Establishment Of High Frequency Regeneration System And Genetic Analysis For Mature Leaf Petioles In Upland Cotton (G. Hirsutum L.)

Huge economic and social benefits have been acquired from the improvement of varieties of transgenic technique in cotton. Agrobacterium-mediated transformation is one of main means in cotton genetic transformation. But there are still a lot of problems to be solved in the development of bio-technology of cotton at present, especially in transgenic technology, witch include the genotype limitation of plant regeneration, the low efficiency of cotton tissue culture with excessive time and labor, the complicacy of transgenic conditions leading to difficult test replication and low transgenic ratio and the instability of agronomic traits. In order to take some measures for solving these problems which have become the bottleneck of transgenic technology, the objective of the current study was to choose the explants for tissue culture, research genotype restriction of embryogenesis callus (EC) and select the optimal cotton line for culture. The further study is to investigate the genetic basis of EC in a high EC Chinese cotton genotype by analyzing the segregation of EC from mature leave's petioles as explants using classical and quantitative genetics, and molecular marker analysis. Important technique supports were provided for the rapid identification of gene function and the creation of higher EC cotton lines, because of the ratio of Agrobacterium-mediated transformation increased by 1-2 times, the period of transformation from aiming at gene to transgenic seeding shortened to 6 months and the transformation efficiency eas raised to 30% steadily.Establishment of mature leaf petioles as explants culture system:A series of experiments of callus induction, SE callus differentiation and germination were conducted to establish a tissue culture system for cotton mature leaf petioles as explants from CCRI24. It showed that no significant difference was detected between the mature leaf petioles and asepsis seedling hypocotyls tissue culture system commonly used in culture cycle, about 6 months, and EC differentiation ratio, around 30% steadily. It also showed that the phenomenon of callus tissue brown was influenced by hormones content, 6-BA 0.08～0.16 mg·L-1 and KT 0.08～0.12 mg·L-1 in KT/6-BA or KT 0.08~0.12mg·L-1 and IAA 0.08~0.12mg·L-1 in KT/IAA, which could be adjusted to SE differentiation early, and the brown of new differentiated callus tissue was resulted in nitrogen ratio(KNO3 3.04g·L-1 and NH4NO3 0.66g·L-1 of MS medium) of the culture medium in subculture. So, the difficulty was overcome of callus differentiating into embryonic callus in a tissue culture system of cotton mature leaf petioles as explants, and the problem was solved of the brown of embryonic callus from cotton mature leaf petioles easier than from cotton asepsis seedling hypocotyls.Inheritance of EC differentiation rate in mature leaf petiole of cotton:The genetic analysis was confirmed by the joint segregation analysis (JSA) based on a mixed major gene plus polygenes inheritance model. It revealed that the variation in cotton EC was best explained by a mixed two major genes plus polygenes model and that EC was governed primarily by the first one in two dominant major gene alleles, tentatively designated Re1 and Re2.The JSA also detected the presence of the epistatic effects between the two major genes. The majority of the variation in EC was accounted for by the two major genes in that heritability (h2mg) due to the major genes ranged from 74.68 percent to 83.22 percent. The contribution of polygenes (h2pg) to the total phenotypic variation was very low, 10.47~16.78%. The average of total heritability form two major genes plus polygenes in F2 population segregations is about 78.85 %.Mapping of QTLs Associated with mature leaf petiole EC in cotton:A high embryogenesis callus (HEC) line, W10, was selected by mature leaf petioles callus culture from a commercial Chinese cotton cultivar CRI24 and crossed with non-embryogenic(NEC)line TM-1 to identify markers associated with EC using mature leaf petioles as explants in F2 population segregations. Of 19 polymorphic SSR loci, 12 SSRs were assembled into 2 linkage groups, LG1 and LG2(LOD≥3.0)with 5 and 7 markers respectively. The length was about 194.3 cM of LG1 and109.1 cM of LG2. The linkage distance of markers ranged from 12.7cM to 43.3cM. the segregation of 9 SSRs among 12 polymorphic SSRs, about 75%, in F2 followed a 3(EC):1(non-EC) or 1:2:1 ratio(χ2=0.02, P>0.05).By the method of composite interval mapping , SSR marker analysis detected 3 QTLs (LOD≥2.5)for EC located on two linkage groups, one of which is a major QTL, accounting for 58.1% of the phenotypic variation in EC. It is reasonable to believe that this major QTL is one of the major genes detected in the mixed major gene plus polygenes genetic analysis. The other two QTLs were 14.4% and 8.4% of the phenotypic variation in EC.Culture characteristics of cotton mature leaf petioles as explants: Using petioles on cotton plant different parts and at squaring stage, peak squaring stage, flowering stage, peak flowering stage, the formation of dedifferentiation callus and the rate of callus differentiation was no significant difference, but the growth speed of callus was different significantly. The status of inducted callus changed with the senescence of cotton mature leaf petioles. Though the callus grew slowly at the later growing stage, The rate of EC differentiation was about 42.8%. It indicated that using cotton mature leaf petioles as explants were suitable for tissue culture.Application of Cotton mature leaf petiole tissue culture system in genetic transformation: Using cotton mature petioles and the asepsis seedling hypocotyls seeding as explants, which were mediated by Agrobacterium transformation and cultured under the selected optimum content of hormone combination, the induction rate of resistant callus were 84.3% and 46.7% respectively, and the differentiation rate of resistant callus were 65.1% and 71.8% respectively. It showed that the transformation efficiency of cotton mature leaf petioles is 1.55 times as much as that of the asepsis seedling hypocotyls. It was possible that the higher induction rate of mature leaf petioles resistant callus were benefited from the bigger cross-sectional area of cotton mature leaf petioles which provided with more cells surrounded by Agrobacterium.The selection of high-differentiation ratio material in cotton: By using this cotton mature leaf petiole culture system, fifteen high differentiation ratio lines, with the EC ratio higher than eighty percent, were selected from three commercial varieties of china CCRI24, Zhong394 and Jihe312. Among which six lines can differentiate as high as 100% of EC differentiation rate with cotton asepsis seedling hypocotyls as explants in tissue culture and the characteristic could be inherited steadily. An efficient and stable cotton tissue culture system with high differentiation ratio was established for the selection of high-differentiation ratio material in cotton.Callus tissue differentiation could be significantly affected by cotton callus induction medium in tissue culture experiment of high-differentiation ratio lines. The optimum content of hormone combination is not the same in callus induction and differentiation medium for different genotypes materials as explants. By Agrobacterium-mediated transformation, the transform efficiency of the selected high-differentiation ratio materials on improved culture medium , was 1.88 times higher than that of CCRI24, ranged from 30 percent to 40 percent.