Capaci Piergiorgio, Barozzi Fabrizio, Forciniti Stefania, Anglana Chiara, Iuele Helena, Accogli Rita Annunziata, Carra Angela, Lenucci Marcello Salvatore, Del Mercato Loretta L, Di Sansebastiano Gian Pietro
Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento, Campus Ecotekne, 73100 Lecce, Italy.
Institute of Nanotechnology-NANOTEC, Consiglio Nazionale delle Ricerche (CNR), Campus Ecotekne, 73100 Lecce, Italy.
Plants (Basel). 2024 Dec 18;13(24):3530. doi: 10.3390/plants13243530.
Erben & Raimondo is a rare and endangered taxon, endemic to a limited area on Mount Pizzuta in northwestern Sicily, Italy. Its population is significantly threatened by anthropogenic activities, including fires, overgrazing, and habitat alterations. Temporary immersion systems (TISs) have proven effective for large-scale propagation in various protected species, offering potential for ex situ conservation and population reinforcement of . This study aimed to establish a bioreactor-based micropropagation protocol for shoot multiplication and compare the efficacy of a TIS with that of conventional solid culture medium (SCM). Three different plant growth regulators (PGRs) were also compared: 6-benzylaminopurine (BA), zeatin, and meta-topolin-9-riboside (TR). The starting material originated from seeds collected from mother plants in their natural environment. The best growth outcomes (in terms of shoot multiplication, shoot length, and relative growth rate) were achieved using THE RITA® TIS, with BA (0.2 mg/L) and mTR (0.5 or 0.8 mg/L) outperforming SCM. Anomalous or hyperhydric shoots were observed with all zeatin treatments (especially with 0.8 mg/L) in both the TIS and SCM, suggesting that this cytokinin is unsuitable for biomass production. The rooting phase was significantly improved by transferring propagules onto rockwool cubes fertilized with Hoagland solution. This approach yielded more robust roots in terms of number and length compared to the conventional agar-based medium supplemented with indole-3-butyric acid (IBA). Flow cytometry analysis confirmed the genetic fidelity of the regenerants from the optimal PGR treatments, showing that all plantlets maintained the diploid ploidy level of their maternal plants. Over 90% of the in vitro derived plantlets were successfully acclimatized to greenhouse conditions. This paper represents the first report of biomass multiplication using a RITA bioreactor. The stability of the regenerants, confirmed by nuclei quantification via cytofluorimetry, provides guidance in establishing a true-to-type ex situ population, supporting conservation and future reinforcement efforts.
埃尔本氏草与雷蒙多草是一种珍稀濒危分类群，仅分布于意大利西西里岛西北部皮祖塔山的有限区域。其种群受到包括火灾、过度放牧和栖息地改变在内的人为活动的严重威胁。临时浸没系统（TISs）已被证明对多种受保护物种的大规模繁殖有效，为迁地保护和种群增强提供了潜力。本研究旨在建立基于生物反应器的微繁殖方案以进行芽增殖，并比较TIS与传统固体培养基（SCM）的效果。还比较了三种不同的植物生长调节剂（PGR）：6-苄基腺嘌呤（BA）、玉米素和间-拓扑替康-9-核糖苷（TR）。起始材料源自从自然环境中的母株采集的种子。使用THE RITA® TIS，添加BA（0.2 mg/L）和mTR（0.5或0.8 mg/L）时获得了最佳生长结果（在芽增殖、芽长度和相对生长率方面），优于SCM。在TIS和SCM中，所有玉米素处理（尤其是0.8 mg/L）均观察到异常或水培过度的芽，这表明这种细胞分裂素不适用于生物量生产。通过将繁殖体转移到用霍格兰溶液施肥的岩棉块上，生根阶段得到了显著改善。与添加吲哚-3-丁酸（IBA）的传统琼脂培养基相比，这种方法在根的数量和长度方面产生了更健壮的根。流式细胞术分析证实了来自最佳PGR处理的再生植株的遗传稳定性，表明所有小植株都保持了其母株的二倍体倍性水平。超过90%的离体再生小植株成功适应了温室条件。本文是关于使用RITA生物反应器进行生物量增殖的首次报道。通过细胞荧光法进行细胞核定量证实的再生植株的稳定性，为建立真实类型的迁地种群提供了指导，支持保护和未来的增强工作。
