Agrobacterium-mediated Transformation of GmWRKY21 into Soybean
|School||Zhejiang Normal University|
|Keywords||soybean GmWRKY21 low temperature Agrobacterium-mediated transformation genotype|
Soybean is an important economic crop in international trade due to its high protein and fat contents. A substantial development in soybean industry will be necessary to remit the shortage of soybean productions and to satisfy the growing consumption demand in China. Breeding of soybean varieties by traditional methods has some disadvantages, such as long breeding cycle and low selection efficiency. Plant transgenic technology, by contrast, provides a direct, simple, efficient, and quick way to improve soybean qualities, such as an enhancement of soybean resistance to biological and abiotic stresses, improvement of nutritional qualities, or increment of seed yield. Gene transformation has become a widely used technology in plant breeding.In this study, both cotyledonary node and embryonic tips jointed with one piece of cotyledon were employed as explants in the transformation. The transcription factor gene GmWRKY21 was introduced into soybeans via Agrobacterium-mediated transformation system to generate new germplasms with improved cold tolerant abilities. The main results were summarized as follows:1. In the experiment of Agrobacterium-mediated cotyledonary node transformation system, effects of 18 soybean genotypes and GA3 content in the restoration culture medium on shoot induction efficiencies were investigated. Significant differences of shoot induction were observed among soybean genotypes. The preferable genotypes include zhechun No.3, H0301, you02-32, jixiaodou No.7, zhechun No.2 and A7601. Protein contents in cluster shoot of five genotypes with different shoot induction efficiencies were analyzed by Kjeldahl determination. Further analysis showed that no significant correlations between protein content and shoot induction or growth situation were detected.Zhechun No.3, which has an excellent shoot induction and elongation ability in repeat trials, was chosen to perform the next hormone experiment. Effects of GA3 on shoot induction during restoration cultivating stage were studied. The optimal concentration of GA3 was 0.5 mg/L for both shoots induction and elongation. Higher or lower GA3 concentration will reduce shoot induction and elongation.2. In the experiment of A grobacterium-mediated embryonic tip (jointed with one piece of cotyledon) transformation system, the transcription factor GmWRKY21 was introduced into’zhechun No.5’ and’zhonghuang 13’. Transformation events were confirmed using gene-specific and construct-specific PCR analysis.Further chilling experiment showed that transgenic plants exhibited significantly lower relative electric conductivities than their non-transgenic controls both before and after chilling treatment. Unlike the relative electric conductivity, no coincident patterns were observed on the proline contents of transgenic lines after chilling treatment. Better low-temperature tolerability of transgenic plants was confirmed after recovered for days. The transgenic plants grew normally, while their controls died in 7 to 10 days after chilling treatment. These results indicate that GmWRKY21 gene has been introduced into the soybean genome and it possibly participates in the response to low-temperature stress, and finally leads to an improved cold tolerance.