In Vitro conservation and genetic diversity analysis of rare species Ribes janczewskii

Abstract

Ribes janczewskii is a rare and valuable plant known for its resistance to spring frosts, pests, and diseases. It is used in hybridization to develop resistant currant varieties but is on the verge of extinction, listed in Kazakhstan Red Book. This study developed a micropropagation and slow-growth storage protocol for conservation. Genotypes were identified through DNA barcode analysis (rbcL, ITS, and matK) and sequences uploaded to the National Center for Biotechnology Information database. Genetic diversity was assessed using iPBS primers, generating 98 fragments with 88-94% polymorphic bands. Biochemical analysis of fruits showed vitamin C content from 4.64 to 5.61 mg/100 g, vitamin E from 2.26 to 3.16 mg/100 g, vitamin B5 from 3.18 to 4.93 mg/100 g, and quercetin up to 12.5 mg/100 g. Micropropagation stages were optimized with 12% hydrogen peroxide for surface sterilization, achieving up to 73.3% explant viability. Effective hormonal combinations for in vitro culture included WPM with BAP 0.2 mg L^-1 and GA 0.5 mg L^-1, and for propagation, BAP 0.25 mg L^-1, GA 0.5 mg L^-1, and IBA 0.5 mg L^-1. Mannitol (20 g L^-1) was used for slow-growth storage, keeping explants viable for 4 months without re-cultivation.

Keywords: Ribes janczewskii; DNA barcoding; Micropropagation; Slow-growth storage; iPBS.

Fig. 1

Electrophoretic pattern of R. janczewskii fragments using iPBS primer 2224, 2228, 2229 and 2393.

Fig. 2

Sterile and viable explants of R. janczewskii.

Fig. 3

Regenerated R. janczewskii shoots on WPM nutrient medium with the addition of BAP 0.2 mg L⁻¹ and GA 0.5 mg L⁻¹.

Fig. 4

The effect of plant growth regulators (PGRs) on shoot multiplication of R.janczewskii in comparing the effect of three cytokinins (a) control; (b) Kin 0.25 mg L⁻¹; (c) Kin 0.5 mg L⁻¹; (d) Kin 0.75 mg L⁻¹; (e) kin 1.0 mg L⁻¹; (f) BAP 0.25 mg L⁻¹; (g) BAP 0.5 mg L⁻¹; (h) BAP 0.75 mg L⁻¹; (i) BAP 1.0 mg L⁻¹.

Fig. 5

The effect of osmotic agents on slow-growth storage of R. janczewskii (a) sucrose 30 g L⁻¹; (b) sucrose 60 g L⁻¹; (c) sucrose 90 g L⁻¹; (d) mannitol 10 g L⁻¹; (e) mannitol 20 g L⁻¹; (f) mannitol 30 g L⁻¹.

Fig. 6

Created in vitro collection of R. janczewskii.

Fig. 7

Locations of sample collection of R. janczewskii in the Kazakhstan Sayram-Ugam National Park territory.

Fig. 8

Berries of Ribes janczewskii.