Establishment Of Plant Regeneration System And Study On Agrobacterium-mediated Gladiolus Transformation Of NPR1 Gene

Gladiolus (Gladiolus hybridus) is one of perennial and monocotyledonous bulbous flowers,which belongs to the gladiolus of iridaceous and is one of the four world-famous cut flowers. Theproduction of corm by asexual reproduction way during cut flower production of gladiolus,so thepathogen on the old corm would infection offspring. The accumulation of pathogen by a few yearscan lead to serious disease in the production of cut flower.Using the disease-resistant varieties is the best way for preventing and curing the plantdiseases. But the disease-resistant resource of gladiolus is so little and the cycle of the generalbreeding is so long that the disease-resistant variety is far from meeting the demand of the gladiolusproduction. With the rapid development of biotechnology, transferring new disease-resistant geneof other sources into the gladiolus by using genetic engineering means brings new hope in thedisease-resistant breeding of gladiolus.This study include that we get embryogenic callus from gladiolus'bud,using it setup upregeneration system by organogenesis and somatic embryogenesis and study the robust culture ofbuds in organogenesis way and the formation of somatic embryo in the embryogenesis way. We usethe callus of gladiolus as the explants of genetic transformation, The broad-spectrum diseaseresistance gene (NPR1 gene) transferred into gladiolus mediated by the agrobacterium, weestablish genetic transformation system of gladiolus. Finally, we get PCR positive plants. The maincontent and result of experiment are as follows:1. The regeneration system by organogenesis: Taking the slice of gladious'corms as explant,MS basal medium supplemented with 2,4-D4mg/L and 6-BA0.5mg/L was advantageous toembryogenic callus induction,The frequency of callus induction was 95%;The combination of6-BA0.5mg/L and NAA 0.1mg/L was the best for callus differentiation with the rate of 86%;100%of the robust and regenerated shoot treated with 1/4MS and PP₃₃₃1mg/L and NAA1mg/L andsucrose50g/L; MS basal medium supplemented with IBA1.5 mg/L could develop their roots,therate of robust plantlets was 100%.The survival ratio of transplanting reach 100%.2. The regeneration system by somatic embryogenesis: Taking the slice of gladious'corms asexplants, MS basal medium supplemented with 2,4-D4mg/L and 6-BA0.5mg/L was advantageousto callus induction,The frequency of callus induction was 95%; MS+2,4-D1.0mg/L+TDZ0.2 rag/L+sucrose30g/L+30g/L was the best culture medium that the induction of somatic embryo,the rate was 57%. After that there was three steps to complete the culture: First, the regenerated somatic embryowere cultured in the MS medium to growth enough.; Second,the growful somatic embryo wascultured in the MS+6-BA2.0mg/L to induce shoots;Third,the regenerated plants were obtained inthe MS medium.The appearance and growth of somatic embryo were observed through paraffinslices and press slices. Histological observations showed that parenchyma cells first experienceddedifferentiation and followed with callus-forming, ensued with embryogenic cell masses which likea tubercular structure.and these embryogenic cell masses experienced globular, scutellate stages anddeveloped into whole plantlets.3. The establishment of genetic transformation system: Taking the slice of gladious'corms asexplants, pre-culture of callus for 14d;the agrobacterium tumefaciens solution wasOD₆₀₀=0.3～0.5,the time of infect was 10～15min, the time of co-culture was 3d,the delayedscreening on regeneration for 20d, during this time,the proliferation of Agrobacterium tumefacienswas selected Cef, the suit concentration was 250mg/L. After 20d, we start to select resistant callus,the select pressure of PPT was 2.0mg/L. The regeneration of resistant callus by organogenesis andsomatic embryogenesis way.4. Detection of the transformed plant: For callus' regeneration by organic regeneration, wegained 69 resistant plants. Three of them is PCR positive plant, positive percentage is 4.3% andtransformation percentage is 0.3%. For callus' regeneration by somatic embryo regeneration,therewas no resistant plants obtioned from it..