Lata H, Uchendu E, Chandra S, Majumdar C G, Khan I A, ElSohly Mahmoud A
National Center for Natural Products Research, School of Pharmacy, University of Mississippi, Mississippi, USA.
National Center for Natural Products Research, School of Pharmacy, University of Mississippi, Mississippi, USA; Department of Agronomy, University of Ibadan, Ibadan, Nigeria.
Cryo Letters. 2019 Sep-Oct;40(5):291-298.
Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of important germplasm. Recent use of the cryo-plate system has proven beneficial in further simplifying the cryopreservation protocols.
Developing an efficient protocol for the cryopreservation of axillary buds of Cannabis sativa elite cultivars (MX and V1-20) by the V-cryoplate droplet-vitrification technique.
Stem segments (5 cm in length) with mature axillary buds collected from indoor-grown plants were surface sterilized and then either precultured on MS basal medium with 0.1 M sucrose (1 step preculture) for 72 h or non-precultured. All mature axillary buds (1 mm) were aseptically excised from stem segments and precultured for an additional 48 h on MS basal medium with sucrose (0.3 M) and 5% DMSO prior to cryopreservation (2 step preculture). Biomass samples of fully mature mother plants and regrown cryopreserved plants were analyzed for Δ-THC and CBD content using gas chromatography-flame ionization detector (GC/FID).
The survival and regrowth rates of cryopreserved axillary buds of cultivar MX following this two-step preculture were 45% and 42% respectively, while those of cultivar V1-20 were 47% and 44% respectively. A direct preculture of axillary buds (2 step preculture) on high sucrose (0.3M sucrose) significantly decreased both the survival and regrowth levels of axillary buds of cultivar MX (5% and 3% respectively) as well as those of cultivar V1-20 (20% and 17% respectively). Δ-THC and CBD content of mother plants and regrown cryopreserved plants were found to be highly comparable to each other.
The resulting plants after cryopreservation appeared normal without any callus formation or morphogenetic variation. On maturity, mother plants and re-grown cryopreserved plants were comparable in terms of Δ-THC and CBD content. This report provides an efficient protocol for cryopreservation of axillary buds of Cannabis sativa cultivars which may be applicable to other important cultivars, plant parts and other related medicinal plants.
冷冻保存是唯一一种能够安全且经济高效地长期保存重要种质的方法。最近使用的冷冻板系统已证明有助于进一步简化冷冻保存方案。
通过V型冷冻板液滴玻璃化技术，开发一种高效的大麻优良品种（MX和V1 - 20）腋芽冷冻保存方案。
从室内种植的植株上采集带有成熟腋芽的茎段（长度约5厘米），进行表面消毒，然后在含有0.1 M蔗糖的MS基本培养基上预培养72小时（一步预培养）或不进行预培养。将所有成熟腋芽（约1毫米）从茎段上无菌切下，并在冷冻保存前（两步预培养），在含有蔗糖（0.3 M）和5%二甲基亚砜的MS基本培养基上再预培养48小时。使用气相色谱 - 火焰离子化检测器（GC/FID）分析完全成熟的母株和冷冻保存后再生植株的生物量样品中的Δ - 四氢大麻酚（Δ - THC）和大麻二酚（CBD）含量。
经过两步预培养后，MX品种冷冻保存腋芽的存活率和再生率分别为45%和42%，而V1 - 20品种分别为47%和44%。在高蔗糖（0.3 M蔗糖）上直接对腋芽进行预培养（两步预培养）显著降低了MX品种腋芽的存活率和再生水平（分别为5%和3%）以及V1 - 20品种的存活率和再生水平（分别为20%和17%）。发现母株和冷冻保存后再生植株的Δ - THC和CBD含量彼此高度可比。
冷冻保存后得到的植株外观正常，没有任何愈伤组织形成或形态发生变异。成熟时，母株和冷冻保存后再生植株在Δ - THC和CBD含量方面具有可比性。本报告提供了一种大麻品种腋芽冷冻保存的高效方案，该方案可能适用于其他重要品种、植物部位及其他相关药用植物。
