Trajković Milena, Antonić Dragana, Cingel Aleksandar, Ghalawenji Nabil, Subotić Angelina, Jevremović Slađana
Department of Plant Physiology, Institute for Biological Research "Siniša Stanković", University of Belgrade, Bulevar Despota Stefana 142, Belgrade, 11060 Serbia.
3 Biotech. 2019 Jan;9(1):17. doi: 10.1007/s13205-018-1540-4. Epub 2019 Jan 2.
The aim of this study was to develop a fast, reliable and true-to-type protocol for in vitro plant regeneration and long-term storage of horned pansy ( L). Seed germination over 60% was recorded after 12 weeks of growth at 10 °C or 4 °C. Calli formation and shoot induction were obtained in petiole and hypocotyl culture on half-strength MS mineral salts with full concentration of Na-FeEDTA and vitamins (½MS medium) with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.1 mg/L) and 6-benzylaminopurine (BAP, 2.0 mg/L) and leaf culture on ½MS medium with thidiazuron (TDZ,1.0 mg/L). The highest frequency of adventitious shoot induction (50%) with six shoots/explant was achieved in hypocotyl culture from top hypocotyl segments, close to epicotyl which was grown 8 weeks at 16 h light/8 h dark photoperiod. Subsequent shoot multiplication was achieved on ½MS medium with α-naphthaleneacetic acid (NAA, 0.1 or 0.5 mg/L) and BAP (1.0 mg/L). Rooting of shoots was obtained on ½MS medium with low concentration (0.1 mg/L) of auxins: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or NAA, or without growth regulators. In vitro-derived plantlets were acclimatized under greenhouse conditions. All plants developed normally, bloomed and set seeds. Shoot tips were cryopreserved succssefully using modified plant vitrification 3 (PVS3-based vitrification procedure). Cold acclimation for 2 weeks significantly improved shoot regrowth (64%) after thawing in comparison to non-acclimated shoots (39%). Clonal fidelity of regenerated plantlets at ploidy level was confirmed by chromosome counting. The presented protocol can be useful for mass propagation, genetic transformation studies and long-term storage of valuable spp.
本研究的目的是开发一种快速、可靠且类型保真的体外植物再生及角堇（ ）长期保存方案。在10℃或4℃生长12周后，种子发芽率记录超过60%。在含有全浓度Na-FeEDTA和维生素的半强度MS矿质盐（½MS培养基）上，用2,4-二氯苯氧乙酸（2,4-D，0.1mg/L）和6-苄基腺嘌呤（BAP，2.0mg/L）进行叶柄和下胚轴培养，以及在含有噻苯隆（TDZ，1.0mg/L）的½MS培养基上进行叶片培养，均可获得愈伤组织形成和芽诱导。在靠近上胚轴的顶端下胚轴切段的下胚轴培养中，在16小时光照/8小时黑暗光周期下生长8周，不定芽诱导频率最高（50%），每外植体有6个芽。随后在含有α-萘乙酸（NAA，0.1或0.5mg/L）和BAP（1.0mg/L）的½MS培养基上实现芽增殖。在含有低浓度（0.1mg/L）生长素（吲哚-3-乙酸（IAA）、吲哚-3-丁酸（IBA）或NAA）或无生长调节剂的½MS培养基上实现芽生根。体外获得的小植株在温室条件下进行驯化。所有植株均正常发育、开花并结籽。使用改良的玻璃化法3（基于PVS3的玻璃化程序）成功地对茎尖进行了超低温保存。与未驯化的芽（39%）相比，2周的冷驯化显著提高了解冻后芽的再生率（64%）。通过染色体计数确认了再生小植株在倍性水平上的克隆保真度。所提出的方案可用于珍贵 物种的大规模繁殖、遗传转化研究和长期保存。
