An Efficient Plant Regeneration System From Young Leaf Materials And Heterologous Synthesis Of EPA In Maize

The effective components of fish oils are very long-chain polyunsaturated fatty acids (LCPUFAs), such as EPA (20:4, eicosapentaenoic acid) and DHA (20:5, docosahexaenoic acid) that are long recognized as beneficial for brian development in infants and for health and disease prevention in adults. The biosynthesis efficiency of EPA/DHA in human body is very lowly, therefore, in order to maintain our body healthy and to prevent certain diseases it is necessary to supplement EPA/DHA in the daily diet. At present, these fatty acids are mainly from deep-sea fish oils. Due to overfishing, the natural fishstocks have declined significantly in recent years. This fish oil source has no longer been able to meet the rapidly increasing market demand. In addition, problems, such as environmental pollution, result in the accumulation of toxic compounds, including heavy metals and organochlorine, in fish oils. Therefore, it is imperative to seek other sustainable, safe and economical sources of EPA/DHA. Alternative production of fish oils from oilseed crops through genetic engineering maybe an ideal solution. Qi et al. (2004) first proved the possibility and feasibility of this alternative solution by producing EPA in the model plant Arabidopsis. Subsequently, the production of EPA and DHA in oilseed crops, such as flax, soybean and mustard has been reported (Kinney et al. 2004; Abbadi et al. 2004; Wu et al. 2005). Althogh the yields in these transgenics were often low it indicated that transgenic plants could indeed provide the alternative, stable and cheap sources for EPA/DHA in the future.Maize (Zea mays L.) is a ternary crop that integrates feed, grain and industrial raw material into one。Corn contains unsaturated fatty acids such as linoleic acid (LA) and linolenic acid (ALA). Like other higher plants, it cannot make long-chain polyunsaturated fatty acids (carbon number≥20). Elongase and desaturase genes related to EPA/DHA biosynthesis can be cloned from marine single-celled microalgae and fungi and these can be transferred to maize through transgenic technology in theory. If successful, this would add high industrial value to this crop. Our main findings are summarized as below.Six maize inbred lines that routinely used in breeding were tested in this study. The initiation, maintenance and differentiation of callus from seedling-derived leaf segments had been compared. The effect of 2,4-D on primary callus induction of leaf materials was studied. The callus induction and differentiation frequency were recorded, and callus morphology was also observed. The result showed that inclusion of 2,4-D into induction medium was a critical factor for inducing primary callus from leaf materials. 3mg/L 2,4-D was found to be the optimum concentration to induce leaf materials-derived to produce callus. The callus morphology was in accordance to genotype. Majority of the callus generated from Qi319, Luyuan92 and Mo17 were embryogenic callus, while that of the other three inbred lines were non-embryogenic callus. The differentiation rate is determined by the quality of the callus. Oue result showed that all six inbred lines could be regenerated, and the regeneration rate was ranged from 27.58% to 8.36%. Through various comparisons of the six genotypes in the regeneration process, we screened out three genotypes that showed high induction rate and good callus morphology, and these were Qi319, Luyuan92 and Mo17. Through this study, we developed an efficient regeneration system for maize using leaf materials as explants. We proved that young leaf materials could substitute immature embryos for callus induction in tissue culture. This laid a foundation for future maize genetic transformation using young leaf materials as receptor.Transgenic maize containing a multigene expression vector for the production of EPA was generated. This was achieved, by the biolistic-mediated transformation method, through the transformation of the embryogenic callus of maize inbred line Qi319. We obtained 25 transgenic plants and the average transformation efficiency was 7.91%. These trnsgenics were obtained by PPT resistant analysis on agar medium plates. Further selection was carried out by painting 200 mg/L PPT on leaves of these transgenics at their seedling stage, and 20 of plants were survived. We also extracted genomic DNA from these 20 plants to amplify the target genes by PCR. Four of the transgenic were confirmed to containall the three transgenes, theΔ9-Elo,Δ8-Des andΔ5-Des. Total fatty acids of these four plants were extracted for analysis by GC and GC-MS. The result showed that two peaks, being identified by GC-MS to be AA and EPA, were present in these plants. The contents of AA and EPA were 0.59% and 1.99%, respectively. These results showed that the heterologous transgenes were transformed into maize and expressed, resulting in the production of EPA. Although the yields of these two fatty acids were low, our research provided a good foundation for further studies for the alternative sources of fish oils in maize.