Studies On Cross-breeding And Agrobacterium-mediated Genetic Transformation Of Lilium Spp.

Lily,a kind of perennial bulb plant with large and colorful flowers,has been one of the most important fresh cut flowers.Recent years,primary study for variety improvement is revolving the breeding objectives,such as ornamental character,fastness, quality and reproductive capacity.This study is a kind of preliminary exploration for variety improvement of lily by approaching of cross breeding and genetic transformation.1.The impact factors including sucrose concentration and storage time for in vitro germination were tested.The results revealed that 100 g/L sucrose liquid medium supplemented with 2 mg/L boric acid and 10 mg/L CaCl₂ was suitable for pollen.culture. Pollens from L.longiflorum and ’Gelria’ belonging to Longiflorum hybrids,’Marco Polo’, ’Bernini’,’Acapulco’ and ’Novatore’ belonging to Oriental hybrids,’Bright Yellow’,’ Yellow Giant’ and L.davidii vat.unicolor belonging to Asiatic hybrids,were tested with high pollen viability and could be the appropriate choice for paternal parents. ’Iceberg’ from Asiatic hybrids and ’Tiber’ from Oriental hybrids were tested with low pollen viability but suitable for maternal parents.The viability of the pollen from L. davidii var.unicolor and L.longiflorum could last for 20 d upon drying preservation at 4℃.2.The development of pollen on stigma surface in different hybrid combinations was observed by fluorescence microscopy.The pollen germinates and grows in stigma in 5 hybrid combinations,including L.longiflorum×’Biarritz’,’Simplon’×’Iceberg’, ’Biarritz’×L.davidii vat.unicolor,’Bright Yellow’×’Gelria’,’Gelria’×L.davidii var. unicolor,which showed compatibleness between the parents.3.The immature embryos of 38 hybrid combinations were cultured in vitro.The top germination rate was found in B5 medium supplemented with 9%sucrose when ovules from inbreeding of the wild lily in Guizhou were cultured.It was also found that B5 media supplemented with 6%or 9%sucrose were sutable for the development of ovule in initial stage of ovary slice culture.The ovary was picked up for slice culture and ovule culture after 25 d of the stub style pollination.One hybrid of ’Simplon’×’Siberia’ was found in the B5 medium supplemented with 9%sucrose,and the hybrid was identified based on sequence-related amplified polymorphism（SRAP） analysis.The hybrid could root directly in the MS medium after subculture.The hybrid was transplanted into vermiculite firstly,and then in the medium made up of peat:perlite（1:1）.4.Using the scale as the explant,we established the regeneration system from ’Siberia’ and L.leucanthum calli.The orthogonal test with four factors and three levels including the concentration of sucrose,2,4-D,NAA and 6-BA showed that MS medium supplemented with 2.0mg/L 2,4-D,2.0 mg/L NAA,0.5 mg/L 6-BA and 9%sucrose was suitable for the induction of callus using small scale of ’Siberia’.The three levels of the concentration of sucrose,2,4-D,NAA and 6-BA had significant impact on the callus induction of ’Siberia’;however,MS medium supplemented with 4.0mg/L 2,4-D,0.5 mg/L NAA,0.5 mg/L 6-BA and 6%sucrose was suitable for the callus induction of L. leucanthum,and the concentration of sucrose,2,4-D,6-BA had no significant impact on the callus induction of L.leucanthum.The medium suitable for callus differentiation is MS supplemented with 1.5 mg/L 6-BA and 0.2 mg/L NAA.5.The system of Agrobacterium-mediated tranfromtion of Lilium spp.was established in this study.In screening culture,the concentration of kanamycin for resistance selection of the small scale from ’Siberia’ was 120 mg/L,the concentration of kanamycin for resistance selection of the callus from L.longiflorum was 50 mg/L.The efficient transformation could be acquired when the callus was infiltrated for 30 min by Agrobacterium.0.4 mol/L mannitol was helpful to the transformation of lily callus.In this study,we obtained the callus of L.longiflorum stably expressing GUS gene,but the resistant callus was difficult to develop into the healthy seedling.Using the scale of ’Siberia’ as the transformation receptor,we obtained five resistant seedlings,but the resistant seedlings were false positive according to PCR analysis.Because the scales had higher false positive than the callus,we found that the callus was more suitable to be used as the transformation receptor.