MICRO-PROPAGATION TECHNIQUE IN PINEAPPLE
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
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.
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| Fig: ex vitro gradual performance of tissue culture grown seedlings of pineapple |