PINEAPPLE RESEARCH STATION (KERALA AGRICULTURAL UNIVERSITY), VAZHAKULAM, MUVATTUPUZHA
Plant tissue culture technology has proven itself to be an effective and viable option for growers to seriously consider in a variety of different situations.
When large-scale propagation of new or superior plant varieties is required for early introduction to market
TISSUE CULTURE PROTOCOLS FOR PINEAPPLE
Tissue culture is a collection of experimental methods whereby fragments of living tissue are isolated from plants and grown respectively on a nutrient rich medium for a definite period. The beginning of plant tissue culture was made as early as 1898 – German Botanist G. Haberland (father of tissue culture). Totipotency is the basis of plant cell and tissue culture. Each cell of a multicellular organisms is capable of independent development when provided with suitable conditions by regeneration. By the development of suitable medium (1940-1970) for plant cells, tissue, protoplasts, anthers, root tips, and embryos In vitro morphogenesis of plants was successfully done.
METHODS OF PLANT TISSUE CULTURE
1. Preparation suitable nutrient medium: Suitable media is prepared for pineapple tissue culture and transferred into suitable containers and autoclaved at 15 psi for 30 minutes.
Four broad classes of growth regulators namely auxins, cytokines, gibberellins and abscisic acid. Differentiation and organogenesis of tissue become feasible only on the addition of growth regulators. Auxins induces cell division, elongation of stem,internodes,tropism,apical dominance and rooting.Auxins used are IAA,NAA,IBA,24-D.Cytokines are mainly concerned with cell division, modification of apical dominance and shoot differentiation. Most frequently used are BAP,BA,2-iP.Gibberellins and abscisic acid enhances callus growth according to the species.
Vitamins and Amino acids
Cultured cells are normally capable of producing vitamins and amino acids for performing various metabolic activities .If the cells are unable to produce, addition of vitamins and amino acids to the medium is recommended.
Addition of antibiotic in the media retards the cell growth. But some cells have systemic infection of microorganisms. To prevent the of microbes we use antibiotics.(gentamycin)
Preparation of stock solution (MS)
A series of standard stock solutions are prepared for the preparation of MS and other media. Specific media are prepared for callus initiation, multiplication, elongation and rooting.
2. Surface sterilization of the explant and inoculation into the medium
Explant (slips, suckers, crown and leaf) obtained directly from the field grown pineapple should be surface sterilized in the laminar air flow chamber using the chemical agent in order to remove the microbial contamination on their surface. These microbes utilize the nutrients faster than the plant due to their brief life cycle and finally kill the plant tissue.
Healthy explants were selected and washed in running tap water for 15 minutes, subsequent step were done in LAF aseptically
Explants were kept in 1% saaf for 20 minutes
The chamber is swabbed with ethanol before starting work
Sterile Petri dishes, 70% ethanol, mercuric chloride were kept on the table of laminar cabinet
Explants were transferred to sterile beaker and treated with 0.1% mercuric chloride for 10 minutes. The explant were surface sterilized by frequently swirling the beaker.
Sterilized explants were then thoroughly washed several times with sterile distilled water.
The explants were then transferred aseptically to basal media with hormones for callus initiation and observed periodically.
To control contamination, fumigate the culture room, laminar air flow chamber and mist chamber using formaldehyde. For fumigating the LAF potassium permanganate is used with formaldehyde.
There are chances for contamination even if each step of plant tissue culture is done aseptically. The contamination may be caused by bacteria and fungi. The bacterial contamination is treated with 0.05% mercuric chloride. Antibiotics (Gentamycin) are also used, they are added in the medium after sterilization. The fungal contamination is treated with 0.5% saaf.
The tissue culture plants that have shoot and root are treated with trichoderma (10g/l) and 0.5-0.6ml any rooting hormone (NAA,IAA & IBA) for 30 minutes. These plants were planted in soil which is mixed with pseudomonas, trichoderma and rock phosphate (each 10g/l). The plants were kept in the mist chamber for one week. These plants were watered regularly. After one week these plants are kept outside the chamber and saaf is sprayed on them. The plants are watered regularly as before. Two weeks later bavastin is sprayed. After 2-3 weeks hilban is used for spraying. Once in every week ash (10g/l) and urea (20g/l) is sprayed.
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