Studies On The Transformation Of Larix Based On The System Of Somatic Embryogenesis

Larix ssp. is one of the species which have great economical and ecological values. However,the existed breeding techniques, such as regeneration of plantlets or the traditionaltime-consuming breeding methods, can not meet our needs. Therefore, establishing an efficienttransgenic system has significant influence for the Larix breeding. In this paper, we used twomethods for the transformation of Larix——Agrobacterium tumefaciens-mediated genetransformation and particle bombardment gene transformation.(1) Based on the system of somatic embryogenesis, we developed an efficient andreproducible procedure for the transformation of Larix and analysed a series of factorsaffecting the Agrobacterium tumefaciens-mediated gene transformation. Callus subculturedover 3 times in the medium containing 9 g·L^-1 cyclohexanhxol and 0.8 mg·L^-16-Benzylaminopurine (BA), and followed by a 4-day-precultivation, it will get higherproportion of embryogenesis and divide rapidly. Compared with other pretreatment methods, itdemonstrated to yield higher frequency of kanamycin-resistant tissues. Co-cultivated withAgrobacterium tumefaciens with the infection medium (OD₆₀₀ value is 4.0) containing 0.1%(v/v) a surfactant (Tween-20) for 20 minutes and then transferred onto the co-cultivationmedium for 3 days has been proved to be a suitable way for the transformation. Bothacetosyringone (AS) and coniferyl alcohol (CA) are beneficial to an efficient transformation,furthermore, adding 50μM CA and 50μM AS to the infection medium and the co-cultivationmedium at the same time could increase the transformation most apparently.(2) Additionally, certain efficient measures to reduce the harm of Agrobacteriumtumenfaciens and the inhibition of antibiosis are as follows: to decrease the maintenance ofcarbenicillin gradually, and to apply selection 7 days after co-cultivation and reduce theselection period to 2 times.(3) Using the improved procedure to transform several cell lines of Larix, the frequency ofkanamycin-resistant tissues is about 10% and we get 42 transformed plantlets. The resultssuggest that the Agrobacterium tumefaciens-mediated transgenic system is applicable to a widerange of Larix embryogenic cell lines.(4) It also proved that during the panicle bombardment, it will produce more Km resistantcalli when bombarded 2 times when the vacuum is 26 cmHg and the target distance is 6 cm.However, the frequency of Km resistant calli is unstable and we did not get any positive resultseventually.