Chapter No. 3 Introduction to Banana Tissue Culture
INTRODUCTION TO BANANA TISSUE CULTURE Contents 3.1 Origin of Banana Fruit 3.2 Commercial Banana Cultivation 3.3 Difference Between Conventional and Scientific Banana Farming 3.4 Brief History of Tissue Culture of Banana 3.5 Banana Tissue Culture Technology 3.6 Merits of Tissue Culture of Bananas 3.7 Scientific Plantation Method 3.8 Cost-Benefit Analysis 3.9 Marltet Potential 3.10 Banana Export 3.11 Conclusion
INTRODUCTION TO BANANA TISSUE CULTURE 3.1 Origin of Banana Fruit Banana is believed to have originated in South Eastern Asia with India as one of the centers of origin apart from Indonesia, Phihppines, and Malaysia etc. Edible banana varieties are mostly hybrids of two wild seeded species namely Musa Acuminata and Musa Balbisiana. i Banana is one of the popular fruits in India because of its low cost and free availability. Banana provides a balanced diet than other fruits. Banana is composed of mainly water and carbohydrates which provides energy (104 Cal. Per 100 g.) It is rich in Minerals, Phosphorus and Calcium. 2 Banana ranks first in production and second in area among the fruits grown in India, accounting for the production of 104 lakh tons annually from an area of 4 lakh hectors. Its share in total fruit production is 32 percent from 12 percent area under fruits. The important banana growing states are Maharashtra, Gujrat, Tamil Nadu, Karnataka, Andhra Pradesh, Kerala, Orissa, Bihar, West Bengal and Assam. 1. Seminar on technological advancement in Banana production handling and processing management - Souvenir 27-28 March 1999 S. Uma, B. Shyam, S. D. Pandey and M. Dayarani. National Research Centre on Banana No. 44, Ramalinga Nagar South Extn. Vayalur Road, Trichy 620 017. Pg. 10 2. Seminar on technological advancement in Banana production handling and processing management - Souvenir 27-28 March 1999 B.M.C. Reddy and Prakash Patil. AICRP on Tropical Fruits. Indian Institute of Horticultural Research. Hessaraghatta, Bangalore 560 089. Pg. 1-3,6 31
Climatic Requirements Banana is strictly a tropical plant, however, it is also grown in subtropical to humid climates. The optimum temperature required for bananas is between 25 to 30 C, with upper and lower limits of about 40 and lo^c respectively. For the optimum growth of the banana plant, the area should have a monthly rainfall of 20-22 mm. distributed evenly. Strom and strong wind of 40 to 60 km/hr can cause damage to leaves and wind velocity of 95km/hr can cause complete destruction of banana plantations. Banana must be protected from strong winds by planting windbreak of tall trees in the windward side. 3 Soil Requirement Banana is grown successfully on a wide range soil. It can be grown on all type of soils except deep black cotton and pure sandy soils where small remedial measures are to be taken. Since banana crop is sensitive to water logging, soil with good drainage with low water table meter should be selected. A soil ph of 5.5 to 8.0 is found to be the optimum. Varieties In Maharashtra different banana varieties like Safed Velchi, Dwarf Cavendish, Robusta, Grand nain, Sindhurni, Hanuman, Ardhapuri, Lalvelchi and Rajeli are grown. 4 3. Seminar on technological advancement in Banana production handling and processing management - Souvenir 27-28 March 1999 B.M.C. Reddy and Prakash Patil. AICRP on Tropical Fruits, hidian Institute of Horticultural Research. Hessaraghatta, Bangalore 560 089. Pg. 1-3,6 4. Seminar on technological advancement in Banana production handling and processing management - Souvenir 27-28 March 1999 Table 4 Banana Cultivars grown in different states. Singh and S. Uma, 1996. Pg. 15 32
Planting For Dwarf Cavendish varieties optimum plantation i.e. higher density plantation of (4444 plants per hector) with closer spacing of (1.5 x 1.5 m.) was recommended. 5 Farmers used the recommended spacing which was calculated as 5'x5' and plantation of 1700 banana plants per acre. Fertilizers The fertilizer recommendation for Cavendish varieties is 200 g. N, 100 g. P 2O5 and 200 g. K2O/PIant/crop. Nitrogen and Potash are to be applied in four equal split doses at 30,75, 120 and 165 days after planting while that of phosphorus cab be applied at the time of planting, g Drip Irrigation Drip irrigation is network of pipes through which water is supplied to the plant. Drip irrigation was used for banana plantation which was also useful in providing liquid nutrients and showed better performance. Fruiting and Covering Banana bunches matures in about 110 to 130 days after flowering. The grown bunches are covered with polythene bags to protect it from the sun. Yield The average yield range of Cavendish is 50-100 tons / hector, however 150 tons/hector can be obtained under good cultural practices with high density planting. 7 5., 6., 7. Seminar on technological advancement in Banana production handling and processing management - Souvenir 27-28 March 1999 B.M.C. Reddy and Prakash Patii. AICRP on Tropical Fruits. Indian Institute of Horticultural Research. Hessaraghatta, Bangalore 560 089. Pg. 1-3,6 33
