Training manual and training scheme for professionals and skilled workers on vegetable seed treatment and conditioning

[For professionals and skilled workers]

TRAINING MANUAL

Vegetable Seed Treatment and Conditioning

Muhammad Boota Sarwar
Edited by

For Facilitation Unit for Participatory Vegetable Seed And Nursery Production Program

Ministry of Food, Agriculture & Livestock, Islamabad

CONTENTS Sr. No .
1 2 3 4 5 6 7 8 9 10 Introduction PART-I: Vegetable Seed Treatment PART-II: Vegetable Seed Coating PART-III: Vegetable Seed Pelleting PART-IV: Vegetable Seed Encrusting PART-V: Vegetable Seed Priming PART-VI: Organic Seed Treatment & Coating PART-VII: Technology Licensing Opportunities PART-VIII: Encapsulation of Embryos Artificial Seeds PART- IX : Art i f i c i a l seed technology: Development of a protoco l Geodorum in 4 5 21 27 80 85 87 103 112

Chapters

Pag e No.
3

densi f l o r um(Lam) Schl t r .An endangered orch id

INTRODUCTION

PART-IX: Encapsulation of Embryos Artificial Seeds

Introduction to Artificial / Synthetic Seeds: Plant propagation using Artificial or Synthetic Seeds developed from somatic and not zygotic embryos opens up new vistas in agriculture. Artificial seeds make a promising technique for propagation of transgenic plants, non-seed producing plants, polyploids with elite traits, plants with high dormancy seeds and plant-lines with problems in seed propagation. Being clonal in nature the technique cuts short laborious selection procedure of the conventional recombination breeding and can bring the advancement of technology to the doorsteps of the farmer in a cost effective manner. Definition of Artificial Seed: SYNTHETIC seeds are def ined as art i f i c i a l l y encapsula ted somatic embryos, shoot buds, cel l aggregates , or any other t i s sue that can be used for sowing as a seed and that possess the abi l i t y to convert in to a plant in vi under t ror o ex
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vi t r ocondi t i ons and that reta in th i s potent ia l also after storage. Ear l i e r , synthet i c seeds were refer red only to the somatic embryos that were of economic use in crop product ion and plant del i very to the f i e l d or greenhouse. In the recent past , however, other micropropagules l i ke shoot buds, shoot t ips , organogenic or embryogenic cal l i , etc . hav also been employed in the product ion of synthet i c 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 organogenesis and enhanced axillary bud proliferation system.

Implementation of synthetic seed technology requires manipulation of in vi t ro cul tu re systems for la rge- sca le product ion of viab le mater ia l s that are able to convert in to plants , for encapsula t i on . Somatic embryogenesis , organogenesis and enhanced axi l l a r y bud pro l i f e r a t i on systems are the ef f i c i en t techniques for rap id and la rge- sca 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 large-scale 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 vi t rder o ived propagules in to synthet i c seeds or synseeds or a r t i f i c i a l seeds . The encapsulation technology has been applied to produce synthetic seeds of a number of plant species belonging to angiosperms and gymnosperms (Table 2). Nevertheless, their number is quite small in comparison to the total number of plant species in which in vi t rregenerat o i on system has been estab l i shed . Production of artificial seeds has unraveled 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 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 handle5. Furthermore, they are generally regarded to lack 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-toplant development.

A number of patents covering the development of seed analogues have been issued7. 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: desiccatedand hydrated. The desiccated synthetic seeds are produced from somatic embryos either naked or encapsulated in polyoxyethylene glycol (Polyox) 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 desiccation tolerant. 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 Janick 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. 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 re-hydrate, and then scored for embryo survival.

In 1984 Redenbaugh et al 11 . developed a technique for hydrogel encapsula t i on of ind iv i dua l somatic embryos of al fa l f a . Since then encapsula t i on in hydrogel remains to be the most stud ied method of art i f i c i a l seed product ion . A number of substances l i ke potass ium alg ina te , sodium alg ina te , carrageenan, agar, gel r i t e , sodium pectate , etc . have been tested as hydrogels but sodium alg inate gel i s the most popular5 . Hydrated art i f i c i a l seeds consis t of somatic embryos ind iv i dua l l y encapsula ted in a hydrogel (F igure 2) . To produce hydrated synthet i c seeds, the somatic embryos are mixed with sodium alg inate gel (0.55 .0% w/v) and dropped in to a calc ium sal t solu t i on [CaCl2 (30100 mM), Ca (NO3)2 (30100 mM)] where ion - exchange react i on occurs and sodium ions are replaced by calc i um ions forming calc i um alg ina te beads or capsules surrounding the somatic embryos. The s ize of the capsule i s contro l l ed by vary ing th inner diameter of the pipet te nozz le . 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 satisfactory. However, Molle et al .found that for the product ion of synthet i c seeds of carro t , 1% sodium alg inate solut i on , 50 mM Ca2+ and 2030 min t ime per iod were sat i s f ac to ry for proper hardening of calc i um alg ina te capsules . They have suggested the use of a dual nozzle pipet te in which the embryos f l ow through the inner pipet te and the alg inate solut i on through the outer pipet te . As a resu l t , the embryos are posi t i oned in the cent of the beads for better protect i on . 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 (Table 2). The technology of hydrogel encapsulation is also favored 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 micro-cuttings (approximately 4 or 5 mm in length) are encapsulated in sodium alginate gel following the method described by Redenbaugh et al .for al fa l f a somatic embryos. However, mulberry and banana plant l e t s were obta ined f rom alg inate encapsula ted shoot buds without any speci f i c root induct i on t reatment. To avoid bacterial contamination Ganapathi et al .added an ant ib i o t i c mixture (0.25 mg/l ) conta in ing rifampicin (60 mg), cefatoxime (250 mg) and tetracyclineHCl (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. Importance of Artificial Seeds: Development of micropropagation techniques will ensure abundant supply of the desired plant species. In some crop species seed propagation is not successful; mainly due to heterozygosity of seed, minute seed size, presence of reduced endosperm and the requirement of seed with micorrhizal fungi association for germination (Orchids etc), and also in some seedless varieties of crop plants like grapes, watermelon, etc. Some of these species can be propagated by vegetative means. However, in vivo propagat ion techniques are t ime consuming and expensive . Development of art i f i c i a l seed product ion technology i s current l y being considered as an ef fec t i ve and ef f i c i en t al te rnate method of propagat ion in severa l commercia l l y important agronomic and hort i cu l t u ra l crops. I t has been suggested as powerfu l too l for mass propagat ion of el i t e plant species with high commercia l value. Artificial seed technology involves the production of tissue culture derived somatic embryos encased in a protective

coating. Artificial seeds have often referred to as Synthetic seed however the term may not be confused with synthetic cultivar which is defined as advanced generation of an open pollinated populations composed of a group of selected inbred clones or hybrids. The Synthetic seed would also be a channel for new plant lines produced through biotechnological advances or hybridization to be delivered directly to the greenhouse or field. Advantages of artificial / synthetic seeds over somatic embryos are listed below in Table-1. This Synthetic seed production technology is a high volume, low cost production technology. High volume propagation potential of somatic embryos combined with formation of synthetic seeds for low cost delivery would open new vistas for clonal propagation in several commercially important crop species.

