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<p>Conventional tissue-culture-based transformation systems are highly hampered by low regeneration, severe contamination, and operational complexity. To overcome these limitations, the present study preliminarily constructed a rapid, convenient, and relatively efficient non-tissue-culture genetic transformation system for pitaya (<i>Hylocereus polyrhizus</i>) using the visual reporter gene RUBY and the root-inducing <i>Agrobacterium rhizogenes</i> strain K599. Through orthogonal experimental design, key parameters for the protocol were optimized as follows: 1–2&#xa0;cm length stem segments from 70-day-old seedlings were used as explants; <i>Agrobacterium</i> was cultured in an induction solution containing 0.5&#xa0;M MES, 1.5&#xa0;M MgCl₂, 50&#xa0;g/L sucrose, and 100 µM acetosyringone (OD₆₀₀ = 1.0); infection duration was set to 30&#xa0;min; and cultivation was carried out in the dark for 2 days, followed by a 35-day incubation at 25&#xa0;°C, 70% humidity, and a light intensity of 1400&#xa0;lx. Using this system, the RUBY-positive red roots were successfully induced, with a 50% RUBY-positive rate. Compared with the tissue-culture-based methodology, the current system gave a 4-fold improvement in transformation efficiency and 70.83% reduction in operational time, which provides a potential technical platform for gene functional analysis and related research on root biology in pitaya.</p>

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An efficient non-tissue-culture root transformation system for pitaya as mediated by Agrobacterium rhizogenes

  • 期刊论文

  • 发表日期：

    2026年7月6日

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  • Qingwei Zeng,
  • Xiaowei Cai,
  • Luonan Shen,
  • Xiaocui Liu,
  • Chao Ma,
  • Aihua Wang,
  • Xiaopeng Wen

摘要

Conventional tissue-culture-based transformation systems are highly hampered by low regeneration, severe contamination, and operational complexity. To overcome these limitations, the present study preliminarily constructed a rapid, convenient, and relatively efficient non-tissue-culture genetic transformation system for pitaya (Hylocereus polyrhizus) using the visual reporter gene RUBY and the root-inducing Agrobacterium rhizogenes strain K599. Through orthogonal experimental design, key parameters for the protocol were optimized as follows: 1–2 cm length stem segments from 70-day-old seedlings were used as explants; Agrobacterium was cultured in an induction solution containing 0.5 M MES, 1.5 M MgCl₂, 50 g/L sucrose, and 100 µM acetosyringone (OD₆₀₀ = 1.0); infection duration was set to 30 min; and cultivation was carried out in the dark for 2 days, followed by a 35-day incubation at 25 °C, 70% humidity, and a light intensity of 1400 lx. Using this system, the RUBY-positive red roots were successfully induced, with a 50% RUBY-positive rate. Compared with the tissue-culture-based methodology, the current system gave a 4-fold improvement in transformation efficiency and 70.83% reduction in operational time, which provides a potential technical platform for gene functional analysis and related research on root biology in pitaya.