3.2 Commercial Banana Cultivation Commercial banana cultivation requires specific climatic conditions, soil, irrigation, fertigation, planting material and post harvest technologies. By following scientific method of plantation, the grower can produce optimum and best qualities of yields and earn more profits. Banana is a crop which can be grown throughout the year. This has least gestation period where the first yield can be expected in just 11 to 12 months from the period of transplanting. Reduced gestation period greatly helps in achieving higher profits. To achieve reduced gestation period, selection of proper planting material is required. Planting Material Banana is commonly propagated through sword suckers having narrow leaves. Three month's old suckers with developed rhizomes are used for plantation. Disease free planting material is made ready for plantation. Though some farmers are using sword suckers for banana plantation, many farmers are now using Micropropagated plants for plantation. Tissue cultured banana plants are getting commercial acceptance due to their qualities viz. superior, disease free varieties and mass production. From tissue culture of bananas no genetic improvement was expected, yet these plants have proved better, compared to traditional planting material, only under a good management system. 34
3.3 Difference between conventional and scientific Banana Farming Conventional Farming: Suckers are used as planting material, which may not be free from any infections and can be of inferior quality. In such case, there can be some mortality. As characteristics of the parent material is not exactly known, yield upto the expectations may not be achieved. The banana plant yields, on an average, about 25 kg per plant. Total plant population is from 1200 to 1380 plants per acre. Water is fed either through flood, furrow or basin system so there is possibility of under or over water resulting yield variation. As solid fertilizers are applied manually, there is high labour cost and the fertilizer is not utilized fully due to either under or over watering. As flowering and fruiting is inconsistent and at indifferent times, market acceptability is moderate. Total profit is not so high in unit area, which is due to less plant population and mortality. Scientific Farming: Tissue cultured plants are used as planting material, which are free from all infections, resulting no mortality of plants. The yields are very high, upto the expectation. Plants yield on an average of about 40 kg per plant. Total Plant population is about 2003 plants per acre according to high-density method of plantation. As water is fed through drip irrigation, the desired amount of water is available directly to the root zone. So higher yield is expected. As water solubleorliquid fertilizers are used through drip irrigation, there is a saving of labour cost and fertilizer is used fully as it is directly available to the root zone. As flowering and fruiting is uniform and timely, market acceptability is very high. Total profit is very high due to high-density i.e. high population and nil or negligible mortality. 35
3.4 Brief history of tissue culture of banana Every plant cell has the potential to regenerate into a single plant. When this regeneration of the plant cell into a single plant rapidly takes place at optimum level, it is called as Micro propagation. When a cell is born, it divides, grows or dies. These cells are kept in a disease free, clean and controlled environment with artificial light and temperature where cells start growing and give desirable results. Soon after, reports of banana plantlets produced by in vitro shoot tip culture came from Taiwan (Ma and Shii, 1974). Nearer home, intense work was initiated at the IIHR by Dr. R. Doreswamy and co-workers (Doreswamy et al., 1983). Eversince, several investigators the world over have reported success (Cronauer and Krikorian, 1986). g It was found that with the help of tissue culture technology constant improvement in the productivity, profitability, stability and sustainability of the banana plantation system could be achieved. 3.5 Banana Tissue Culture Technology Scientists experimented on the different nutrient medias and observed where the plant showed best results. These results were recorded and when standardized, these medias were called as the Protocols to produce those particular plants. The media for each and every stage of growth was different. For Initiation, Muhiplication, Rooting different medias were prepared. 8. Tissue culture propagation of Banana. Dr. R. M. Pandey, Director, Indian Institute of Horticultural Research. Hessaraghatta Lake P.O. Bangalore 560 089 Pg. 2 36
Banana tissue culture involved following steps: Procurement of mother plants Disease free banana plantation areas are located. From these areas, the best yielding farms are chosen. The elite mother plants are selected from the disease free farms and maintained under hygienic conditions by spraying fungicides, bactericides and insecticides. At the time of use, these plants are sterilized and then used for initiation. In the laboratory five major steps are involved in the production process. Stage I: Initiation In the laboratory, after the surface sterilization of the plant part, the innermost tissue (ex-plant) is dissected in sterile conditions and put onto the initiation medium for growth. Initiation medium contains micro and macro elements, vitamins, irons and growth promoting hormones, solidified by agar agar. (Figure 1.) Stage II : Multiplication This is the next stage to multiply the plants in sterile conditions. When the tissue starts growing and forms a shoot, it is transferred to another medium containing growth-promoting hormones that enhance the cell division. The growing shoot multiplies and forms a cluster of three or four shoots. Same cycles are repeated for ten to twelve times to reach to the optimum production. (Figure 2.) 37
