How to raise the citrus (Kaghzi lemon) rootstock in vitro?
Set up the rootstock of this very valuable planting fabric, tissue lifestyle strategies, specially micropropagation assume significance and remains the handiest feasible opportunity. There are special strategies for micropropagation of citrus, viz. Somatic embryogenesis, adventitious shoot bud production and axillary enhancement that are routinely used. Amongst those, axillary enhancement the usage of the nodal segment as explant is taken into consideration pleasant because it does no longer involve a callus phase, consequently, reduce the hazard of somaclonal variant and it offers economically premier multiplication rate. However, efforts have no longer yet been made to expand away for axillary enhancement in citrus. Efforts have also been made to place regenerated number one branches to a couple of shooting. Although this technique gives the least threat of somaclonal variation, nevertheless, versions may additionally stand up throughout successive culture cycles mainly because of prolonged publicity on plant growth regulators. In view of the above, the existing investigation becomes completed to broaden an efficient regeneration device for multiplication of citrus. The genetic constancy of the micropropagation system needs to be ascertained earlier than the use of it at a business stage.
Plant material and explant preparation
Young tender pencil-thick shoot pieces of seedless lemon) were collected from 2–3 years old plants growing in the orchard of Agricultural Research Station, Rajasthan Agriculture University, Bikaner during the month of November to March. In order to remove the micro-flora present on surface, shoot pieces were thoroughly washed under running tap water for 15–20 min, and then immersed in an aqueous solution of liquid detergent containing 2–3 drops of Tween 20 250 mg l−1cefotaxime and 1 % Bavistin for 15 min followed by three to four rinses in sterile distilled water. The nodal segments of 2.5–3.0 cm containing at least one node from these shoot pieces were used as explant. Finally, inside a laminar air-flow cabinet, the nodal explants were surface sterilized with 0.1 % (w/v) mercuric chloride (HgCl2) for 4–5 min followed by 5–6 rinses with sterile double distilled water.
Culture media and growth conditions
MS (Morishige and Skoog 1962) medium containing 3 % sucrose and 0.8 % agar was used throughout the experiments; different growth regulators were supplemented as per the requirement of the experiment. The pH of the medium was adjusted to 5.8 prior to autoclaving for 15 min at 121 °C temperature and 15 psi for shoot proliferation, surface sterilized explants were inoculated onto MS medium supplemented with different concentration of growth regulators. Cultures were incubated in a growth room at 25 ± 2 °C 16/8 h (light/dark) photoperiod at a photon flux of 50–70 mmol m−2 s−1 from cool white fluorescent tubes (Philips, India). Subculture was done at an interval of 20–25 days. Shoots initiated on each treatment in each culture were cultured for further shoot proliferation and root induction. Data was recorded on (I) frequency of responding cultures, (ii) days to shoot induction, (iii) number of shoots per explant cultures, and (iv) shoot length (cm), after 60 days after culture.
For root induction, three different media, each having MS medium supplemented with 1.0 mg/l of either of IBA, IAA or NAA was tested. Data was recorded on (i) frequency of cultures showing root induction, (ii) days to root induction, (iii) numbers of roots per explant, and (iv) root length (cm), 60 days after culture.
Hardening of plantlets and transfer to soil
For hardening, healthy plantlets obtained from shoots rooted on agar medium were removed from culture tubes, washed under running tap water to remove agar sticking to the roots and transplanted into plastic cups filled with sterilized soil rite and covered with polyethylene bags. The pots were then kept in a culture room at 25 ± 2 °C, and 16-h light and 8 h dark period. The pots were irrigated every alternate day with distilled water. After 7 days, the polyethylene bags were gradually removed over a period of 15 days. The plantlets were kept in the culture room for 10 days and then transferred to room temperature for another 10 days before they were transferred to a glasshouse. After 2 to 3 months in the greenhouse, the plants were transplanted under the field conditions. The genetic integrity of the in vitro regenerated citrus plants was checked using random amplified polymorphic DNA (RAPD) analysis.
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