Monthony Adrian S, Jones Andrew Maxwell P
Département de Phytologie, Université Laval, Québec, QC G1V 0A6, Canada.
Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec, QC G1V 0A6, Canada.
Plants (Basel). 2024 Jan 2;13(1):130. doi: 10.3390/plants13010130.
De novo regeneration of L. (cannabis) using tissue culture techniques remains unreliable and infrequent. Conventional methods for the regeneration and transformation of cannabis have not achieved the reliability and replicability that need to be integrated into research and breeding programs. Protoplast systems are effective for gene expression studies and transformation and genome-editing technologies and open the possibility of somatic hybridization to create interspecific hybrids. To date, leaf-derived protoplasts have been isolated for transient gene expression studies, but protoplast-to-plant regeneration has not been reported. The present study aims to evaluate the efficacy of using a callus culture system as an abundant tissue source for protoplast isolation and lays the groundwork for a protoplast-to-plant regeneration system. Using hypocotyl-derived callus cultures, which are known to have relatively greater regenerative potential, the efficacy of protoplast isolation and initial cell division were assessed. In this study, the effect of 2-aminoindane-2-phosphonic acid (AIP), a competitive inhibitor of phenylalanine ammonia lyase (PAL), in callus culture media and the effect of subculture frequency on protoplast yield were assessed. This study found that inclusion of AIP at 1 mM resulted in a 334% increase in protoplast yield compared with AIP-free medium, representing the first known use of AIP in cannabis tissue culture. Inclusion of AIP led to a 28% decrease in total soluble phenolics and 52% decrease in tissue browning compared with the control medium. Lastly, a two-phase culture system for protoplast regeneration was tested. At a concentration of 2.0 × 10 protoplasts per mL, cell wall reconstitution and cell division were observed, providing one of the first know reports of cell division from cannabis protoplasts and setting the stage for the future development of a protoplast-to-plant regeneration system.
利用组织培养技术从头再生大麻仍然不可靠且不常见。大麻再生和转化的传统方法尚未达到可纳入研究和育种计划所需的可靠性和可重复性。原生质体系统对于基因表达研究、转化和基因组编辑技术是有效的，并为体细胞杂交创造种间杂种开辟了可能性。迄今为止，已分离出叶源原生质体用于瞬时基因表达研究，但尚未报道原生质体到植株的再生。本研究旨在评估使用愈伤组织培养系统作为原生质体分离丰富组织来源的效果，并为原生质体到植株的再生系统奠定基础。使用已知具有相对更大再生潜力的下胚轴来源的愈伤组织培养物，评估原生质体分离和初始细胞分裂的效果。在本研究中，评估了愈伤组织培养基中苯丙氨酸解氨酶（PAL）的竞争性抑制剂2-氨基茚-2-膦酸（AIP）的作用以及继代培养频率对原生质体产量的影响。本研究发现，与不含AIP的培养基相比，在培养基中添加1 mM的AIP可使原生质体产量提高334%，这是AIP在大麻组织培养中的首次已知应用。与对照培养基相比，添加AIP导致总可溶性酚类物质减少28%，组织褐变减少52%。最后，测试了用于原生质体再生的两相培养系统。在每毫升2.0×10个原生质体的浓度下，观察到细胞壁重建和细胞分裂，这是大麻原生质体细胞分裂的首批已知报道之一，为原生质体到植株再生系统的未来发展奠定了基础。
