Banana tissue culture and synthetic seeds: Banana is a globally important fruit crop with 97.

5 million tones of production. In India it supports livelihood of millions of people. With total annual production of 16.91 million tons from 490.70 thousand ha. with national average of 33.5 T/ha. Maharashtra ranks first in production with 60 T/ha. Banana contributes 37% to total fruit production in India. Banana is one of the major and economically important fruit crops of Maharashtra. Banana occupies 20% area among the total area under crop in India. Maharashtra ranks second in area and first in productivity in India. Jalgaon is a major Banana growing district in Maharashtra which occupies 50,000 hectares area under Banana. But most of Banana is grown by planting suckers. The technology development in agriculture is very fast, it results in developing Tissue Culture Technique. Tissue culture means cloning and micro-propagation of tissues of the selected Elite plants and daughter suckers. The process consists of five important steps: Initiation, Multiplication, Shooting & rooting, Primary Hardening in green houses and Secondary Hardening in shade houses. Strict adherence to aseptic standards and micro-climatic conditions and care during the hardening process alone can ensure success. Banana is basically a tropical crop, grows well in temperature range of 13C 38C with RH regime of 75-85%. In India this crop is being cultivated in climate ranging from humid tropical to dry mild subtropics through selection of appropriate varieties like Grandnaine. Chilling injury occurs at temperatures below 12C. The normal growth of the banana begins at 18C, reaches optimum at 27C, then declines and comes to a halt at 38C. Higher temperature causes sun scorching. High velocity wind which exceeds 80 km phrs damages the crop. Soil for banana should have good drainage, adequate fertility and moisture. Deep, rich loamy soil with pH between 6-7.5 are most preferred for banana cultivation. Ill drained, poorly aerated and nutritionally deficient soils are not suitable for banana. Saline solid,

calcareous soil are not suitable for Banana cultivation. Avoided soil of low laying areas, very sandy & heavy black cotton with ill drainage. Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part of the group of technologies called plant biotechnology. Tissue culture has been exploited to create genetic variability from which crop plants can be improved, to improve the state of health of the planted material and to increase the number of desirable germplasms available to the plant breeder. Tissue-culture protocols are available for most crop species, although continued optimization is still required for many crops, especially cereals and woody plants. Tissue culture techniques, in combination with molecular techniques, have been successfully used to incorporate specific traits through gene transfer. In vitro techniques for the culture of protoplasts, anthers, microspores, ovules and embryos have been used to create new genetic variation in the breeding lines, often via haploid production. Cell culture has also produced somaclonal and gametoclonal variants with crop-improvement potential. The culture of single cells and meristems can be effectively used to eradicate pathogens from planting material and thereby dramatically improve the yield of established cultivars. Large-scale micropropagation laboratories are providing millions of plants for the commercial ornamental market and the agricultural, clonally-propagated crop market. With selected laboratory material typically taking one or two decades to reach the commercial market through plant breeding, this technology can be expected to have an ever increasing impact on crop improvement as we approach the new millenium. A soil that is not too acidic & not too alkaline, rich in organic material with high nitrogen content, adequate phosphorus level and plenty of potash are good for banana. Prior to planting banana, grow the green manuring crop like daincha, cowpea etc. and burry it in the soil. The land can be ploughed 2-4 times and leveled. Use ratovator or harrow to break the clod and bring the soil to a fine tilt. During soil preparation basal dose of FYM is added and thoroughly mixed into the soil. A pit size of 45cm x 45cm x 45cm is normally required. The pits are to be refilled with topsoil mixed with 10 kg of FYM (well decomposed), 250 gm of Neem cake and 20 gm of

conbofuron. Prepared pits are left to solar radiation helps in killing the harmful insects, is effective against soil borne diseases and aids aeration. In saline alkali soil where PH is above 8 Pit mixture is to be modified to incorporate organic matter. Addition of organic matter helps in reducing salinity while addition of purlite improves, porosity and aeration. Alternative to planting in pits is planting in furrows. Depnding on soil strata one can choose appropriate method as well as spacing and depth at which plant is required to be planted. Sword suckers weighing approximately 500-1000 gm are commonly used as propagating material. Suckers generally may be infected with some 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 are recommended for planting. They are healthy, disease free, uniform and authentic. Properly hardened secondary seedlings are only recommended for planting Planting of tissue culture Banana can be done throughout the year except when the temperature is too low or too high. Facility of drip irrigation system is important. There are two important seasons in Maharashtra, India;

Mrig

Baug

(Kharif)

Month

of

planting

June

July.

