信息摘要：
2026年，华中农业大学园艺林学学院国家柑橘育种中心团队在Horticulture Advances期刊发表研究论文《A rapid and broadly applicable in planta transformation and genome editing system involving wounded epicotyls in citrus》(DOI: 10.1007/s44281-026-00135-5)，研究中使用LUYOR手持荧光检测器对柑橘创伤上胚轴再生的转基因组织进行GFP/RFP荧光观察与筛选，用于快速、广泛适用的柑橘in planta转化与基因组编辑系统的建立及多基因型验证，为柑橘功能基因组学研究与遗传改良提供了高效平台。
华中农业大学将LUYOR手持荧光检测器用于柑橘创伤上胚轴in planta转化GFP/RFP荧光观察实验
2026年，华中农业大学园艺林学学院国家柑橘育种中心团队在Horticulture Advances期刊发表研究论文《A rapid and broadly applicable in planta transformation and genome editing system involving wounded epicotyls in citrus》，研究中使用LUYOR手持荧光检测器用于柑橘创伤上胚轴in planta转化GFP/RFP荧光观察实验，对转化后再生芽进行每周的GFP/RFP荧光成像与筛选，并依据荧光信号移除非荧光芽，从而富集荧光阳性芽并获得非嵌合转基因植株。LUYOR手持荧光检测器在基于GFP/RFP荧光标记的柑橘in planta转化体系中承担关键的荧光筛选环节，通过手持式激发光源对再生组织进行绿色与红色荧光双色观察，快速区分荧光阳性转基因组织与阴性组织，确保非嵌合转化效率统计和转基因植株获得的准确性与可重复性，为快速、广泛适用的柑橘in planta转化体系的建立提供了可靠的实验数据支撑。
华中农业大学园艺林学学院位于武汉，是国家柑橘育种与生物技术研究的重点单位，依托国家园艺种质创新与利用重点实验室等平台开展柑橘遗传改良研究。该研究团队围绕柑橘遗传转化与基因组编辑技术开展研究，结合创伤上胚轴再生体系与组织培养中常用的植物激素处理，建立了快速、广泛适用的in planta转化系统：通过移液枪头构建的局部琼脂糖腔室，依次向创伤上胚轴递送农杆菌、植物生长调节剂（6-BA、NAA）和抗生素（卡那霉素），实现转化、再生与筛选的一体化操作。激素组合为3.0 mg/L 6-BA与0.05 mg/L NAA，在Carrizo枳橙中获得13.92%的非嵌合转化效率；系统进一步在甜橙、柚、柠檬、香橙和金橘等5个柑橘基因型中验证，非嵌合转化效率为2.58%~18.68%，播种后约40天即可获得转基因植株。LUYOR手持荧光检测器用于的GFP/RFP荧光观察实验作为快速鉴定转基因再生组织的关键手段，为上述转化体系的建立和验证提供了重要实验证据。
本实验采用LUYOR手持荧光检测器作为柑橘创伤上胚轴in planta转化GFP/RFP荧光观察实验的核心筛选设备，用于对再生芽进行明场与GFP/RFP荧光双色观察与筛选。实验将携带目标载体（含GFP或RFP标记）的农杆菌EHA105菌液接种于创伤上胚轴切口，依次经共培养和抗生素筛选后转入光照条件培养，每周使用LUYOR手持荧光检测器对再生组织进行荧光成像，移除非荧光芽以促进荧光阳性芽生长，经2~3轮荧光引导修剪后回收非嵌合荧光阳性芽。LUYOR手持荧光检测器用于GFP/RFP荧光观察结果表明，激素组合3.0 mg/L 6-BA与0.05 mg/L NAA在Carrizo枳橙中产生11株非嵌合GFP阳性植株（79株处理幼苗），非嵌合转化效率达13.92%；该体系在甜橙、柚、柠檬、香橙和金橘中非嵌合转化效率为2.58%~18.68%；101株转基因植株中80株在CsZFP7位点发生编辑，并成功获得无转基因的CsLMI1编辑植株，为证实该in planta转化体系的高效性和广泛适用性提供了直接实验证据。
该研究证实，LUYOR手持荧光检测器可用于柑橘等多年生木本植物转基因再生组织GFP与RFP荧光信号的高通量观察与筛选，适用于基于荧光标记的植物遗传转化、基因组编辑、嵌合体鉴定与无转基因编辑植株筛选等应用场景。LUYOR手持荧光检测器在国内外高校和科研院所得到广泛好评，欢迎私信或留言联系上海路阳了解详情！
原文段落
柑橘创伤上胚轴in planta转化GFP/RFP荧光观察实验方法原文
The seedlings were transversely cut below the first true leaf using a blade, leaving an epicotyl of approximately 2–3 cm. A 20-µL pipette tip was filled with the Agrobacterium suspension using a dropper, supplemented with the indicated concentrations of 6-BA and NAA. Following inoculation, the pipette tip containing the bacterial suspension was replaced with a pipette tip containing agarose coculture medium. The pots were covered with opaque black plastic film to maintain high humidity and darkness during incubation at 26 °C for 3 d. Subsequently, the coculture tip was then replaced with a tip containing agarose selection medium, and the pots were covered again with opaque black plastic film and incubated at 26 °C for an additional 5 d. After selection, the pipette tips were removed, and the epicotyl wounds were gently rinsed with water using a spray bottle to remove any residual medium. Afterward, the pots were covered with transparent plastic film and