Barrera Marion, Olmedo Blanca, Narváez Matías, Moenne-Locoz Felipe, Rubio Anett, Pérez Catalina, Cordero-Lara Karla, Prieto Humberto
Biotechnology Laboratory, La Platina Research Station, National Institute of Agriculture (INIA), Santiago 8831314, Chile.
Natural Sciences, Mathematics, and Environment Faculty, Metropolitan Technological University, Santiago 8330526, Chile.
Plants (Basel). 2025 Jul 5;14(13):2059. doi: 10.3390/plants14132059.
Rice ( L.), a staple food for over half of the global population, plays a pivotal role in food security. Among its two primary groups, and , temperate varieties are particularly valued for their high-quality grain and culinary uses. Although some of these varieties are adapted to cooler climates, they often suffer from reduced productivity or increased disease susceptibility when cultivated in warmer productive environments. These limitations underscore the need for breeding programs to incorporate biotechnological tools that can enhance the adaptability and resilience of the plants. However, New Genomic Techniques (NGTs), including CRISPR-Cas9, require robust in vitro systems, which are still underdeveloped for temperate genotypes. In this study, we developed a reproducible and adaptable protocol for protoplast isolation and regeneration from the temperate cultivars 'Ónix' and 'Platino' using somatic embryos as the starting tissue. Protoplasts were isolated via enzymatic digestion (1.5% Cellulase Onozuka R-10 and 0.75% Macerozyme R-10) in 0.6 M AA medium over 18-20 h at 28 °C. Regeneration was achieved through encapsulation in alginate beads and coculture with feeder extracts in 2N6 medium, leading to embryogenic callus formation within 35 days. Seedlings were regenerated in N6R and N6F media and acclimatized under greenhouse conditions within three months. The isolated protoplast quality displayed viability rates of 70-99% within 48 h and supported transient PEG-mediated transfection with GFP. Additionally, the transient expression of a gene editing CRISPR-Cas9 construct targeting the () gene confirmed genome editing capability. This protocol offers a scalable and genotype-adaptable system for protoplast-based regeneration and gene editing in temperate rice, supporting the application of NGTs in the breeding of cold-adapted cultivars.
水稻（Oryza sativa L.）是全球半数以上人口的主食，在粮食安全中起着关键作用。在其两个主要类别粳稻和籼稻中，温带粳稻品种因其优质的谷粒和烹饪用途而备受重视。尽管其中一些品种适应较凉爽的气候，但在温暖的高产环境中种植时，它们往往会出现生产力下降或疾病易感性增加的情况。这些限制凸显了育种计划需要纳入生物技术工具，以提高植物的适应性和恢复力。然而，包括CRISPR - Cas9在内的新基因组技术（NGTs）需要强大的体外系统，而对于温带粳稻基因型来说，这些系统仍未充分发展。在本研究中，我们以体细胞胚作为起始组织，开发了一种可重复且适应性强的方案，用于从温带粳稻品种“Ónix”和“Platino”中分离原生质体并使其再生。通过在0.6 M AA培养基中于28°C下用酶（1.5%纤维素酶Onozuka R - 10和0.75%离析酶R - 10）消化18 - 20小时来分离原生质体。通过包裹在藻酸钠珠中并与饲养层提取物在2N6培养基中共培养来实现再生，在35天内形成胚性愈伤组织。幼苗在N6R和N6F培养基中再生，并在三个月内在温室条件下驯化。分离出的原生质体质量在48小时内显示出70 - 99%的活力率，并支持用绿色荧光蛋白（GFP）进行瞬时聚乙二醇（PEG）介导的转染。此外，针对Waxy（Wx）基因的基因编辑CRISPR - Cas9构建体的瞬时表达证实了基因组编辑能力。该方案为温带粳稻基于原生质体的再生和基因编辑提供了一个可扩展且适应基因型的系统，支持新基因组技术在耐寒品种育种中的应用。
