Module 1 : APPLICATIONS OF PLANT BIOTECHNOLOGY IN CROP IMPROVEMENT

Lecture 5 : Micropropagation by Adventitious Shoot Proliferation

     

EXPERIMENT

AIM: Micropropagation by adventitious shoot proliferation from leaf-disc explants of Neem (Azadirachta indica A. Juss)

EQUIPMENTS: Autoclave, pH-meter, Magnetic stirrers, Magnetic beads, Weighing balance, Laminar-air-flow

 

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, aluminum 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). Black markers, micropipette, micropipette-tips, test-tube stands, autoclavable baskets, plastic trays, fresh leaves of Azadirachta indica .

 

Plant Growth regulators (Sigma):

Benzyl amino purine (BAP)

6-furfuryl amino purine (Kinetin)

Thidiazuron (TDZ)

6-γ γ –Dimethylallylamino purine (2-iP)

Indole-3-acetic acid (IAA)

Naphthalene acetic acid (NAA)

 

PROTOCOL:

  1. 1. Surface sterilize the freshly plucked leaves by using 0.1% HgCl2 for 10 min and rinse thrice with sterile distilled water.

    2. 2. Bring sterilized leaf samples under sterile conditions inside the Laminar-air-flow.

    3. Make leaf-disc of uniform size by using cork borer of 5mm diameter

    4. Using sterile forceps inoculate the explants horizontally on either of the following media in Petriplates or test-tubes -

    MS + TDZ (5µM) / MS + Kinetin (5µM) / MS+BAP (5µM)

    MS + BAP (5µM) + IAA (0.1µM) / MS +BAP (5µM) + NAA (0.5µM)

    MS +BAP (5µM) + 2iP (5µM)

    5. Seal the Petriplates with parafilm or test-tube with cotton plug.

    6. Label each Petriplate/test-tube with date and media combination used.

    7. Incubate the cultures at 25 ± 2°C temperature and 50-60% relative humidity under a 16/8 hour (light/dark) photoperiod with diffuse light (1000-2000 lux).

    8. Observe the cultures at regular intervals for adventitious shoot proliferation directly or indirectly via callus proliferation, and remove the contaminated culture, if any. Record the time of shoot proliferation, number of shoots per culture and the size of each shoot in single culture. If required use the Stereo zoom microscope to see shoot development and take photographs for the record.