Studies On Transformation Of The Choline Oxidase Gene CodA And Physiological Mechanism In Tropical Waterlily

Waterlily is an aquatic perennial herb plant,belonging to genus Nymphaea in the family Nymphaeaceae,which was divided into two kinds of ecological types,tropical waterlily and hardy waterlily.The tropical waterlily has higher ornamental and economic value than the hardy waterlily.However,the application value of the tropical waterlily was affected seriously because it could not overwinter in north subtropical and northern areas.So the primary breeding objective of the tropical waterlily is to improve cold tolerance,prolong the flowering period and improve the survival rate in winter.It is difficult to enhance cold tolerance of tropical waterlily by traditional cross-breeding.Fortunately,the development of molecular biology and genetic engineering techniques provides new ideas and methods for it.Firstly,efforts have been made to investigate various factors and the problems encountered in the nursery,especially in the aspects of substrate treatment,pest control and weed removal.A perfect seed breeding system was established,which greatly improved the seedling rate emergency of tropical waterlilyThe transgenic system of pollen-tube-pathway was established in tropical waterlily.The Agrobacterium was suspended to OD600=0.7 with the stigmatic secretions and used to infect the flower on the first day of flowering at 9:30 to 11:00 on the first day of flowering.Seeds were selected for germination in water with 10mg· L-1 Hygromycin B.Through the pollen-tube-pathway,CodA gene encoding choline oxidase was transformed into the genome of tropical waterlily.Seedlings selected by Hygromycin were planted in the field.Twelve transgenic lines were able to overwinter from 2015 to 2016(minimum temperature-8.9?)in Hangzhou.It was confirmed that the CodA gene had been integrated into the tropical waterlily genome by PCR detection.The results of RT-PCR showed that CodA gene could be expressed under low temperature stress.According to physiological investigation,we found that both the conductivity and MDA content of transgenic plants were lower than those of non-transgenic plants after low temperature treatment,whereas the content of betaine and the activity of SOD,CAT and POD were higher than those of non-transgenic plants.The results suggested that the expression of exogenous CodA gene could increase the betaine content and improve the cold tolerance of transgenic plants.Four transgenic plants and 40 excellent individual plants from Zhejiang Humanities Landscape Co.,LTD.were selected as parents and 164 hybrid combinations were designed,79 of which obtained seeds.144 seedlings of 10 combinations were planted in the open field in spring of 2016 and then five plants could overwinter in Hangzhou.In this study,the transgenic system of pollen tube pathway of tropical waterlily was established.The transgenic plants and their hybrids were obtained,some of which could overwinter in the open field.It has laid a solid foundation for solving the overwintering problem of tropical waterlily in the temperate area,which will also promote the development of genetic engineering in tropical waterlily.