Kande Baug (Rabi) Month of planting October - November. 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 2cm below the ground level. Soil around the plant is gently pressed. Deep planting should be avoided.

Banana, a water loving plant, requires a large quantity of water for maximum productivity. But Banana roots are poor withdrawal of water. Therefore under Indian condition banana production should be supported by an efficient irrigation system like drip irrigation. Water requirement of banana has been worked out to be 2000mm per Annum. Application of drip irrigation and mulching technology has reported improved water use efficiency. There is saving of 56% of water and increasing yield by 23-32% under drip. Irrigate the plants immediately after planting. Apply sufficient water and maintain field capacity. Excess irrigation will lead to root zone congestion due to removal of air from soil pores, thereby affecting plant establishment and growth. And hence drip method is must for proper water management in Banana Month Baug) June July August September October November December January February March April May (MaugQty. (lpd.) 06 05 06 08 10-12 10 10 10 12 16-18 20-22 25-30 Month baug) October November December January February March April May June July August September (KandeQty. (lpd.) 04-06 04 04 06 08-10 10-12 16-18 18-20 12 12 14 14-16

Banana requires high amount of nutrients, which are often supplied only in part by the soil. Nutrient requirement has been worked out on all India basis is to be 20 kg FYM, 200gm N; 60-70gm P; 300gm K/plant. Banana requires heavy nutrition. Banana crop requires 7-8 Kg N, 0.7- 1.5 Kg P and 17-20 Kg K per metric tonne yield. Banana responds well to application of nutrients. Traditionally farmers use more of urea and less of phosphorous and potash. In order to avoid loss of nutrients from conventional fertilizers i.e. loss of N through leaching, volatilization, evaporation and loss of P and K by fixation in the soil, application of water soluble or liquid fertilizers through drip irrigation (fertigation) is encouraged. A 25-30% increase in yield is observed using fertigation. Moreover, it saves labour and time and the distribution of nutrients is uniform

Water Soluble Solid fertilizers Schedule of water soluble fertilizer application. Qty. per 1000 Period Grade plants basis 4.13 (Kg)Total Qty. every 4th day (Kg.) 82.60

After planting uptoUrea

12:61:003.00 60.00 00:00:505.00 100.00 Urea 6.00 120.00 65 to 135 days 12:61:002.00 40.00 00:00:505.00 100.00 Urea 6.50 65.00 135 to 165 days 00:00:506.00 60.00 Urea 3.00 150.00 165 to 315 days 00:00:506.00 300.00 Schedule is directive only and may change according to planting season and soil fertility 65 days status (soil analysis). Interculture Operations The Root system of banana is superficial and easily damaged by cultivation, use of intercrop which is not desirable. However short durational crops (45-60 days) like mung, cowpea, daincha are to be considered as green manuring crops. Crops from cucurbitaceous family should be avoided as these carry viruses.

Weeding Spraying of Glyphosate (Round up) before planting at the rate of 2 lit/ha is carried out to keep the plantation weed free. One or two manual weedings are necessary. Micronutrient Foliar Spray Combined foliar application of ZnSo4 (0.5%), FcSo4 (0.2%), CuSo4 (0.2%) and H3Bo3 (0.1%) can be adopted to improve morphological, physiological and yield attributes of banana. The micronutrient spray solution is prepared by dissolving the following in 100 lit. of water. Zinc sulphate Ferrom sulphate Copper sulphate Boric acid 500 gm 200 gm 200 gm 100 gm For every 10 litre of mixture 510ml of sticker solution such as Teepol should be added before spraying.

