Cryopreservation of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall. ex Lindl. protocorm-like bodies by encapsulation-vitrification

Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured in liquid Murashige and Skoog (MS) medium containing 0.2 mg l⁻¹ α-naphthalene acetic acid and 0.5 mg l⁻¹ 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 μmol m⁻² s⁻¹) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped in a 0.5 M CaCl₂ solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then, these were dehydrated with a plant vitrification solution 2 (PVS₂) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8, for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots, and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum.     

Simplified cryopreservation of sweet potato [<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.] by optimizing conditions for osmoprotection

Shoot tips of sweet potato were successfully cryopreserved using an encapsulation vitrification method. Encapsulated shoot tips were pre-incubated in liquid Murashige-Skoog medium containing 30 g/l sucrose for 24 h, then precultured in sucrose-enriched medium (0.3 M sucrose) for 16 h. Shoot tips were osmoprotected with a mixture of 2 M glycerol and 1.6 M sucrose for 3 h before being dehydrated with a highly concentrated vitrification solution (PVS2) for 1 h at 25 degrees C. The encapsulated and dehydrated shoot tips were transferred to a 2 ml cryotube, suspended in 0.5 ml PVS2, and plunged directly into liquid nitrogen. Rapidly warmed shoot tips developed normal shoots and roots in 21 days without any morphological abnormalities after plating on a recovery medium. High levels (average of about 80%) of shoot formation were obtained for three cultivars of sweet potato. This encapsulation vitrification method appears promising for cryopreservation of sweet potato germplasm.     

Cryopreservation of an endangered <Emphasis Type="Italic">Hladnikia pastinacifolia</Emphasis> Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification

The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.     

Droplet-vitrification cryopreservation of <Emphasis Type="Italic">in vitro</Emphasis>-grown shoot tips of grapevine (<Emphasis Type="Italic">Vitis</Emphasis> spp.)

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.     

Cryopreservation of shoot tips from the endangered endemic species Tuberaria major

Tuberaria major is an endangered endemic species from the Algarve, in the south of Portugal. We investigated two techniques for the cryopreservation of T. major shoot tips, namely vitrification and encapsulation-dehydration. Before the cryopreservation trials, shoot tips were precultured for 1 day on liquid Murashige and Skoog (MS) medium containing 0.3 M sucrose. For the vitrification method, shoots tips were exposed for 0, 30, 60, 90 and 120 min to plant vitrification solution 2 (PVS2). As for the encapsulation-dehydration method, shoot tips were dried inside a laminar air flow cabinet for 0, 1, 2, 3, 4, 5 and 6 h at room temperature. The highest regrowth percentages were approximately 60 and 67 % for vitrification and encapsulation-dehydration, respectively. The best times were 60 min exposure to PVS2 for vitrification and 3 h desiccation for encapsulation-dehydration. Though these are preliminary results, the use of the cryopreservation techniques tested here proved to be an important asset in the conservation of this endangered species and will complement the conservation strategies previously developed.     

The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification

This report highlights the first successful cryopreservation protocol for shoot tips of Garcinia mangostana L. achieved by using vitrification technique. We investigated the effects of different temperatures and exposure periods to a plant vitrification solution 2 (PVS2), sucrose concentrations and preculture periods, and unloading treatments in steps of the vitrification protocol on the survival of G. mangostana shoot tips after cryopreservation. Exposure to PVS2 for 25 min gave beneficial effects with 10.4 ± 1.8 % survival at 0 °C with average water content of 1.1 ± 0.3 g g^?1 dry mass. Survival was 13.7 ± 5.5 % when using preculture medium with full-strength Murashige and Skoog (MS) medium supplemented with 0.6 M sucrose for 2 days. A significant difference was observed in survival of shoot tips when treated with various sucrose concentrations in preculture which strengthens their importance towards enhancing survival of shoot tips after cryopreservation. MS with 0.4 M sucrose and 2 M glycerol applied as an unloading solution increased the survival of shoot tips to 44.1 ± 6.5 %. Experiments on the effect of ascorbic acid were also conducted for each step of vitrification. Our results showed higher survival of 45.8 ± 3.8 % but there were no significant effects compared with the control (without ascorbic acid). Further study on the recovery dark/light period was conducted. Survival of shoot tips significantly increased to 50.0 ± 16.7 % when subjected to 7 days in the dark before transferring to 16 h/8 h light/dark photoperiod. These studies strengthen suggestions that cryopreservation through vitrification is possible for ex situ conservation of germplasm of this tropical recalcitrant species.     