transferred to light conditions at 26 °C under a 16 h light/8 h dark photoperiod. Green fluorescent protein (GFP)- or red fluorescent protein (RFP)-positive regenerated tissues were screened approximately 2 weeks after transfer to light. During regeneration, fluorescence screening was performed weekly using a handheld fluorescence detector (Luyor Instrument, Shanghai), and nonfluorescent shoots were removed to promote the growth of fluorescence-positive shoots. After 2 to 3 rounds of fluorescence-guided pruning, the number of fluorescence-positive shoots stabilized.
柑橘创伤上胚轴in planta转化GFP/RFP荧光观察结果原文
After the outer seed coats were removed, the citrus seeds were sown in soil and grown at 26 °C under a 16 h light/8 h dark photoperiod. After approximately 10–15 d, the seedlings reached a height of 5–7 cm and were used for in planta transformation. The combination of 3.0 mg/L 6-BA and 0.05 mg/L NAA produced 11 nonchimeric GFP-positive plants from 79 treated seedlings, corresponding to the highest transformation efficiency of 13.92%. In addition, 8 chimeric GFP-positive plants were obtained under this treatment. When the 6-BA concentration was increased to 6.0 mg/L, the regeneration efficiency decreased markedly, ranging from 42.37% to 56.67%, and the transformation efficiency ranged from 0 to 5.98%. Large-scale transformation experiments in Carrizo citrange consistently produced numerous GFP- or RFP-positive plants, further demonstrating the reliability of the optimized in planta transformation system. Nonchimeric GFP-positive plants were successfully recovered from all tested genotypes, with the transformation efficiency ranging from 2.58% in ‘Shatian’ pummelo to 18.68% in ‘Ziyang Xiangcheng’. Furthermore, 29 of the 101 plants flowered within 3 months after transformation, and a putative transgene-free CsLMI1-edited plant exhibiting an oil glandless phenotype was recovered.
研究结论原文
In this study, we established a rapid and broadly applicable citrus in planta transformation system by integrating hormone treatments commonly used in conventional tissue culture with an in planta transformation approach. The optimized combination of 3.0 mg/L 6-BA and 0.05 mg/L NAA achieved a nonchimeric transformation efficiency of 13.92% in Carrizo citrange. Transgenic plants were successfully generated from all 6 tested citrus genotypes within 40 d after seed sowing, demonstrating the broad applicability and efficiency of this system. Furthermore, the optimized platform supported CRISPR/Cas9-mediated genome editing, with editing events detected in nearly 80% of the analyzed transgenic plants, and enabled the recovery of a putative transgene-free CsLMI1-edited plant. This method provides a simple and efficient platform for functional citrus genomics and genome editing, with potential applications in citrus breeding and genetic improvement.
DOI:doi.org/10.1007/s44281-026-00135-5