Desuckering Removal of unwanted suckers is a critical operation in banana for reducing internal competition with mother plant. Desuckering should be done regularly until shooting. However in areas where ratoon is also taken for the second crop, a follower is allowed after inflorescence has appeared and this should be managed that planting space is not disturbed. Follower should be opposite to the inflorescence. It should not be far apart from the main plant. Deflowering It consists of removal of the withered style and perianth. This is generally not practiced. Therefore, they remain attached to the fruit bunch & then removed after harvesting which is damaging to the fruits. It is therefore suggested that you remove them just after flowering. Pruning of leaves Rubbing leaves damages the fruit, therefore, such leaves should also be pruned during regular check. Older leaves and infected leaves also be pruned as required. Green leaves should not be removed. Earthing up Keep the soil loose by harrowing from time to time. Earthing up should be done at 3-4 months after planting i.e. raising the soil level around the base of the plant by 10-12. It is better to prepare a raised bed and keep the drip line on bed 2-3 away from the plant. It also helps to protect plants from wind damage and production losses to some extent.. Removal of male buds (Denavelling) Removal of male buds helps fruit development and increases bunch weight. Male buds are removed from the last 1-2 small hands with a clean cut keeping a single finger in the last hand. Bunch Spray Spray of monocrotophos (0.2%) after emergence of all hands takes care of the thrips. Thrips attack discolors the fruit skin and makes it unattractive. Bunch Covering

Covering bunch using dried leaves of the plant is economical and prevents the bunch from direct exposure to sunlight. Bunch cover enhances quality of fruit. But in rainy season this practice should be avoided. Sleeving of bunch is done to protect fruits against dust, spray residue, insect and birds. For this blue plastic sleeves are preferred. This also increases temperature around developing bunch and helps in early maturity. Dehandling of false hands of bunch In a bunch there are some incomplete hands which are not fit for quality produce. These hands should be removed soon after bloom. This helps in improving the weight of other hands. Sometimes the hand just above the false hand is also removed. Propping Due to heavy weight of bunch the plant goes out of balance and the bearing plant may lodge and production and quality are adversely affected. Therefore they should be propped with the help of two bamboos forming a triangle by placing them against the stems on the leaning side. This also helps in uniform development of bunch. Pest and disease management A large number of fungal, viral and bacterial diseases and insect pests and nematodes infest the banana crop and reduce production, productivity and quality. Summary details of major pest and diseases of banana along with control measures are given herewith: Sno.Name Pest i) Rhizome Sordidus) Symptoms weevila) creates network galleries rhizome weakens plant. Control measures

Largea) Use healthy planting ofmaterial b) Sanitation in in orchard and c) Trapping of the adult weevils using pseudostem or

(Cosmopolites

rhizome pieces and d) application carbufuran @.2gm/plant ii) Pseudostem weevila) Small holesa) Management (Odaiporous longicolis) on pseudostem with exudation transparent gummy substance b) Existenceb) tunneling leaf Secondly, approach identical of is to Soil of

rhizome weevil

ininjection of lime

sheathsolution 150 ml in 350 ml water) using stem injector 4 ft. 30 above angle the is ground level at

and inner core(Monocrotophos of the stem

recommended. c) Abortion ofc) Use bunches longitudinal split (30cm length) or disc on stump traps @ 100/ha. Keep

the split portion of tray facing the ground. Collected weevils are then iii) Thrips killed. a) They scrapa) Spray or

(Chaetanaphotrips from attackedinject & signipennis &plant Heliaothrips kodaliphilus) and them organsMonocrotophos render@ 0.05% on the browninflorescence the

and discoloredbefore

especially theunfurling of top fruits. most bract. iv) Fruit scarring battlea) Adults feeda) Sanitation (Besilepta subcostatum) on leaves fruits tenderspray of 0.05% moncrotophos andor of 0.1% the andcarbaryl on the unfolded

cause scarringheart

of skin plants b) Plant losses immediately its vigour and after the quality of emergence of bunch is poor) new foliage and during season fruiting is

recommended. v) Aphids (Pentaloniaa) They area) Spray of nigronervosa) vecturs banana of0.1% monocrotophos

bunchy and seen can

topor

0.03%

visus (BBTV)phosphonidon beon the leaves is aseffective

congregation under the leaf base vi) Nematodes of Apply pseudo stem a) Stunteda)

growth corbofuron @40 b) Small gm per plant at leaves planting & 4 month c) roots d) after planting. Cuttedb) Use neem cake as organic manure. Purplec) Use merigold

black lesionsas trap crop. on roots and their splitting. Fungal Diseases vii) Panama (Furarium oxysporium) wilta) Yellowinga) Cultivation of of old leavesresistant cultivar progressing towards younger leaves. (Covendish b) group) Affectedb) Trim and 0.1%

leaves petiole

treat the suckers andBavistine before

collapse nearin

hang. plant. c) Pseudoc) stem splittingbioagents is common.

