Establishment Of Regeneration System, Molecular Cloning Of HNL24b And Induced Mutation Breeding Of Cassava

Cassava as food, feed and raw materials of processing industry, its share of the market has been growing. However, due to its characteristics, the development of conventional breeding is limited. Cassava have some important traits, such as physiological post harvest deterioration, Vulnerable to virus damage, prematurely leaves senile and plants containing cyanide and so on. It is hard to modify these traits through traditional breeding, so scientists are paying attention to mutation breeding, genetic engineering, molecular markers assisted selection. In this paper, an efficient plant regeneration and transformation system had been established. In order to more efficiently express HNL and reduce the cyanide content in the plants, the full length DNA of HNL24b was cloned and over expressed in cassava. In order to make the roots and leaves have different cyanide content, a root specific promoter patatin had been cloned from potato and used for activating HNL24b gene expression specifically in roots. Also, the irradiation technique for inducing mutants with fine quality traits of cassava was investigated. The main results briefly were as follows:1. Adding 0.5 mg/L of alpha-naphthalene acetic acid (NAA) to the MS based medium was most favorable to the in vitro growth of cassava stem and fibrous roots. Combination of 0.5 mg/L BA and 0.5mg/L NAA was most favorable to the growth of micro-tubers. BA (0 mg/L-2.0 mg/L) showed effective on shoot regeneration. NAA (0 mg/L-2.0 mg/L) proved to be effective on root development. Plantlets with fibrous roots turned out to be easy to generate microtubers in vitro. The microtubers were inducted only when both BA and NAA were used in combination. MS medium supplemented with sucrose at 6%(w/v) resulted in the highest frequencies of shoot induction (62.34%), average shoots height (5.6044 cm), microtuber induction (71.42%) and average fresh weight of microtubers (0.6129 g), all superiors to other treatments, when BA (0.5 mg/ L) and NAA (0.5 mg/L) were also added to the MS medium.2. Addition of 1 mg/L KT and 5 mg/L NAA to MS medium showed effective on formation of cassava leaf callus. Early culture with 8 h/d light was beneficial to the growth of callus. Early culture with 0 h/d light was beneficial for the callus to grow into green. Early culture with Oh/d light and 1 mg/L KT and 2 mg/L NAA was beneficial to rooting, with the rooting rate being 97.51%.3. Addition of 12 mg/L of picloram resulted in the highest rate of somatic embryogenesis (97.53%), cotyledon regeneration (98.72%) and plant regeneration (44.16%). It indicated that picloram was more suitable than 2,4-D and NAA for cassava somatic embryogenesis.10 mg/L of picloram is more suitable for induction of secondary embryoids (95.76%). Low concentration of BA was most favorable to the regeneration of embryogenic cotyledons, while high concentration of BA will lead to the growth of non-embryonic cotyledon. As addition of BA at 0.1 mg/L, frequency of embryogenic cotyledon regeneration reached the highest at 95%.Addition of 1 mg/L of BA and 1 mg/L of IBA resulted in the highest frequencies of plant regeneration (47.6%). 1mg/L of BA and 1 mg/L of NAA showed effective on rooting of embryogenic cotyledons.4. The CTAB method combined application of LiCl turned out to be a good protocol for cassava RNA extraction with good quality.5. The full length cDNA of HNL24b gene was obtained by RT-PCR cloning. The sequencing result for the DNA showed that the sequence encoding for the HNL was not fully consistent with those already published. The full sequences analysis demonstrated that the highest homology of amino acid sequence about 87.72% to MeHNL4 genes, about 86.32% to HbHNL and about 42.81% to MeHNL24. The cDNA was cloned into expression vectors pBI121, pCAMBIA 1323, PET32a(+) by PCR and recombinant DNA technique. Prediction of the encoding protein indicated that the 1-15 amino acids were signal peptide region,31-252 amino acids wereα/βfold domain region. Predicted elements composition of this protein is as follow:C (1344), H (2091), N (343), O (379), S(7).6. The Solanum tuberosum tuber specific promoter Patatinb was cloned. The full sequences analysis demonstrated that the highest homology of sequence was about 95% to Patatin genes from Solanum tuberosum.7. After the transformation of PET32a(+), screening for high copy transformants, induction with SDS-PAGE analysis, enzymatic activityassay, the HNL24b efficiently expressed in BL21 and the enzyme activity reached over 2978 units/L of culture product.8. HNL24b gene was inserted into expression vector, antisense expression vector, GUS expression vector and transformants of those vectors were obtained. A root-specific expression vector was constructed. Expression of HNL24b gene in transgenic plants can effectively reduce the cyanide content in plants.9. By Gama ray irradiation, cassava mutants with one or more good traits, such as high yield, high content of lysine, high HNL expression, lower post-harvest physiological deterioration were obtained.