Micro propagation of Banana
Banana is a globally important fruit crop with 97.5 million tones of production. In India, it supports livelihood of millions of people. The banana plant (Musa sps) is a large perennial herb with leaf sheaths that form trunk-like pseudostems. The plant has 8 12 leaves that are up to 9 ft long and 2 ft wide. Root development may be extensive in loose soil in some cases up to 30 ft laterally.
Banana is a globally important fruit crop with 97.5 million tones of production. In India, it supports livelihood of millions of people. The banana plant (Musa sps) is a large perennial herb with leaf sheaths that form trunk-like pseudostems. The plant has 8 12 leaves that are up to 9 ft long and 2 ft wide. Root development may be extensive in loose soil in some cases up to 30 ft laterally.
The ovaries contained in the first (female) flowers grow rapidly, developing parthenocarpically (without pollination) into clusters of fruits, called hands. Fruits mature in about 60 90 days after flowers first appear. Each bunch of fruits consists of variable numbers of "hands" along a central stem. Each "hand" consists of two transverse rows of fruits fingers"). The fruit quality is determined by size (finger length and thickness), evenness of ripening, freedom from blemishes and defects, and the arrangement of the clusters. Edible bananas do not produce seeds.
The main method of vegetative propagation in banana is by means of daughter suckers formed at the base of the pseudo stem suckers (5 to 10 in number depending on the variety). Traditionally, sword suckers with narrow leaves, weighing approximately 500-1000 gm are the preferred planting material for vegetative propagation. The major constraint for conventionally propagating banana is the lack of ready availability of large quantities of sword suckers at an given time. The problem is felt more acutely in non-availability of sword suckers consistently. Besides, suckcrs generally may be infected with tome pathogens and nematodes. Similarly due to the variation in age and size of sucker, the crop is not uniform, harvesting is prolonged and management becomes difficult.
Therefore, in vitro clonal propagation i e. tissue culture plants (properly hardened secondary seedlings
are recommended for planting as they are healthy, disease free uniform and authentic. Banana plants produced from tissue culture are free from diseases at the time of supply and they give high yields since they are made from selected high yielding mother plants.
are recommended for planting as they are healthy, disease free uniform and authentic. Banana plants produced from tissue culture are free from diseases at the time of supply and they give high yields since they are made from selected high yielding mother plants.
The process of tissue culture consists of five important steps:
1. Initiation,
2. Multiplication,
3. Shooting & rooting,
4. Primary Hardening in green houses
5. Secondary Hardening in shade houses.
1. Initiation,
2. Multiplication,
3. Shooting & rooting,
4. Primary Hardening in green houses
5. Secondary Hardening in shade houses.
The tissue culture process involves the micro-propagation of a sucker growing point under sterile conditions. A sucker is detached from the nursery parent plant and brought to a laboratory where the outside tissue is pared away until only the growing point remains inside a plug of 10mm This is placed in a jar on agar containing a nutrient solution in a sterile environment and under controlled conditions of temperature and light. The growing point subdivides into several shoots, which are subdivided and re-established on fresh agar. This process is called sub-culturing.
The shoots are every day checked for contamination and the contaminated shoots are transferred to a fresh medium. Meanwhile a set of well grown healthy shoots are taken for rooting.
MASS MULTIPLICATION
Contamination free explants are further cultured on multiplication media supplemented with plant growth hormones which help in proliferation of auxiliary buds (cytokinins) into multiple shoots. These shoots are divided and multiplied to bulk up the multi culture stock The multiplication cycles are restricted to 8 because beyond that banana is genetically highly unstable.
Contamination free explants are further cultured on multiplication media supplemented with plant growth hormones which help in proliferation of auxiliary buds (cytokinins) into multiple shoots. These shoots are divided and multiplied to bulk up the multi culture stock The multiplication cycles are restricted to 8 because beyond that banana is genetically highly unstable.
SHOOTING
Multi cultures are further divided and transferred to shooting media which is composed of uxins (PGR) to get the elongation. In this stage, leaves will develop and the whole plant will grow up to 4 to 5 cm.
Multi cultures are further divided and transferred to shooting media which is composed of uxins (PGR) to get the elongation. In this stage, leaves will develop and the whole plant will grow up to 4 to 5 cm.
ROOTING
Plantlets from shooting media are separated and single plant let's are transferred to media containing charcoal and auxins or medium without any growth regulators. It will take 2-3 weeks for rooting and fresh roots arise at the base of the shoot. In this stage, roots will develop and plants will be ready for dispatch from. laboratory.
Plantlets from shooting media are separated and single plant let's are transferred to media containing charcoal and auxins or medium without any growth regulators. It will take 2-3 weeks for rooting and fresh roots arise at the base of the shoot. In this stage, roots will develop and plants will be ready for dispatch from. laboratory.
