PJB-2019-616
CRYOPRESERVATION OF IN VITRO GROWN SHOOT TIPS OF EMMENOPTERYS HENRYI BY DROPLET VITRIFICATION
Yanli Niu
Abstract
The genus Emmenopterys only comprises one species, E. henryi, belonging to the family Rubiaceae, which is an extremely rare and endangered species due to artificial destruction of its natural habitat such as overexploitation and lack of nature regeneration. In this study, we established a rapid and reliable method for in vitro cryopreservation of E. henryi About 2 mm shoot tips with 2-3 leaf primordial were excised from plantlets then cultivated on common Murashige and Skoog (MS) medium for producing stable explants. We further performed the main steps of cryopreservation in E. henryi as follows: 1) shoot tips were precultured in hormone free MS medium containing 0.5 M sucrose for 3 days and then incubated in PVS2 for 1 h at 0℃, followed by a direct immersion in liquid nitrogen for 1h; 2) after rapid thawing in a warm water bath at 40 °C and rinsed 5 to 6 times with sterile-distilled water, the shoot tips were plated on the regeneration medium fortified with 1.0 mg/L TDZ, 1.0 mg/L ZT, 0.6 g/L PVP, 30 g/L sucrose and 6.8 g/L agar for one week in dark prior to exposure in normal conditions; 3) SRAP marker was employed to assess the genetic diversity of E. henryi using DNA samples from crucial cryopreservation steps. Our results showed that the survival and regrowth percentage of E. henryi shoot tips after in vitro cryopreservation could respectively reach up to 88.9% and 79.2% and no significant variation from cryopreserved shoots was observed by using SRAP markers. Take together, we have developed an efficient method for germplasm conservation of E. henryi and this strategy provides a new insight on in vitro cryopreservation of other endangered specieses.
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