Table-1: Characteristics of Clonal Propagation # Micropropagation Greenhouse Cuttings Low volume, small scale propagation method Artificial Seeds High volume, large scale propagation method

1 Low volume, small scale propagation method 2 Maintains genetic purity of plants 3 Acclimatization of plantlets required prior to field planting 4 High cost per plantlet 5 Relatively low

Maintains genetic Maintains genetic purity of plants purity of plants Rooting of plantlets required prior to field planting High cost per plantlet Multiplication Direct delivery of propagules to the field, thus eliminating transplants Low cost per plantlet Rapid
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multiplication rate

rate limited by mother plant size

multiplication of plants

The concept of artificial or synthetic seed is shown in figure 1 below.

Artificial seed Somatic embryo Artificial endosperm Figure 1: Concept of Artificial Seed

Figure 2: Somatic embryos of mangoMangife ( ra ind ica L. ) encapsulated in calc ium alg inate capsule (embryos are approx imate ly 35 mm long) .

Table-2: Some Plant Species with Encapsulation Methodology.

Plant
Actinidia deliciosa Propagule used for encapsulation

Plant
Arachis hypogaea

Propagule used for encapsulation Somatic Embryo

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(Kiwifruit) Asparagus cooperi Betula pendula (B i rch) Daucus carota (Carrot )

Shoot Bud Somatic Embryo Shoot Bud Somatic Embryo

(Groundnut) Brass i ca campestr i s (Mustard) Cymbidium giganteum (Orchid ) Dendrobium wardianum (Orch id) Shoot Bud protocorm-like bodies protocorm-like bodies

Eleus ine coracana Somatic Gaertn. (F inger mil l e t ) Embryo Malus pumilaMil l . (Apple roots tock M.26) Medicago sat i va (Al f a l f a ) Musa (Banana cv. Basrai ) Shoot Bud

Eucalyptus ci t r i odora Somatic Embryo (Eucalyptus ) Mangife ra ind i ca L. (Mango cv. Amrapal i ) Morus ind i ca (Mulberry ) Pis tac i a vera L. (P i s tach io ) Somatic Embryo

Somatic Embryo Shoot Bud

Shoot Bud Somatic Embryo & Embryogenic Masses

Psid ium guajava (Guava) Rubus (B lackberry cv. Jumbo, Veten) Solanum melongena (Eggplant ) Zingiber of f i c i na l e Rosc. (Ginger)

Somatic Embryo Shoot Bud Somatic Embryo Shoot Bud

Rubus idaeusL. (Raspberry ) Santalum album (Sandalwood) Vi t i s vin i f e (Grape) ra

Shoot Bud Somatic Embryo Somatic Embryo

Advantages of Artificial or Synthetic Seeds over Somatic Embryos for Propagation: 1. Ease of handling while in storage 2. Easy to transport 3. Has potent ia l for long term storage without loos ing viab i l i t y 4. Mainta ins clona l of the resu l t i ng plants 5. Serves as a channel for new plant lines produced through biotechnology advances or hybridization to be delivered directly to the greenhouse or field 6. Allows economical mass propagation of elite plant varieties

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What are Somatic Embryos? Somatic embryos are bipolar structures with both apical and basal meristematic structures, which are capable of forming shoot and root, respectively. A plant derived from a somatic embryo is sometimes referred to as an embling. Somatic embryos are structurally similar to zygotic embryos and possess many of their useful features including the ability to grow into complete plants. However, somatic embryos differ in that they develop from somatic cells instead of zygote (fusion product of male and female gametes) and thus potentially can be used to produce duplicates of a single genotype the mother plant. This characteristic of somatic embryos allows not only clonal propagation but also specific and directional to be introduced into desirable elite individuals by inserting isolated gene sequences into somatic cells. This bypass genetic recombination and selection inherent in conventional breeding technology. Basic Requirements for Production of Artificial Seeds Recently, production of synthetic seeds by encapsulating somatic embryos has been reported in few species; as it requires inexpensive production of high-quality, vigorous somatic embryos that can produce plants with frequencies compatible to natural seeds. Inability to recover such embryos is often a major limitation in the development of synthetic seeds. Encapsulation and coating systems, though important for delivery of somatic embryos, are not limiting factors for development of synthetic seeds. At present the characteristic lack of developmental synchrony in embryogenic systems stymies multi-step procedures for guiding somatic embryos through maturation; and is single most hurdle to be overcome before advances leading to wide-spread commercialization of synthetic seeds can occur.

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Procedure for Production of Artificial Seeds: Establish somatic embryogenesis Mature somatic embryos Synchronize and singulate somatic embryos Mass production of somatic embryos Standardization of encapsulation Standardization of artificial endosperm Mass production of synthetic seeds Greenhouse and field planting Types of Gelling Agents used for Encapsulation: Several gels like agar, alginate, polyco-2133 (Bordon Co.), carboxy methyl cellulose, carrageenan, gelrite (Kelko Co.), guar-gum, sodium pectate, tragacanth gum etc were tested for synthetic seed production, out of which alginate hydrogel encapsulation was found most suitable and practicable. Alginate hydrogel is frequently selected as a matrix for synthetic seed because of its moderate viscosity and low spin-ability of solution, low toxicity for somatic embryos and quick gellation, low cost and bio-compatibility characteristics. The use of agar as gel matrix was deliberately avoided as it is considered inferior to alginate with respect to long term storage. Alginate was chosen because it enhances capsule formation and also the rigidity of alginate beads provides

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better protection (than agar) to the encased somatic embryos against mechanical injury. Principles and Conditions for Encapsulation with Alginate Matrix: Alginate is a straight chain, hydrophilic, colloidal polyuronic acid composed primarily of hydro--D-mannuronic acid residues with 1-4 linkages. The major principle involved in the alginate encapsulation process that the sodium alginate droplets containing the somatic embryos when dropped into the CaCl2.2H2O solution form round and firm beads due to ion exchange between the Na+ in sodium alginate with Ca2+ in the CaCl2.2H2O solution. The hardness or rigidity of the capsule mainly depends upon the number of sodium ions exchanged with calcium ions. Hence the concentration of the two gelling agents, sodium alginate and CaCl2.2H2O, and the complexing time should be optimized for the formation of the capsule with optimum bead hardness and rigidity. In general, 3% sodium alginate upon complexation with 75 Mm CaCl2.2H2O for half an hour gives optimum bead hardness and rigidity for the production of viable synthetic seeds. Artificial Endosperm: Somatic embryos lack seed coat (testa) and endosperm that provide protection and nutrition for zygotic embryos in developing seeds. To augment these deficiencies, addition of nutrients and growth regulators to the encapsulation matrix is desired, which serves as an artificial endosperm. These synthetic seeds can be stored for a longer period of time even up to 6 months without loosing viability, especially when stored at 4OC.

