Studies On Regeneration System Establishment Of 21 Wild Lilium Species And Expression Analysis Of GA20ox Gene In Lilium Oriental Hybrids ‘Sorbonne'

Lily has wide market demands and prospects due to its important ornamental,edible and medicinal values.Despite aboundant and precious germplasm resources of wild lily in china,the protection and exploition in Lilium was inadequate,owing to the lack of systematic reproduction system and independent intellectual property rights varieties.Tissue culture has been widely used for disease elimination and plant improvement in the propagation of lily,which can not be achieved by traditional methods.Meanwhile,it provides a fast and stable propagation route for the modern production of lily,which could be beneficial to the resource conservation of wild Lilium and further genetic engineering research.Plant height is an important factor affecting the quality of cut flowers and the application form of potted plants,and has attracted the attention of breeding workers in recent years.The development of genetic engineering breeding technology provides an alternative way for lily breeding in addition to the traditional methods.To develop lily molecular breeding about plant height,the separation and identification of valuable genes related to plant height are required.In the present study,the effects of sterilizated time,plant growth regulator,explant types and culture conditions during the establishment of regeneration system of bulbs in 21 wild Lilium species and‘Sorbonne'were investigated.Additionally,in order to investigate the mechanism of plant height regulation,GA20ox gene were cloned from the leaves of‘Sorbonne'and its expression patterns were studied.The main results were as follows.1.The optimal sterilization method to the bulb scales of L.lancifolium was 75%ethanol for 30 s,followed by 0.2%HgCl₂ for 7 min.Taken both the immature degree of explants and the optimal sterilization time in L.lancifolium into account,the sterilization time of explants in other 21 Lilium species was estimated and conducted for callus or adventitious shoot induction.Although regeneration of all species was achieved,some species showed poor regeneration ability,including L.wenshanense,‘Sorbonne',L.leucanthum and L.davidii var.Willmottiae,suggesting that the improvement of explant regeneration situation should not rely solely on the adjustment of disinfection time.2.For bulb scales of L.lancifolium,the optimal medium for bud induction was MS+1.0mg·L^-1 6-BA+0.1 mg·L^-1 NAA,with the number of buds/explant 2.09;the optimal medium for callus induction was MS+1.5 mg·L^-1 PIC,with the rate of callus induced 86.66%.For bulb scales of L.martagon,the optimal medium for bud induction was MS+2.0 mg·L^-1 6-BA+0.1 mg·L^-1 NAA,with the number of buds/explant 2.50;the optimal medium for callus induction was MS+0.2 mg·L^-1 TDZ+0.5 mg·L^-1 NAA,with the rate of callus induced77.14%.Factors influencing regeneration of 20 Lilium species were analyzed by three factor analysis of variance,which indicated that the effect of 6-BA concentration on regeneration ability of lilies is different among multiple genotypes and explants,regeneration ability of different genotypes was greatly different,and bulb scales had stronger regeneration ability than leaves.3.Compared to light condition,dark condition was more appropriate for callus proliferation and differentiation in L.martagon,with the proliferation coefficient 1.86 and number of buds/callus 5.43.Its optimal medium for callus proliferation was MS+0.5 mg·L^-16-BA+0.1 mg·L^-1 NAA,with a promoted proliferation coefficient 2.93.In contrast,light condition was more appropriate for callus proliferation and differentiation of L.sargentiae,with the proliferation coefficient 3.10 and number of buds/callus 2.67.The optimal medium for callus proliferation was MS+1.0 mg·L^-1 6-BA+0.1 mg·L^-1 NAA.Factors affecting the callus proliferation and differentiation of 11 Lilium species were analyzed by two factor analysis of variance,which indicated that genotypes and light/dark culture conditions had interaction and single factor effect on callus proliferation and differentiation and had different effect on the growth status of callus and buds.4.A 100 percent of rooting rate from in vitro cultured buds of all tested 18 species was obtained in medium of MS+0.1 mg·L^-1 NAA+6%sucrose,with well-developed roots in most species.The aseptic seedlings of 18 species grew well after transplant,with the survival rate above 90%.5.A cDNA encoding GA20ox was cloned from‘Sorbonne'using rapid amplification of cDNA ends?RACE?.Sequences analysis indicated that the full-length of GA20ox was 1 502bp containing an open reading frame?1 167 bp?,which encoded 388 amino acids.Amino acid sequence alignment showed that GA20ox of‘Sorbonne'was similar to other GA20ox proteins.They all shared a PLN02276 binding domain.Phylogenetic analysis indicated that GA20ox protein of‘Sorbonne'was highly homologous to other GA20ox proteins,such as that of Phalaenopsis equestris and Dendrobium catenatum.6.Quantitative Real-time PCR?qRT-PCR?analysis showed that the expression of GA20ox was closely related to growth and development,exhibiting temporal and spacial regulation.The application of 200 mg·L^-1 GA₃ in germination stage made a significant increase in plant height and inhibited the expression of GA20ox in multiple tissues.These results showed that the expression of GA20ox may be received feedback regulation by active gibberellins,so GA20ox may play a key role in the maintenance of endogenetic active gibberellins homeostasis.