Pineapple is very easy to propagate vegetatively. Suckers arising in the axil of the leaves on the main stem form roots and can be used for propagation. Even the crown of leaves above the fruit and parts of the stem itself can be used. Another method of propagation is by slips, which are the suckers, arising immediately below the fruit. Suckers and slips should be preferred for planting as they come to bearing earlier than the crown and produce larger fruits. Plants from crown bear flowers after 3 to 20 months later than suckers and slips depending on the climatic conditions. Therefore, crowns are not normally used. Suckers should be planted within 2 weeks after removing from the mother plant. The planting material should be selected from healthy disease free plant.
Micropropagation
Conventionally the average production is 4-5 propagules per year and it takes considerable time to produce enough planting material. Large-scale production of planting material can be achieved by using in vitro micropropagation techniques. A protocol for large-scale multiplication has been established using dormant auxiliary buds from pineapple crowns with a capacity of producing 1000-1200 plants in a year from a single crown. The protocol has been standardized for the establishment of cultures, multiplication, rooting and hardening of the plants in the field. Tissue cultured plants have been field planted at the BARC campus and the Rashtriya Chemicals Fertilizers (RCF) Experimental Field at Alibagh.
Pineapple is very easy to propagate vegetatively. Suckers arising in the lower axils of the leaves on the main stem form roots and can be used for propagation. Even the crown of leaves above the fruit and parts of the stem itself can be used. Another method of propagation is by slips, which are the propagules arising from the base the fruit. Suckers and slips should be preferred over the crown for planting as they come to bearing earlier and produce larger fruits.
TISSUE CULTURE PROTOCOLS FOR PINEAPPLE
INTRODUCTION
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
Basic Steps:
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.
Growth Regulators
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.
Antibiotics
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.
3. Fumigation
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.
4. Contamination
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.
5. Hardening
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.
Selection of suckers
Suckers are selected from disease and pest free healthy plants. Suckers are to be graded into those having less than 500g, 500-750 g and more than750g in weight to avoid competition between plants of different sizes. Suckers weighing 400-500g or slips of 350-450g are considered ideal for planting. The graded suckers are planted in different blocks or plots, to get uniformity in growth and flowering. Bigger suckers give early yield. Prior to planting curing of slips and suckers for 8-10 days in shade is necessary as fresh suckers planted in moist soil begin to decay. Before planting some of the lower leaves are removed from the sucker to facilitate the formation and entry of roots into the soil. After removing scaly leaves, planting material should be treated with Monocrotophos (0.15%) or 0.05% quinalphos and Carbendazim (0.1%) solution to protect against mealy bugs and heart rot, respectively. Alternatively, a solution of Hilban (2.5ml/l) and Indofil (2.5g/l) can be used for dipping of suckers.