Cryopreservation ofRosa hybridacv. Helmut Schmidt by PVS2 vitrification method usingin vitrofragmented explants (IFEs)

Abstract

1415 This is the first report on cryopreservation via PVS2 vitrification method on roses using in vitro 16 fragmented explants (IFEs) as the starting material. The aim of this study is to optimize the efficient 17 plant recovery and regeneration system for cryopreservation of Rosa hybrida cv. Helmut Schmidt 18 using IFEs. Some important parameters have been optimized in this study are the effect of ascorbic 19 acid (0.3 mM) examined separately and in combination at all steps in cryopreservation procedure 20 (preculture, loading, unloading and growth recovery), loading type, loading duration, and PVS2 21 duration. The highest growth recovery of 43.33% was obtained when 3-4 mm size IFEs precultured 22 on 0.25 M sucrose media supplemented with full-strength MS for one (1) day, followed by loading 23 treatment supplemented with 1.5 M glycerol + 0.4 M sucrose + 5% DMSO + 0.3 mM ascorbic acid 24 for 20 minutes, dehydration with PVS2 solution for 30 minutes and then treated with unloading 25 solution supplemented with 1.2 M sucrose + 0.3 mM ascorbic acid for 20 minutes. This finding 26 implies that long-term storage of Rosa Hybrida cv. Helmut Schmidt by PVS2 vitrification method 27 was successful with essential biomolecules. 28 29 30 Keywords: In vitro fragmented explants (IFEs), PVS2 vitrification, ascorbic acid, Biomolecules, 31 Rose 32 34 Roses are one of the most important ornamental plants in many parts of the world. This 35 is because they bloom beautiful flowers and as such, they are widely used in the horticultural 36 industry. Cultivation of rose plants via tissue culture micropropagation has disadvantages in 37 terms of contamination, somaclonal variations and also due to biotic stresses [1]. Hence, 38 cryogenic storage offers a stable and safe method for long term storage of plant materials 39 while safeguarding genetic stability [2]. Cryopreservation at temperatures of -196°C has been 40 considered as an ideal means for long term storage of plant germplasm. Through 41 cryopreservation, hybrids that are in danger of being extinct can be conserved with low 42 maintenance apart from being easy for international exchange. Cryopreservation through 43 vitrification reduces injury caused by intracellular crystallization and provides high 44 regeneration rates prior to immersion into liquid nitrogen. Basically, the most appropriate 45 plant part utilized is the meristem culture since it generates virus free lines [3]. For PVS2 46 cryopreservation study in roses, in vitro fragmented explants (IFE) represent a new source of 47 explant as starting material. IFEs were first introduced by authors [4] who conducted research 48 on Vanilla planifolia "Andrews". The study discovered that IFEs allows the production of a 49 larger number of shoots than micro cuttings after 4 months in culture. However, every 50 species of plants has different genetic makeup and respond differently to various treatments. 51 Therefore, improvement of the cryopreservation method is crucial in each step for growth 52 after recovery. Vitrification ...

Effect of Cryopreservation Method Supported With Biochemical Analyses in the Axillary Bud of Jewel Orchid, Ludisia Discolor

“…Scavenging these surplus radicals may achieve tolerance to such harmful effects (Halder et al 2018). Published reports have employed antioxidants such as ascorbic acid, glutathione, and melatonin to directly remove the ROS and protect cellular functions, thus improving the chances of survival (Uchendu et al 2013;Uchendu et al 2014;Diengdoh et al 2019;Mubbarakh et al 2019;Khor et al 2020;Soonthornkalump et al 2020). Present ndings showed that melatonin-supplemented during the recovery stage did not accelerate the growth of cryopreserved axillary buds of L. discolor (Fig.…”

“…Axillary buds were collected for enzyme extraction at various cryopreservation stages adapted from Mubbarakh et al (2019) as follows:…”

Cryo-derived plants through embryogenesis showed same levels of vinblastine and vincristine (anticancer) in Catharanthus roseus and had normal genome size

“…In this study, the suspended cells of C. roseus were treated with various concentrations of osmotica and cryoprotective agents, the moderate level was noted to be more efficient in promoting viability and regrowth than the higher levels, very consistent with earlier observation 30 , 31 . Similarly earlier research indicated that the plant vitrification solution 2 (PVS2)—a mixture of compounds containing 30% (w/v) glycerol + 15% (w/v) PEG + 15% (w/v) DMSO + 0.4 M sucrose (osmoticum) added medium improved preservation of cells and promoted culture regrowth 19 , 32 . The extra diffusion/removal of water, however, damages the prospect of survivability and in such a situation, the slow gradual cooling and inclusion of cryoprotective agents like DMSO have been noted to be successful in several investigated plants genera 33 .…”

Effect of Cryopreservation Method Supported with Biochemical Analyses in the Axillary Bud of Jewel Orchid, Ludisia discolor

“…Scavenging these surplus radicals may achieve tolerance to such harmful effects [ 65 ]. Published reports have employed antioxidants such as ascorbic acid, glutathione, and melatonin to directly remove the ROS and protect cellular functions, thus improving the chances of survival [ 35 , 36 , 62 , 66 , 67 , 68 ]. Studies describing the protective role of melatonin and enhancement in the recovery of cryopreserved shoot tips of American elm and yam were documented [ 66 , 69 ].…”

“…Axillary buds were collected for enzyme extraction at various cryopreservation stages adapted from Mubbarakh et al [ 67 ] as follows: Axillary buds excised from in vitro plants (control); Preculture on medium containing 0.2 M sucrose; Osmoprotection by loading solution; Dehydration with PVS2 for 10 min; Rapid cooling and rewarming; Recovery after 1 day on growth medium with 0.05 µM melatonin (Recovery 1); Recovery after 3 weeks on growth medium with 0.05 µM melatonin (Recovery 2). …”