Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, 28 Jagiellonska st, Katowice, 40-032, Poland.
Department of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, al. Mickiewicza 21, Krakow, 31-120, Poland.
BMC Plant Biol. 2023 Aug 10;23(1):385. doi: 10.1186/s12870-023-04402-9.
Fagopyrum tataricum (Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of Fagopyrum. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat.
In this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 10 protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts.
This study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of Fagopyrum tataricum used as source material. These findings suggest that Tartary buckwheat's protoplast cultures have potential implications for the species' somatic hybridization and genetic improvement.
苦荞（鞑靼荞麦）是一种具有重要营养价值的宝贵作物，因为它含有高水平的生物活性化合物。通过体外培养获得具有高价值代谢物水平或所需特征的植物可能会有很好的机会。在已知的体外技术中，原生质体技术是遗传操作的一种令人兴奋的工具，可用于改善作物特性。在这种情况下，原生质体融合可用于在不同苦荞物种之间产生杂种细胞。为了将原生质体培养应用于本研究中的上述方法，我们在鞑靼荞麦中建立了原生质体-植物系统。
在这项工作中，纤维素酶和果胶酶的活性使非形态发生和形态发生愈伤组织（MC）中的原生质体得以分离，平均每克鲜重达到 2.3×10 个原生质体。然而，为了从下胚轴中释放原生质体，关键步骤是在酶混合物中应用 driselase。我们表明，与藻酸盐基质相比，将原生质体嵌入琼脂糖中可以诱导集落形成。在基于 Kao 和 Michayluk 并添加植物磺基肽（PSK）的培养基中培养的原生质体重建细胞壁，经历了多次有丝分裂分裂，形成了聚集体，随后导致愈伤组织的形成。在 10 天龄的原生质体培养物中，集落形成的表达细胞聚集体数量的接种效率，对于形态发生的愈伤组织为 14%，而对于用作原生质体来源的下胚轴为 30%。然而，仅在 MC 衍生的原生质体培养物的培养过程中才发生体细胞胚胎发生和器官发生的植株再生。
本研究表明，应用的原生质体分离方法有助于恢复有活力的原生质体。此外，将原生质体嵌入琼脂糖基质中，并在培养基中添加 PSK，可有效刺激细胞分裂和进一步发育鞑靼荞麦原生质体培养物，并进行植物再生。总之，这些结果首次证明了从作为起始材料的 Fagopyrum tataricum 的 MC 中建立原生质体-植物系统的可行性。这些发现表明，苦荞的原生质体培养物可能对该物种的体细胞杂交和遗传改良具有重要意义。
