Cryopreservation of orchid seeds through rapid and step freezing methods

Abstract

Ecuador has a great variety of climatic regions that potentiate biodiversity. The family Orchidaceae constitutes one of the most important of the country, having identified about 4032 species with a high degree of endemism, therefore the development and research of alternative methods of storage and conservation of species is a strategy of primary interest for researchers and for society in general. In cryopreservation, temperatures reach below -190°C in order to paralyze the chemical reactions and keep the plant material viable for long periods. The present research focuses on the development of protocols for cryopreservation of seeds, aimed at the preservation of biodiversity, focusing on the family Orchidaceae, for the subsequent generation of a seed bank. The assays were performed on seeds of Epidendrum quitensium, Sobralia rosea, and Epidendrum anderssonii. Two freezing rates were tested: rapid freezing at -196°C; and step freezing at -22°C, -60°C to 196°C, further analyzed four combinations from Dimethylsulfoxide DMSO, glycerol and sucrose (DMSO 1M; DMSO 1M + glycerol 1M; DMSO 1M + sucrose 1M; DMSO 1M + glycerol 0,5M + sucrose 0,5M). The best results were obtained both in rapid and stepped freezing without the use of cryo-protective substances, by introducing the seeds directly into liquid nitrogen. Species of the genus Epidendrum presented a more efficient response in comparison to Sobralia. The viability of the seeds was evaluated by the tetrazolium test.

Cryopreservation of orchid seeds through rapid and step freezing methods

“…The use of cryopreservatives is recommended for seeds with a high moisture content, as stated by Reed and others 12 – 14 . Furthermore, Harding 15 states that it is necessary to demonstrate the genetic stability of plants regenerated from cryopreserved plant material to approve their release and reintroduction into the environment; but to date, there have been no reports showing changes at the phenotypic, biochemical, chromosomal or molecular levels attributed to storage systems by cryoconservation 14 . The cryoconservation method that gave the best results was the “Rapid” freezing without the addition of any cryopreservative substance.…”

“…Dataset 3: TTC-stained seeds subjected to the “Rapid” cryopreservation process: Epidendrum anderssonii . 10.5256/f1000research.13622.d194235 17 …”

Temperature conditions optimization for storing tropical orchid seeds on the example of <i>Eulophia streptopetala</i> and <i>Stanhopea tigrina</i>

“…Большинство видов орхидей имеет ортодоксальные семена, и содержание влаги в зрелых семенах орхидей обычно составляет менее 13% [9, p. 122]. Проблема надежного и длительного сохранения ценного генофонда на данный момент решается путем хранения семян, клеток и микроорганов в жидком азоте [10] Для культивирования Eulophia streptopetala и Stanhopea tigrina использовали агаризованную питательную среду [21]. В течение месяца растения культивировались в условиях темноты при температуре +10°C, после чего были перенесены на освещаемые стеллажи с температурой +20°C для дальнейшего культивирования.…”

Efficiency of cryoprotectors for cryopreservation of two orchid species from Americas

“…Cryopreservation of orchid seeds of the genera Encyclia and Epidendrum has been previously studied for Encyclia pygmea and Encyclia odorantissima (Pardo-Alvarez and Ferreira 2006); Encyclia cochleata Lemée (Nikishina et al 2001); Epidendrum quitensium Rchb.f. and Epidendrum anderssonii Hágsater & Dodson (Cerna et al 2018); Encyclia tampensis, Epidendrum amphytomum, Epidendrum nocturnum and Epidendrum rigidum (Hughes & Kane 2018).…”