In vitro conservation of Dendrobium germplasm

Dendrobium is a large genus in the family Orchidaceae that exhibits vast diversity in floral characteristics, which is of considerable importance to orchid breeders, biotechnologists and collectors. Native species have high value as a result of their medicinal properties, while their hybrids are important as ornamental commodities, either as cut flowers or potted plants and are thus veritable industrial crops. Thus, preservation of Dendrobium germplasm is valuable for species conservation, breeding programs and the floriculture industry. Cryopreservation represents the only safe, efficient and cost-effective long-term storage option to facilitate the conservation of genetic resources of plant species. This review highlights 16 years of literature related to the preservation of Dendrobium germplasm and comprises the most comprehensive assessment of thorough studies performed to date, which shows reliable and reproducible results. Air-drying, encapsulation–dehydration, encapsulation–vitrification, vitrification and droplet-vitrification are the current cryopreservation methodologies that have been used to cryopreserve Dendrobium germplasm. Mature seeds, pollen, protoplasts, shoot primordia, protocorms and somatic embryos or protocorm-like bodies (PLBs) have been cryopreserved with different levels of success. Encapsulation–vitrification and encapsulation–dehydration are the most used protocol, while PLBs represent the main explant explored.     

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).     

Low-temperature preincubation enhances survival and regeneration of cryopreserved Saussurea involucrata callus

Saussurea involucrata Kar. et Kir. is one of the most well-known Chinese medicinal plants, and it is utilized for a variety of medical conditions. Due to the overexploitation of this endangered species, it is crucial to develop methods for both conservation and propagation. To address this issue, we have developed and optimized a simple and effective vitrification process for the cryopreservation of S. involucrata callus tissue. The optimized method consisted of a 3-d incubation period on medium containing 0.3 M sucrose, transfer to a plant vitrification solution (PVS2) containing 30% (v/v) glycerol, 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, and 0.4 M sucrose first at 60% PVS2 for 40 min, then at 100% PVS2 for 60 min, followed by immediate immersion and storage in liquid nitrogen. To thaw the tissue, tissues were rewarmed at 40°C for 2 min. This method resulted in a survival rate of approximately 56% and a regrowth rate of approximately 40%. Survival and regrowth were significantly improved by the addition of a low-temperature preincubation step. Incubating the calli at 4°C for 12 d prior to initiating the optimized cryopreservation protocol increased the survival rate of the tissue to 75%, increased the regrowth rate to 60%, and more than doubled the number of regenerated shoots per explant. Following cryopreservation, greater than 90% of the regenerated shoots formed complete plantlets, and 81% of the regenerated plantlets survived and grew vigorously under greenhouse conditions.     

Antioxidants and improved regrowth procedure facilitated cryoconservation of Paphiopedilum insigne Wall. Ex. Lindl. - An Endangered Slipper Orchid

