The Wild And Artificial Germplasm Assessment By Cytogenetics And Mass Propagation System Establishment In Lilium

Lily is one of the five fresh cutting flower species. With its big and colorful flowers, beautifulstature and fragrance, it is cultured extensively worldwide and occupies very important position inthe floral market. In China, there are abundant wild resources of lily with a total of 47 species, inwhich 36 species and 15 variants are native species.Based on Giemsa C-banding analysis and Fluorescence in situ Hybridization (FISH) using45S rDNA as probe, we studied all collected wild lily provenances. The standard C-banding wasset up for most wild lilies. The number and position of 45S rDNA genes were identified on thechromosomes of wild lilies, which build standard mode and fast and accurate technical platformfor the analysis and identification of lily resources, provide valuable reference for the study of lilyrelatives. By crossing between cultured lily and wild lily, we analyzed the Giemsa C-banding ofthe parents and the hybrid offspring, the 45S rDNA in the parents and the offspring using FISH.The difference of Giemsa C-banding and FISH using 45S rDNA as probe between the parents andoffspring verified the existence of the hybrid. This provided fast and accurate method for theidentification of hybrid offspring and early selection.In this study, we used scale segments and callus as explant, studied the effects of variousfactors on organogenesis and somatic embryogenesis, such as cytokines, auxin, solid media, pH,sugar and inorganic salts. The effect of sugar, active carbon, inorganic salts and Paclbutrazol onthe growth of tuber and its diameter were also studied using in vitro cultured lily as explant. This isthe first time to induce the somatic embryo of 'Tiber' and regenerate the plantlets. We observedthe generation and development of adventitious buds and somatic embryo using paraffin section.The indoor cellular engineering preservation system of wild lily was set up. And the fast culturalpropagation of oriental lily and the industrial system of large cultured tuber were established whichwere further industrialized. We gained preliminary knowledge of the flowering mechanism of invitro cultured lily.Using improved HBSG procedure to analyze Giemsa C-banding of the collected lilyprovenances, 13 standard C-banding of wild lilies were built based on C-banding pattern,chromosome length and arm ratio. The karyotypes of chromosome are all 3B. The total bands for ahyploid chromosome are 15-33. By the comparison of the C-bandings on the triploidchromosomes of both edible 'Yi Xing' lily and the wild 'JuanDan' lily, high stability of C-bandingwas found within the specie, which could be used to identify lily Germplasm. We used FISH technique to study 37 species of lily (including wild lilies, hybrid parental lilyand hybrid offspring) with 45S rDNA as the probe. The result indicated that most of thehybridization signal of 45S rDNA was distributed at the chromosome centrimere and a few at thelong arm and short arm. There is 4-10 hybridization signals of 45S rDNA in 33 diploid lilies, while12-14 existed for 3 triploid lilies, 19 existed in 1 tetraploid lily.We analyzed the C-banding and FISH with 45S rDNA as probe of the parents of 2 cross'sand reverse cross, 1 single hybrid and their hybrid offspring. According to the C-banding ofchromosomes and the hybridization signals of 45S rDNA, chromosomes from both parents wereobserved in the root tip chromosomes of hybrid offspring, which further confirmed the existence ofhybrid.Using in vitro cultured lily scale segment as explant, we studied the effect of plant growthregulators, sugar concentration, media, pH on the adventitious buds differentiation of lily scalesegments, and the next-generation culture of in vitro cultured plantlets. The results showed that theoptimal cultural condition to differentiate adventitious bud for oriental lily 'Sorbonne' and'Siberia' is 1/2MS, 0.2mg/L 6-BA, 0.5mg/L 2,4-D, 20g/L sugar and a pH of 5.8. MS with 30g/Lsugar is suitable for the long-term preservation of wild lily. The result of paraffin section indicatedthat adventitious buds could be generated in two different ways, in which one type is developedfrom the epidermal cells or the cells of 3-4 layers under the epidermal cells; the other type wasdeveloped from the small vascular bundle inside the explant. Both belong to direct organogenesis.Using callus of 'Tiber' as explant, we studied the effect of plant growth regulators on thegeneration of somatic embryo and adventitious buds. The result showed that 2,4-D and darkculture are the key factors in the somatic embryogenesis. MS with 0.1mg/L 6-BA, 0.1mg/L KT.0.2mg/L 2, 4-D is a proper media for the next generation propagation of callus. MS with 0.1mg/L2, 4-D is optimal for somatic embryogenesis. Somatic embryogenesis through the phase of callusis an indirect somatic embryogenesis.Using in vitro cultured lily as explant, we studied the effect of sugar, active carbon, inorganicsalts, Paclbutrazol on the growth of tuber and its diameter of in vitro cultured lily. The resultindicated that 2MS, 80g/L sugar, 2.0g/L AC, 3.2mg/L PP₃₃₃ is the optimal media to produce'Sorbonne' cultured tuber with large diameter and so is 2MS, 80g/L sugar, 4.0g/L AC, 1.6mg/LPP₃₃₃ for 'Siberia'.