| Common wheat(Triticum aestivum L.) is one of the most important members of cereal family with the most widely cultivated area and the highest yield in the world. Improving the wheat yield and quality may have a crucial effect on the global grain problem, which is becoming more and more serious.To breed varieties with broader adaptability, resistant to diseases and pests, higher yield and better quality, it is an important research direction of wheat breeding to apply genetic engineering technology. Gene gun bombardment has been widely used in wheat genetic transformation, for the virtues of no host-limit, wide target receptors, controllable and easy to operate. In this study, we deliveried a transcription factors gene, AtMYB44of Arabidopsis thaliana, into wheat variety Yangmai158via gene gun bombardment method. The results are as follows:1. An efficient regeneration system for Yangmai158was established and optimized.Plant hormone2,4-D, KT and IAA at different concentrations were added into the induction medium and the differentiation medium. The calli were100%induced from wheat immature embryos on the induction medium with2mg/L2,4-D and showed vigorous growth.The calli produced more green points and then more buds on the differentiation medium contained1.0mg/L KT and0.5mg/L IAA. The calli differentiation rate reached the highest93.3%. The optimum culture condition for further study was determined as MS+2mg/L2,4-D for induction medium and MS+1.0mg/L KT+0.5mg/L IAA for differentiation medium.2. The main parameters of gene gun bombardment for wheat callus was optimized with green fluorescent protein (GFP) as a marker gene.The results showed that the temporary expression rate of GFP gene reached the highest37.1%at8cm bombardment distance and35.4%for600μg/1μg of tungsten powder/DNA. The average fluorescence cell numbers were13.5and12.8, respectively. The optimum bombardment conditions for Yangmai158calli was firmed:,8cm bombardment distance,600μg/1μg for tungsten powder/DNA, helium pressure of7.5MPa, rupture of the membrane for7MPa, a vacuum degree of0.095MPa,1μm diameter of the tungsten powder.3. The gene AtMYB44of Arabidopsis thaliana was successfully delivered into Yangmai158calli and transgenic plants were obtained with a transformation rate of0.67%.Among1044calli bombarded and transformed,314calli produced green bud on selective differentiation medium and then297plants were regenerated on rooting medium. The differentiation rate and regeneration rate was30.1%and28.4%, respectively. The seedlings were transplanted after plantlet hardening and oversummering processing,198regenerated plants ultimately survived and the survival rate of transplant was66%. Genomic DNA of198regenerated plants (To generation) were extracted and the target gene AtMYB44and the reporting gene Bar were detected by PCR. Positive PCR results were generated from7plants. One amplified band of AtMYB44was recovered and cloned for DNA sequencing, the result indicated the successful transformation.4. Seven offsprings of transgenic plant M-141were performed PCR detection, and the results showed that the expected DNA fragments were amplified from seven plants.In this study, the target gene AtMYB44was transferred into the calli of wheat variety Yangmai158, the regenerated plants were obtained after resistance screening and differentiation culture. The regenerated plants (To) and future generations of transgenic plants (T1) were identified by PCR, the results showed that the transgenic wheat plants were attained. It laid the foundation for further studies on both gene expression of AtMYB44in transgenic wheat and stress resistance of transgenic plants. Further studies on genetic stability and field resistance of transgenic plants should be carried out to select those with drought-resistant, cold-resistant and salinity-resistant traits. It will have an important significance on improving the utilization of saline lands and shoals and the total production of wheat. |
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