Orchids and their sustainability are very important issues that need global conservation efforts. Paphiopedilum insigne (an endangered orchid), is one of the most excessively exploited species of orchids and is mentioned in the IUCN Red List and Appendix I of CITES. The prospect for conservation and commercialization of this species would be strengthened with the development of improved cryopreservation techniques. This study reports on successful cryopreservation of protocorms of P.insigne after cryopreservation using vitrification (Vit) and encapsulation-vitrification (E-Vit) techniques. The study compared the addition of four antioxidants to the pretreatment and recovery stages, three growth media, and agitated vs. semisolid culture medium for initial recovery. Recovery after cryopreservation for the control was 27% for Vit and 37% for E-Vit. In both cases agitated culture produced improved recovery by about 10%, but with significantly better recovery with E-Vit. The best recovery (51.2 ± 0.9%) was recorded for 0.5 M sucrose precultured encapsulated protocorms treated for 45 min with PVS2 and recovered in ½ MS (L/S) liquid medium for 10 days under agitation, followed by transfer to semi-solid medium. This recovery was further enhanced (62.7 ± 0.5%) with the incorporation of 30 μM glutathione in both liquid preculture and the liquid and semisolid regrowth medium. This new protocol improved the E-Vit cryopreservation recovery from the initial 37%–63%, providing a suitable technique for storage of this threatened orchid.     

Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5–1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l^?1 N ⁶-benzyladenine, 0.5 mg l^?1 indole-3-acetic acid, 0.2 mg l^?1 zeatin, 20 g l^?1 glucose and 6 g l^?1 Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %.     

Cryopreservation of protocorm-like bodies (PLBs) of <Emphasis Type="Italic">Phalaenopsis bellina</Emphasis> (Rchb.f.) christenson by encapsulation-dehydration

Protocorm-like bodies (PLBs) of Phalaenopsis bellina were successfully cryopreserved by the encapsulation-dehydration approach. Various stages in obtaining successful cryopreservation using this method were optimized. Encapsulated PLBs precultured in half-strength MS medium supplemented with 0.75 M sucrose for 3 days exhibited the highest viability in terms of 2,3,5-triphenyltetrazoliumchloride (TTC) reduction. The amount of sucrose in the PLBs after incubation in different concentrations of sucrose for different periods of time determined by HPLC. The highest sucrose concentration was 7 mg/g of PLBs for the PLBs treated with 0.75 M sucrose for 3 days as compared to the control which had only 1 mg/g sucrose. After sucrose preculture, the PLBs were subjected to desiccation using one of two methods. Desiccation using silica gel was more efficient in reducing PLBs moisture content. After 6 h of desiccation, PLBs desiccated using laminar air flow had 43.5% moisture content while for those desiccated using silica gel had 32% moisture content. PLBs desiccated to different moisture contents were plunged into LN. After storage in LN the encapsulated PLBs were re-warmed. Two weeks after re-warming PLBs viability was determined by TTC reduction and re-growth assessed. Encapsulated PLBs precultured with 0.75 M sucrose for 3 days followed by desiccated using silica gel for 5 h resulting in a moisture content of 39% lead to the highest post re-warming viability in terms of TTC reduction (46.6% of control PLBs) and 30% re-growth.     

Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification

The seeds of a Japanese terrestrial orchid (Bletilla striata Rchb.f.) were germinated and cultured on solidified new Dogashima (ND) medium for 10 days. These embryos were then precultured on ND medium supplemented with 0.3 m sucrose for 3 days at 25°C in continuous dark. The embryos were then overlaid with a mixture of 2 m glycerol and 0.4 m sucrose for 15 min at 25°C and finally dehydrated with highly concentrated vitrification solution (PVS2) for 3 h at 0°C prior to immersion into liquid nitrogen for 30 min. After rapid warming, the embryos were washed with liquid ND medium supplemented with 1.2 m sucrose for 20 min and then plated on ND medium. Successfully vitrified and warmed embryos developed into normal plantlets. The rate of plant regeneration amounted to about 60%. This vitrification method appears to be a promising technique for cryopreservation of orchids. Received: 19 September 1996 / Revision received: 3 January 1997 / Accepted: 24 February 1997     

High-efficiency cryopreservation of the medicinal orchid <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl.

