Cryopreservation of in vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant: effect of cryogenic procedure and storage duration

In vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant, were successfully cryopreserved using the vitrification and the encapsulation-dehydration techniques with subsequent high frequency plant regeneration. Using vitrification, post-liquid nitrogen (LN) shoot regeneration up to 83% was recorded when excised shoot tips were pretreated overnight on MS medium containing 0.3 M sucrose followed by loading with MS containing 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degree C, dehydration with PVS2 for 90 min at 0 degree C and quenching in LN. After 1 h of storage in LN, the shoot tips were rewarmed in a water-bath at 40 degrees C, unloaded with 1.2 M sucrose solution for 20 min and cultured on recovery growth medium. While using encapsulation-dehydration, the highest regeneration frequency recorded was 76% when sucrose-pretreated shoot tips were encapsulated with 3% calcium alginate, precultured in 0.75 M sucrose for 3 days, dehydrated to 25% moisture content (FW basis) under the laminar air flow, stored in LN for 1h and rewarmed at 40 degree C. The cryopreserved shoot tips maintained their viability and an unaltered level of regeneration capability after up to one year of storage in LN.     

Cryopreservation of shoot tips of blackberry and raspberry by encapsulation-dehydration and vitrification

Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for the long-term conservation of a diverse group of Rubus germplasm. Cold acclimation for a 4-week period prior to cryopreservation was necessary for regrowth of shoot apices from blackberry and raspberry genotypes. For the encapsulation-dehydration protocol, encapsulated apices were pretreated in 0.75 M sucrose for 20 h, desiccated 6-h under laminar flow to c. 20 percent moisture content, then plunged in liquid nitrogen (LN) and rapidly warmed. The PVS2-vitrification protocol included pretreating shoot tips on 5 percent dimethyl sulfoxide (DMSO) medium for 48 h, exposure to loading solution (LS) and PVS2 for 20 min each at 25 degree C , followed by immersion in LN and rapid warming. Shoot tips of 25 genotypes in 9 Rubus species and 9 Rubus hybrids were successfully cryopreserved with recovery of 60 to 100 percent using the encapsulation-dehydration protocol. Four genotypes of 3 species were tested using the vitrification protocol with 71 percent average regrowth. The present results indicate that both of these improved cryopreservation protocols can be applied to a diverse range of Rubus genetic resources.     

Cryopreservation of in vitro-grown shoot tips of Crateva nurvala Buch. Ham, an important medicinal tree

Cryopreservation of in vitro axillary shoot tips of Crateva nurvala Buch. Ham, an important medicinal tree, was investigated. Axillary buds (c. 1mm in length) excised from 4-week-old in vitro cultures, were pre-cultured on liquid MS medium supplemented with 0.4 M sucrose for 16 h. These were incubated in 2 M glycerol+0.4 M sucrose for 20 min at 25 degree C before being dehydrated with PVS2 solution for 40 min The dehydrated shoot tips were directly immersed in LN. Following cryopreservation and after rapid warming at 40 degree C, shoot tips were quickly washed with MS+1.2 M sucrose solution for 20 min and then plated on top of filter paper placed on MS medium supplemented with 0.1 mg l-1 BAP, kept in darkness for one day followed by placement of shoots directly on the medium and incubation in darkness for a day more, before transfer of cultures to light. Average survival in terms of normal shoot formation after 4 weeks of plating was 56.6 percent. The rescued shoot tips were bulked up by subsequent nodal cultures and when put onto 0.02 mg l-1 NAA showed a rhizogenic response. Thus, in vitro-grown shoot tips of Crateva nurvala were successfully cryopreserved following the optimization of the PVS2-vitrification protocol.     

Cryopreservation of garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions

This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92 % regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 degree C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/L zeatin and 0.3 mg/L gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95 %.     

Cryopreservation of cold-acclimated mint (Mentha spp.) shoot tips using a simple vitrification protocol

Accessions of Mentha x piperita, M. x villosa, and M. spicata were evaluated for regrowth after cooling in liquid nitrogen using shoot tips from in-vitro grown plantlets and a simple vitrification protocol with aluminium foil as a carrier. The influences of plant preculture, loading solution and loading time and of the effects of the cryoprotectant PVS 2 on plant re-growth after re-warming were investigated. Nodal segments were cultivated at constant temperatures of 20 or 25 degree C or in alternating temperature regimes (25/15C or 25/-1C). The illumination was always 16 h per day. The re-growth levels after re-warming were significantly higher in plants pre-cultured at 25/-1C regime than in plants cultivated at 20C or 25C or at 25/15C regime for all nine tested accessions. The mean re-growth levels increased from 36 percent at 20C to 69percent at alternating temperatures, respectively. The maximum of plant re-growth after re-warming was 89 percent. A pre-culture at alternating temperatures of 25/15C did not increase the recovery of plants. Loading in sucrose solutions with different dehydration capacities did not alter the plant re-growth. Differences in the loading time between 20 min and 2 h were not important for re-growth either. No significant differences were found between freezing without and with PVS 2 droplets on the aluminium foil. Re-grown shoots rooted easily on the re-growth medium and plantlets were successfully transferred to soil.     

