In Vitro Multiplication and Cryopreservation of Penthorum chinense Shoot Tips

Abstract

This study provides alternative approaches toward ex situ conservation by means of in vitro seed germination and the multiplication of Penthorum chinense Pursh using nodal explants. An overlay of a liquid medium on top of a gelled medium significantly increased the growth of shoots and roots, while the presence of activated charcoal or growth regulators (benzyl adenine and α-naphthaleneacetic acid) decreased the growth. Shoot tips of in vitro plantlets were cryopreserved using a droplet-vitrification method. The standard procedure included preculture with 10% sucrose for 31 h and with 17.5% sucrose for 17 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 20 min, cryoprotection with alternative plant vitrification solution (PVS) A3-70% (29.2% glycerol + 11.7% DMSO + 11.7% EG + 17.4% sucrose, w/v) at 0 °C for 30 min, cooling the samples in liquid nitrogen using aluminum foil strips and rewarming by plunging into pre-heated (40 °C) unloading solution (35% sucrose) for 40 min. A three-step regrowth procedure starting with ammonium-free medium followed by ammonium-containing medium with and without growth regulators was essential for the regeneration of cryopreserved shoot tips. The species was found to be very sensitive to the chemical cytotoxicity of permeating cryoprotectants during cryoprotection and to ammonium-induced oxidant stress during initial regrowth steps. Improvement of donor plant vigor by using apical sections and liquid overlay on top of the solid medium for propagation, improved shoot tip tolerance to osmotic stress and increased post-cryopreservation regeneration up to 64% were observed following PVS B5-85% (42.5% glycerol + 42.5% sucrose) treatment for 60 min. The systematic approach used in this study enables fast optimization of the in vitro growth and cryopreservation procedure for a new stress-sensitive wild plant species.

Optimizing the Droplet-Vitrification Procedure by Balancing the Cryoprotection and Cytotoxicity of Alternative Plant Vitrification Solutions Based on the Nature of Donor Plant Vigor

“…Therefore, a lower-concentration PVS A3-70% (52.3%) produced the most increased LN regeneration compared to PVS2 (41.1%), A3-80% (36.8%), and B5-80% (40.9%) in the first round of studies. But after the rejuvenation of donor plants by inoculation of apical sections and overlay of the liquid medium on top of the Gellan gum-gelled medium (liquid-overlay), another variant of PVS B5-85% resulted in the highest LN regeneration (64.2%), which was higher than the A3-80% (53.6%) and PVS2 (45.5%) [48]. Recently, in chrysanthemum shoot tips, A3-80% (85.4%) showed the highest LN regeneration, followed by, in descending order, B5-85% (78.8%) > PVS2 (66.2%) > A3-90% (56.7%) > PVS3 (43.9%) with their optimized conditions [7].…”

“…[64], and Cymbidium kanran [52], and also for materials susceptible to biochemical toxicity, such as Chrysanthemum morifolium [44,59] and Solanum tuberosum [61]. The sensitive and soft materials, both biochemically and osmotically with smaller sizes, respond well with a diluted PVS3, i.e., B5-80%; for example, hairy roots of R. akane [45] and shoot tips of Aster altaicus [70] and Penthorum chinense [48].…”

“…Since A3-90% contains a higher concentration of glycerol and sucrose than PVS2, facilitating the efflux of water (dehydration) and the influx of CPAs (loading), it has a higher glass-forming tendency but drives the plant material into severe cytotoxicity, even when cryoprotected at 0 • C. Later, hence, less-concentrated PVSs A3-80%, A3-70%, and B5-80% (less toxic with mild chemical toxicity and osmotic stress) were applied successfully to shoot tips of the sensitive species L. rosmarinifolia [62], C. morifolium [69], A. altaicus [70], Fragaria × ananassa [72], Postemon yatabeanus [74], P. chinens [48], and Dendrathema grandiflourum [7].…”

“…With insufficient cryoprotection using diluted PVSs due to their cytotoxicity, using AFS over cryovials is more critical in hairy roots of R. akane (86.3% vs. 58.8%) [65], shoot tips of A. altaicus (56.1% vs. 4.5%) [70] and P. yatabeanus (88% vs. 11%) [74], and shoots of P. chinense (35.0% vs. 12.5%) [48] and D. grandiflourum (86.7% vs. 55.6%) [7].…”

Shoot Tip Cryopreservation and Elimination of SMV and CMV Virus from Infected Stocks of Pinellia ternata (Thunb.) Breit.

“…Vitrification relies on exposure of explants with a highly concentrated cryoprotectant solutions to dehydrate materials, and thus all extraneous water that would otherwise freeze is removed from the cells, and both intra-and extra-cellular vitrify while avoiding the growth of intracellular ice crystals when directly immersion in LN (Benson and Reed 2008;Lambardi et al 2000;Pawłowska and Szewczyk-Taranek 2014;Pennycooke and Towill 2000). High regeneration rates (52.9 to 90.0%) were yielded from the shoot tips of Colocasia esculenta (L.) Schott (Takagi et al 1997), Mint (Hirai and Sakai 1999), Penthorum chinense (Zilani et al 2022), Musa and Ensete , and Oil palm (Gantait et al 2015). In the past 20 years, vitrification-based cryopreservation has been developed and using for long-term storage of various plant species (Zamecnik et al 2021).…”