Transformation Of Antimicrobial Peptide Genes AlfAFP And SpCEMA Into Wheat

Wheat is the main grain in China, and its yield needs to be improved along with theeconomic development and the rapid population growth. Wheat disease is one of the mostserious factors affecting both the yield and the quality of wheat. As conventional breeding hasseveral deficiencies in meeting the requirements of economic and social development,biotechnology breeding becomes an important supplementary means of breeding for wheatdisease resistance, with more widely available gene resource and shorter time cycle comparedwith the general means.The exogenous gene is an important prerequisite for biotechnology breeding. The alfalfaantifungal peptide gene alfAFP used in this study is a plant defensin gene isolated from theseeds of Medicago sativa and prior studies have already demonstrated that it could displaystrong activity against the agronomically important fungal pathogen Verticillium dahliae intransgenic potato. The other gene used in this research is an antimicrobial peptide genespCEMA which is modified from CEMA, a cation antimicrobial peptide with highantimicrobial activity. These two genes are used in this wheat transformation study usingmainly biolistics, as well as the Agrobacterium-mediated transformation and the pollen-tubepathway. Serial problems were studied in order to construct a stable and efficient wheattransformation system and obtain transformed plants.1. Transformed wheat with fusion gene via biolisticsIn this study, the alfalfa antifungal peptide gene alfAFP and the antimicrobial peptidegene spCEMA were linked by the linker peptide2A sequence via Overlapping PCR and thefusion gene alfAFP:2A:spCEMA connected to the plant expression vectors pWMB002andpRTL2was used as the target gene in the following co-transformation process with pAHC20containing bar as a selective marker. Regenerated plants were obtained and PCR array wasdone to detect both bar and the target gene alfAFP:2A:spCEMA.In all,7transformed plants containing both bar and the target gene alfAFP:2A:spCEMAwere obtained and the frequency of co-integration was0.1%; a total61transformed plantswere regenerated and the overall transformation frequency was0.86%. Several transformationand regeneration related factors were studied during the screening and regenerated processand the co-transformation system of wheat cultivar XiNong1376was established.2. Agrobacterium-mediated transformation of wheat Different Agrobacterium strains were used to transform various portions of wheatincluding shoot tip, undifferentiated spike, floral organs as well as callus. Serial differentresults were obtained and transient expression were demonstrated via GUS histochemicalstaining. However no adult plants were regenerated due to the root developing problemcaused by the inappropriate screening methods.3. Pollen-tube pathway transformation of wheatWheat cultivars XiNong1376, XiaoYan22and XiaoYan6were transformed afterartificial pollination and cutting off the stigma and bar gene was used as the marker gene.Transformed offspring in T1generation of the regenerated plants with alfAFP as the targetgene were obtained after continuous spraying Basta and PCR array proved this screeningprocess is effective with a5%frequency of the detection of bar gene.In all, wheat transformation using biolistic co-transformation, Agrobacterium mediatedtransformation as well as the pollen tube pathway was done and transgenic wheat plantletswere obtained. Several influential factors of these conducting process were discussed in orderto improve each method.