Studies On Technological Systems Of Plant Regeneration And Genetic Transformation In Soybean [Glycine Max (L.) Merr.]

Cultivated soybean [Glycine max (L.) Merr.] has its origin from wild soybean [Glycine soja Sieb. et Zucc.] in China and has been a major source of edible vegetable protein and oil supplement for livestock for several thousand years. The importance of the crop has led efforts to improve its production through conventional breeding, and more recently it has integrated with modern genetic engineering technologies to avoid some of the limitations in traditional breeding procedures. Application of biotechnology is effective way for soybean improvement, for example the establishment of highly efficient regeneration system and transformation system. In the past, the scientists have paid more attention to study how to develop and optimize the methods on plant regeneration and transformation procedures, but the molecular mechanism and genetic basis of tissue culture response has not been well examined. Genetic studies of tissue-culture traits, such as callus growth, embryogenesis and differentiation, will make it possible to transfer genes controlling desirable tissue-culture traits into recalcitrant cultivars or species. With the development of molecular biology, the traditional quantitative genetics has run to the stage of molecular quantitative genetics, which provides a new route to study the complicated quantitative traits. Therefore, detailed genetic studies are required to identify the genes or QTLs associated with the tissue culture response. The present research was intended to study the plant regeneration system, genetic basis and genetic transformation system of organogenesis and somatic embryogenesis, but there was no good results obtained for partial experiments because of the restriction of time and energy. Our studies involved organogenesis, somatic embryogenesis and plant regeneration in soybean; optimization of Agrobacterium-mediated cotyledonary nodes transformation system and its utilization on transgenic soybean; isolation and functional characterization of a novel SERK gene (somatic embryogenesis receptor kinase) from soybean; mapping QTLs for tissue culture response in soybean. The main results of this research were listed as follows:1. Plant regeneration system:(1) Twenty five varieties mainly from Jiangsu province were screened for their regeneration ability. Significant differences in regeneration frequency of these soybean varieties were found. Among them, the regeneration frequencies of N23676, N25277 and N00060 were relatively higher with 90.35%,86.53% and 84.48%, respectively. High shoot induction frequency was found when the cotyledonary node explants were incubated in the SIM only supplemented with 1.67 mg/L 6-BA.(2) Ninety eight Chinese soybean varieties were screened for their capacity to generate somatic embryos. From these 12 varieties were selected for further study to enhance the efficiency of somatic embryogenesis and plant regeneration. The effects of different mannitol concentrations, abscisic acid (ABA) and embryo explant age size were investigated. The results showed that all three factors were relevant for raising rates of callus initiation and somatic embryogenesis, but with differential responses among the genotypes. The three soybean varieties N25281, N25263 and N06499, all agronomically important to the Lower and Middle Changjiang Valleys, were found to have a high somatic embryogenic capacity and therefore recommended in cultivar development through genetic engineering. The treatment of 3.0% w/v mannitol,5 mg/L ABA and a 4-5 mm sized explant was found to be optimal for somatic embryogenesis generating the highest regeneration rate at 82.95%. The greatest average number of plantlets regenerated per explant (1.35) was observed in N25281. The above results provide a basis for efficient regeneration of soybean, and are informative for the development of genetic transformation systems in Chinese soybean germplasms.2. Genetic basis of the regeneration trait:(1) Using a RIL population (NJRIKY) provided by the National Center for Soybean Improvement, the identification of QTLs for tissue culture response in soybean were conducted. The linkage map of NJRIKY contained 834 molecular markers on 24 molecular linkage groups and spaned 2307.83 cM of the soybean genome with an average interval distance of 2.77 cM, the average markers per group of 34.75. Two characters (callus induction and somatic embryogenesis ability) were chosen to evaluate the tissue culture response of soybean. With the method of composite interval mapping (CIM) described in Windows QTL Cartographer Version 2.5,3 quantitative trait loci (QTL) were identified for the frequency of callus induction, on chromosomes B2 and D2, accounting for phenotypic variation from 5.84% to 16.60%; 4 QTLs located on chromosome G were revealed for the frequency of somatic embryo initiation and explained the phenotypic variation from 7.79%-14.16%. The present results will be contributing for genetic improvement for regeneration traits with marker-assisted selection (MAS) in soybean.(2) A novel gene designated GmSERK1 was isolated from soybean [Glycine max (L.) Merr.]. Sequence and structural analysis determined that the GmSERK1 protein, which encodes 624 amino acids, belongs to the SERK gene family. GmSERK1 shared all the characteristic domains of the SERK family, including five LRRs, a SPP motif, TM, and kinase domains. GmSERK1 showed very high homology to rice OsSERK1 (87%), maize ZmSERK1 (85%), alfalfa MtSERKl (94%), and Arabisopsis AtSERKl (88%). As expected from the multiple deduced amino-acid sequences alignment, the result from phylogenetic analysis also revealed that GmSERK1 is closest to MtSERK1 from Medicago, the model plant of legume. DNA gel blot analysis indicated that a single copy of the GmSERKl gene resides in the soybean genome. We also explored GmSERK1 tissue-specific and induced expression patterns using quantitative real-time PCR. The analysis revealed dissimilar expression levels in various tissues and under different treatments. It is obviously that GmSERK1 shows lower expression level in roots and stems, but higher level in leaves and floral buds. For the immature zygotic embryos, there was no significant difference among immature zygotic embryos of <3mm,4-5mm and 6-8mm, while the visible decline occurred for immature zygotic embryos of>8mm in length. An increase in the transcript accumulation could be observed at the early period. The expression level of GmSERK1 reached the maximum at 15d, when the globular-stage embryos firstly occurred. Past 15d of culture, the GmSERK1 transcript level decreased. Meanwhile, GmSERK1 was highly expressed in embryogenic cultures, but no expression was seen in the non-embryogenic cultures. In addition, transient expression experiments in onion epidermal cells indicated that the GmSERK1 protein was located on the plasma membrane. The results of this study suggested that GmSERK1,a member of the SERK gene family, exhibits a broader role in various aspects of plant development and function, in addition to its basic functions in somatic embryogenesis.3. Genetic transformation system:The factors influencing the transformation frequency were investigated, such as the infection time on T-DNA delivery into explants, the co-culture time, the sonication-assisted treatment. The results indicated that infection time of 30 min, co-cultivation time of 3 days, the sonication-assisted treatment can enhance the transformation frequency.2 mg/L AgNO3 supplemented to SIM enhanced the shoot induction frequency of soybean explants. The TaNHX2 gene (GenBank Accession No. AY040246) encoding a Na+/H+ antiporter was transferred to soybean using the above transformation system. The results of PCR detection showed that the transgenic soybeans were obtained. At the same time, the salt tolerant character of transgenic soybean was determined under NaCl treatment. Transgenic soybean could normally grow under the treatment of 250 mmol/L NaCl, while the growth of control soybean was seriously inhibited. From the results, it concluded that transgenic soybean plants were acquired by transformation of a vacuolar Na+/H+ antiporter gene, and TaNHX2 confers soybean resistant against salt stress. Meanwhile, the vector GmFAD2-3-RNAi was produced using the Gateway Technology.