Establishment Of Two Transformation Systems Based On Tissue Culture And On Pistal Drip Following Pollination In Cotton

Agrobacterium-mediated transformation is the main method used in the field of biotechnology. It is attractive because of the ease of the protocol coupled with minimal equipment costs. Moreover, transgenic plants developed by this method often contain simple copy insertions. These advantages were a driving force to adapt the method to many different crops including cotton. But cotton as well as many economically important plant species, or elite varieties of particular species, remain highly recalcitrant to Agrobacterium-mediated transformation. In this study, we endeavored to extend the host range and transformation efficiency of cotton by Agrobacterium, to seek a novel mothed of in planta transformation avoiding from the process of tissue culture.Xinjiang is an excellent cotton growing region in China, producing more than one-third of Chinese cotton production. But worsening conditions of cotton diseases and insect pests as well as the dry condition in Xinjiang had caused a bottleneck in sustainable development of cotton in this region. Cultivars with resistance to abiotic or biotic stresses, such as salt tolerance, cold resistance, and disease resistance, as well as good quality and high yield are needed in Xinjiang. Genetic improvement through genetic transformation can be useful in fulfilling this demand. An efficient somatic embryo production and maturation procedure was developed to regenerate plantlets from hypocotyls of four cotton cultivars grown in Xinjiang. Calli were effectively produced by0.01-0.10mg l-1kinetin (KT) and0.10mg l-12,4-dichlorophenoxyacetic acid (2,4-D), with a better result by0.02mg l-1KT+0.1mg l-12,4-D and0.10mg l-1KT+0.1mg l-12,4-D. Split hypocotyl segments and double amounts of KNO3during induction of calli were beneficial to the emerge of embryogenic calli. Embryogenic calli and globular-stage somatic embryos were effectively initiated with high concentration of KT and low concentration of2,4-D in ECM media (0.05mg l-1KT 0.01mg l-12,4-D and0.10mg l-1KT+0.01mg I-12,4-D). Embryos were further developed into plantlets in MSBF medium under the conditions of dehydration and ventilation, which were achieved using filter paper on medium and cotton tampon sealing flask. Using this protocol, normal plantlets with strong roots were developed from these four cotton cultivars in6-8months.Embryogenic calli of Xinluzhong20were transformed with Agrobacterium carrying drought resistant gene bcp or disease resistant gene hcml under the selection by Phosphinothricin (PPT) or Kanamycin, respectively. Low density of transformed embryogenic calli in Petri dish provided more effective contact between embryogenic calli and selective agents, and increased the selective efficiency with less escape. Transformed embryogenic calli could regenerate into transgenic plantlets after6months. And the number of transgenic plantlets obtained from9Petri dishes of transformed embryogenic calli may reach as many as100independent transgenic lines in8months, tremendously improving transformation frequency and reducing the overall time period significantly. A frequency of100%somaclonal variation was observed among the regenerated plants. PCR tests of plantlets regenerated under selection by100mg l-1kanamycin or1.5mg l-1PPT showed a positive rate of90%in terms of nptⅡ or bar gene, while75%positive rate in terms of hcml gene or bcp gene. The resistance test with Kanamycin or Basta proofed the expression of the integrated nptll or bar gene. And Southern blot analysis confirmed the integration of hcml gene or bcp gene into the cotton genome of some of PCR positive plants, suggesting that Xinluzhong20was efficiently susceptible to Agrobacterium-mediated transformation.Simple incubation of Agrobacterium with cotton pistil after pollination could produce transformed seeds, enabling transformed plants to be produced without the necessity of a tissue culture system.10%sucrose inoculation medium containing0.05%(v/v) Silwet L-77and40mg l-1acetosyringone facilitated the entrance of Agrobacterium into interior of germ line and transformation of cotton. The importance of Agrobacterium inoculation time and pistil position (stigma, style and stigma excisions) for inoculation was evaluated in terms of transgenic plant production. No transformants produced from pistil drip during9:00～11:00on the first day of flowering. Pistil drip during17:00～19:00on the first day of flowering resulted in0.07-0.17%Basta resistant plants/number of viable seeds generated, and stigma excision prior to pistil drip during this time period gave rise to a transformation efficiency of0.46-0.93%, in contrast with0.04-0.06%generated from pistil drip during9:00～11:00on the second day of flowering. Among four experiments with seven indepentant treatment days, three experiments produced transformed plants from two or three indepentant treatment days. The transformation efficiency ranged from0.04%to0.93%, with the highest rate of transformation from the treatment time period during the afternoon of flowering. PCR and Southern blot analysis consistently confirmed the integration of bar gene into the TO cotton genome, and herbicide resistance test of TO and T1generations showed bar gene expressed and functioned well in these transgenics. Transgenic cotton plants produced from two or three independent treatments of these experiments demonstrated the reliability and reproducibility of this simple transformation procedure by drop dip of Agrobacterium-sucrose onto cotton pistil. Compared with traditional tissue culture based transformation procedures, this Agrobacterium-mediated in planta transformation is technically easier and generally faster to obtain transgenic plants, and it overcomes the problems of somaclonal variation caused by tissue culture.