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Addition of Adjuvant to the Matrix: In addition to preventing the embryo from desiccation and mechanical injury, a number of useful materials such as nutrients, fungicides, pesticides, antibiotics and microorganisms (eg. rhizobia) may be incorporated into the encapsulation matrix. Incorporation of activated charcoal improves the conversion and vigor of the encapsulated somatic embryos. It has been suggested that charcoal breaks up the alginate and thus increase respiration of somatic embryos (which otherwise lose vigor within a short period of storage). In addition, charcoal retains nutrients within the hydrogel capsule and slowly releases them to the growing embryo. Utilization of Artificial Seeds: The artificial seeds can be used for specific purposes, notably;

1. Multiplication of non-seed producing plants 2. Multiplication of ornamental hybrids, currently propagated by cuttings 3. Propagat ion of polyp lo id plants with el i t e tra i t s ; or plants with meiot i ca l l y - unstable el i t e genotypes. 4. Propagation of male or female sterile plants for hybrid seed production 5. Cryo- preserved art i f i c i a l seeds may also be used for germplasm preservat i on , part i cu l a r l y in reca lc i t r an t species (such as mango, cocoa and coconut) , as these seeds wi l l not undergo desiccat i on . 6. Transgenic plants which requi re separate growth fac i l i t i e s to mainta in or ig i na l genotype may also be preserved using art i f i c i a l seeds. Somatic embryogenesis i s a potent ia l too l in the genet i c engineer ing of plants . Potent ia l l y , a s ing le gene can b inser ted in to a somatic cel l . In plants that are regenerated by somatic embryos f rom a s ing le transgenic cel l , the progeny wi l l not be chimer ic .

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7.

Multiplication of elite plants selected in plant breeding programs via somatic embryos avoids the genetic recombination, and therefore does not warrant continued selection inherent in conventional plant breeding, saving considerable amount of time and other resources. Art i f i c i a l seeds produced in t i s sue cul tu re are f ree of pathogens; thus , another advantage i s the t ranspor t of pathogen f ree propagules across the in te rnat i ona l borders avoid ing bulk t ranspor ta t i on of plants , quarant ine and spread of diseases .

Achievements and prospects: 1. Somatic embryos Although various micropropagules have been considered for synthetic seed production, the somatic embryos have been largely favored (Table 2) as these structures possess the radicle and plumule that are able to develop into root and shoot in one step, usually without any specific treatment. The advantages of preparing synthetic seeds from somatic embryos have been discussed by Redenbaugh. The use of somatic embryos as artificial seeds is becoming more feasible as the advances in tissue culture technology define the conditions for induction and development of somatic embryos in an increasing number of plant species. Various types of artificial seeds have been prepared using somatic embryos which have been either dried or maintained fully hydrated, these may or may not be encapsulated. However, if the somatic embryo is dried to moisture content of approximately 10%, as in a number of true seeds, the propagation system has the additional advantage of serving as a germplasm storage system, which maintains the propagule in a quiescent state for extended periods of time. Dried somatic embryos would also provide a more efficient use of space and labor in a commercial production system and storage for planting in the future. Attempts have been made to desiccate somatic embryos with or without

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encapsulation to exploit this potential, but success has been relatively limited except for Medicago sat i va . In alfalfa (M. sat i va ) desiccat i on - to le rance of somatic embryos was induced by exogenous appl i ca t i on of abscis i c acid (ABA) by Senaratna, Mckers ie and Bowley. Subsequent ly , the embryos were dr ied to 1015% moisture and stored for at leas t 3 weeks in the dry state . Under appropr ia te t reatment condi t i ons , 65% of these somatic embryos surv ived and germinated in a manner analogous to a true seed. Desiccat i on - to le rance has also been induced in al fa l f a somatic embryos by exposure to sub- le tha l leve l s of low temperature , water , nutr i en t or heat st ress . However, these pret reatments had deleter i ous ef fec t s on embryo maturat ion and plant l e t vigor . Onishi, Sakamoto and Hirosawa have demonstrated a protocol for the production of synthetic seeds involving automation at the production and encapsulation stages. These authors have emphasized that high and uniform conversion of synthetic seeds under a practical sowing situation, such as, nursery bed in a greenhouse or in the field, is an essential requirement for their use in clonal propagation of plants. They found that conversion of celery and carrot embryos produced in bioreactors, could be raised to 5380% from 0% by three sequential treatments:
(i)

(ii)

Cultur i ng the embryos for 7 days in a medium of high osmolar i t y (wi th 10% mannito l ) less than 16 h photoper iod with 300 lux of i l l uminat i on for promoting embryo development. This treatment increased the s i ze of embryos f rom 13 mm to 8 mm and the i r chlorophy l l content . ( i i ) Dehydrat ion of embryos to reduce thei r water content f rom 9599% to 8090% by keeping them for 7 days on 27 layers of

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(iii)

filter paper under a 16 h photoperiod of 14 mE m2 s1 irradiance. Post-dehydration culture on SH medium containing % sorbitol, 0.01 mg/l BAP and 0.01 mg/l GA3, in air enriched with 2% CO2 under a 16 h photoperiod at 20C for 14 days to acquire autotrophic nature and reserve food. The bead quality was also modified by impregnating them with 3% sucrose, by coating the microcapsules with a fungicidal mixture comprising 8% Elvax 4260 and beeswax, and 0.1% Topsin M.

