Plant Regeneration From Somatic Embryogenesis Of Chrysanthemum (Yuukou) And The Establishment Of Genetic Transformation System

Chrysanthemum (Yuukou) was used as experimental material. The objective of this study is to transfer the LcFER gene to chrysanthemum (Yuukou) in order to establish regeneration system and transformation system of chrysanthemum mediated by Agrobacterium tumefaciens and obtain new species of chrysanthemum with stress resistance. The research contents and the results obtained are stated in details as follows:1. Chrysanthemum Leaf was used for explants to induce callus and somatic embryos.6-BA, NAA and2,4-D were tested in order to screen out the optimal medium for initiation callus and embryogenesis and setting up an effective chrysanthemum regeneration system. The results showed that callus and somatic embryogenesis were induced in MS medium supplement with0.5mg/L6-BA and1.5mg/L NAA, yielding100%and93%respectively. The obtained plantlets were transferred onto the MS medium without any growth regulators. The regenerated roots showed the rapid growth and the rooting percentage was98%.2. On the basis of establishing chrysanthemum regeneration system, morphological and histological observation were proceeded during the different development stages of leaf explants via microscopic observation and paraffin section, which was in order to reveal the process of the somatic embryo formation. The results showed that callus were induced from leaf explants, then differentiated into proembryo followed by the formation of globular structures and cotyledon structure and then developed into complete plantlets. Histological observation showed that the new regenerated embryoid was formed without vascular connections with the original tissue. So we can confirm that the regeneration system from chrysanthemum leaf explant was set up through somatic embryogenesis.3. The optimal system for genetic transformation mediated by Agrobacterium tumefaciens was studied based on transient expression frequency of Gus gene. The results showed the optimal transformation system was pre-culture for3d, infection for25min when OD6oo was0.5, co-culturation for2d and the concentration of Cef was300mg/L, selective concentration of mannose and xylose for transgenic plants were25g/L and28g/L respectively.4. The obtained transgenic chrysanthemum was confirmed by using PCR amplication. Target gene was detected in parts of transgenic plants, which identified that the target gene has been transferred into chrysanthemum genome.