Polyploid Induction Techniques And Polyploidization Effects In Lilium

Lilium spp.plays an important role in the world’s flower industry.Polyploid breeding is an effective way to obtain new varieties with desirable traits and restore fertility of F1 distant hybrid.Among them,asexual and sexual polyploidization are the main ways of polyploid breeding.China has a large number of lily resources,but the research on lily breeding started late.So far there is market recognition of the desirable cultivars are still less.In this study,polyploid breeding techniques such as in vitro chromosome doubling,2n gametes induction,occurrence of natural fertile gametes in triploid and their cytological mechanisms,and determination of ploidy level were studied.The main results were as follows:(1)Chromosome doubling protocol in vitro of bulb explants were established.According to the orthogonal design analysis of Lg(34),the effects of different pre-culture time,colchicines concentration and treatment time on the tetraploid induction rate were compared.The preferred procedure of allotetraploid induction was to pre-culture segments 6 days and then treat with 0.05%colchicine for 24h.In order to analyze the karyotypic stability of tetraploid plants induced by colchicine,karyotype analysis was carried out combined 45S-FISH and GISH techniques,the results showed that tetraploid had stable karyotype structure.There were significantt differences in phenotypic traits between diploid and tetraploid plants,tetraploid plants exhibit most of the characteristics of polyploid plants.A total of 258 genotype backcross triploid hybrids were obtained following the pollen fertility test,which indicating the F1 hybrid restored fertility at tetraploid level.(2)Chromosome doubling protocol in vitro of seed explants and the rapid ploidy identification process were established.Under the in vitro culture condition,the most efficient procedure for chromosome doubling was to treat germinated seeds with 0.10%colchicine solution for 36h,tetraploid induction rate was 44.43%.According to correlation analysis between the number of swollen-hypocotyl plants and tetraploid plants,the correlation was 73.97%.Thus,the swollen-hypocotyls of L.concolor var.pulchellum seeds could be used as an early screening of polyploid effective indicator.Diploid plants and tetraploid plants showed significant phenotypic differences,it was also found that the morphological changes of the plants were not only between diploid and tetraploid,but also within the different tetraploids genotype groups.(3)A protocol for Asian lily 2n pollen artificial induction were established.A method for instant identification of the meiosis process of Asian lily with reference to the development of the largest flower buds of single plant.Based on the detection of meiosis of different size buds of the same plant,it was found that the meiosis of microspore mother cells in the maximal flower bud length was at metaphase I stage,while most of microspore mother cell meiosis were between prophase I and metaphase I stage,this phase was the optimum treatment period of 2n pollen induction by colchicine.The optimal colchicine treatment concentration was 0.2%,and the highest 2n pollen induction rate was 18.3%.A total of 40 genotypes of triploid flowering plants were obtained by 2n pollen hybridization.(4)The fertility of the gametes and the cytological mechanism of the male gametes were verified by the triploid OT lily cultivars of fertile male and female gametes.Pollen staining and germination test showed that the genotype can produce viable single pollen grains and adherent pollen grains,and most of the germinated adherent pollen grains had two pollen tubes.A total of 275 genotypes of regenerated plants were obtained after reciprocal crosses.Cytological observations of microsporogenesis revealed that the functional male gamete production could mainly be attributed to disoriented spindles,abnormal cytoplasmic division and cytomixis.The ploidy level of some progenies was tested,and the chromosome number is varied from 24 to 36,these findings demonstrated that the allotriploid cultivar produces viable haploid(x),diploid(2x)and aneuploid gametes.