Studies On Efficient Regeneration System And Regulation Of Callus Proliferaton In Wild Lilies

Lilium spp. is known as ’the king of the bulbous flowers’, which is one of the five fresh cut flowers around the world. Although China is the main natural distribution center of wild lilies, breeding and reproduction lag comparatively because of the late beginning of the commercialization of ornamental lily cultivation. At present, the domestic lily bulbs still rely on the import from Netherlands and other countries, which leads to that lily bulb localization problem has always been a bottleneck problem of commercialization of lily cut flower production in China. Therefore, studies on lily efficient breeding and modern breeding techniques such as genetic transformation aiming to improve its properties, were of important practical value.L. brownii var. giganteum is a newly discovered variety of L. brownii in Zhejiang Province, which is characterized by super-large bulbs, numerous scales, beautiful and large flowers. Besides, it is a good transgenic breeding material, which is of research and development value.Taking the wild lilies resources as materials, the efficient regeneration system were set up and optimized. Taking L. brownii var. giganteum as materials, the genetic transformation receptor system were established and optimized. Besides, the effects of internal and external hormones on callus were also discussed preliminarily. All above lead to the conservation and efficient breeding of the wild lily germplasm resources and the establishment of genetic transformation system of the excellent wild lily material, the main conclusions were as follows:1. Peeling treatment can improve the speed of seed germination and germination rate. With peeling treatment, the optimum culture medium of L. brownii var. giganteum and L. leucanthum Baker was MS medium supplemented with6-BA1.0mg/Land NAA1.0mg/L, L. speciosum Thunb. var. gloriosoides Baker was MS medium supplemented with6-BA2.0mg/Land NAA1.0mg/L. This culture medium formula can effectively increase the germination and induction rate of seeds. Besides, the plantlets grew well. The results showed that the seeds of L. brownii var. giganteum had a certain high viability, which was suitable for sterile sowing and also an excellent wild lily germplasm resource.2. For L. brownii var. giganteum, L. speciosum Thunb. var. gloriosoides Baker and L. lancifolium, the optimum medium of bulblet induction was MS+1.0mg/L6-BA+l.0mg/LNAA, MS and MS+1.0-2.0mg/L6-BA+0.1-0.1mg/L NAA, respectively. The optimum medium formula can improve the induction rate of bulblet and the No. of bulblets effectively. Besides, the optimum medium formula of bulblet induction is the same as the sterile sowing for L. brownii var. giganteum.3. In three kinds of explants (blade, petiole and small bulb), the optimum explants were blades for embryonic callus induction of/,, brownii var. giganteum. The optimum callus induction medium was MS+0.4mg/L6-BA+0.5mg/L NAA+1.0mg/L2,4-D+30g/L sucrose+8g/L agar. In this culture, the callus induction rate was up to92.50%, the browning rate of explants was only5.00%. Besides, the callus inducted was majority of embryogenic callus.4. For/,, brownii var. giganteum, the results demonstrated that the most appropriate condition for proliferation of embryogenic callus was using MS medium supplemented with8g/L agar,30g/L sucrose, O.lmg/L2,4-D,0.5-1.0mg/LNAA and0.1mg/L6-BA/TDZ/ZR and culture in dark. The callus grew well, with drying surface, good graininess, and rare induced shoots and roots. Using PAS staining to observe different states of callus, we found that embryogenic callus in active metabolism was always light yellow or yellow, with small and numerous sugar granules around the cell nuclei. The optimum plant regeneration medium was MS+30g/Lsucrose+8g/L agar.5. Taking the callus of L. brownii var. giganteum cultured in different culture medium for60d as materials, the endogenous hormone levels were studied. We found that as the diameters of callus treated by exogenous hormones (except ZR) increases, the corresponding endogenous ABA/IAA also increased gradually. Combining the previous research results, the conclusion that there was a close relationship between the ratio of callus endogenous ABA/IAA and its embryogenic ability was speculated.