| In the present study,Lilium brownii F.E.Brown ex Miellez var.giganteum G.Y.Li&Z.H.Chen was used as the main research material and L.brownii F.E.Br.ex Miellez and commercial cultivar L.Oriental Hybrids 'Sorbonne' as comparative material.We compared the in vitro regeneration capacities of these three lilies to identify genotypic differences as well as histological observations.The aim of this study was to explore the advantages of the specific material L.brownii var.giganteum^ and to provide fundmental information for further bulb development research.The establishment and optimization of the genetic transformation system of L.brownii var.giganteum might help to realize molecular assisted breeding.The main results were as follows:1.Exploring the advantages of L.brownii var.giganteu,m in vitro(1)Studies on direct organogenesis and bulblet proliferationThe optimal medium for shoot induction of three lilies was identified.For L.brownii var.giganteum,shoots emerged early,and shoots were vigous,the average rate of shoot induction was 100%and the average number of shoots induction was highest(2.96).Shoot lenfgth was also high,significantly higher than that of 'Sorbonne' and L.brownii.Histological observation showed that there were more small cells in the mother scale of L.brownii var.giganteum,and the number of starch granule in each cell was high.After 10 d,the cell division in the mother scale of L.brownii var.giganteum was very active,and the cell size gradually exceeded 'Sorbonne,and L.brownii.The results indicated that L.brownii var.giganteum performed well under in vitro condition with best direct organogenesis ability.The optimal medium for shoot proliferation of three lilies was confirmed.For L.brownii var.giganteum,the factorial increase in bulblet height and diameter,scale multiplication coefficient kept high,bulblet swelled faster and the number of scales increased more.At the early stage of proliferation(35 d),the bulblet multiplication coefficient of L.brownii var.giganteum was the lowest while the bulblcts mainly expanded in size.After 35 d to 90 d,clustered bulblets formed,and bulblet multiplication coefficient of L.brownii var.giganteum was higher than L.brownie but slightly lower than 'Sorbonnc'.(2)Studies on indirect organogenesis and callus statusThe optimal medium for callus induction from scale and leaf of three lilies was determined.The induced callus status of L.brownii var.giganteum was best both of scale and leaf among three lilies.Nevertheless,the callus induction rate of L.brownii was the highest,whether scale or leaf was used as explant.Dedifferentiation ability of 'Sorbonne' was relatively weak as only PIC was added could induce callus successfully.The diameter growth rate of L.brownii var.giganteum was the highest in all the five mediums when scale was used as callus induction explant,and the embryogenic status remained the best during the subculture process followed by L.brownii and'Sorbonne'.There were no significant differences in callus histological observation of three lilies.Each callus contained both embryogenic cells and non-embryogenic cells.The histological observation of two-year callus and fresh callus of L.brownii var.giganteum showed that there were more starch granules in the the two-year callus,and active cell division both existed in the old and the new callus,indicating that the two-year callus also maintained embryogenic status as well.The histological observation of 'Sorbonne' callus induced both from scale and leaf explants showed that the callus was prone to embryogenic.2.Establishment and optimization of genetic transformation system of L.brownii var.giganteum(1)Establishment of embryogenic callus efficient regeneration systemFor the regeneration system,7 d of dark transition before light had a significant effect on callus regeneration with the regeneration rate doubled to 100%,and the number of regenerated shoots of each callus increased by 16 times,each 0.5 mm3 callus induced 14.33 shoots on average and shoots grew well.The optimal regeneration medium was MS + 30 g/L sucrose + 8 g/L agar,without any hormones.After 90 d,the shoots were transferred to the medium containing high concentration of sucrose(60 g/L)and 16.87 small bulblets on average for each callus in 0.5 mm3 were obtained for 60 d.(2)Antibiotic concentration determinationWe failed to determine the kanamycin screening concentration of embryogenic callus.The reason was that the embryogenic callus response to kanamycin from 0 to 150 mg/L did not show any regularity.Small grain callus also survived when kanamycin concentration was even at 200 mg/L,and almost all died when 300 mg/L,Kanamycin was not suitable for used as the screening antibiotic in transformation when embryogenic callus was used as the receptor.The suitable kanamycin screening concentration for scale thin slice was 75 mg/L.When callus size was 0.5 mm3,hygromycin concentration was 60 mg/L,and callus was completely brown and dead.When callus size was 0.2 mm3,callus was completely browned at 50 mg/L of hygromycin.Therefore,callus size had a certain effect on the results,and the suitable hygromycin concentration for callus resistant screening was 50?60 mg/L.Hygromycin concentration suitable for screening scale thin slice was 40 mg/L.(3)Genetic transformation system optimizationThe REM 1.3 gene was transformed with embryogenic callus and scale thin slice as receptors.We found that embryogenic callus was better receptor compared with scale thin slice.The optimal infection time of both receptors was 20 min.The suitable concentration of OD600 for embryogenic callus infection was 0.4 while 0.8 for the scale thin slice.The co-culture method(solid or liquid medium)had little effect on the transformation efficiency.Infection and co-culture medium components significantly affected the transformation rate.When all major elements,micronutrient,ferric salt,organic component were removed,the percentage of resistant tissue reached to the highest,both embryogenic callus and scale thin slice,reached to 30.67%and 15.00%,respectively.Noteworthy,shoots regenerated from resistant callus were albinism which need further investigation. |
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