Research On Induction Of Cassava Polyploid Through2n Gametes

Cassava (Manihot esculenta Crantz) is one of the three tuber crops in the world, with the biological characteristics of outstanding high photosynthetic efficiency and high starch yield, drought resistance, barren and so on, is an important food crops and emerging energy crops in tropical and subtropical.cassava sexual polyploidization through dose-response of Chromosome polyploidization and Restructuring effects of genetic material has a good application prospect for cassava genetic improvement.In this research, Excellent cassava cultivars of SC5, SC7and SC10as the main test material, systematically carried out the study of cassava megaspores and microspore chromosome doubling technology, the hybrid effect of male and female2n Gamete and immature embryo rescue technique in triploid, reveals that how cassava2n pollen occurrenced include its cellular mechanisms.cassava pollen germination characteristics and triploid embryo abortion phenomenon provides technical theoretical basis for cassava sexual polyploidization. The main findings are as follows:(1) The meiosis of microspore mother cells of cassava inflorescence were observed by acetic acid-iron vitriol hematoxylin staining method, a clear correlation between the meiotic process of cassava microspore mother cells and the external morphological characteristics of inflorescence and stamens have been found, applicat colchicine induced the cassava inflorescence through cotton leaching method, obtain the doubles2n pollen. The results show that:Using0.15-0.45%Colchicine and1%DMSO handle the inflorescence3-5days when cassava microspore mother cell enter the meiosis prophase I to the meiosis metaphase I during which the length of Young inflorescence is about1.5-2.5cm,the Lateral pedicel begin to appear and the stamens diameter is about1.0-1.5cm,can get the2n pollen. The0.3%Colchicine is the best inducer, the average of the induction date is10.42%,and the highest induction date is12.56%.(2)Researching the abnormal behavior of chromosomes of the microspore mother cell during the meiosis reveals the main cytology mechanism and the genetic effect of the2n gamete of cassava. Observations indicated that the main approach to produce the2n gamete is the spindle’s abnormal behavior of microspore mother cell during the meiosis. During the meiosis metaphase II,Parallel spindle and figure-eights spindle appear, homologous chromosome recombinate during the meiosis telophase II, leads to the appearance of dyad and triad during the cytokinesis. As the recombination spore contains all the non-sister chromatid of the complete parental set of chromosome, belong to FDR genetic factor in the genetic effect. (3)The whole development process of cassava ovule from differentiation to mature embryo sac have been for a detailed cytological observation by the method of paraffin section, and have a correlate analysis for the external morphology of female flower, presented a method for real-time identification Megasporogenesis process of cassava which regard inflorescence morphology and pistil development as a reference.Using Colchicine induce the megaspore mother cell of flower buds during the meiosis prophase I by the method of cotton dip.Through the survival rate of the inflorescence and the mutation rate of the pistil after the induction indicates the appropriate handle level to the double of megaspore chromosome, gains mutation rate of the pistil reached88.9%.(4)Pollen germination in situ technology is used to observed and analyzed the pollen germination and pollen tube growth characteristics Within selfing species or cross-pollination between varieties in cassava, First verified the differences pollination affinity between Selfing and outcrossing. The result of the stigma pollination rate that detected by the method benzidine-hydrogen peroxide indicates that the best pollination period of the stigma is2-3h after the female flower blooming. Propose2points of critical hybridization technique of artificial pollination those are choose the last1-2times top branch Inflorescence in a year and shorten isolation bagging time. Those techniques give a significant technical guidance to improve the breeding efficiency of Sexual hybridization breeding and Sexual polyploid breeding.(5)The cassava pollen of In and2n both can normal germinate after fertilization.The rate of germination at the stigma and the growth of pollen tube of large pollen is equal to the In pollen,indicates that the2n pollen of cassava has the same competitive ability to inseminate with the In pollen.Use the mixed pollen of In and2n to fertilize, the fertility rate and fruit-set rate were all significantly lower than that natural pollen pollination,this phenomenon indicates the sexual hybridization by mixed pollen of different ploidy may exist obstacle, and the phenomenon of embryo abortion is obvious,also failed to detect the triploid in hybrid seed progeny. But from the progeny of hybrid seed of sc5variant pistillate female flower that after induced, we get2plants of sexual tetraploid, Show the effectiveness of2n female gamete induction and indicates that the cassava embryo of tetraploid has the ability of fertility.(6)Through cassava ovules and immature embryos in vitro culture, proposed the immature embryo culture effect was better after flowering30-40d, and the seedling rate can reached68.4%-77.0%. First applied the embryo rescue technique to get cassava triploid plants, using cassava female flower after induction hybridization with normal male flower, get118strains of tissue culture seedlings from hybrids immature embryo culture by the parental combination SC5(♀) xSC7(♂) and SC10(♀) xSC7(♂), the seedling rate was64.1%, of which19strains of variant plants, seedlings mutation rate was16.1%, and identification of triploid plants through the chromosomes in root tip cells.Therefore, embryo rescue technique can be effectively applied in triploid breeding of cassava.