Studies On Polyploid Breeding Technology Of Amorphophallus Konjac

Amorphophallus konjac is one of main konjac varieties in China. The bulbs of A. konjac contain more than55%glucomannan. In recent years, plantation areas of A. konjac continued expansion because of great economic value. But in return there are many problems, such as the shortage of seed resource, deteriorates of germplasm. poor disease resistance, low output etc.,which gradually became the limiting factors for the development of konjac industry.For the healthy development of konjac cultivation, it is very important to breed new varieties and to modify konjac varieties. Polyploid plants have many advantages such as gigantic organs, good resistance performance, and wide adaptability. So that polyploid breeding is one of important ways of breeding high-yield konjac varieties. In recent years, with the development of plant tissue culture technique, artificial chromosome doubling in vitro is an efficient approach to produce polyploidy plants.In the study, based on previous research work, the regeneration program of A. konjac was established to induce polyploidy konjac with colchicines in vitro and in vitro. And then, the root tip dyeing technology was built to investigate the chromosome of A. konjac. The main results can be summarized as follows:1. Tissue culture regeneration system of A.konjac was established for inducing polyploidy plants with colchicines in vitro. The corm of A.konjac was used as explants for callus induction, bud differentiation and plant regeneration. The results indicated that the corms give a callus induction frequency of85.71%when chultured on MS medium supplemented with6-BA1.0mg/L and NAA0.5mg/L. And when cultured on MS medium supplemented with6-BA2.0mg/L and NAA0.5mg/L, the highest bud differentiation rate of76.09%was obtained. The optimum media for rooting is1/2MS+NAA0.2mg/L with96.30%rooting ratio.2. In vivo, it was applied to induce polyploidy of A. konjac by soaking bulbs and dropping apical buds with different concentrations of colchicines for different treatment time. The results showed that the induction efficiency of soaking bulb was superior to that of dropping apical buds, but the survival rate is relatively low. The treatment of soaking bulb3d with0.20%colchicine solution had the best induction effect, having relative high survival rate with33.67%and mutation rate with54.55%. And the method of dropping buds by0.20%colchicine solution for7days, were best than the others, the survival rate and mutation rate were43.33%and23.08%.3. In vitro conditions, it was applied to induce polyploidy of A. konjac by soaking adventitious buds and callus with different concentrations of colchicine solution for different treatment time. Compared with adventitious buds inducing, soaking callus0.02%colchicine were increased callus browning efficiently and reduced ability to differentiate. In order to ensure that the callus have high differentiation rate and more variant material, the callus were treated with0.01%colchicines for12h had the best survival rate with56.67%. Colchicine has also some toxic for adventitious buds. Usually adventitious buds have the high survival rate with50%and induction rate with33.33%when they deal with0.2%colchicine treatment for5days.4. The biological characteristics of tetraploid A. konjac were investigated in this experiment. Compared with diploid A. konjac, tetraploids had typical polyploidy morphological characteristics, including leaf wider and thicker. The leaf width of A.konjac with double genome increased1.47times, while diameter of petiole is1.1times of the contrast, but leaf index (length/width) is only0.69times of that of diploids. The leaf color of polyploidy A. konjac became dark green. The content of leaf chlorophyll reach29.94in contrast with the control25.41. And the stomata of leaf became bigger evidently. Its length/width are respectively1.37times and1.34times of the control plant. While the density of stomatas decreased, only0.62times of that of diploids. So that both morphological characteristics and stomata density can be used as important characters to identify polyploids.5. The root tip dyeing technique of A. konjac were tested in this research. The results showed that the roots were frozen in the refrigerator at4℃for24h. and then pretreated with the equal solution volume of0.2%colchicine mixing with the0.002mol/L8-hydroxyquinoline for3h. Next they were fixed in Kano-solution for24h. This process could achieve the better result. Root tips were then disintegrated with the solution of1mol/L HCl for20min at60±0.5℃. Finally, the root tips were stained with modified Carbol-fuchsin solution for15min. With this method the best effect of chromosome squashing could be obtained.This research provide theoretical basis for rengeneration system of A. konjac and laid foundation for polyploid breeding of Amorphophallus plant.