Studies on Genetic Diversity, Al Tolerance Selection Method and Effectiveness of Al Tolerance Breeding Program in Sorghum (Sorghum bicolor (L.) Moench)
There are four important things for effective Al-tolerant sorghum breeding program, (i) well understanding in genetic diversity and genetic background of the intended germplasm; (ii) accurate Al screening technique; (iii) understanding how the Al tolerance is inherited; and (iv) efforts to try the new breeding method of genetic engineering.
High phenotypic and genotypic variations were observed among the Japanese cultivated sorghum and sorghum breeding germplasm used in this study. Generally, harvest index, 1000-grain weight, length of head, plant height, stalk diameter and dry weight showed high variation. The highest phenotypic and genotypic variations were shown by 1000-grain weight, with harvest index of next magnitude, then plant height, length of head, dry weight and stalk diameter. High phenotypic variation among plants in germplasm will provide an opportunity in selection of superior plants that can be used as a parent in a cultivar development program.
High phenotypic and genotypic variations in field experiments were also supported by their genetic diversity assessed using SSR markers. Phenotypic or genotypic variation that closely related to the gene diversity would increase the effectiveness of selection. All breeding materials were distinctly placed at different clusters in a dendogram analysis. Japanese and almost USA populations were separately clustered from ICRISAT populations. These populations were basic populations for development of sorghum tolerance to Al toxicity in this study. Further crossings between the materials and germplasm of separated groups were proposed for high yield progenies.
From the markers technology side, using nine SSR markers out of 15 SSR markers, which were usually used for genetic diversity study in multiplex PCR process, satisfied enough for genetic diversity study in sorghum. It was shown by relatively high diversity indices of each SSR marker and polymorphism of all SSR markers. Diversity indices of each locus ranged from 0.70 (Sb6-34) to 0.94 (Sb1-10).
Hematoxylin staining screening method was applied to select Al-tolerant genotypes in this study because it was simpler than other Al screening techniques and had a significant correlation with Al screening technique in Al-added soil in pot method. Hematoxylin staining screening method was also in agreement with performance information at field with high Al saturation. This gave a basis of accurate Al tolerance evaluation and was used all through this study.
Rapid evaluation of Al-tolerant genotypes at the cellular level could be detected by tissue culture. The differences in callus growth and the percentage of callus formation among genotypes were also in agreement with those in the hematoxylin staining screening method, indicating that cell culture should be feasible to develop a strategy based on the use of callus culture to assist in identification of Al-tolerant or susceptible genotypes for use as parents in a breeding program.
Crossings of breeding lines that genetically showed a different genetic background were conducted for development of Al-tolerant genotypes and for genetic study of Al tolerance. Some breeding lines were chosen as parents in the cultivar development program based on the results revealed in this study: (i) their performance in field trials; (ii) their relationship in diversity study using SSR markers technology and (iii) their performance in hematoxylin staining screening technique.
Moderately low heritability (H = 0.35 and H = 0.43) of Al tolerance was observed in two crosses of sorghum and low genetic gain of Al tolerance was also observed. It shows that high allocation of resources in early generations for Al tolerance must be applied if higher gain for Al tolerance is to be obtained.
Indirect selection, which is believed to be effective for improvement of Al tolerance, might be possible to achieve more rapid progress in breeding of Al-tolerant genotypes. Based on the overall correlation data and path analysis data, dry weight was the important component in the relationship with Al tolerance. Therefore, it was suggested that increasing of dry weight and selection of Al tolerance in early generations were more appropriate to maximize the Al tolerance in sorghum.
As it was mentioned above that cell culture was possible to assist in identification of Al-tolerant genotypes, direct gene transfer into sorghum callus using particle bombardment should be an alternative for development of Al-tolerant cultivars through gene transformation. Transient expressions of the GUS gene were observed in floret-derived sorghum calli bombarded with three DNA plasmids (pAct1-D, pWI-GUS and pEX7113). The pWI-GUS construct carrying double CaMV 35S promoter gave the highest gene transformation in floret-derived sorghum calli. The young and vigorously growing fresh callus was more suitable for direct gene transfer using particle bombardment. However, more studies are necessary for optimization of bombardment parameters; i.e. vacuum condition, distance between microparticles and target cell, size and density of the microparticles.
Finally, since the gene control of Al tolerance in sorghum is not well understood, the conventional plant breeding program is still important for development of Al-tolerant sorghum. Improvement of efficiency of the conventional plant breeding program that was judged from results obtained in this study might be gained through: (i) precise selection of parents that will be used for development of Al-tolerant population; (ii) adequate number of plants in early generations for selection of Al tolerance; (iii) use of indirect selection via increasing of dry weight that was closely correlated with Al tolerance; (iv) use of cell culture assay procedure to assist identification of Al-tolerant genotypes. Further development of DNA markers, including SSR markers for Al tolerance and understanding of gene control of Al tolerance that can be utilized in direct gene transfer will more improve the efficiency of the conventional plant breeding program.
This study includes new findings about: (i) genetic relationship and genetic background of the Japanese cultivated sorghum and the elite breeding germplasm; (ii) association between SSR markers and agronomic traits in sorghum; (iii) efficient and reliable Al screening technique in sorghum; (iv) genetic gain of Al tolerance and direct or indirect effect of agronomic traits on Al tolerance. All of this information, which I believe, should contribute to the improvement of efficiency in the sorghum breeding program and support to the gain in world food production for mankind welfare.