Apply like

trichoderma and Pseudomonas fluorescence with organic

d) brown in

manure Reddishd) Keep good drainage lot and of

discoloration apply section of rootin field.

cross-organic manure

& rhizome viii Head rot (Erwiniaa) Rotting ofa) Use healthy carotovora) collar and regionplanting epinastymaterial

of leaves) b) On pulling out of affected plant, plant from collar leaving the topples the region the

corn with root in soil c) On openingb) Drench plants up of collarwith region affected ofEmison followed by 0.1%

plants, yellowish reddish

another todrenching after ooze3 months. Avoid in and in drained

can be seen. d) In earlyc) stage brown soaked in ofplanting orpoorly areas critical infection, darkrocks yellow, watersoils.

region which may decay to form cavities surrounded by dark spongy isa) Remove tissues. ix) Sigatoka leaf spota) It (Mycospharella spp) by leaves,

characterized infected leaves smalland destroy the lesions on the lesion become pale yellow to greenish yellow streaks visible both surfaces from the of

leaf b) Thereafter

linear brownish blackish streaks appear. c) The centreb) Keep proper of the streakdrainage eventually give appearance of eye spot. d) Some timesc) Spray dithane premature ripening observed M-45 isg/ha) Bavistine g/ha. Viral Diseases i) Banana Bunchya) Appearancea) Top Virus(BBTV) of dark 'Morse irregular,free greenmaterial Use virus planting i.e. (1250 or 500 avoid dries up andlogging. and water to

code'Tissue Culture. b) Survey and streaks along eradicate secondary infected plants veins on regularly. leaves on underside of the leaves. b) Leaf size isc) Control insect reduced andvectors leaves remainsespecially

abnormally erect,

aphids

and

brittlemealy bugs. d) Indexing and results. should be followed in the case c) each and of mass multiplication Leavese) Prohibit of other,any plant part bunchedfrom diseased area to healthy

short, close tomovement

at the top

area. d) The tips off) Use resistant the bracts incultivar. male buds Avoid of have greenish. e) Virus isg) spread through aphids. ii) Banana Mosaica) with chlorotic growing

alternate lost as mixed crop or in

near by areas. Chlorosisa) Elimination mildof plants affected and free

Virus (BMV)

streaks alongmaintenance of the veins theydisease never BSV. turnplantation of disease free planting

necrotic as inthrough the use

material Tissue iii) Banana Mosaic (BBMV) seeding. Bracta) Presence ofa) Use Virusspindle shapeddisease pinkish reddish streaks mid petioles iv) Banana Virus (BSV) toplanting material onTissue ribs, and Use

i.e. culture of free i.e. culture

pseudo stem,seeding.

lamina. Streaka) Presence ofa) chlorotic flecking small systematic includes yellow, brown and cigar necrosis, based pseudo stem splitting internal internal pseudo stem black leaf streaking,

of free i.e.

inconspicuous disease planting tomaterial lethalTissue seedings.

culture

necrosis, and

necrosis formation small deformed bunches. Harvesting

and of

Banana should be harvested at the physiological maturity stage for better post harvest quality. The fruit is climacteric and can reach consumption stage after ripening operation Maturity indices These are established on the basis of fruit shape, angularity, grade or diameter of the median figure of the second hand, starch content and number of days that have elapsed after flowering. Market preferences can also affect the decision for harvesting a slight or full mature fruit. Removal of bunch Bunch should be harvested when figures of second hand from top are 3/4 rounded with the help of sharp sickle 30cm above the first hand. Harvest may be delayed upto 100-110 days after opening of the first hand. Harvested bunch should generally be collected in well padded tray or basket and brought to the collection site. Bunches should be kept out of light after harvest, since this hastens ripening and softening. For local consumption, hands are often left on stalks and sold to retailers. For export, hands are cut into units of 4-16 fingers, graded for both length and girth, and carefully placed in polylined boxes to hold different weight depending on export requirements. Post harvest operations At collection site injured and over mature fruits are discarded and for local market bunches should be delivered through lorries or wagons. However, for more sophisticated and export market where the quality is predominant, bunches should be dehanded, fruits are cleared in running water or dilute sodium hypochlorite solution to remove the latex and treated with thiobendasole; air dried and graded on the basis of size of fingers as already stated, packed in

ventilated CFB boxes of 14.5 kg capacity or as per requirement with polythene lining and pre-cooled at 13-15C temperature and at 80-90% RH. Such material should than be sent under cool chain at 13C for marketing Yield The planted crop gets ready for harvest within 11-12 months of planting. First ratoon crop would be ready by 8-10 month from the harvesting of the main crop and second ratoon by 8-9 months after the second crop. Thus over a period to 28-30 months, it is possible to harvest three crops i.e. one main crop and two ratoon crops. Under drip irrigation combined with Fertigation yield of Banana as high as 100 T/ha can be obtained with the help of tissue culture technique, even similar yield in the ratoon crops can be achieved if the crop is managed well.

