Patent ID: 7402433
Filing Date: 2008-07-22
Classification: A01H

Abstract:
1. A method of in-vitro micropropagation of Piper longum by direct rhizogenesis from a lateral bud explant, said method comprising the steps of: i. cutting the lateral buds from stem segments under laminar flow benches (LF) and treating them with hydrogen chloride (HCl2) (0.002-0.2%) for 3 minutes and rinsing the treated lateral buds with autoclaved distilled water; ii. subjecting the lateral buds to a surface sterilization by dipping into sodium hypochloride (NaOCl) (0.5-3%) for 4 minutes and again treating the dipped lateral buds with three additional thorough rinses with autoclave distilled water; iii. inoculating the surface sterilized lateral buds in a culture initiation medium supplemented with auxin; iv. incubating the inoculated explants in dark at 23° C.±1° C., for 7-15 days to produce 6-7 root primordia from basal cut ends of all the lateral buds; v. transferring the incubated cultures of step iv, into a light intensity of 2000-4000 lux and providing a photoperiod of 16 hours followed by a dark period of 8 hours daily; vi. allowing each root to grow to an average size of 1.5 cm in 45 days; through direct rhizogenesis; vii. cuting each root of step vi, into 0.5 cm long segments aseptically under LF; viii. sub-culturing the 18 segments procured from a single explant separately onto a shoot induction medium supplemented with the auxin to produce cultures; ix. keeping the cultures of step viii, at 23° C. under a light intensity of 2000-4000-lux for a photoperiod of 16 hours followed by a dark period of 8 hours to produce 50-55 adventitious shoot buds and 900 shoot buds (18×50); x. cutting each culture clump into two pieces after a 45 day 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 in step ix, for a period of 45 days for shoot elongation; xi. separating 2.5-3 cm long shoots with 2-3 nodes from the culture clumps using a sterile scalpel under LF; xii. sub-culturing 10 separated shoots of step xi, in a wide mouth bottle containing rooting medium supplemented with the auxin; xiii. rooting the regenerated shoots of step xii, under a reduced light intensity of 1000-3000 lux for a period of 28 days, with a 16 hour photoperiod and temperature 23° C.±1° C.; xiv. removing the micropropagated plants carefully from the agar contained in the sub-culturing medium; xv. washing the roots of the micropropagated plants of step xiv, 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; and xvi. planting the micropropagated plantlets in 1:1 ratio of soil and cocopeat.