phenotypically, thus avoiding Ri syndrome. Here we report a stable and reproducible method for A. rhizogenes-mediated transformation of cotton using cotyledon as explants, which provides a useful and reliable platform for gene function analysis of cotton.
Highlights
Cotton is an economically important crop well known for providing natural fibers
Hairy roots induced by A. rhizogenes are a visible and simple marker that permits the selection of transformed roots
Two-week-old cotyledons were inoculated with A. rhizogenes strain AR1193, and after 2 days of co-cultivation, the cotyledons were transferred onto hairy root induction medium (RIM)
Summary
Cotton is an economically important crop well known for providing natural fibers. It is a producer of seed oil which ranks third in oil production globally, after soybean and canolaAbbreviations: MS, Murashige and Skoog medium; B5, Gamborg B5-medium; GFP, green fluorescent protein gene; KT, kinetin; NAA, 1-Naphthaleneacetic acid; IAA, Indole-3-acetic acid; 2.4-D, 2, 4-Dichlorophenoxyacetic acid; NPT II, neomycin phosphotransferase gene; CTAB, hexadecyltrimethylammonium bromide; PCR, Polymerase Chain Reaction.Plant-Regeneration From Cotton Hairy Roots (Shang et al, 2017). Cotton is an economically important crop well known for providing natural fibers. It is a producer of seed oil which ranks third in oil production globally, after soybean and canola. Traditional breeding attempted to improve some agronomic traits of cotton, such as fiber length and quality, disease resistance, and oil yield. In contrast with classical breeding, genetic transformation is a powerful research tool for use in gene discovery and crop improvement. Many genes have been identified which have putative function in fiber development and the seed oil biosynthesis pathway (Walford et al, 2011; Liu et al, 2017; Shang et al, 2017). Previous cotton transformation protocols have key limitations, including the inefficient regeneration of transformed shoots, with successful transformants obtained through time-consuming and labor intensive methods (Sunilkumar and Rathore, 2001; Leelavathi et al, 2004; Zhao et al, 2006)
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