Establishment Of Genetic Transformation System Of Special Economic Plant Sauropus Androgynus And Pelargonium Graveolens And Analysis Of Characteristics Of The Transgenic Plants With Insect Antifreeze Protein(AFP) Gene

Temperature is a major factor which restricts plants geography distribution and influences the growth of plants. Low temperature is one of the abiotic stresses limiting the geographical location suitable for growing crops and periodically account for significant losses in plant productivity,so it is important for agriculture to improve the cold resistance of crops.Many plants can acquire increased frost tolerance after a period of exposure to low, non-freezing temperature through a complex adaptive process called cold acclimation.Plants grown in the frigid zone can generally acquire stronger antifreeze capability to cold harm than that of living in the tropical zone and subtropical zone. Between different varieties of the same crop has alreadly shown obvious difference with antifreeze ability. Recently, much progress has been made in understanding the mechanism in the field of plant physiology and adaptation under the adverse circumstances, ,especially in developing the cold-tolerant crop varieties by traditional ways.But there always exists some problem with the traditional way, for example, it takes a longer time, the range of temperature adaptation for crops is narrow, etc., there is a long way to go to be applied. In the past ten years,with the great advance in the research of molecular mechanisms of cold acclimation,the studies of genetic engineering for cold resistance in plants have also been carried out extensively. Currently, there are two types of genes being used in plant cold-resistant genetic engineering,which are protective genes and regulatory genes.Many studies indicated that both kinds of genes had good effects for improving the cold resistance of plants.The plant antifreeze genetic engineering has become one of the important ways to resolve the cold harm for crops. In present study, we used a plant expression vector pCAMBIA-1302-Mpafp149 in which harbored an anti-freeze protein gene Mpafp149, which is from a desert insect Microdera punctipennis dzungarica, and transformed two special economic plants Sauropus androgynus and Pelargonium graveolens by Agrobacterium-mediated method, and hope to improve the cold tolerance properties of both plants by genetic engineering means.First of all, high efficient genetic transformation system of Sauropus androgynus was established. Stems were used as explants, infected with Agrobacterium tumefaciens EHA105 strain which harbored plasmid pCAMBIA-1302-Mpafp149.. Various factors which likely influence the transformation efficiency were examined and optimized, such as Agrobacterium concentration, pre-culture period, co-culture period, immersion time and concentrations of antibiotics (hygromycin). The results showed that conditions of pre-culture and co-culture for 2 days respectively, immersion in OD600 0.2~0.25 bacterium solution for 8 min had higher transformation rate. Positive signals in PCR of the antibiotic plantlets preliminarily indicated that the Mpafp149 was integrated into the genome of Sauropus androgynus, and the T0 transgenic Sauropus androgynus plants were obtained successfully.Based on the established regeneration system, an efficient transformation system of P. graveolens mediated by A. tumefaciens was also developed.The factors affecting the transformation frequency,such as the types and concentrations of antibiotics inhibiting Agrobacterium Agrobacterium strains growth, preculture time of explants,concentrations of Agrobacterium,infection time and co-culture time, were also discussed. The results showed that conditions with pre-culture for 3 days, co-culture for 2 days, immersion in OD600 0.2~0.25 bacterium solution for 8 min were better for transformation. Results of PCR and RT-PCR with the antibiotic plantlets preliminarily indicated that the Mpafp149 was integrated into the genome of Pelargonium graveolens, and the T0 transgenic Pelargonium graveolens plants were obtained successfully. Transgenic plants were then exposed under low temperature of -1℃,the relative leakage of electrolytes (RLE) and malondialdehyde (MDA) content of leaves were measured and analyzed. The results showed that the RLE increased with increasing of the exposing time; MDA descended first then increased with increasing exposing time.