Vitrification Cryo-Foil for Apple Cryopreservation and the Seesaw Effect Between Shoot Recovery and Virus Eradication

Improvements of existing cryopreservation protocols are necessary to facilitate long-term preservation of plant germplasm and the cryotherapy-effect of pathogen eradication. This study reported a vitrification (V) cryo-foil/plate methods for cryopreservation of shoot tips and cryotherapy effect in ‘Pink Lady’ apple. In V cryo-foil/plate protocols, shoot tips were first attached onto aluminum foils/plates using calcium alginate before other procedures. Shoot tips cryopreserved by V cryo-foil required 6.1 weeks to fully recover and 53% of shoot regrowth was obtained, comparable to the Dv cryopreservation. Similar regrowth levels were produced between applying V cryo-foil and Dv cryopreservation to another 4 Malus genotypes. Histological observations in shoot tips cryopreserved by Dv and V cryo-foil found only those with surviving apical dome and leaf primordia (LPs) could recover after cryopreservation. In apical meristem of shoot tips cryopreserved by Dv and V cryo-foil, higher surviving probability was detected from the V cryo-foil protocol, and the young LPs showed the highest level of surviving. Virus detection in cryo-derived plants showed apple stem grooving virus and apple chlorotic leaf spot virus were all preserved after cryopreservation, and higher eradication efficiency of apple stem pitting virus (70%) was produced by Dv than the 55% of V cryo-foil. These results supported applying V cryo-foil as an improvement to the widely applied Dv method in shoot tip cryopreservation, and also revealed a seesaw mode between shoot recovery and cryotherapy effect. Once the seesaw moves to increase the recovery after cryopreservation, the cryotherapy-effect on the other side would be decreased.

Effect of Poultry and Goat Manures on the Nutrient Content of Sesamum alatum Leafy Vegetables

Sesamum alatum Thonn. is one of the less-popular but nutritious leafy vegetables that is still collected from the wild or as weeds among crops in South Africa. The plant is also used in medicines and cosmetics in Africa and elsewhere. Despite its importance, the cultivation of S. alatum under different agronomic systems for improved harvestable yield and nutrient content is still lacking. The study aimed to determine the response of S. alatum nutrient content to the application of poultry and goat manures. Plants were grown in pots under rain-fed shade cloth conditions, with poultry and goat manures applied at 0, 1, 2, and 3 t ha–1 each, and they were laid in a completely randomized design. Shoot tips were harvested at 60 days after planting and analyzed for nutrient content. Shoots contained better nutrients in S. alatum plants grown during the first than the second season, with minor exceptions. Poultry and goat manure application led to an increase in Ca, Mg, K, P, and micro-nutrients. Goat manure had potential to increase the nutrient content in S. alatum than poultry manure, although differences were not substantial. Therefore, both manures could be equally used to improve nutrient content of S. alatum.

Cryopreservation of aromatic ginger Kaempferia galanga L. by encapsulation-dehydration

Kaempferia galanga L. is an endangered multi-purpose medicinal plant in Family Zingiberaceae, the rhizomes of which are used for several ayurvedic formulations. Encapsulation-dehydration (ED) method was optimized for cryopreservation of shoot tips of K. galanga. Shoot tips (STs) bearing the apical meristem dissected from the established in vitro shoot cultures were preconditioned in MS+0.4 M sucrose prior to encapsulation in calcium alginate and the beads subsequently transferred to MS liquid+0.3 M sucrose for 3 days afterward dehydration inside the laminar airflow for 4 hours upon rapid freezing in LN and rapid thawing produced maximum 62.2% survival and 46.7% regeneration rates. Shoot regeneration was observed from the apical meristems exclusive of intermediary callus phase. The plantlets regenerated from cryopreserved STs transferred to the field were phenotypically analogous with the mother plant.

New Approaches for Micropropagation and Cryopreservation of Agave Peacockii, An Endangered Species

More than 50% of Agave species are endemic to Mexico. Among them, Agave peacockii is listed within the list of threatened species that require special protection. In this work, we aimed at developing new supplementary strategies to achieve micropropagation and perform cryopreservation of in vitro-grown shoot-tips of A. peacockii. For multiplication, the addition of two cytokinins, 6-benzylaminopurine (26.6 μM) and kinetin (27.84 μM) to MS semisolid medium significantly favoured the morphogenetic response and produced the highest shoot generation (87.00±17.18) after 60 d of culture. This interaction was more effective than using the same growth regulators separately. Propagated and rooted plantlets were successfully acclimated with 100% survival and a normal morphological development during greenhouse performance. For cryopreservation, an optimized protocol following droplet-vitrification approach allowed obtaining 98% and 96% regrowth before and after cryopreservation, respectively. Shoot-tips were excised of in vitro-propagated plants, subjected to preculture on MS semisolid medium with 0.3 M sucrose for 1d, loaded in solution with 0.4 M sucrose and 1.6 M glycerol for 20 min, exposed to vitrification solution PVS2 for 15 min, and then, immersed in liquid nitrogen in droplets of PVS2 placed on aluminium foil strips. The vegetative growth of cryo-derived plants and of the in vitro propagated plants was compared under greenhouse culture conditions. No significant differences were detected in most assessed characteristics after 120 d of acclimatization. The results presented here constitute new viable biotechnological approaches for the in vitro propagation and long-term conservation of endangered Agave germplasm.