stage III : Shooting and rooting When the plant is ready, it has to be transferred to the rooting medium. The single shoots are separated and placed onto a shooting and rooting medium. At this stage the hormones may or may not be required. The shoot elongates and new roots come up. Rooting takes about three or four weeks and the plant becomes ready for hardening. (Figure 3.) Stage IV : Semi-Hardening In the semi-hardening process, the plants are made ready to sustain in the natural farm conditions. Hardening is done in the controlled conditions of the green house. The plants are taken out of the bottle and the media adhering to the root system is washed fiilly. Afterwards, the plants are graded as per their size and then transferred singly into the seed tray containing sterile, soil-less medium (a mixture of peat moss, soilrite, sand or perlite). These trays are kept in the humidity chambers for six weeks and thereafter they are kept in open in the Green House. Regular spraying of fungicides, bactericides and insecticides is done to achieve good hygienic condition of the plants. (Figure 4.) Stage V : Hardening In the hardening process, the plants from the seed tray are separated and transferred into polythene bags preferably black coloured, containing a mixture of sand, soil, peat moss, soilrite, perlite or compost. These plants are kept in the shade-house where Fifty to Seventy-five percent of the sunlight is reduced through the nets and entry of insects is also eliminated. Irrigation is done by drip in each polybag and sprinklers or misters maintain humidity. This hardening also takes another six weeks and the plants get fully acclimatized to environmental stresses. These plants are directly used for planfing into the field. (Figure 5.) 38
Figure 1. Laboratory technicians at initiation stage of banana Figure 2. Laboratory technicians at banana multiplication 39
Figure 3. Grown banana plants at rooting stage Figure 4. Semi-hardened banana plants in humidity chambers 40
Figure 5. Hardened banana plant ready for sale Flow Chart No. 2 Process Flow Chart For Tissue Culture of Banana Selection of Elite Plants ight Selection of Daughter Sword Sucker i Surface Sterilization of Sucker and dissection of the shoot tips under aseptic condition i Inoculation of shoot tip on nutrient media and incubation in controlled microclimatic conditions i Establishment of cultures and origin of new shoots I Multiplication of Shoots Elongation or Shooting / Rooting Primary hardening or rooted plants in green house Planting in the field 41
Recent experiments conducted to evaluate the performance of tissue cultured seedlings v/s normal suckers planting in Basrai Banana: 9 A study was conducted to evaluate the performance of tissue culture seedlings V/s normal suckers planting during 1994-95 at Banana Research Station, Jalgaon, Maharashtra. The results of this experiments revealed that tissue culture seedlings planting was found significantly superior over normal sucker planting for all growth, yield and yield attributing characters studied. The commercial method of propagation of banana is by rhizome planting. Now a days, use of tissue culture seedlings as a planting material in banana is becoming popular in this region. To evaluate the prospects and performance of tissue cultured seedlings in comparison with normal suckers as planting material, a comprehensive study was made. Materials and methods: The experiment was conducted about materials and methods used in banana tissue culture during 1994-95. Two treatments viz., use of tissue culture seedling and normal sucker as planting material in randomized and block design replicated six times. Almost uniform suckers of Basrai and tissue culture seedlings were planted at the spacing of 1.5 m x 1.5 m and all plant received uniform dose of NPK. Observations on plant characters; crop, duration, yield and yield attributes were recorded. 9. Seminar on technological advancement in Banana production handling and processing management - Souvenir 27-28 March 1999 CD. Badgujar, S. S. Deshmukh, R. M. Birhade, C. R. Palwe and N. M. Patil. Banana Research Station, M.P.K.V., Jalgaon -425001. Pg. 23 42
The results obtained in this experimented were as follows: The observations on growth and yield parameters were recorded and presented. From the data it revealed that, all the growth and yield characters were found significantly superior in tissue culture planting over normal sucker planting. Average height and girth of pseudostem and average number of leaves per plant 157.35 cm, 63.28 cm. and 36.48 respectively were noticed with tissue culture plant treatments, which was significantly superior over normal sucker planting treatment. Least days for flowering (343.00) and harvesting (438) were noticed with tissue culture planting treatment, which was superior over normal sucker planting treatment. The corresponding figures were 380.13, 477 respectively under normal sucker planting. Thus tissue culture seedlings flowered 37 and matured 39 days earlier than normal sucker planting. The yield and yield parameters viz., average number of hands, fingers per bunch, length and girth of fingers were found significantly superior in tissue culture seedling treatment. (8.59, 142.07, 2034 cm and 11.46 cm) respectively. The corresponding figures in normal sucker planting treatments were 6.55, 91.60, 19.60 cm and 10.89 cm. Maximum average bunch weight (12.69 kg) was noticed under tissue culture planting which was significantly superior over normal sucker planting treatment (9.66 kg). 43