To facilitate the emergence of shoot and root meristems during embryo germination, Onishi, Sakamoto and Hirosawa have made the gel capsule self-breaking under humid conditions. It involved rinsing the beads thoroughly with running tap water, followed by immersion in a 200 Mm solution of KNO3 for 60 min and, desalting them by rinsing in running tap water for 40 min. Such synthetic seeds showed 50% conversion in two weeks after sowing in a greenhouse. In tree species like Santa lum album 24,26, Pis tac i a vera22 and Mangifera ind i ca also the somatic embryos have been encapsulated to produce synthetic seeds. However, further research is needed to optimize protocols for production of viable synthetic seeds that could be stored for longer periods and could be commercially viable. 2. Axi l l a r y shoot buds and apica l shoot t ips In many plant species (Table 2) the unipolar axillary shoot buds and/or apical shoot tips which do not have root meristem, have also been encapsulated to produce synthetic seeds. Since these structures do not have root meristems they should be induced to regenerate roots before encapsulation. Different authors have described how

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encapsulated buds of banana and mulberry converted into plantlets without specific root induction treatments. In different reports Piccioni28 and Capuano, Piccioni and Standardi1 have described conversion of shoot buds of apple clonal rootstock M.26 encapsulated after an appropriate root induction treatment with IBA (24.6 mM) for 36 days. Capuano and coworkers1 have found different conversion behavior of the synthetic seeds made of axillary and apical micro-cuttings. They have reported that conversion of the synthetic seeds obtained with axi l l a r y microcut t i ngs of M.26 apple rootstock always occurred at a very low rate (only 25%) following 6 days of root primordial initiation (RPI) culture and cold storage. In contrast, apical micro-cuttings reached 85% conversion with a 24.6 mM IBA treatment and 3 days of RPI culture without cold storage. These results confirm the suitability of such explants towards encapsulation and synthetic seed production. Besides, the results encourage the use of encapsulated unipolar explants, such as micro-propagated buds for the synthetic seed technology. This kind of capsule could be useful in exchange of sterile material between laboratories due to small size and relative ease in handling these structures, or in germplasm conservation with proper preservation techniques29, or even in plant propagation and nurseries, if the development of the plant could be properly directed towards proliferation, rooting, elongation, etc. 3. Embryogenic masses Stable and regenerative embryogenic masses make an attractive tool for the production of clonal plants and for studies of genetic transformation. However, long-term maintenance of embryogenic masses in culture tubes or mechanically stirred bio-reactors requires frequent transfer of tissue to fresh media which is both labor-intensive and costly. To cope up with these difficulties, the embryogenic masses of Pistacia vera have been encapsulated in sodium

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alginate gel using the method of Redenbaugh et al .and stored at C 4 after treatment with BAP. Onay, Jeffree and Yeoman22 have reported that the encapsulated embryogenic masses recovered their original proliferative capacity after two months storage following two subcultures. Nevertheless, it remains to be established whether the storage period can be extended further, and also if the efficiency of embryogenic masses for production of somatic embryos declines during the long-term storage. 4. Protocorms or protocorm- l i ke bodies In orchids such as Cymbidium giganteum , endrobium wardianum, Geodorum densi f l o r um , Phaius tonkervillae and Spathoglottis plicata synthetic seeds have been produced by encapsulating the protocorm or protocorm-like bodies (PLBs) in sodium alginate gel. Corrie and Tandon30 have reported that the encapsulated protocorms of C. giganteum gave rise to healthy plantlets upon transferring either to nutrient medium or directly to sterile sand and soil. They found that conversion frequency was high in both in vitro (100%) and in vivo (88% in sand, 64% in sand and soil mixture) conditions. These techniques have made it possible to transplant the aseptically grown protocorms directly in the soil, cutting down the cost of raising in vitro plantlets and their subsequent acclimatization. Use of synthetic seeds appears to be particularly promising. The encapsulation, storage and re-growth of homogeneous material allow the possibility of automated mass production of elite plant species. There are several potential uses of synthetic seeds of those crop plants that are vegetatively propagated and have long juvenile periods, e.g. citrus, grapes, mango, etc. The planting efficiency of such crops could theoretically be increased by the use of synthetic seeds instead of cuttings. Synthetic seeds have been found highly advantageous for germplasm conservation in grape and other similar crops.

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Limitations Although results of intensive researches in the field of synthetic seed technology seem promising for propagating a number of plant species (Table 2), practical implementation of the technology is constrained due to the following main reasons:

Limi ted product ion of viab le micropropagules usefu l in synthet i c seed product ion . Anomalous and asynchronous development of somatic embryos. Improper maturat ion of the somatic embryos that makes them inef f i c i en t for germinat ion and convers ion in to normal plants . Lack of dormancy and st ress to le rance in somatic embryos that l imi t the storage of synthet i c seeds. Poor convers ion of even apparent l y normal ly matured somatic embryos and other micropropagules in to plant l e t s that l imi t the value of the synthet i c seeds an ul t imate ly the technology i t se l f .

Development of artificial seeds requires sufficient control of somatic embryogeny from the explants to embryo production, embryo development and their maturation as well. The mature somatic embryos must be capable of germinating out of the capsule or coating to form vigorous normal plants. A number of researchers have tried to improve the quality and quantity of somatic embryos via modification of culture conditions, such as, medium composition, growth regulators (types and concentrations), physical state of the medium, as well as incubation conditions like temperature, illumination, etc. Although large quantities of somatic embryos can be rapidly produced in many plant species, normal plants are difficult to obtain due to their improper or asynchronous maturation.

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Hence, maturation of somatic embryos, which eventually controls germination and conversion rate, is one of the major bottlenecks for synthetic seed production. While studying the effects of different types of osmotica on maturation of somatic embryos of spruce, Attree and Fowke39 and Fowke and Attree44 have described that inclusion of high levels of sucrose (i.e. permeating osmotica) in the standard medium containing ABA (which is associated with water stress), prevents maturation while inclusion of PEG (non-permeating osmotica) with ABA dramatically improves the frequency and synchrony of the somatic embryo maturation. Biochemical analysis of these somatic embryos showed a striking increase in storage lipids and proteins compared to the embryos matured without PEG. For commercial applications, somatic embryos must germinate rapidly and should be able to develop into plants at least at rates and frequencies more or less similar if not superior to true seeds. To achieve conversion of somatic embryos into plantlets and to overcome deleterious effects of recurrent somatic embryogenesis as well as anomalous development of somatic embryos on their conversion, it is necessary to provide optimum nutritive and environmental conditions. Maltose has been found valuable for improving alfalfa somatic embryo conversion7. From a synthetic seed perspective, addition of sucrose in the medium is necessary for viability of somatic embryos, their subsequent development, maturation and germination in many plant species. In an in vi t ro cul tu re system the somatic embryos show great divers i t y in the i r morphology and accord ing ly in the i r response which great l y l imi t s the use of synthet i c seed technology. Lee and Soh have ind i ca ted that cont inuous ABA t reatment inc reases the format ion of somatic embryos with anomalous coty ledons , whi le in some ins tances ABA has been found to promote the normal development of both somatic and zygot i c embryos in vitro. Cytokinin treatment

