EXPERIMENT
AIM: To raise cell suspension culture of Lantana camara .
EQUIPMENTS: Autoclave, pH-meter, Magnetic stirrers, Magnetic beads, Weighing balance, Laminar-air-flow, Microsopes, Incubator shaker.
MATERIALS REQUIRED : Salts and vitamins of Murashige and Skoog's (MS; 1962), sucrose, agar, conical flasks, measuring cylinders and beakers of various sizes. Reagent glass bottles for storage, spatula, tissue rolls, distilled water. Cotton plugs, aluminium foils, muslin cloth, scissor, media stocks, 1N NaOH, 1N HCl, myo-inositol. Autoclavable polybags, rubber bands. Borosil glass test-tubes (150mm x 25mm without rim) and Erlenmeyer flasks. Black markers, micropipette, micropipette-tips, test-tube stands, autoclavable baskets, plastic trays, cork borer, Leaves from healthy L. camara plants bearing pink-yellow colored flowers.
Plant Growth regulators (Sigma):
Benzyl amino purine (BAP), Naphthalene acetic acid (NAA) , 2-4-dichlorophenoxyacetic acid.
METHODS
i. Initiation and establishment of leaf-disc cultures
Leaves were washed with 1% Tween-20 (Merck, India) for 15 min, followed by three rinses in sterile distilled water (SDW). Thereafter, the remaining steps were carried out inside the laminar-air-flow cabinet (Saveer Biotech, India). Leaves were surface sterilized with 0.1% mercuric chloride solution (HgCl2) for 10 min and rinsed thrice with SDW. Leaf-disc explants were prepared by punching the sterilized leaves with 5 mm sized cork-borer before being cultured with the adaxial side in contact with the media.
ii. Establishment of callus cultures
Leaf disc explants were incubated on MS (with 3% sucrose) basal medium consisting of macro and microsalts, vitamins, iron, 100 mgl-1 myoinositol and solidified with 0.8% agar (HiMedia, India). The medium was enriched with combination of cytokinin and auxins, BAP (5μM) + NAA (1 μM) + 2,4-D (1 μM) for induction and multiplication of callus from leaf-disc explants. After adjusting the pH to 5.8, 20 ml of the medium was dispensed into each 150 x 25 mm Borosil rimless glass tubes. The culture tubes were plugged with nonabsorbent cotton wrapped in cheesecloth and autoclaved at 121°C at 15 psi for 15 min. All the cultures were maintained in diffuse light (1000-1600 lux) and 16 h photoperiod at 25 ± 2°C and 50-60% relative humidity. Observations were recorded at weekly intervals. After callus induction, the biomass was multiplied constantly by inoculating 0.2 g of calli onto fresh medium at every 4-week intervals.