Seed Cryopreservation and Germination of Rhus glabra and the Critically Endangered Species Rhus michauxii

Rhus michauxii is a perennial rhizomatous shrub native to the southeastern United States that is found mainly in sunny, dry, open rocky or sandy woodlands. Moreover, it is found on ridges or river bluffs in the inner coastal plane and lower piedmont of Virginia, Georgia, and the Carolinas. Habitat conversion to agriculture, suppression of fires, and low reproduction have caused R. michauxii to become rare and it is now federally listed as threatened. Methods are needed to multiply and conserve R. michauxii. Protocols were developed for seed cryopreservation, in vitro germination, and micropropagation for R. glabra and R. michauxii. Seed scarification in concentrated sulfuric acid for 6 h and germination on ½ MS medium resulted in germination up to 96% for control and cryopreserved seeds of R. glabra and 70 and 40% for control and cryopreserved seeds of R. michauxii. Shortly after germination in vitro, young seedlings were established in a greenhouse potting mix providing new plants from the endemic Georgia R. michauxii populations. Several of the findings meet goals within the R. michauxii recovery plan by providing methods for sexual and asexual multiplication and long-term seed storage under cryogenic conditions. The protocols developed will assist in the safeguarding and conservation of dwindling natural R. michauxii populations.

Seed Cryopreservation, Germination, and Micropropagation of Eastern Turkeybeard, Xerophyllum asphodeloides (L.) Nutt.: A Threatened Species from the Southeastern United States

Xerophyllum asphodeloides (Xerophyllaceae), known as eastern turkeybeard, is an herbaceous perennial found in eastern North America. Due to decline and destruction of its habitat, several states rank X. asphodeloides as “Imperiled” to “Critically Imperiled”. Protocols for seed cryopreservation, in vitro germination, sustainable shoot micropropagation, shoot establishment in soil, and seed germination are presented. Seeds from two tested sources were viable after 20 months of cryopreservation. Germination of isolated embryos in vitro was necessary to overcome strong seed dormancy. Shoot multiplication and elongation occurred on ½ MS medium without PGRs. Shoots rooted in vitro without PGRs or with 0.5 mg/L NAA or after NAA rooting powder treatment and placement in potting mix. When planted in wet, peaty soil mixes, shoots grew for two months and then declined. When planted in a drier planting mix containing aged bark, most plants continued growth. In the field, plant survival was 73% after three growing seasons. Safeguarding this species both ex situ and in situ is possible and offers a successful approach to conservation. Whole seeds germinated after double dormancy was overcome by incubation under warm moist conditions for 12 weeks followed by 12 weeks cold at 4 °C and then warm.

Conservation of Hibiscus acetosella germplasm by seed cryopreservation

Hibiscus acetosella (Malvaceae) is a shrub of great importance for landscaping, food and medicinal purposes. The objective of this study was to preserve H. acetosella germplasm by seed cryopreservation. Half of the seed batch was scarified and the other half was kept intact. Cryopreservation occurred by immersion in liquid nitrogen for 1 hour. Moisture content (MC%), germination percentage (G%), germination speed index (GSI), normal seedling formation (NS%), shoot length (SL), dry matter (DM), biometry and plant survival were evaluated after treatment. MC% ranged between 7.7% and 6.65% in intact and scarified seeds, respectively. Scarification raised G% and GSI compared to intact seeds. Intact and scarified seeds had 100% and 70% NS%, respectively, when not cryopreserved. Cryopreservation reduced NS% to 62% and 12.75%, respectively. The highest SL was observed in intact and non-cryopreserved seeds, with an average of 10.21 cm in height. However, the cryopreservation of intact seeds reduced SL by about 50%, and scarification led to a further reduction, either with (3.32 cm) or without (2.47 cm) cryopreservation. Seedlings from intact and non-cryopreserved seeds showed higher DM in relation to seedlings from cryopreserved seeds. The association of cryopreservation and scarification further reduced DM. The cryopreservation of intact seeds yielded 100% survival at the end of the acclimatization process. However, cryopreservation of scarified seeds reduced the survival percentage to 15%. Changes in color were observed for seeds scarified and subjected to cryopreservation. Thus, cryopreservation is considered an efficient technique for the conservation of intact H. acetosella seeds in the long term.

Cryopreservation of Dendrobium cruentum Rchb. f. Seeds by D Cryo-plate and V Cryo-plate Techniques

Dendrobium cruentum Rchb. f. is a native Thai orchid species that has faced extinction because of its attractive characteristics. Consequently, conservation of this species is urgently needed. In this study, cryopreservation technique was applied to D. cruentum Rchb. f. seeds for long-term conservation. A successful protocol for D. cruentum Rchb. f. seed cryopreservation was developed by using D cryo-plate and V cryo-plate techniques. Seed viability was tested by TCC solution and 93.8 % of dyed seeds were shown. For cryo-plate technique, seeds were encapsulated over the cryo-plate by using 2 % (w/v) sodium alginate and polymerized with 100 mM CaCl2. Encapsulated seeds were desiccated by using a laminar airflow and PVS2 solution treatment with the same exposure time (0, 30, 60, 90, and 120 min). After cryopreservation, encapsulated seeds were cultured on modified VW agar medium. From the results, the maximum germination and regrowth percentage were observed; D cryo-plate technique with 60 min of dehydration time gave the highest germination (68.9 %) and regrowth (57.8 %). Thus, the excess of dehydration may cause the reduction of germination and plant regeneration. In conclusion, D cryo-plate technique proved to be appropriate for D. cruentum Rchb. f. seed cryopreservation.