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also increases the number of somatic embryos with multiple cotyledons. It is suspected that the morphological development of somatic embryos is regulated by endogenous hormones. Liu, Xu and Chua have described the effect of anti-auxins on polar auxin transport which controls embryo development. For initiation of the two cotyledons, a polar auxin transport in the embryo is needed for a short period during the globular stage and developmental abnormalities occur due to cell divisions in the meristematic areas prior to differentiation of the shoot apex and cotyledons. The developmental anomalies, however, are not intrinsic to somatic embryos, because immature zygotic embryos can also exhibit similar irregularities when removed from the seed and allowed to develop in vi t r . o Choi et al. have suggested that unbalanced endogenous hormone distribution by exogenous hormone treatment may result in the abnormal somatic embryos. In many plant species the somatic embryos have been found to be sensitive to desiccation. Desiccation damages the somatic embryos and inhibits their germination and conversion into plants in desiccation-sensitive plant species. Nevertheless, desiccation and subsequent rehydration have been found useful in inducing a high frequency conversion of somatic embryos into plantlets in some species. Gradual drying of alfalfa somatic embryos with progressive and linear loss of water gave better response and improved the quality of embryos in comparison to uncontrolled drying7. Similarly, desiccation improved the germination frequency in soybean also. Senaratna et al. have reported that desiccation tolerance can be induced in somatic embryos of alfalfa by external stimuli such as ABA, exposure to cold, heat, water and osmotic stress at sub-lethal levels or increasing the sucrose content in the medium. Attree et al. and Fowke and Attree have reported that somatic embryos of spruce matured in the presence of PEG and ABA were very tolerant to low moisture levels. According to them, such somatic embryos had less than 50% moisture content which was further reduced to less than 10% following desiccation.

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These embryos were stored at 20C for a year and thereafter successfully germinated following imbibition with no loss in viability. The coating material may also limit success of the synthetic seed technology, and at present none of the embryo encapsulation methods described earlier is completely satisfactory. The hydrated capsules are more difficult to store because of the requirement of embryo respiration6. A second problem is that capsules dry out quickly unless kept in a humid environment or coated with a hydrophobic membrane. Calcium alginate capsules are also difficult to handle because they are very wet and tend to stick together slightly. In addition, calcium alginate capsules lose water rapidly and dry down to a hard pellet within a few hours when exposed to the ambient atmosphere. These problems can be offset by coating the capsules with Elvax 4260 (ethylene vinyl acetate acrylic acid terpolymer, Du Pont, USA). Redenbaugh, Fujii and Slade have reported that the limitations caused by coating materials can be overcome by selecting appropriate coating material for encapsulation. According to them, the coating material should be nondamaging to the embryo, mild enough to protect the embryo and allow germination and be sufficiently durable for rough handling during manufacture, storage, transportation and planting. The concentration of the coating material is also an important limiting factor for the synthetic seed technology. The coat must contain nutrients, growth regulator(s) and other components necessary for germination and conversion and it should be transplantable using the existing farm machinery. Though many coating materials have been tried for encapsulation of somatic embryos, sodium alginate obtained from brown algae is considered the best and is being popularly used at present. Alginate has been chosen for ease of capsule formation as well as for its low toxicity to the embryo. The rigidity of the gel beads protects the fragile embryo during handling. According to Redenbaugh et al , .the

25

capsule gel can potentially serve as a reservoir for nutrients (like an artificial endosperm) that may aid the survival and speed up the growth of the embryo. Conclusions Despite considerable research input into artificial seed production during the last fifteen years, several major problems remain with regard to its commercialization. The first requirement for the practical application of the artificial seed technology is the large-scale production of high quality micropropagules, which is at present a major limiting factor. Additional factors responsible for poor germination of synthetic seeds are the lack of supply of nutrients and oxygen, microbial invasion and mechanical damage of somatic embryos. In fact, conversion is the most important aspect of the synseed technology, and still remains one of the factors limiting commercial application of this technology. Until recently, most reports on somatic embryogenesis focused only on the production of embryos and recovery of a few plants. Among tree species, regeneration of viable plantlets from somatic embryos is a frequently encountered problem. The bottleneck may occur at any of a number of stages including maturation, germination, shoot apex elongation, rooting of shoots or acclimatization. While treatments to overcome these bottlenecks vary with the plant species, one general approach can be to simulate the conditions experienced by zygotic embryos in seeds prior to germination. The desiccation process, which damages the embryo, and other problems associated with desiccated artificial seeds need resolution. Occurrence of high levels of somaclonal variations in tissue culture is another aspect to be considered seriously while recommending the use of artificial seeds for clonal propagation. One of the future usage of synthetic seeds would be in germplasm conservation through cryopreservation.

26

Either hydrated calcium alginate-based or desiccated polyoxyethylene glycol-based artificial seeds might be used, but it is likely that some degree of drying before cryopreservation would be beneficial. The synthetic seed technology offers tremendous potential in micropropagation and germplasm conservation; however further research is needed to perfect the technology so that it can be used on a commercial scale. References:
1. 2.

3. 4.

5.
6. 7. 8. 9. 10. 11. 12. 13.

Akhtar , N., Ph D thes i s , Banaras Hindu Univers i t y , Varanas i , 1997 Ammirato , P. V. , Handbook in of Plant Cel l Culture (eds Evans, D. A. , Sharp, W. R. , Ammirato , P. V. and Yamada, Y. ) , Macmil l an Publ i sh ing Co, New York, 1983, vol . 1, pp. 82123 Ammirato , P. V. Plant , Physio . ,l 1977, 59 , 579586 Ara, H., Jaiswal, U. and Jaiswal, V. S., Plant Cel l Rep ., 1999, 19 , 166170 Ara, H., Ph D thesis, Banaras Hindu University, Varanasi, 1998 Arya, K. R., Beg, M. U. and Kukreja, A. K., Ind ian J . Exp. Bio l ., 1998, 36 , 11611164 Attree, S. M., Moore, D., Sawhney, V. K. and Fowke, L. C., Ann. Bot. , 1991, 68 , 519525 Attree, S. M., Pomeroy, M. K. and Fowke, L. C., J . Exp. Bot., 1995, 46 , 433439 Attree, S. and Fowke, L., Plant Cel l Tissue Org.., Cult 1993, 35 , 135 Attree, S. M., Pomeroy, M. K. and Fowke, L. C., Plant Cel l Rep ., 1994, 13 , 601606 Bapat, V. A. and Rao, P. S., Plant Cel l Tissue Org.., Cult 1990, 20 , 6970 Bapat, V. A. and Rao, P. S., J . Plant Biochem. Biotechnol ., 1992, 1, 109113 Bapat, V. A. and Rao, P. S., Plant Cel l Rep ., 1988, 7, 434436

27

14. Bapat, 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

31.

