Autopolyploid Induction Of Gossypium Herbaceum L. And Hybrid Identification Between G. Herbaceum And G. Raimondii

This research included two sections,Autopolyploid Induction of Gossypium herbaceum L.and Identification of a Hybrid between G. herbaceum and G. raimondii.On the one hand,the diploid Gossypium herbaceum L. was used to induce autotetraploid with colchicine technique, and the comparison of morphology,anatomy,physiology and cytology characters between obtained tetraploid and diploid control was studied in this paper. On the other hand,an obtained hybrid plant was identified through morphological,cytological and molecular biological methods. The main results were showed as follows:1. Results of the induce and identification of diploid G. herbaceum.(1) Polyploid inducing conditions:The seedling tip bud of diploid G. herbaceum were induced by colchicine solution at the concentration of 0.2% under three treatment time of 24h,48h and 72h. Statistic analysis showed that the treatment group of 0.2% and 24h was the best and the mutant rate was up tol6.67%(2)Results of the identification of mutants:Comparing with contrast, there were many morphology changes of mutant plant. When the mutant was seedling after induce, many strumaes produced at mesocotyl,cotyledon became bigger and shriveled, parts of the euphyllas grown in cluster,the number of cracks were different,leaves became rugged. The growth rate of mutated plant in different period were all slower than contrast and the strain height in different period were all lower than contrast. The functional leaf area of mutated plant in different period were all bigger than contrast. The number of the cracks on function leaf increased while the cracks were deeper than the contrast. The leaf indexes were bigger than the contrast during the seedling and bug stage,and developed smaller at flower and boll setting stage,as well as boll opening stage. The internode length of mutated plant became smaller than contrast during different periods,while its diameter changed obviously.The radio of length to width, j aggies number of mutated bract and the anther number per flower of the mutated plant decreased,bract area increased.Seeds number per boll, boll number each plant,single seed weight and fiber weight of single seed of the mutant all decreased.Anatomical changes of the mutant were mainly as to the subepidermal stoma,compared to the control, the mutant had lower stomatal density, higher length-width ratio, higher approximate area and higher chloroplast number in each stomatal. Physical index,SPAD value also increased.There were many cytology differences between mutated plant and contrast:By chromosome observation of root tip cells, it showed that the chromosome number of a part of mutated cells was 2n=4x=52, but it was 2n=2x=26 in all of contrast tip cells. The karyotype formula of contrast and mutated were K=2n=2x=26=16m+4sm+6sm(6SAT), K=2n=4x=52=36m+4sm+12sm(12SAT)and belonged to 2A and 2B karyotype respectively. Moreover,The mitosis behavior of the induced was abnormal,appearing as some phenonenon,such as lagged and dissociative chromosome at Metaphase,lagged chromosome,chromosome bridge and unequal separation at Anaphase.And there appeared two types of spindle,vertical spindle and"八"like spindle.The results of meiosis behavior showed that the induced was much more complex than that of the diploid.The pollen grains of mutants developed uneven with specially big and extremely small ones. The forms varied in many styles,such as hollow,black heart,lack of spike and irregularity pollen grain.But the pollen grains of control were regular in plump. The average diameter of mutant pollen grains was bigger than that of control.During the meiosis of the mutant,there appeared univalent and polyvalent at Metaphase I,unbalance separation of chromosomes existed during anaphase Ⅰ and anaphase Ⅱ,dyad, triad, polyad and micronucleus were also observed. Cytoplasm division was aberrant,it was divided into two or three parts. However,in miosis of contrast,chromosomes and cytoplasm were divided into four parts equally.2.Identification of the obtained hybrid between Gherbaceum and Graimondii.(1) Morphology differences:Both of vegetative growth and reproduction of the hybrid plant were exuberant,the plant flowing all year round,these may be heterosis phenomenon.most of its characters were middle types. When it was seedling,all the leaves were belong the same shape,area was between its parents,both the shape and area was similar with G. Hirsutum and Gdarwinni.With the growing and development,there appeared many different shapes later.But the number of cracks were increased,the crack was deeper than parents.The flower size was between Gherbaceum and G.raimondii,smaller than allotetraploid cotton;The bract was bigger than both of the parents,morphology was similar with the 5 allotetraploid cotton;Petal size and colour were between G.herbaceum and G.razmondii,morphology was similar with Gherbaceum and Gbarbadense; The colour of its stylet and stamen were the same with Graimondii.The length of stylet and bulgy height were also between Gherbaceum and Graimondii.The number of anther decreased seriously;Anthocaulus length was longer than parents.(2)Cytogenetical analysis showed that miosis of the bybrid was abnormal,resulted in many aborted pollen grains.Dissociative chromosomes exited during metaphase I and metaphase II,unequal separation of chromosomes were observed at anaphase I and anaphase Ⅱ.At Telophase II of miosis,there were Asynchronous dyad division,multipartite chromosomes,tripartite cytoplasm,Dyad,Triad,hexad and micronucleus.(3)In the last of this study,two polymorphic primers were screened from 27 pairs of SSR primers,that were NAU1355 and NAU2026.In amplification products of the former,the hybrid Amplification bands were consisted of two parents Specific bands;The latter amplified new bands except the Specific of Gherbaceum and Graimondii.Conclusion:From this study, we successfully obtained tetraploid of G. arboreum L. We revealed the reason of the hybrid infertility and proved it was an really hybrid from Gherbaceum and G.raimondii;Genetic relationship was mentioned by comparing the characters among the hybrid and 5 allotetraploid cottons.