Cassava Autopolyploid Induction And Mesophyll Protoplast Isolation

Cassava(Manihot esculenta Crantz), which originates in Latin America, were introduced to China in the 1820s. Nowadays, cassava is planted in large areas of Guangxi and Hainan provinces owing to its superior agricultural characteristics, such as having high content of starch, being drought and barren tolerance. Cassava was used as animal feed, food for human, and inductrial material for ethanol production. For cassava, the conventional cross breeding was confronted with obstacles like heterozygous genotypes, low pollen viability and segregating progenies. Application of transgene technique on cassava was hindered by its single and inefficient transformation/regeneration system. However, by polyploid breeding, these short- comings can be avoided. Ployploidy plants were proven to possess the following advanced characteristics:stronger growth vigor, bigger flowers, fruits and leaves, significantly increased accumulation of intracellular effective components. Good properties of cassava can be stably maintained by asexual reproduction approaches, which also can be applied in industry. In this study, axillary buds were treated by colchicine to induce polyploidy cassava, effect of enzymolysis time on protoplasts isolation were also explored. The main results were as follows:1. Axillary buds of five cassava cultivars (SC8, SC5, M.Co122, NZ188, NZ199) were used as explants, six buds were induced in a single group. Combinations of colchicine concentration (0.1 g/L or 0.3 g/L or 0.5 g/L) and time for treatment (3 h or 6 h or 9 h) were applied on one group of explants respectively. After moved to the basic culturing medium, the treated buds were cultivated into shoots. Autotetraploids were then identified via both flow cytometer and root tip squash technique. Stable autotetraploids were obtained from all the five cultivars of cassava after subcultured for multiple generations, including seven plants from SC8, five from SC5, three from M.Co122, five from NZ188, and two from NZ199.2. Autotetraploidy plantlets originated from SC8 and M.co122 and corresponding parental plants were transplanted into greenhouse. After growth for five month, it was observed that leaves of autotetraploid became sufficiently wider and thicker relative to parental plants, also length/width and density of stomata had significant difference between the control and polyploid. 3. Effect of enzymolysis time on isolation of mesophyll protoplasts from cassava diploid SC8 and M.col22 were explored, and the optimum time was shown to be 15 h.In summary, we have got autotetraploids from five cultivars of cassava. In addition, the optimal incubation conditions were determined for mesophyll protoplasts isolation, which would provide a good basis for cassava protoplasts fusion.