Establishment Of Regeneration System And Primary Study On Genetic Transformation In Four Kind Of Rose Cultivars

Rose is one of the most important ornamental plants in the world,and it is also a good object to study the gene function of woody horticultural plants.Establishing an efficient regeneration system and genetic transformation system is necessary for transgenic breeding.In this experiment,the leaflets of Rosa chinensis var.spontanea,R.chinensis ‘Old Blush',R.hybrida ‘Coral Babylon Eyes' and R.hybrida ‘Samantha' were used as explants,screen relevant factors affecting regeneration,and the regeneration system of ‘Coral Babylon Eyes' was established through the organogenesis direct pathway and somatic embryogenesis pathway,and the regeneration system of 'Coral Babylon Eye'and 'Samantha' was established through the soamtic embryogenesis pathway,and the 'Samantha'genetic transformation system was preliminarily established using somatic embryos as receptors.The main research results are as follows:1.Direct organogensis of rose is affected by genotype and dark culture time.'Coral Babylon Eyes' was cultured in MS + 1.5 mg/L TDZ + 0.05 mg/L NAA + 30 g/L Sucrose + 7.0 g/L Agar culture medium for 12 days and then transferred to differentiation medium.The adventitious shoot induction rate reached 31.33 %.2.Somatic embryos can be induced in direct or indirect ways,but they are also affected by the genotype.Transfer the callus of ‘Coral Babylon Eyes' induced by callus induction culture medium(MS + 3.0 mg/L 2,4-D + 0.05 mg/L KT + 30g/L Glucose + 3.0 g/L Gel)to culture medium(MS + 4.0mg/L KT + 30 g/L Glucose + 3.0 g/L Gel),somatic embryos can be induced,the induction rate was about 3.81%;‘Samantha' had the highest somatic embryos induction rate on MS + 0.05 mg/L KT +2.0 mg/L 2,4-D + 30 g/L Glucose + 3.0 g/L Gel,which was about 24.61%.3.Adding a low concentration of DNA methylation inhibitor to the medium can increase the somatic embryos induction rate,while a high concentration significantly inhibits the occurrence of somatic embryos.Adding 0.01 ?mol/L 5-Azad C or 0.1 ?mol/L Zebularine to the culture medium can significantly increase the somatic embryos induction rate of ‘Samantha'.4.Somatic embryos of ‘Coral Babylon Eyes' can only grow slowly on the medium of MS + 1.5mg/L ZT + 0.25 mg/L NAA + 0.1 mg/L GA3 + 30 g/L Glucose + 3.0 g/L Gel.Somatic embryos of‘Samantha' had the best proliferation efficiency on on MS + 1.0 mg/L 2,4-D + 0.1 mg/L TDZ + 30g/L Glucose + 3.0 g/L Gel culture medium,and the proliferation coefficient was about 3.57.At the same time,the use of ABA can promote the maturation of somatic embryos and transform somatic embryos into shoot-like embryos.5.Somatic embryos of ‘Coral Babylon Eyes' had the highest differentiation efficiency of 14.81%on 1/2MS + 1.0 mg/L 6-BA + 0.005 mg/L NAA + 0.1 mg/L GA3 + 30 g/L Sucrose + 3.0 g/L Gel;while ‘Samantha' had the highest differentiation efficiency of 81.63% on MS + 1.0 mg/L 6-BA + 0.1mg/L TDZ + 0.005 mg/L NAA + 0.1 mg/L GA3 + 30 g/L Sucrose + 3.0 g/L Gel.6.Using somatic embryos of ‘Samantha' as receptor,infected with Agrobacterium liquid with an OD value = 0.6 for 30 min,and then co-cultured for 3 days,added 150 ?mol/L AS to the co-culture medium,and the transient expression rate of GUS gene could achieve 47.22%.