An efficient protocol for cryopreservation of protocorm like bodies (PLBs) of Dendrobium nobile, based on encapsulation–dehydration (ED) and encapsulation–vitrification (EV), was established. In both cryogenic procedures, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix [comprising 3% (w/v) sodium alginate and 0.1 M CaCl₂]. Out of the two methods, EV resulted in higher survival (78.1%) and regrowth (75.9%) than ED (53.3 and 50.2% respectively). Incorporation of 0.4 M sucrose and 2 M glycerol in the encapsulation matrix resulted in higher survival percentage after cryopreservation. In both the cases (ED and EV), shoots regenerated from cryopreserved PLBs with an intermediary PLB formation. Regenerated shoots were successfully rooted in the medium containing 1.5 mg/l Indole-3 butyric acid. Successful acclimatization of plantlets was obtained in the compost containing brick pieces and charcoal chunks (1:1) + a top layer of moss with a maximum survivability (82%). EV method proved to be most appropriate way to cryopreserve the PLBs of D. nobile. Regenerated plantlets showed normal morphology as that of control plants.     

Cryoprotectants and components induce plasmolytic responses in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) suspension cells

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.     

Cryopreservation of somatic embryos from <Emphasis Type="Italic">Petiveria alliacea</Emphasis> L. by different techniques based on vitrification

Petiveria alliacea L. is a medicinal plant originating from the Amazon region. This study describes an efficient cryopreservation protocol for somatic embryos (SEs) produced from roots of P. alliacea based on the comparison of vitrification, encapsulation-dehydration, and D cryo-plate techniques. With the vitrification technique, SEs treated with PVS2 solution (0.4 M sucrose, 3.3 M glycerol, 2.4 M ethylene glycol, and 1.9 M DMSO) for 30 min displayed high viability (85%) and intermediate proliferation recovery (about 12 adventitious SEs produced from original SEs [SEs/SE] after 90 d of culture). With the encapsulation-dehydration technique, lower viability (70%) and very low proliferation recovery (about two SEs/SE) were achieved with cryopreserved SEs dehydrated for 10 min in a laminar air flow cabinet. The D cryo-plate technique led to high viability (85%) and proliferation recovery (19 SEs/SE) of cryopreserved SEs after 90 min dehydration. In the experimental conditions tested, the D cryo-plate method was the most efficient technique for cryopreservation of P. alliacea SEs.     

Cryopreservation of ex-vitro-grown Rosa chinensis ‘Old Blush’ buds using droplet-vitrification and encapsulation-dehydration

Axillary buds from greenhouse-grown plants of Rosa chinensis ‘Old Blush’ were successfully used to establish cryopreservation protocols using both droplet-vitrification and encapsulation-dehydration methods. In droplet vitrification, regrowth occurred after exposure to liquid nitrogen even without pre-culture in the loading solution (LS) before immersion in the plant vitrification solution 2 (PVS2). However, a 20–80 min LS step followed by a short immersion in PVS2 for 3 or 15 min, at 0 °C gave the best regrowth rates (82–86 %). In encapsulation dehydration, the level of dehydration significantly influenced shoot regrowth. The best regrowth rate, 60 %, was obtained at a bead water content of 0.35 g water per g dry weight. These results demonstrate the possibility of using greenhouse plants of rose for cryopreservation by droplet vitrification and encapsulation dehydration.     

Cryopreservation of hybrid Cymbidium protocorm-like bodies by encapsulation–dehydration and vitrification

Cryopreservation can be a stable, long-term method of germplasm conservation, but successful application can be challenging for tropical material. To optimize survival and re-growth from cryopreserved tissues derived from protocorm-like bodies (PLBs) of hybrid Cymbidium Twilight Moon ‘Day Light’, the effects of explant type (intact PLBs, half-PLBs, or PLB longitudinal thin cell layers) and various explant treatments were studied. Encapsulation in alginate beads was essential, and intact PLBs were best for cryopreservation, based on survival and ability to form neo-PLBs and/or percentage re-growth. Osmotic hydration of intact PLBs in 2% sucrose for 24 h increased neo-PLB formation and re-growth, with the best responses seen when PLBs were excised from alginate beads prior to re-growth after cryopreservation. Both non-transgenic and transgenic PLBs were amenable to cryopreservation for up to 1 year using these methods. This optimized protocol will improve the viability of hybrid Cymbidium germplasm after long-term cryopreservation.     

