Development Of Transgenic Wheat Germplasm With Low Seed Lipoxygenase Activity

Wheat is one of the most important food crops, its yield and quality are directly related tothe food security and people’s living standards.Transgenic technology has gradually becomeone of the effective ways to improve the quality of wheat. Lipoxygenase (LOX) can catalyzethe oxidation of fatty acids. The storage period of seed with high LOX activity will beshortened. Thus, development of wheat varieties (lines) with low seed LOX activity isimportant for wheat breeding. The Screening and detection of exogenous gene is a key aspectin the process of transgenic wheat, and the marker removal is popular concerned. The copynumber of exogenous gene in transgenic plants is an important factor influencing theexpression and genetic stability of transgene. Real-time PCR has been used as a new, simple,high-throughput and sensitive approach to analyze the copy number of exogenous gene intransgenic plants.Researchers in the Institute of Genetics and Developmental Biology,Chinese Academy,co-transformed wheat cultivar Longchun23with RNAi sequence of LOX gene (Loxi）andselection marker gene bar by biolistic particle, obtaining positive transgenic T0plants. Thepositive transgenic T0plants were used as the male parent, crossed with agronomic parentXinong889, QinMai8918, QinMai3and YanMai8911and Shanyou225, respectively, toobtain TF1plants of the five cross combinations. All the TF1plants were selfinged to get theTF2populations of the five cross combinations. In this study, these transgenic TF2progenywere used as material，to select continuously in each of the TF2～TF6generations for getinggeneticly stable transgenic lines without selectable marker gene as well as with low seed LOXactivity. Another purpose is to explore the feasibility of the method combining the transgenictechnology and conventional cross to improve wheat traits in wheat breeding program. Theresearch work and the main results obtained were as follows:（1）All seeds of individual TF2plants from five cross combinations were screened for thosewith LOX activity decreased by50%to selfed continuly in progeny. TF3~TF6transgenicwheat lines were identified by gene-specific PCR testing and its seed LOX activity. Onlythose without select gene marker transgenic lines and with low seed LOX activity as well aswith good agronomic traits are retained continue to self breeding in each generation. Based on this detection and selection,16transgenic wheat lines of TF5or TF6with significantly lowseed LOX activity mostly and without selection gene were obtained in the present study,including5transgenic lines of TF5in Xinong889background,2in Qinmai8918,3in Yanmai8911,6in Shaanyou225. The seed LOX activity of these transgenic wheat lines with differentgenetic background decreased to20.71%～32.76%,56.48%～77.65%,28.98%～54.08%, and26.96%～95.47%of their no transgenic parent, respectively.（2）Taqman real-time quantitative PCR was performed to estimate the copy number of Loxiin6transgenic TF5wheat lines with good agronomic traits including3TF5lines ofXinong889(Xinong889T-Loxi-1, Xinong889T-Loxi-2, Xinong889T-Loxi-3) and3TF5lines ofShaanyou225(Shaanyou225T-Loxi-1, Shaanyou225T-Loxi-2, Shaanyou225T-Loxi-3), usingan endogenous single copy gene Pinb (puroindoline b) as a reference gene. The resultshowed that the copy number of the Loxi in6transgenic wheat lines was very different, withthe minimum copy number1and the maximum8. The seed LOX activity of two parentcultivars Shanyou225and Xinong889was860±28.20U and1132±59.80U, respectively.Compared to that of their parent, the seed LOX activity of the transgenic wheat lines weredecreased by various degree (approximately from21%to95%of their parents). Thetransgene copy number for3transgenic wheat lines of Xinong889T-Loxi-1,Xinong889T-Loxi-2and Xinong889T-Loxi-3in Xinong889background was2,6and8,respectively, and the corresponding lipoxygenase activity was32.76%,26.39%and21.05%oftheir parent, respectively. While, the transgene copy number for3transgenic wheat lines ofShaanyou225T-Loxi-1, Shaanyou225T-Loxi-2and Shaanyou225-Loxi-3in Shanyou225background was1,3and6, respectively, and the corresponding lipoxygenase activity was95.47%,51.06%and37.73%of their parent. The copy number of exogenous Loxi in thetransgenic wheat lines was negatively correlated with the seed LOX activity.In general, the transgenic wheat lines with low seed LOX activity were developed, andtheir exogenous gene copy number were detected. These marker-free genetically modifiedwheat germplasm can be used as the parent material in wheat breeding program. This studyalso demonstrated the method combining transgenic technology and conventional cross iseffective in improving wheat quality traits.