Synthetic seeds
SYNTHETIC seeds are defined as artificially encapsulated somatic embryos, shoot buds, cell aggregates, or any other tissue that can be used for sowing as a seed and that possess the ability to convert into a plant under in vitro or ex vitro conditions, and that retain this potential also after storage1. Earlier, synthetic seeds were referred only to the somatic embryos that were of economic use in crop production and plant delivery to the field or greenhouse. In the recent past, however, other micropropagules like shoot buds, shoot tips, organogenic orembryogenic calli, etc. have also been employed in the production of synthetic seeds. Thus, the concept of synthetic seeds has been set free from its bonds to somatic embryogenesis, and links the term not only to its use(storage and sowing) and product (plantlet) but also to other techniques of micropropagation like organogenesisand enhanced axillary bud proliferation system. Implementation of synthetic seed technology requires manipulation of in vitro culture systems for large-scale production of viable materials, that are able to convert into plants, for encapsulation. Somatic embryogenesis, organogenesis and enhanced axillary bud proliferation systems are the efficient techniques for rapid and largescale in vitro multiplication of elite and desirable plant species. Through these systems a large number of somatic embryos or shoot buds are produced which are used as efficient planting material as they are potent structures for plant regeneration either after having minor

treatment or without any treatment with growth regulator(s). Because the naked micropropagules are sensitive to desiccation and/or pathogens when exposed to natural environment, it is envisaged that for largescale mechanical planting and to improve the success of plant (in vitro derived) delivery to the field or greenhouse, the somatic embryos or even the other micropropagules useful in synthetic seed production would necessarily require some protective coatings. Encapsulation is expected to be the best method to provide protection and to convert the in vitro derived propagules into synthetic seeds or synseeds or artificial seeds The encapsulation technology has been applied to produce synthetic seeds of a number of plant species belonging to angiosperms and gymnosperms . Nevertheless, their number is quite small in comparison to the total number of plant species in which in vitro regeneration system has been established. Production of artificial seeds has unravelled new vistas in plant biotechnology. The synthetic seed technology is designed to combine the advantages of clonal propagation with those of seed propagation and storage. Despite the fact that the technology is an exciting and rapidly growing area of research in plant cell and tissue culture, there are many limitations for its practical use. The purpose of this review is to present a report on prospects and limitations of synthetic seed production. The subject has been earlier reviewed in a different context by various researchers The technology Basic hindrance to synthetic seed technology was primarily based on the fact that the somatic embryos lack important accessory tissues, i.e. endosperm and protective coatings, that make them inconvenient to store and handle. Furthermore, they are generally regarded tolack a quiescent resting phase and to be incapable of undergoing dehydration. The primary goal of synthetic seed research was, therefore, to produce somatic embryos that resemble more closely the seed embryos in storage and handling characteristics so that they can be utilized as a unit for clonal plant propagation and germplasm conservation. In achieving such a goal the technology of encapsulation has evolved as the first major step for production of synthetic seeds. Later it was thought that the encapsulated synthetic seed should also contain growth nutrients, plant growth promoting microorganisms (e.g. mycorrhizae), and/or other biological

