In vitro cloned micropropagation and conservation for two cultivars of Diospyros kaki (Diospyros kaki Thunb.)

The study is aimed at improving the microsprout regeneration method for the 'Suvenir Oseni' and 'Yuzhnaya Krasavitsa' Diospyros kaki (persimmon) cultivars of the Nikitsky Botanical Gardens. The breeding of and optimising the conditions for non-stop 12-month in vitro deposition of kaki persimmon was carried out for the purpose of creating a gene bank of valuable subtropical crops. The explanting of in vitro culture and the regeneration of persimmon microsprouts was carried out in the laboratory of plant biotechnology and virology of the "Nikitsky Botanical Gardens" National Scientific Centre RAS. For regeneration of microsprouts, Murashige and Skoog medium (MSO) containing 6-benzylapinopurin (BAP) and 3-(1,2,3-Thiadiazolin-5)- 1-phenylurea (TDZ) growth regulators was used. In order to ensure in vitro preservation, microsprout segments were placed in a nutrient medium composed by ¼ of normal MS, sucrose and chlorocholinchloride (CCC). The culture vessels were placed in refrigerators to maintain a low positive temperature (4–14 °С). In the course of the experiments, the inducing role of BAP (2–4 mg/L) in the MS nutrient medium at the induction stage of spout formation was established to ensure stable direct regeneration of microsprouts from the vegetative burgeons of kaki persimmon. The maximum number of normal microsprouts having no visible changes was obtained in MS medium having a BAP concentration of 4 mg/L and equal to 2.0±0.4 and 2.7±0.4 pcs. for the 'Suvenir Oseni' and 'Yuzhnaya Krasavitsa' cultivars, respectively. The average length of microsprouts reached 1.90±0.04 cm for the 'Suvenir Oseni' cultivar, while, for 'Yuzhnaya Krasavitsa', this value was equal to 3.1±0.07 cm. The presence of TDZ in the MS medium facilitated the formation of microsprouts through indirect organogenesis in the leaf cutting culture of the studied cultivars. The frequency of spout formation from leaf cuttings reached 65–79 % following 6 weeks of cultivation on media with 1.1 and 1.7 mg/L concentration of TDZ. Differences in the morphogenetic potential of explants were traced throughout all stages of development. Data from histological callus studies confirm the presence of proliferatively active cells giving rise to microsprout meristems. The concentration of 60.0 g/L and 0.2–0.4 g/L for sucrose and CCC, respectively, contained in ¼ MS medium was shown to stabilise the viability of persimmon explants at a storage temperature of 8–10 °С for 12 months.

Diospyros kaki, micropropagation, in vitro, organogenesis, deposition

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