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Acta Prataculturae Sinica ›› 2023, Vol. 32 ›› Issue (1): 165-177.DOI: 10.11686/cyxb2022189

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

Kai JIANG(), Xue-li WU(), Yi-jun LIU, Yue MA, Yang SONG, Wen-jie LU, Zeng-yu WANG   

  1. College of Grassland Science,Qingdao Agricultural University,Qingdao 266109,China
  • Received:2022-04-27 Revised:2022-06-16 Online:2023-01-20 Published:2022-11-07
  • Contact: Xue-li WU

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