AGAR WEANING OF PLANTS
Well developed single plantlets need to be removed from the culture incubation room end exposed to ambient conditions in the culture vessel for four to five days. The plantlets are then carefully removed and the roots washed in running tap water. Depending on the parameters such as location the site of planting, soil quality and the climatic conditions defined by the customer, the ex-agar plant for sale could be in vitro rooted plants or only the shoots.
Well developed single plantlets need to be removed from the culture incubation room end exposed to ambient conditions in the culture vessel for four to five days. The plantlets are then carefully removed and the roots washed in running tap water. Depending on the parameters such as location the site of planting, soil quality and the climatic conditions defined by the customer, the ex-agar plant for sale could be in vitro rooted plants or only the shoots.
When the tissue culture piants are sold at this stage, the plants are washed in sterilized water to rentowe the agar medium. The plants after being removed from nutrient media should preferably be transplanted within 72 hours. Polybags is separated from the plant without disturbing the root ball of the plant and then plants are planted in the pits keeping the pseudo-stem 2 cm below the ground level soil around the plant is gently pressed. Deep planting should be avoided.
PRIMARY HARDENING
A quick dip in 0.5% Bavistin solution follows and finally in-vitro plants are transferred to trays containing sterilized coco peat. These trays are kept under tunnels made of transparent PP Plastic sheets to maintain the humidity above 80%. These tunnels should be under 50% to 75% shade nets. Primary hardening will take at least 4 weeks depending upon the climatic conditions. In final week, these trays are gradually exposed to 50% shade by removing plastic sheets. These plantlets are sprayed with fungicides, bactericide, and water soluble fertilizers as per schedule.
A quick dip in 0.5% Bavistin solution follows and finally in-vitro plants are transferred to trays containing sterilized coco peat. These trays are kept under tunnels made of transparent PP Plastic sheets to maintain the humidity above 80%. These tunnels should be under 50% to 75% shade nets. Primary hardening will take at least 4 weeks depending upon the climatic conditions. In final week, these trays are gradually exposed to 50% shade by removing plastic sheets. These plantlets are sprayed with fungicides, bactericide, and water soluble fertilizers as per schedule.
SECONDARY HARDENING
Primary hardened plants after 4 to 5 weeks are transferred to Poly bags (Nursery Rags) of suitable size. Soil mixture is prepared by mixing sand, soil and farm yard manual into 12:1 ratio. The plants are kept in these Poly bags for 6 to 8 weeks under 50% shades. Humidity is maintained around 60% to 70% and regular foliar sprays of plant protection chemicals and water soluble fertilizers are given regularly. Any possible variation if observed is discarded at this stage. The plant ready for sale will be having 5 to 6 opened leaves and almost 1 feet in height. The plantlets after acclimatization should be transported to the required place. Normal transportation is done where the plants are placed and grown in plastic bags. Well grown plants are removed to provide space in green house for the next cycle of plants and also to lower the cost of storage
Primary hardened plants after 4 to 5 weeks are transferred to Poly bags (Nursery Rags) of suitable size. Soil mixture is prepared by mixing sand, soil and farm yard manual into 12:1 ratio. The plants are kept in these Poly bags for 6 to 8 weeks under 50% shades. Humidity is maintained around 60% to 70% and regular foliar sprays of plant protection chemicals and water soluble fertilizers are given regularly. Any possible variation if observed is discarded at this stage. The plant ready for sale will be having 5 to 6 opened leaves and almost 1 feet in height. The plantlets after acclimatization should be transported to the required place. Normal transportation is done where the plants are placed and grown in plastic bags. Well grown plants are removed to provide space in green house for the next cycle of plants and also to lower the cost of storage
Problem of Banana micro propagation
Banana tissues often suffer from excessive blackening caused by oxidation of ployphenolic compounds released from wounded tissues Therefore, during first 4-6 weeks, fresh shoot tips are transferred to new medium every 1-2 weeks. Alternatively, freshly initiated cultures can be kept in complete darkness for one week. Anti oxidants such as ascorbic acid or citric acid in concentrations ranging from 10-150 mg/L, are added to the growth medium to reduce blackening or the explants are dipped in anti oxidant solution (Cysteine 50 mg/L) prior to their transfer to culture medium. Tissue culture is the most rapid method of propagation of valuable disease-free material
Banana tissues often suffer from excessive blackening caused by oxidation of ployphenolic compounds released from wounded tissues Therefore, during first 4-6 weeks, fresh shoot tips are transferred to new medium every 1-2 weeks. Alternatively, freshly initiated cultures can be kept in complete darkness for one week. Anti oxidants such as ascorbic acid or citric acid in concentrations ranging from 10-150 mg/L, are added to the growth medium to reduce blackening or the explants are dipped in anti oxidant solution (Cysteine 50 mg/L) prior to their transfer to culture medium. Tissue culture is the most rapid method of propagation of valuable disease-free material