V. A. , Synseeds in (ed. Redenbaugh, K.) , CRC Press , Boca Raton, 1993, pp. 381407 Burns, J . A. and Wetzste in , Plant H. Y. , Cel l Tissue Org. Cult. , 1997, 48 , 93102 Capuano, G., Piccioni, E. and Standardi, A., J . Hort i c . Sci . Biotechnol ., 1998, 73 , 299305 Capuano, M. and Debergh, P. C., Plant Cel l Tissue Org. Cult., 1997, 48 , 2329 Choi, Y. E., Kim, H. S., Soh, W. Y. and Yang, D. C., Plant Cel l Rep ., 1997, 16 , 738744 Compton, M. E., Benton, C. M., Gray, D. J. and Songstad, D. D., In vi t ro Cel l Dev.., Bio 1992, l 28 , 197 201 Corrie, S. and Tandon, P., Ind ian J . Exp. ., Bio 1993, l 31 , 6164 Crouch, M. L. and Sussex, I. M., Planta , 1981, 153 , 64 74 Datta, K. B., Kanjilal, B. and Sarker, D., Curr . Sci ., 1999, 76 , 11421145 Fowke, L. and Attree, S., Plant Tissue Cult . Biotechnol ., 1996, 2, 124130 Ganapathi, T. R., Suprasanna, P., Bapat, V. A. and Rao, P. S., Plant Cel l Rep ., 1992, 11 , 571575 George, L. and Eapen, S., Ind ian J . Exp. ., Bio 1995, l 33 , 291293 Ghosh, B. and Sen, S., Plant Cel l Rep ., 1994, 13 , 381 385 Gray, D. J. and Purohit, A., Cri t . Rev. Plant., 1991, Sci 10 , 3361 Gray, D. J., HortSc i ., 1987, 22 , 810814 Hammatt, N. and Davey, M. R., J . Plant Physio ., 1987, l 128 , 219226 Gupta, P. K., Shaw, D. and Durzan, D. J., in Cel l and Tissue Culture in Forest (eds ry Bonga, J. M. and Durzan, D. J.), Martinus Nijhoff Publishers, Dordrecht, 1987, pp. 101108 Hussain Ara, Uma Jaiswal and V. S. Jaiswal; Synthetic seed: Prospects and limitations; CURRENT SCIENCE , VOL. 78, NO. 12, 25 JUNE 2000

28

32. I .

33.

34.

35. 36.

37.

38. 39. 40. 41. 42. 43. 44.

45. 46. 47.

Kinoshi ta , The Product ion and use of art i f i c i a l seed Research Journa l of Food and Agr icu , l t15(3) u re , 6- 11, 1993. J . A. Fuj i i , D. T. Slade, K. Redenbaugh and K. A. Walke Art i f i c i a l Seeds for Plant Propagat Trends ion , in Biotechnology , 5, 335- 339, 1987. Ja in , S. M., Gupta, P. K. and Netwon, Somatic R. J . , Embryogenesis in Woody Plants , Kluwer Academic Publ i shers , Dordrecht , 1995 Janei ro , L. V. , Bal les te r , A. and Vie i Plant t ez , A. Cel M., l Tissue Org. Cult . , 1997, 51 , 119125 Janick, J., Kim, Y.-H., Kitto, S. and Saranga, Y., in Synseeds (ed. Redenbaugh, K.), CRC Press, Boca Raton, 1993, pp. 1234 Jeong, R. B., Fujiwara, K. and Kozai, T., in Hort i cu l t u ra l Reviews (ed. Janick, J.), Wiley, New York, 1995, vol. 17, pp. 125172 Kitto, S. K. and Janick, J., HortSc i ., 1982, 17 , 488 Kitto, S. and Janick, J., J . Am. Soc. Hort i c . ., 1985, Sci 110 , 277, 283 K. Redenbaugh; Synthet i c Seeds, CRC Press, Boca Raton, 1993. Lakshmana Rao, P. V. and Singh, B., Plant Cel l Rep ., 1991, 10 , 711 Lee, K. S. and Soh, W. Y., Korean J . Plant Tissue., Cult 1994, 21 , 287291 Liu, C.-M., Xu, Z.-H. and Chua, N.-H., Plant Cel , 1993, l 5, 621630 Lulsdorf, M. M., Tautorus, T. E., Kikcio, S. I., Bethune, T. D. and Dunstan, D. I., Plant Cel l Rep ., 1993, 12 , 385 389 Malemngaba, H., Roy, B. K. Bhattacharya, S. and Deka, P. C., Ind ian J . Exp. ., Bio 1996, l 34 , 801805 Marsolais, A. A., Wilson, D. P. M., Tsujita, M. J. and Senaratna, T., Can. J . Bot ., 1991, 69 , 11881193 Mckersie, B. D. and Bowley, S. R., in Synseeds (ed. Redenbaugh, K.), CRC Press, Boca Raton, 1993, pp. 231255

29

48. Michler , 49.

C. H. and Bauer, E. Plant O., Sci . , 1991, 77 ,

50.

51. 52. 53. 54. 55.

56. 57. 58.

59. 60.

61. 62. 63. 64.

111118 Molle, F., Dupius, J. M., Ducos, J. P., Anselm, A., Crolus, S. I., Petiard, V. and Freyssinet, G., in Synseeds (ed. Redenbaugh, K.), CRC Press, Boca Raton, 1993, pp. 257270 Muralidharan, E. M. and Mascarenhas, A. F., in Somatic Embryogenesis in Woody Plants (eds Jain, S., Gupta, P. and Newton, R.), Kluwer Academic Publishers, Dordrecht, 1995, pp. 101108 Norstog, K., Am. J . Bot ., 1965, 52 , 538546 Onay, A., Jeffree, C. E. and Yeoman, M. M., Plant Cel l Rep ., 1996, 15 , 723726 Onishi, N., Sakamoto, Y. and Hirosawa, T., Plant Cel l Tissue Org. Cult ., 1994, 39 , 137145 Padmaja, G., Reddy, L. R. and Reddy, G. M., Ind ian J . Exp. Bio .,l 1995, 33 , 967971 Parrott, W. A., Dryden, G., Vogt, S., Hildebrand, D. F., Collins, G. B. and Williams, E. G., In Vit ro Cel l Dev. Bio l , . 1988, 24 , 817820 Piccioni, E., Plant Cel l Tissue Org.., Cult 1997, 47 , 255 260 Piccioni, E. and Standardi, A., Plant Cel l Tissue Org. Cult., 1995, 42 , 221226 Redenbaugh, K., Fujii, J. A. and Slade, in Synseeds (ed. Redenbaugh, K.), CRC Press, Boca Raton, 1993, pp. 3846 Redenbaugh, K., HortSc i ., 1990, 25 , 251255 Redenbaugh, K. and Walker, K., in Plant Tissue Culture : Appl i ca t i ons and Limi ta (ed. t i ons Bhojwani, S.), Elsevier, Amsterdam, 1990, pp. 102135 Redenbaugh, K., Nichol, J., Kossler, M. E. and Paasch, B., In vi t r ,o 1984, 20 , 256257 Redenbaugh, K. (ed.), Synseeds, CRC Press, Boca Raton, 1993 Redenbaugh, K., Slade, D., Viss, P. and Fujii, J., HortSc i ., 1987, 22 , 803809 Refouvelet, E., LeNours, S., Tallor, C. and Daguin, F., Sci . Hort., ic 1998, 74 , 233241

