Liquid culture for efficient in vitro propagation of adventitious shoots in wild Vaccinium vitis-idaea ssp. minus (lingonberry) using temporary immersion and stationary bioreactors

Abstract

Efficient systems to multiply adventitious shoots regenerated from excised leaves of greenhouse-grown wild Vaccinium vitis-idaea L. ssp. minus (Lodd.) Hult. (lingonberry) clones were developed in vitro. Shoot regeneration took place when the leaf explants were cultured on a semi-solid gelled medium, positioned with the adaxial or abaxial side touching the culture medium and maintained in darkness for 2 weeks followed by culturing for another 6 weeks under light. Although zeatin (9.1 μM) or TDZ (1.8 μM) were efficient to induce adventitious shoots from leaf explants, shoot elongation was better on a medium with zeatin. Nodal segments of regenerated shoots were evaluated for shoot proliferation on a semi-solid and in a liquid medium using stationary (SB) and temporary immersion bioreactors (TIB). After 8 weeks of culture, shoot multiplication was 2–3 times more in liquid medium than on a semi-solid medium. Although shoots proliferated in a liquid medium containing 9.1 μM zeatin or 1.8 μM TDZ showed 10 % and 25 % hyperhydricity in SB and TIB respectively, rooting was not affected when regenerated shoots were dipped into 39.4 mM indole-3-butyric acid powder and planted in a 2:1 of peat: perlite medium. Liquid culture-derived plants when transferred to a greenhouse for acclimatization, 90–95 % plants survived. Micropropagated plants were phenotypically similar to the donor mother plants although the contents of chlorophyll a and b were more in the leaves of cutting propagated plants compared to micropropagated ones. Expressed sequence tag - simple sequence repeat (EST-SSR), EST- polymerase chain reaction (PCR) and inter simple sequence repeat (ISSR) banding profiles of the regenerated plants were monomorphic like those of the donor plants that confirmed clonal fidelity of tissue culture plants derived from in vitro leaf culture in V. vitis-idaea ssp. minus.