components necessary for optimal embryo-to-plant development. A number of patents covering the development of seed analogues have been issued. However, success of the synthetic seed technology is constrained due to scarcity and undesirable qualities of somatic embryos making it difficult for their development into plants. The choice of coating material for making synseeds is also an important aspect for synseed production. Based on technology established so far, two types of synthetic seeds are known: desiccated and hydrated. The desiccated synthetic seeds are produced from somatic embryos either naked or encapsulated in polyoxyethylene glycol (Polyoxr) followed by their desiccation. Desiccation can be achieved either slowly over a period of one or two weeks sequentially using chambers of decreasing relative humidity, or rapidly by unsealing the petri dishes and leaving them on the bench overnight to dry. Such types of synseeds are produced only in plant species whose somatic embryos are desiccationtolerant. On the contrary, hydrated synthetic seeds are produced in those plant species where the somatic embryos are recalcitrant and sensitive to desiccation. Hydrated synthetic seeds are produced by encapsulating the somatic embryos in hydrogel capsules. The production of synthetic seeds for the first time by Kitto and Janick8 involved encapsulation of carrot somatic embryos followed by their desiccation. Of the various compounds tested for encapsulation of celery embryos, Kitto and Janick810 selected polyoxyethylene which is readily soluble in water and dries to form a thin film, does not support the growth of micro-organisms and is non-toxic to the embryo. Janick et al.3 have reported that desiccated artificial seeds were produced by coating a mixture of carrot somatic embryos and callus in polyoxyethylene glycol. The coating mixture was allowed to dry for several hours on a Teflon surface in a sterile hood. The dried mixture was then placed on a culture medium, allowed to rehydrate, and then scored for embryo survival. In 1984 Redenbaugh et al.11 developed a technique for hydrogel encapsulation of individual somatic embryos of alfalfa. Since then encapsulation in hydrogel remains to be the most studied method of artificial seed production. A number of substances like potassium alginate, sodium alginate, carrageenan, agar, gelrite, sodium pectate, etc. have been tested as hydrogels butsodium alginate gel is the most popular. Hydrated artificial seeds consist of somatic embryos individually encapsulatein a hydrogel To produce hydrated synthetic seeds, the somatic embryos are mixed with sodium alginate gel (0.55.0% w/v) and dropped

into a calcium salt solution [CaCl2 (30100 mM), Ca(NO3)2 (30100 mM)] where ionexchange reaction occurs and sodium ions are replaced by calcium ions forming calcium alginate beads or capsules surrounding the somatic embryos. The size of the capsule is controlled by varying the inner diameter of the pipette nozzle. Hardening of the calcium alginate is modulated with the concentrations of sodium alginate and calcium chloride as well as the duration of complexing. Usually 2% sodium alginate gel with a complexing solution containing 100 mM Ca2+ is used and is found to be satisfactory5,7,12. However, Molle et al.13 found that for the production of synthetic seeds of carrot, 1% sodium alginate solution, 50 mM Ca2+ and 2030 min time period were satisfactory for proper hardening of calcium alginate capsules. They have suggested the use of a dual \nozzle pipette in which the embryos flow through the inner pipette and the alginate solution through the outer pipette. As a result, the embryos are positioned in the centre of the beads for better protection. For the past several years other unipolar structures such as apical shoot tips and axillary shoot buds as well as apolar protocorms or protocorm-like bodies and even Undifferentiated embryogenic calli are also being employed in synthetic seed production The technology of hydrogel encapsulation is also favoured for synthetic seed production from these micropropagules. For production of synthetic seeds from apical shoot tips and axillary shoot buds, these organs are usually first treated with auxins for root induction and then their microcuttings (approximately 4 or 5 mm in length) areencapsulated in sodium alginate gel following the method described by Redenbaugh et al. for alfalfa somatic embryos. However, mulberry and banana plantlets were obtained from alginate-encapsulated shoot buds without any specific root induction treatment. To avoid bacterial contamination Ganapathi et al.15 added an antibiotic mixture (0.25 mg/l) containing rifampicin (60 mg), cefatoxime (250 mg) and tetracycline- HCl (25 mg) dissolved in 5 ml dimethyl sulphoxide to the gel matrix. Activated charcoal (0.1%) was also added to the matrix to absorb the polyphenol exudates of the encapsulated shoots of banana.

Banana Tissue culture and Synthetic Seeds

Progress in biotechnological research during the last two decades has opened up unprecedented opportunities in many areas of basic and applied biological research.Plant tissue culture, which is an important component of plant biotechnology, presents new strategies for the improvement of cereals, legumes, forest trees, plantation crops and ornamental plants. Besides, plant cell cultures provide a good system for many basic studies in plant breeding, plant physiology, genetics and cell biology. Cell manipulations through the sophisticated methods of genetic engineering for plant quality and product improvement has to rely on plant tissue culture for the final goal. Micro propagation is an area of plant tissue culture which has received maximum attention of researchers for its potential commercial applications.

