Efficient method of in-vitro micropropagation of piper longum

An efficient method of in-vitro micropropagation of Piper longum from lateral bud explant, said method comprising the steps of:—

• collecting the lateral bud explant from the mother plant by cutting twig with a sharp scalpel before 8.00 am and dipping the cut end into a medium comprising effective amounts of sterilants to keep the plants free from fungus and insect attack and transferring these plants under Laminar Flow benches (LF) for final surface sterilisation;
• cutting the lateral buds from the stem segments under LF and treating them with hydrogen chloride (HCl2) (0.002-0.2%) for 3 minutes and rinsing it with autoclaved distilled water;
• subjecting the lateral buds to surface sterilisation by dipping in sodium hypochloride (NaOCl) (0.5-3%) for 4 minutes and again giving three thorough rinses with autoclave distilled water;
• inoculating the surface sterilised lateral buds in a culture initiation medium supplemented with growth hormones;
• autoclaving the medium of step iv, at 15 psi for 20 minutes and cooling it to solidify at room temperature;
• incubating the inoculated explants in dark at 23° C.+1° C., for 7-15 days to produce 6-7 root primordia from the basal cut end of all the lateral buds;
• transferring the incubated cultures of step vi, in light intensity of 2000-4000 lux and providing a photo period of 16 hours followed by a dark period of 8 hours daily;
• allowing each root to grow to an average size of 1.5 cm in 45 days;
• through direct rhizogenesis (organogenesis);
• cutting each root of step viii, into 0.5 cm long segments aseptically under LF;
• sub-culturing the 18 segments procured from a single explant separately onto a shoot induction medium supplemented with the growth hormone;
• keeping the cultures of step x, at 23° C. under light intensity of 2000-4000-lux for a photo period of 16 hours followed by a dark period of 8 hours to produce 50-55 adventitious shoot buds and 900 shoot buds (18×50);
• cutting each culture clump into two pieces after 45 days of interval and sub culturing each piece in a separate culture bottle containing 25 ml of the same medium, and growing them under similar physical conditions as mentioned step xi, for 45 days for shoot elongation;
• separating 2.5-3 cm long shoots with 2-3 nodes from the culture clumps using a sterile scalpel under LF;
• sub-culturing 10 separated shoots of step xiii, in a wide mouth bottle containing rooting medium supplemented with growth hormone;
• rooting the regenerated shoots of step xv, under reduced light intensity of 1000-3000 lux for 28 days, with 16 hour photoperiod and temperature 23° C.+1° C.;
• removing the micropropagated plants carefully from the agar contained in the sub-culturing medium;
• washing the roots of the micropropaged plants of step xvi, under tap water to remove any trace of agar adhered to the roots and dipping the roots in Bavistin (0.05-1%) for half an hour;
• planting the micropropagated plantlets in 1:1 ratio of soil and cocopeat;
• growing the above micropropagated plantlets in green house having 80% relative humidity (RH) and temperature below 28° C. and gradually increasing the temperature to ambient temperature of 30-32° C. and reducing the RH to 40% over a period of 4 weeks for hardening the plants;
• transferring the hardened plants for acclimatization to plantation site conditions, to partial shade under shade-net for a week prior to transferring the plants to the field;
• and
• transplanting the micropropagated hardened and acclimatized plants to the field for cultivation in a conventional manner.