Research On Key Technology Of Cassava Tetraploid Induction

Cassava(Manihot esculenta Crantz)is the important food and energy crop in the tropical and subtropical area of the world,especially in developing country.Some cassava cultivars of South-China 205(SC205),South-China 5(SC5)and South-China8(SC8)were employed in this experiment to gain the best method of induction of somatic embryo and friable embryonic callus(FEC)based on the former work of our laboratory.And then the cultural method of suspension embryonic cell cultural system was established.Colchicine was used to treat the buds of field growing cassava and cultured seedlings in vitro to get multiploid cassava.And the method of protoplast isolation,culture and protoplast electrofusion were studied that were helpful to gain multiploid cassava by the hybrid of somatic cell.The main results were as follow.1.The suitable somatic embryogenesis induction mediums from young leaves and axillary buds of SC205 was cassava basic medium(CBM)+6~8 mg/L 2,4-D and CBM+12 mg/L picloram and the induction rate was 28.89%and 28.89%,re-spectively.Using the two kinds of suitable somatic embryogenesis induction medium to induce SC8 and SC5,the induction rate of somatic embryogenesis in SC8 from young leaves and axillary buds were 70%and 74%,respectively,whereas in SC5,they were 32%and 42%,respectively.The suitable FEC induction medium was Gresshof and Doy Basal Medium(GD)+12 mg/L picloram.2.The friable embryogenesis callus was usd to establish the suspension culture system in this experiment.The results indicated that it was suitable to suspend 0.7 g callus in 30 ml aquatic medium.The best aquatic cultural medium was SH medium plus 10 mg/L picloram and 40 g/L sucrose with pH5.8.The medium must be renewed every 6 days.3.The lateral buds of the field growing cassava and the cassava seedlings in vitro were treated by colchicine.The experiment gained some putative chromosome multiplied cassava by morphological screening.After several screening including flow cytometer test and and chromosome counting,the tetraploid cassava seedlings were gained.The longitude and the width of stomata of the tetraploid cassava are larger than those of the diploid cassava.But the density of stomata on the leaf lobe of tetraploid cassava is smaller than that of the diploid.4.The effects of different enzymes and the treatment methods to isolate proto-plast from leaf and embryonic suspension cells were studied.The results indicated that the best combination of enzymes to isolate protoplast from leaf of cassava was0.75%cellulase R-10,0.75%Macerozyme R-10 and 0.05%pectinase enzyme Y-23,while the best method for suspension cell was 1.0%cellulase R-10,0.2%Macer-ozymeR-10,0.05%pectinase enzyme Y-23.The co-cultivation period was 14h under28?.The protoplast of cassava can grow better using liquid thin layer culture method with basic culture medium of TM2G.The suitable sugar is glucose with con-centration of 55~60 g/L.The cells can grow better if the cell concentration is 5×10~5cell/ml.5.The method of protoplast electrofusion were studied by Eppendorf multipora-tor electric apparatus.The results showed that the best method to make cassava pro-toplast fusion was to treat protoplast with alternative current(AC)of 100 v/cm for 50s,and then treat them with direct current(DC)of 1500 v/cm for 45?s with twice times.