Cryopreservation of cultivated and wild potato varieties by droplet vitrification: effect of subculture of mother-plants and of preculture of shoot tips

In this paper, we studied the effect of subculture of mother-plants and of preculture of shoot tips of two potato varieties (Dejima, cultivated and STN13, wild) cryopreserved using the droplet-vitrification technique. The subculture conditions (light intensity, aeration and planting density) significantly affected survival of both non-cryopreserved and cryopreserved shoot-tips in both varieties. The subculture duration and the position of the shoot tips on the axis of the in vitro plantlets had a significant (P<0.0001) effect on survival of cryopreserved shoot tips. The optimal subculture duration was 7 and 5 weeks and the optimal size of shoot tips was 1.5-2.0 and 1.0-1.5 mm for var. Dejima and STN13, respectively. Survival of cryopreserved shoot tips was influenced by the sucrose concentration in the preculture medium and the preculture duration. The highest survival of cryopreserved shoot tips was observed after preculture with 0.3 M sucrose for 8 h followed by 0.7 M sucrose for 18 h. These results indicate that the parameters of the subculture of mother-plants and of preculture of shoot tips should be carefully optimized, especially in the case of wild species.     

Cryopreservation of spores of Dicksonia sellowiana: an endangered tree fern indigenous to South and Central America

Spores of Dicksonia sellowiana (Presl.) Hook., an endangered tree fern, were stored in liquid nitrogen. Surface sterilized spores were placed in 1 ml sterile polypropylene cryotubes and were plunged into liquid nitrogen cryo-cans for 15 minutes, 15 days, 1 month and 3 months. In all, of the treatments the percentage of germination was higher than the control (fresh spores). Germination in Dyer and MS media supplement with 10 (-7) M and 5 x 10(-7) M BA was also promoted as comparing to control. There was no difference between the germination of spores thawed rapidly in a water bath at 45 degree C during 5 minutes or slowly at room temperature. Cryopreservation seems to promote germination of some dormant spores of D. sellowiana. The pre-treatment in cryoprotective solution of dimethyl sulphoxide 15%(v/v) in 1 M glycerol inhibited the germination of cryopreserved spores     

Effect of preculture, pvs2 and vitamin C on survival of recalcitrant Nephelium ramboutan-ake shoot tips after cryopreservation by vitrification

This paper reports the cryopreservation of Nephelium ramboutan-ake shoot tips derived from in vitro shoot multiplication and in vitro seed germination using vitrification. Preculture with either 0.5 M sucrose for 2 days or a combination of 0.3 M sucrose and 0.5 M glycerol for 3 days enhanced dehydration tolerance and resulted in the highest survival of shoot tips; however, none of the shoot tips withstood liquid nitrogen (LN) exposure. The use of a lower temperature (0 degree C) during exposure to plant vitrification solution (PVS2) led to higher survival of shoot tips, compared to exposure at 25 degree C. The survival percentage of shoot tips exposed to PVS2 for up to 20 min at 0°C was 83.3 percent. It was only 53.3 percent when shoot tips were exposed to PVS2 at 25 degree C for 5 min. The importance of vitamin C for reducing oxidative stress in shoots tips was demonstrated. The addition of 0.28 mM vitamin C during critical steps of the vitrification process resulted in a high survival (96.7 percent) without LN exposure, compared to 73.3 percent for shoot tips not treated with vitamin C. Moreover, 3.3 percent shoot tips withstood LN exposure when vitamin C was added during the loading step. This result suggests that cryopreservation is possible for this tropical, recalcitrant seeded tree species.     

PIXE‐based detection of elemental accumulation during direct organogenesis in Blepharispermum subsessile DC.: An endangered medicinal plant of Odisha,India

Particle‐induced X‐ray emission was used to investigate mineral accumulation during different developmental stages of direct organogenesis from cotyledon explants of Blepharispermum subsessile beginning from shoot bud initiation and formation to in vitro regenerated roots. Mineral uptake and accumulation appeared selective and varied between different stages of shoot bud initiation and formation (Stage 1), proliferation of leafy shoots (Stage 2), and in vitro regenerated roots (Stage 3). The concentrations of 2 macro elements, K and Ca, were found in higher quantity during proliferation of shoot buds to leafy shoots stage suggesting their role in cell division, bud formation, and multiplication of the plant. Most of the micronutrients such as Mn, Fe, Ni, Cu, and Zn were found to be accumulated in higher quantities in in vitro regenerated roots, as they provide the plant with a larger surface area and hence a greater potential for mineral uptake. The results of particle‐induced X‐ray emission test suggest that the information on the accumulation of elements during developmental stages in vitro could be useful for formulating a media for the induction of high‐frequency regeneration of in this important endangered medicinal plant species for its ex situ conservation.