Studies On In Vitro Bublet Formation Of Lycoris Sprengeri

Lycoris sprengeri Comes ex Baker is one of bulb flowers of the Lyricis family of Amaryllidaceae.Lycoris spp.has great ornamental and application value because of its beautiful flower shape and rich color.However,bulbs of Lycoris have not been artificially propagated due to their low natural regeneration rate,long juvenile period,and slow expansion speed.In recent years,the amount of excavation of wild resources has increased,with more and more landscape applications,resulting in the reduction of its resources and the need for protection.Moreover,as no variety of Lycoris in China,and it takes a long time for hybrid Lycoris seeds to grow into mature bulbs,which is the main limiting factor for breeding of Lycoris.In the present study,wild seeds of L.sprengeri collected from Zhoushan Island,Zhejiang Province were used as materials to establish an aseptic germination and bulblet formation system without destroying the wild resources.Meanwhile,we induced bulblets of hybrid Lycoris in vitro and obtained optimum cutting proliferation system,by changing cutting method,hormone ratio and sucrose concentration,which providing an experimental basis for accelerating the breeding process of Lycoris.The effects of exogenous sucrose and 6-Benzylaminopurine(6-BA)on bulblet formation of L.sprengeri in vitro were explored from several aspects,including morphology,histocytological,physiological,biochemical and molecular level.This research provided an experimental basis for exploring the mechanism of the bulblet formation in Lycoris,as well as the action of exogenous sucrose and 6-Benzylaminopurine in this process.The main results were as follows:1.Aseptic germination and bulblet formation of L.sprengeriProgram for the aseptic germination and bulblet formation of L.sprengeri could be described as follows:Natural dry or stored seeds pretreated with warm water(40 ℃)for 5 h,then,sterilized with 75%ethanol for 30 s,0.1%HgCl2 for 10 min and 2%NaCIO for 5 min.Then,sowing seeds in the MS + 30 g/L sucrose + 0.2 mg/L NAA medium,were cultured at 25 ℃ for 3 months.At the end,we obtained in vitro bulblets.This method could also applied to L.aurea,and other plants of the genus Lycoris.2.in vitro cutting proliferation system of hybrid LycorisThree kinds of cutting methods were used to induce bulblet formation of hybrid Lycoris in vitro.The results showed that the best cutting method was keep the leaves 1.5 cm,cross cutting from the bottom of basal plate to 1/2 depth of the bulb.At the same time,MS + 60 g/L sucrose + 1 mg/L NAA + 5 mg/L 6-BA was the best medium for inducing bulblets,from which the average proliferation number was 47.86,and status of bulblets,roots and mother scales were good.3.Effects of exogenous sucrose on bulblet formation of L.sprengeri in vitro(1)bulblet formation stages of L.sprengeri in vitroBased on morphology and histocytological observations,bulblet formation of L.sprengeri in vitro belongs to the axillary bud formation in the direct organogenesis pathway.The propagation process within 0-30 days after bulb cutting was divided into three typical stages:0-6 days(axillary meristem formation),6-15 days(axillary bud formation),15-30 days(bulblet formation).(2)Exogenous sucrose determined whether bulblets could form in vitroExogenous sucrose determined the formation of bulblets,while sucrose concentration only affected the status of bulblets and mother scales.Histocytological observations and carbohydrate determination results showed that exogenous sucrose was a key factor for bulblet formation acting as both carbon and energy source.When exogenous sucrose is present,sucrose and soluble sugar accumulated before the formation of axillary buds,and the accumulation of starch granules at the base of the scale provided a material basis for bulblet formation here.When exogenous sucrose was absent,carbohydrate content and the number of starch granules decreased continuously,and bulblet could not form eventually.In addition,the presence of sucrose promoted the accumulation of MeJA in plants and inhibited the abnormal increase in the expression of LsWIP1,LsERS2 and LsEIN2 genes levels,which may mean that sucrose initiated a damage protection mechanism as a signaling molecule and promoted the formation of bulblet.4.Effects of exogenous 6-BA on bulblet formation of L.sprengeri in vitroThe addition of exogenous 6-BA rapidly inhibited the content of abscisic acid at 1 day after cutting,aggravated the division of the meristematic meristem,accelerated the accumulation of sucrose and soluble sugar before the formation of axillary bud,and finally increased the number of axillary buds between middle and outer scales.At the molecular level,the addition of exogenous 6-BA significantly promoted the expression of sucrose synthase genes LsSuSyl,LsSuSy2,auxin response factor LsARF and cytokinin receptor gene LsAHK at 6 h,and the differential expression of these genes mainly concentrated before 6 days.It can be speculated that 6-BA mainly acts on the formation stage of axillary meristems formation.