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65. Saiprasad G. V. S; Resonance, May 2001, page 39-47 66. Sharma, A. , Tandon, P. and Kumar,Ind A.ian , J . Exp. Bio l . , 1992, 30 , 747748 67. Sharma, T. R., Singh, B. M. and Chauhan, R. S., Plant Cel l Rep ., 1994, 13 , 300302 68. Senaratna, T., Mckersie, B. D. and Bowley, S. R., Plant Sci., 1989, 65 , 253259 69. Senaratna, T., Saxena, P. K. Rao, M. V. and Afele, J., Plant Cel l Rep ., 1995, 14 , 375379 70. Singh, F., Lind leyana , 1991, 6, 6164 71. T. Senaratna, Artificial Seeds, Biotechnology Advances , 10, 379-392, 1992. 72. Wetzstein, H. Y. and Baker, C. M., Plant Sci ., 1993, 92 , 8189

Artificial seed technology: Development of a protocol in Geodorum densif lorum (Lam) Schltr . An endangered orchid
# K. B. Datta*, B. Kanji la l and D. De Sarker

Molecular Cytogenetics and Tissue Culture Laboratory, Department of Botany, University of North Bengal, Raja Rammohunpur 734 430, India # Department of Botany, Raiganj Univers i t y Col lege , Raiganj 134, 733 Ind ia (Received 10 August 1998; rev i sed accepted 29 January 1999)

The research communication reports the production of artificial seeds through encapsulation of protocorm-like bodies (PLBs) of Geodorum densif lorum (Lam) Schltr. an endangered orchid taxon of Terai Hil ls , North- eastern Himalaya. 30-day-old PLBs were encapsulated in sodium alginate. Germination and regeneration capacity of the encapsulated seeds were tested by germinating such seeds in modified Knudson C (KnC) medium supplemented with coconut milk 15% (v/v) , peptone (2 g l1), 6-benzyl-aminopurine (2 mg l1), and a -napthaleneacetic acid (1 mg l1). 88% Germination was recorded. Artificial seeds stored at 4 C for 120 days showed no reduction in viability. Non-encapsulated PLBs showed no viability after 30 days at 4 C. Artificial seeds showed 28% viability when directly transferred to non-sterile soil condition

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after incorporating food preservative and fungicide in its encapsulating gel. PRODUCTION of artificial seeds has unraveled new vistas in plant biotechnology. The artificial seed technology is an exciting and rapidly growing area of research in plant cell and tissue culture. The idea of artificial seeds was first conceived by Murashige1 which was subsequently developed by several investigators. Initially, the development of artificial seeds had been restricted to encapsulation of somatic embryos in a protective jelly. It had been considered that the induction of somatic embryogenesis (SE) and/or pollen embryogenesis which genetically differs from zygotic embryogenesis is the prerequisite for the preparation of artificial seeds. Their induction has been reported in a number of cereals, millets, tuberous plants, vegetables, and other commercially important plants like soybean, mustards, coffee, tobacco, and cotton. However, because of certain inherent problems, the rate of production of uniform and high quality embryos is much lower as a result of which the preparation of efficient and quality seeds has been successful in only a few crop plants like carrot3 and alfalfa4. Recent advances in the area have revealed that besides somatic embryos, encapsulation of cells and somatic tissues obtained following tissue culture techniques has become popular as a simple way of handling cell and tissue, protecting them against strong external gradients, and as an efficient delivery system4,9. A number of encapsulating agents have been tried out of which agar, agarose, alginate, carragenan, gelrite, and polyacrylamide are important2. Recently, nitrocellulose and ethylocellulose have also been tried out for encapsulation11. However, the present investigation by us on Geodorum densi f l o rum (Lam) Schltr., as well as by those of a few others79, suggests the that most suitable encapsulating agent for orchid protocorm-like bodies (PLBs) is sodium alginate, due to its solubility at room temperature and its ability to form completely permeable gel with calcium chloride (CaCl2 2H2O). Our findings have revealed that this method provides an efficient mechanism for handling and storage of orchid PLBs. To date, encapsulation methodologies have met with success in only a few non-orchid angiosperms. Orchids, the most precious and costly ornamentals, are one of the few flowering plants to be propagated in vi t ro , both through seed and tissue culture. The most sensational development has been the use of Cymbidium apical shoot meristem as a means of clonal propagation by Morel5,6 which revolutionized the 32

orchid industry and triggered the global explosion of tissue culture for rapid clonal propagation of other ornamentals as well. Uptil now, synthetic seed production by encapsulating PLBs has been achieved in only a few orchids like Dendrobium wardianum7, Phaius tankervillae8, and Spathoglottis plicata9. The present communication of successful artificial seed production in G. densiflorum, an endangered terrestrial orchid, will be a further addition. Attempts have also been made for direct transfer of the artificial seeds to the field following treatments with antifungal and antibacterial agent in the encapsulating matrix. For production of PLBs, mature undehisced capsules of G. densiflorum were collected and washed thoroughly with Tepol (BDH) under running tap water. They were then surface-sterilized with 3% sodium hypochlorite solution (v/v) for 15 min and were subsequently rinsed in sterilized double-distilled water. The capsules were cut longitudinally with the help of a sharp sterilized surgical blade and the seeds were inoculated in modified Knudson C (KnC) medium10 supplemented with 2 mg l1 of 6-benzylaminopurine (BAP), 1 mg l1 a -napthaleneacetic acid (NAA), 2 gl1 peptone and 15% coconut milk (CM). The pH of the medium was adjusted to 5.65.8. The cultures were maintained at 25 2 C under 16 h photoperiod from cool-white-light giving 1000 lux at culture level. After 5 weeks following inoculation, green pin-headlike PLBs appeared. For encapsulation of PLBs, 30 days following seed germination the PLBs were collected and washed in liquid KnC medium. Sodium alginate solution (4%; w/v) was prepared by mixing with liquid KnC medium. PLBs were mixed with sodium alginate solution and were subsequently singly dropped into an autoclaved-50 mM solution of calcium chloride (CaCl2 2H2O). Calcium alginate beads were formed within 1520 min on a rotary shaker moving at 80 rpm. Beads were taken out by decanting off the CaCl2 solution, washed with sterilized double-distilled water, and surface-dried with sterilized blotting paper. Freshly prepared beads were directly inoculated in KnC medium; the organic additives and the concentrations of growth regulators being the same as used during seed germination. The cultures were kept at same conditions as before. A set of 150 artificial seeds was stored in dark at 4 C in sterile petri dishes, sealed with parafilm. They were taken out at regular intervals of 30 days, and inoculated to see their germination percentage. Another set of 150 PLBs was kept at room temperature (25 2 C). Non-encapsulated PLBs were kept both at room temperature and at 4 C. Each treatment had 10 replicates and was repeated at least thrice.

