Comparative study on transformation systems of seashore paspalum using hpt and bar genes as selection markers

doi:10.11686/cyxb2022189

Abstract

Abstract:

Seashore paspalum is a warm-season turfgrass with strong salt tolerance， and it has a wide range of potential applications. To establish an efficient and stable genetic transformation system for this plant， we used its mature seeds as explants， and determined the optimal selection pressures of hygromycin and glufosinate during callus subculture and the regeneration stage. After optimizing the genetic transformation conditions， we compared the transformation efficiency of hpt and bar genes as selection markers in Agrobacterium-mediated transformation of seashore paspalum. The results showed that the regeneration rate of callus decreased significantly after subculture for 36 weeks， and the highest regeneration rate was 67.9% after subculture for 48 weeks. The optimal concentration of hygromycin for screening was 120 mg·L^-1 at the subculture stage and 80 mg·L^-1 at the regeneration stage. The optimal concentration of glufosinate for screening was 1.2 mg·L^-1. Calli subcultured for 24 weeks were selected for Agrobacterium-mediated genetic transformation. The optimal transformation conditions were as follows： ultrasonic treatment of the explant for 20 min with bacterial solution （concentration OD₆₀₀=0.6） containing 100 μmol·L^-1 acetosyringone and 0.01% Silwet L-77， followed by vacuum treatment for 10 mins， then soaking for 30 mins and co-culturing for 2 days. Through screening multiple resistant calli and resistant regenerated seedlings， we obtained regenerated transgenic plants expressing each vector. Analyses by PCR confirmed that hpt and bar genes were successfully expressed in the respective transformed seashore paspalum materials. The average efficiency of GUS stable expression and PCR detection with hygromycin as the screening agent was 18.9%， and the highest transformation efficiency was 20.3%， which were higher than those obtained using glufosinate as the screening agent. Our results indicate that the hpt gene is more suitable as a selectable marker gene for the genetic transformation of seashore paspalum， as it achieves a higher transformation efficiency.

Key words: seashore paspalum, explants, in vitro regeneration, Agrobacterium-mediated, transgenic plants

Kai JIANG, Xue-li WU, Yi-jun LIU, Yue MA, Yang SONG, Wen-jie LU, Zeng-yu WANG. Comparative study on transformation systems of seashore paspalum using hpt and bar genes as selection markers[J]. Acta Prataculturae Sinica, 2023, 32(1): 165-177.

Figures/Tables 9

References 44

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培养基类型Medium type	培养基配方Formula of medium	其他Others
诱导和继代培养基Induction and subculture media	MS基本培养基+2.5 mg·L^-1 2，4-二氯苯氧乙酸+0.6 mg·L^-1 CuSO₄ MS basic medium+2.5 mg·L^-1 2，4-dichlorophenoxy acetic acid （2，4-D）+0.6 mg·L^-1 CuSO₄	添加30 g·L^-1蔗糖，调pH值为5.8，加入8 g·L^-1琼脂，121 ℃高温灭菌20 min。Added 30 g·L^-1 sucrose， adjusted the pH value to 5.8， add 8 g·L^-1 agar， and sterilized at 121 ℃ for 20 min.
选择继代培养基Selective subculture medium	MS基本培养基+2.5 mg·L^-1 2，4-D+0.6 mg·L^-1 CuSO₄+200 mg·L^-1头孢噻肟+适宜浓度筛选剂MS basic medium+2.5 mg·L^-1 2，4-D+0.6 mg·L^-1 CuSO₄+200 mg·L^-1cefotaxime+suitable concentration screening agent
再生培养基Regeneration medium	MS基本培养基+0.05 mg·L^-1激动素+8.0 mg·L^-1 6-苄氨基嘌呤+10 mg·L^-1 CuSO₄ MS basic medium+0.05 mg·L^-1 kinetin （KT）+8.0 mg·L^-1 6-benzylaminopurine （6-BA）+10 mg·L^-1 CuSO₄
选择再生培养基Select regeneration medium	MS基本培养基+0.05 mg·L^-1激动素+8.0 mg·L^-1 6-苄氨基嘌呤+10 mg·L^-1 CuSO₄+200 mg·L^-1头孢噻肟+适宜浓度筛选剂MS basic medium+0.05 mg·L^-1 KT+8.0 mg·L^-1 6-BA+10 mg·L^-1 CuSO₄+200 mg·L^-1cefotaxime+suitable concentration screening agent
壮苗培养基Strong seedling medium	1/2 MS基本培养基1/2 MS basic medium
选择壮苗培养基Select seedling growth medium	1/2 MS基本培养基+适宜浓度筛选剂1/2 MS basic medium+suitable concentration screening agent

培养基类型Medium type	培养基配方Formula of medium	其他Others
诱导和继代培养基Induction and subculture media	MS基本培养基+2.5 mg·L^-1 2，4-二氯苯氧乙酸+0.6 mg·L^-1 CuSO₄ MS basic medium+2.5 mg·L^-1 2，4-dichlorophenoxy acetic acid （2，4-D）+0.6 mg·L^-1 CuSO₄	添加30 g·L^-1蔗糖，调pH值为5.8，加入8 g·L^-1琼脂，121 ℃高温灭菌20 min。Added 30 g·L^-1 sucrose， adjusted the pH value to 5.8， add 8 g·L^-1 agar， and sterilized at 121 ℃ for 20 min.
选择继代培养基Selective subculture medium	MS基本培养基+2.5 mg·L^-1 2，4-D+0.6 mg·L^-1 CuSO₄+200 mg·L^-1头孢噻肟+适宜浓度筛选剂MS basic medium+2.5 mg·L^-1 2，4-D+0.6 mg·L^-1 CuSO₄+200 mg·L^-1cefotaxime+suitable concentration screening agent
再生培养基Regeneration medium	MS基本培养基+0.05 mg·L^-1激动素+8.0 mg·L^-1 6-苄氨基嘌呤+10 mg·L^-1 CuSO₄ MS basic medium+0.05 mg·L^-1 kinetin （KT）+8.0 mg·L^-1 6-benzylaminopurine （6-BA）+10 mg·L^-1 CuSO₄
选择再生培养基Select regeneration medium	MS基本培养基+0.05 mg·L^-1激动素+8.0 mg·L^-1 6-苄氨基嘌呤+10 mg·L^-1 CuSO₄+200 mg·L^-1头孢噻肟+适宜浓度筛选剂MS basic medium+0.05 mg·L^-1 KT+8.0 mg·L^-1 6-BA+10 mg·L^-1 CuSO₄+200 mg·L^-1cefotaxime+suitable concentration screening agent
壮苗培养基Strong seedling medium	1/2 MS基本培养基1/2 MS basic medium
选择壮苗培养基Select seedling growth medium	1/2 MS基本培养基+适宜浓度筛选剂1/2 MS basic medium+suitable concentration screening agent

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