Studies On The Transformation Of Cold Resistance Genes Into Cassava (Manihot Esculenta Crantz)

Cassava(Manihot esculenta Crantz) is one of the most important food and economic crops in tropic and subtropic areas of the world.The traditional breeding method has greatly improved cassava characteristics.As the higher heterozygosity trait and the limitation of cassava germplasm resource, it becomes much difficult to further improvement. With the development of molecular biology, and the mature technologies of gene manipulation, it becomes a useful method to increase cassava resistance to disease, insects and environment stress, or enhance cassava "storage roots" production, starch quality, or decrease harmful matter content in cassava. Cassava has a good ability of stress resistances, but it is short to cold tolerance. In this research, some of cold related genes have been transformed to three cassava cultivars through Agrobacterium to improve its cold resistance.In this thesis, four cassava cultivars (SC5,SC6, SC7, SC8) were used for the following researches:establishment of cassava in vitro culture system; researches on cassava somatic embryo cycle induction culture and somatic embryo cotyledon organogenesis;the effects of sterilants, sample collecting time and conditions on the cassava sterilization; the effects of 6-BA on growth of cassava seedlings; the effects of 2,4-D and picloram on cassava somatic embryogenesis;the effects of CaCl2 on cassava somatic embryo activity; the effects of mature time of somatic embryo on cotyledon organogenesis; establishment of gene transformation systems for the three cassava cultivars; transformation of six vector into three cassava cultivars by Agrobacterium; research the effects of co-culture time on gene transformation; selection and detection the transgenic cassava plants;transplant the transgenic cassava into pots and detection their ability to low temperature in both in vitro plants and pot plants.The main results are as follows:All factors such as sample collecting time, area and sterilants affected cassava sterilization. The best method for cassava sterilization was:cassava materials were collected from the room cultured plants;foam water to wash 10 min; fresh water to wash 20 min; 75% ethanol for 40 s;0.1% HgCl2 or 10 min. The sterilization ratio was up to more than 90%.6-BA can obviously inhibit the growth of cassava seedling tips, which was more serious in higher concentration. The inhibition was on the growth of radial buds and roots. However, suitable content on medium could induce radial buds to form thickly growth buds.For seedling faster growth in cassava in vitro culture system,the MS media with no hormone was the best one.Both 2,4-D and picloram could induce cassava primary somatic embryos, which was the similar to the 4 cassava cultivars. When the primary somatic embryos were continually subcultured, only three of them (SC5,SC6 and SC8)could obtain 100% secondary somatic embryos.The somatic embryos of SC7 became decline after subculture.The effects of CaCl2 on cassava primary somatic embryo induction was not obviously clear, but after subculture for 30 days, high concentration of CaCl2(15 mmol/1) could improve somatic embryogenesis, and keep the activity till 100 days.The mature time of somatic embryos affected the rate of the cotyledon regeneration.10～15 d mature somatic embryo cotyledon regenerated well and rate of the organogenesis was up to 70%.The suitable gene transformation system for cassava cultivars (SC) was:little pieces of 10～15 d mature somatic embryo cotyledon were infected by Agrobacterium which OD600 was around 0.9～1.1,for 45 min, then were co-cultured on organogenesis media for 3 d and washed away the bacterium; the transferred plantlets were firstly cultured on the low stress selection organogenesis media for 1 week, then on the higher stress selection organogenesis media with 500 mg/1 carbenicillin and 20 mg/1 hygromycin for anyone 2-3 weeks. After selection, many resistant shoot clusters were induced. The resistant shoot clusters were placed on the shoot elongation media till the shoots grew to 1-2 cm in length, then cultured them on the media without any hormone to regenerate plants.For root selection,1-2 cm tip part of the plants were places on the selection media with 10 mg/1 hygromycin for root growth. Finally, the plants with roots were detected by molecular methods. This transformation system was suitable for SC5,SC6 and SC8,while the transgenic ratio was a little different. It was best for SC8,and the same for SC5 and SC6.After detection of the putative transgenic plants by PCR,198 positive transformants of pVKH-35S-AtGolS2-pA were selected, in which 81 transformants developed roots on stress medium; 203 positive transformants of pVKH-35S-AtGol52-ipt-pA were selected, in which 79 transformants developed roots on stress medium; 167 positive transformants of pVKH-35S-CSF3-pA were selected, in which 50 transformants developed roots on stress medium; 123 positive transformants of pCA-CP-CSF3-pA were selected, in which 78 transformants developed roots on stress medium; 309 positive transformants of pVKH-35S-AtGolS3-pA were selected, in which,93 transformants developed roots on stress medium; 104 positive transformants of pCA-CP-CBF3-35S-AtGolS3-pA were selected, in which 25 transformants developed roots on stress medium.The positive transformants,which developed roots on stress medium and identified by PCR, were detected by RT-PCR. The result:65 positive transformants from 81 of pVKH-35S-AtGolS2-pA were identified by RT-PCR; 48 positive transformants from 79 of pVKH-35S-AtGolS2-ipt-pA were identified by RT-PCR; 39 positive transformants from 50 of pVKH-35S-CBF3-pA were identified by RT-PCR; 57 positive transformants from 78 of pCA-CP-CBF3-pA were identified by RT-PCR; 64 positive transformants from 93 of pVKH-35S-AtGolS3-pA were identified by RT-PCR; 20 positive transformants from 25 of pCA-CP-CBF3-35S-AtGolS3-pA were identified by RT-PCR.Parts of the positive transformants were detected by PCR-southern, All of them were showed positive results.After the serials of detection, the cold related genes have been transformed into the 3 cassava cultivar genome (SC5,SC6, SC8).The most economic methods for cassava transplanting was:The ground substance:red soil+sand(+decay soil);Time:Feb.-Mar. in a year; Plants:1 month old in vitro plants were cultured in water 3 d under closed conditions, then for another 4 d under open conditions;finally, these plants were transferred into the pots with soil., By this method we have obtained 600 transgenic plants.In the spring, clip the transplants which transferred in the winter of last year to 10 cm of stem in length. They grew better.The low temperature treatment on transgenic cassava in vitro plants and pot plants indicated that:after 4℃treatment for 10 days and recovery for 20 days, all control plants were died, while some of AtGolS2,AtGolS2-ipt、CBF3 transgenic plants were still alive. For the pots plants,after 4℃treatment for 36 h, SOD activity was higher than controls in some of the AtGolS2, AtGolS2-ipt, CBF3 transgenic plants, which the highest was 3 folds of the control plants;meanwhile MDA content was lower than controls in these transgenic plants at natural temperature. Thus, it indicates that the transgenic cassava plants will be better to low temperature.