Selection of optimal stage for protocorm-like bodies and production of artificial seeds for direct regeneration on different media and short term storage of Dendrobium Shavin White

Micropropagation is currently the most popular method for orchid propagation through the production of protocorm-like bodies (PLBs). It is suggested that converting the PLBs into artificial seeds by encapsulation with sodium alginate can be useful for short-term preservation and distribution to the laboratories and commercial nurseries. Prior to the production of artificial seeds, the best developmental stage of PLBs based on sizes for increased conversion to plantlet was determined. PLBs were categorized based on size and presence of shoot namely ≤2 mm (S1), >2–4 mm (S2), >4–6 mm (S3), >2–4 mm with shoot (S4) and >4–6 mm with shoot (S5). S4 and S5 gave significantly higher conversion percentage (85 and 90 %, respectively) as compared to the PLBs without shoot (S1, S2 and S3). Thus, for uniformity PLBs of 3–5 mm with shoot were used for encapsulation with sodium alginate to form artificial seeds. The feasibility of germinating artificial seeds of Dendrobium Shavin White in different substrates namely; M1 (semi-solid ½ Murashige and Skoog (1962) basal medium), M2 (cotton irrigated with sterilized liquid ½ MS basal medium), M3 (cotton irrigated with sterilized distilled water) and M4 (cotton irrigated with non-sterilized distilled water) was tested. The encapsulated PLBs regenerated well in M1 where 96 % of encapsulated PLBs germinated after 12 days of inoculation and 76 % of them converted into plantlet after 37 days of inoculation while PLBs subjected to sterile distilled water gave 56 % germination and 44 % conversion after 42 and 167 days of inoculation respectively. The ability to store encapsulated PLBs would be advantageous for transport of planting materials. Encapsulated PLBs survived longer when stored at 25 ± 2 °C compared to 4 °C, 10 °C and 30 ± 2 °C whereby storage up to 75 days retained 80–92 % survival. Further storage up to 135 days retained 52 % survival. All plantlets survived after acclimatization when transferred to charcoal media under shade.     

Cryopreservation of in vitro Grown Shoot Tips of Red Bud Taro by Encapsulation-Vitrification

A simple procedure for cryopreservation of in vitro grown shoot tips of red bud taro (Colocasia esculenta L. Schott var. cormosus‘Hongyayu’) by encapsulation vitrification is investigated. Shoot tips were excised from 8 week old stock shoots and encapsulated into alginate gel beads. Encapsulated shoot tips were precultured in liquid MS medium supplemented with 35mg·L-1 6 BA, 05mg·L-1 IBA, 01mg·L-1 GA3 and 03mol·L-1 sucrose for 24h, then they were loaded with a mixture of 2mol·L-1 glycerol plus 04mol·L-1 sucrose for 30min at 25℃. After dehydration with PVS2 at 25℃ for 20min, the encapsulated and dehydrated shoot tips were plunged directly into liquid nitrogen. After rapidly rewarming in a 40℃ water bath for 3min, PVS2 was drained from the cryotubes and replaced third with liquid MS medium supplemented with 35mg·L-1 6 BA, 05mg·L-1 IBA, 01mg·L-1 GA3 and 12mol·L-1 sucrose and each kept for 10min at 25℃and then post cultured on solidified MS medium supplemented with 35mg·L-1 6 BA, 05mg·L-1 IBA and 01mg·L-1 GA3 in the dark for 3 days and then transferred to the light conditions. The average survival rate amounted to about 80%. Plantlets regenerated from cryopreserved shoot tips were morphologically uniform. This encapsulation vitrification procedure promises to become a routine method for the cryopreservation of shoot tips of Chinese genuine red bud taro.