Rapid In vitro Micro propagation of Sugarcane through Callus Culture.
Plant tissue culture techniques have become a powerful tool for studying and solving basic and applied problems in plant biotechnology. During the last thirty years, micropropagation and other in vitro techniques like somatic clones, somatic embryogenesis, seed hybridization, haploid plants, this all is become more widely used in commercial horticulture and agriculture for the mass propagation and storage of crop plants.
Sugarcane is an important agricultural cash crop in tropical and subtropical region of the world and is the major source of sugar with respect to export product in many developing countries that accounts for more than 60% of the world’s sugar production. It is the only member of the family belong to genus in which, in vitro propagation are standardized and commercially viable. Varieties of sugarcane are highly heterogeneous and generally multiplied vegetative by stem cutting. In tropical countries nodal sections of sugarcane with 2 or 3 nodes are commonly, used as a planting material.
Lack of suitable multiplication procedure and contamination by systemic diseases are the serious problem to multiply an genotype of sugarcane in the open field.
MATERIALS AND METHODS
Explant source: Healthy young meristems were collected by removing the leaf sheath from field grown plants of sugarcane. These young meristems were cut into thin, smaller pieces of 1.0 to1.5 cm length. The explants were washed thoroughly under running tap water for 20-30 minutes and then washed with sterile distilled water and transferred to a laminar air flow cabinet. The young meristem explants were treated with 70 % alcohol for 30 second to one minute. Finally, washed with distilled water. The young meristem cuttings before the inoculation in to sterilized nutrient agar media pre-packed in culture tubes. All the above operations were performed under aseptic conditions in a laminar airflow cabinet.
Culture medium and condition: The young meristem cutting explants was inoculated on to sterilized semi solid basal MS medium in to the sterilized test tubes.
Supplemented with different concentrations and combinations of different plant growth regulators.
Callus induction: For callus induction different range of concentration of auxins are tried individually such as 2,4-D, IBA with MS basal medium.
Shoot regeneration Medium: White friable calli were cultured on MS medium supplemented with different combinations and concentrations of BAP, and IBA, for
Multiple shoot regeneration either individually or in combination.
Rooting medium: Elongated micro shoots measuring about 5-6 cm in length were excised from culture tube and transferred to half-strength (1/2 MS) MS medium supplemented with different concentrations of IBA, and IAA either individually or in combinations.
Environmental condition: The pH of the medium was adjusted to 5.8 before gelling with Agar and prior to autoclaving for 20 min at 120° C and at 15 lbs psi pressure.
Sugar was added at the concentration of 30 gm/l. Molten medium of 20 ml was dispensed into the culture tube and plugged with non absorbent cotton wrapped in one layer of cheesecloth. All the cultures were incubated in a growth room with a 16h photoperiod except callus culture cool, white fluorescent light and the temperature was maintained at 25° ± 30 with 70-80% relative humidity in the culture room.
Each treatment consisted of 10 replicates and repeated thrice. For better callus induction just after autoclaving the culture tube containing semisolid sterilized media should positioned , such a manner as if the inside semisolid media in the culture tube should spread to maximum to wards one side only.
At the time of inoculation 2 to 3 explant per culture tube was used. For callus induction experiment culture tubes were kept under 20, 25 and 30°C in complete dark or light/dark condition with 16/8 h light/dark photoperiod with 140μmol m-2S-1 light from cool, white fluorescent lamps. Acclimatization and transfer of plantlets to soil: Plantlets with well-developed roots were removed from the culture medium. Washed the roots gently under running tap water and were transferred to plastic trays for hardening which contain autoclaved garden soil, farmyard manure and sand (2:1:1). The harden plantlets in the plastic trays were covered with porous polyethylene sheets for maintaining high humidity and were kept under shade in a net house for further growth and development. After 30 days, the plant lets were transplanted in to the soil in field condition.
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