Plant tissue culture refers to growing and multiplication of cells, tissues and organs of plants on defined solid or liquid media under aseptic and controlled environment. The commercial technology is primarily based on micropropagation, in which rapid proliferation is achieved from tiny stem cuttings, axillary buds, and to a limited extent from somatic embryos, cell clumps in suspension cultures and bioreactors. The cultured cells and tissue can take several pathways. The pathways that lead to the production of true-to-type plants in large numbers are the preferred ones for commercial multiplication. The process of micropropagation is usually divided into several stages i.e., prepropagation, initiation of explants, subculture of explants for proliferation, shooting and rooting, and hardening. These stages are universally applicable in large-scale multiplication of plants. The delivery of hardened small micropropagated plants to growers and market also requires extra care. The regeneration of plants through the techniques of plant tissue culture and their subsequent acclimatization and delivery to the field poses many problems to make tissue culture technology a viable alternative proposition. The successful demonstration of encapsulation of tissue culture derived propagules in a nutrient gel has initiated a new line of research on

synthetic seeds. Synthetic seeds are basically defined as, "encapsulated somatic embryos which functionally mimic seeds and can develop into seedlings under sterile conditions". In a broader sense, it would also refer to encapsulated buds or any other form of meristems which can develop into plants. Many plant systems are known to produce abundant number of embryos in culture which share many properties similar to natural embryos including germination leading to plant production. To mimic the natural seeds, embryos from cultures are encapsulated in a nutrient gel containing essential organic/inorganic salts, carbon source, plant hormones and antimicrobial agents and coated completely to protect the embryos from mechanical damages during handling and to allow the development and germination to occur without any undesirable variations. Several agents have been attempted for encapsulation and sodium alginate complexing with calcium chloride is found to be the most suitable. Bythis method, two types of synthetic seedsare prepared: hydrated and desiccated.Hydrated synthetic seeds consist ofembryos individually encapsulated in ahydrogel, whereas in desiccated type thecoating mixture is allowed to dry for severalhours in a sterile hood.The Plant Cell Culture Technology Group ofNuclear Agriculture and BiotechnologyDivision had initiated research on syntheticseeds in the late 1980s working withsandalwood and mulberry. Eventually other crop systems such as banana, cardamomand rice have also been taken up for theproduction of synthetic seeds.

I (1-3) : Synthetic seeds and plantlets in mulberry and banana. 1- synthetic seeds of mulberry planted in soil; 2- mulberry synthetic seeds germinating into plantlets in soil; 3-complete plantlets of banana obtained from synthetic seeds (arrow indicates portion of the synthetic seed still attached to the plantlet)

Banana is an economically profitable cropwith a large consumption in the country and a considerable export potential. Ediblebananas are vegetatively propagated by suckers as viable seeds are generally notproduced in these cultivars. New and effective means of propagating banana would be advantageous over conventional use of sucker material for germplasm maintenance, exchange and transportation. Shoot tips excised from the aseptically raised shoot cultures were excised and encapsulated to prepare synthetic seeds. High percent germination of these synthetic seeds was achieved on a very simple nutrient medium. Addition of the extract of blue green algae to the nutrient matrix enhanced germination frequency. A cell mass (callus) initiated from male flower buds produced embryos which have been successfully encapsulated and germinated. Hence, a twin facility is available in banana to either encapsulate shoot apices or embryos. The examples presented above suggest that, by employing synthetic seeds, the tissue culture raised plants can be regenerated on a simplified medium eliminating subcultures, thus reducing the cost of operation. Development of protocols for direct recovery of plants from synthetic seeds under non sterile conditions may havea greater impact. Although large number of plants can be produced in tissue cultures through embryogenesis / multiple shoot cultures, their delivery is cumbersome. Embryos or shoots have to be separated singly and transferred for rooting to achieve root shoot balance, and the plants have to be hardened in the green house before field planting. Direct sowing of synthetic seeds in the soil does not need acclimatization often required for the tissue cultured plants. It thus provides an ideal delivery system enabling easy flexibility in handling and transport as compared to large parcels of seedlings or plants. For large scale commercialization in synthetic seeds technology, enhanced production of propagules is necessary. Current tissue culture methods do not generate adequate propagules and are not sufficient to meet the demands of commercial exploitation of synthetic seeds technology. Standardization of methods for synchronization of developing propagule followed by

automation of the whole process of sorting, harvesting, encapsulation and germination of the coated propagules can enhance the pace in the production of synthetic seeds.

Reference
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