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Fungicide bavastin was incorporated into the nutrient gel at a constant concentration of 4 mg l1, as higher concentrations of fungicide were found to inhibit the growth of PLBs (data not shown). Sodium bicarbonate was used as food preservative, and was incorporated in the same way. The concentrations of sodium bicarbonate used were 5, 10, 15, 20, 25, 30 and 40 mg l1. The encapsulated PLBs were washed with sterile double-distilled water, and finally placed in autoclaved soil in petri dishes. The petri dishes were covered with lid to maintain the required humidity. Tap water was sprayed at regular intervals to keep the soil moist. Freshly encapsulated PLBs (without storage; control) (Figure 1 b) when directly inoculated on KnC medium supplemented with CM (15% v/v), peptone (2 g l1), BAP (2 mg l1) and NAA (1 mg l1), showed induction of growth after second week. Subsequently, they emerged out by rupturing the alginate matrix and established contact with the media. These were then sub-cultured on the same media, and within 68 weeks well-developed plantlets were obtained (Figure 1 c). As the PLBs (Figure 1 a) were randomly selected for encapsulation, the time requirement for the encapsulated PLBs to come out through the matrix was different. 88% germination was noted at this stage, which is quite high (Table 1). Encapsulated PLBs stored at 4 C for 120 days showed 86% viability, but the same when stored at room temperature showed only 44% viability (Table 1). The germination percentage decreased gradually with increase in storage time. The non-encapsulated PLBs on the other hand showed no viability or regeneration after a storage of only 30 days at 4 C. The germination percentage of artificial seeds stored at room temperature was always much lower in comparison to those stored at 4 C (Table 1). The encapsulated PLBs containing fungicide and different concentrations of food preservative did not show any contamination up to 8 weeks in soil. With varying concentrations of food preservative used, optimum frequency of germination (28%) was at 20 mg l1 concentration, which was followed by emergence of plantlets through the matrix (Figure 1 d). Although in all concentrations of sodium bicarbonate, some degree of germination was noted, the continued growth of PLBs at lower concentrations ceased due to desiccation (Table 2). Besides rapid and mass propagation of plants, the artificial seed technology has added new dimensions not only to handling and transplantations but also for conservation of endangered and desirable genotypes. Many facets of artificial seed production by encapsulating the PLBs have been intensively investigated in the terrestrial and fast disappearing orchid, namely, G. densiflorum. With the ultimate 34

objective of conservation of orchid taxon; PLB production, encapsulation, in vi t ro and in vivo germination of the artificial seeds (Tables 1 and 2) had been achieved. The primary goal of artificial seed system was to recover whole plantlets from artificial seeds under in vitro as well as under field conditions. On immediate transplantation of artificial seeds to the same medium, a high percentage of emergence of well-developed plantlets had been noted within 68 weeks following subculturing. High viability percentage of stored artificial seeds at 4 C in comparison to room temperature indicated the efficiency of low temperature for storage of artificial seeds. The retention of high viability percentage up to 120 days may be due to the availability of nutrients within the gel matrix. It may be noted that while in SE-derived artificial seeds of Santalum album12, only 17% germination could be obtained under nonsterile soil condition, in the present investigation an encouragingly high percentage of germination (28%) under natural condition was achieved in G. densiflorum, following supplementation of the encapsulating matrix with suitable food preservative and fungicide. It was interesting to note that besides preventing desiccation, the food preservative sodium bicarbonate alone was effective in checking contamination. However, in the long run, presence of a fungicide appears to be crucial to resist contamination.

35

Figure- 1: Protocorm- l i ke bodies (PLBs) , encapsulated PLBs and the i r regenerat i on under in vi t ro and natura l condi t i on . The bar represents 10 mm for al l photographs. a, PLBs selected for encapsulation. b, Encapsulated PLBs. c, In vitro regeneration of PLBs. d, Regeneration and emergence of artificial seeds under in vivo conditions.

Development of the protocol for artificial seed production in G. densi f l o rumsuggests that the lengthy and empir i ca l process of hardening could be avoided for transplanta t in i on vitro of -grown plantlets from laboratory condition to natural conditions (Table 2).

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Moreover, the development of the protocol for the endangered orchid G. densi f l o rum may be an usefu l addit i on to in the vivo germination and regeneration of plantlets for storage and transplantation of precious and costly hybrid orchids as well as for conservation of endangered germplasm. The judicious and intelligent coupling of artificial seed technology with that of microcomputer in achieving automated encapsulation and regeneration of plantlets would tremendously increase the efficiency of encapsulation and production of homogeneous and high quality artificial seeds, and will thus revolutionize the current concept of commercial micropropagation method by the beginning of twenty-first century.

References: 1. Murashige, T., in Frontiers of Plant Tissue Culture (ed. Thorpe, T. A.), 1978, p. 15. 2. Kitto, S. L. and Janick, J., J. Am. Orchid Soc. Hortic. Sci., 1985, 110, 277288. 3. Fujii, J, A. Slade and Redenbough, K., In vitro Cell Dev. Biol., 1989, 25, 1179. 4. Datta, S. K. and Potrykus, I., Theor. Appl. Genet., 1989, 77, 820 824. 5. Morel, G. M., Am. Orchid Soc. Bull., 1960, 29, 495497. 6. Morel, G. M., Am. Orchid Soc. Bull., 1964, 31, 473477. 7. Sharma, A., Tandon, P. and Kumar, A., Indian J. Exp. Biol., 1992, 30, 744748. 8. Malemngaba, H., Roy, B. K., Bhattacharya, S. and Deka, P. C., Indian J. Exp. Biol., 1996, 34, 801805. 9. Singh, F., Lindleyana, 1991, 6, 6164. 10. Knudson, L., Am. Orchid Soc. Bull., 1946, 15, 214217. 11. Wadhwa, M. K., Verma, K. L. and Singh, R., Seed Sci. Technol., 1989, 17, 99105. 12. Fernendez, P. C., Bapat, V. A. and Rao, P. C. Indian J. Exp. Biol., 1992, 30, 839841.

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38