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

TRAINING MANUAL[For professionals and skilled workers]

Vegetable Seed Treatment and Conditioning

Edited by

Muhammad Boota Sarwar

For

Facilitation Unit for Participatory Vegetable SeedAnd Nursery Production Program

Ministry of Food, Agriculture & Livestock, Islamabad

1

CONTENTS

Sr.No.

Chapters Pag

e No.

1 Introduction

3

2 PART-I: Vegetable Seed Treatment 4

3 PART-II: Vegetable Seed Coating 5

4 PART-III: Vegetable Seed Pelleting 21

5 PART-IV: Vegetable Seed Encrusting 27

6 PART-V: Vegetable Seed Priming 80

7 PART-VI: Organic Seed Treatment & Coating 85

8 PART-VII: Technology Licensing Opportunities 87

9 PART-VIII: Encapsulation of Embryos – Artificial Seeds

103

10 PART-IX: Artificial seed technology: Development of a protocol in Geodorum

112

2

densiflorum (Lam) Schltr. – An endangered orchid

INTRODUCTION

3

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 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

4

vitro conditions and that retain this potential also after storage. 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 or embryogenic 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 organogenesis and 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 large-scale 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

5

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 (Table 2). 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 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-to-plant development.

6

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: desiccated and 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.

7

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 but sodium alginate gel is the most popular5. Hydrated artificial seeds consist of somatic embryos individually encapsulated in a hydrogel (Figure 2). To produce hydrated synthetic seeds, the somatic embryos are mixed with sodium alginate gel (0.5–5.0% w/v) and dropped into a calcium salt solution [CaCl2 (30–100 mM), Ca (NO3)2 (30–100 mM)] where ion-exchange 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 satisfactory. However, Molle et al. found that for the production of synthetic seeds of carrot, 1% sodium alginate solution, 50 mM Ca2+ and 20–30 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 (Table 2). The technology of hydrogel encapsulation is also favored for synthetic seed production from these micropropagules.

8

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 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. added an antibiotic mixture (0.25 mg/l) containingrifampicin (60 mg), cefatoxime (250 mg) and tetracycline-HCl (25 mg) dissolved in 5 ml dimethylsulphoxide 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 propagation techniques are time consuming and expensive. Development of artificial seed production technology is currently being considered as an effective and efficient alternate method of propagation in several commercially important agronomic and horticultural crops. It has been suggested as powerful tool for mass propagation of elite plant species with high commercial value.

Artificial seed technology involves the production of tissue culture derived somatic embryos encased in a protective

9

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

# Micro-propagation

Greenhouse Cuttings

Artificial Seeds

1 Low volume, small scale propagation method

Low volume, small scale propagation method

High volume, large scale propagation method

2 Maintains genetic purity of plants

Maintains genetic purity of plants

Maintains genetic purity of plants

3 Acclimatization of plantlets required prior to field planting

Rooting of plantlets required prior to field planting

Direct delivery of propagules to the field, thus eliminating transplants

4 High cost per plantlet

High cost per plantlet

Low cost per plantlet

5 Relatively low Multiplication Rapid

10

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 mango (Mangifera indica L.) encapsulated in calcium alginate capsule (embryos are approximately 3–5 mm long).

Table-2: Some Plant Species with Encapsulation Methodology.Plant Propagule

used forencapsulation

Plant Propaguleused for

encapsulation

Actinidia deliciosa (Kiwifruit) Shoot Bud

Arachis hypogaea (Groundnut)

Somatic Embryo

11

Asparagus cooperi Somatic Embryo

Brassica campestris (Mustard)

Shoot Bud

Betula pendula (Birch) Shoot Bud Cymbidium giganteum (Orchid)

protocorm-like bodies

Daucus carota (Carrot)

Somatic Embryo

Dendrobium wardianum (Orchid)

protocorm-like bodies

Eleusine coracana Gaertn. (Finger millet)

Somatic Embryo

Eucalyptus citriodora (Eucalyptus)

Somatic Embryo

Malus pumila Mill. (Apple rootstock M.26)

Shoot Bud Mangifera indica L. (Mango cv. Amrapali)

Somatic Embryo

Medicago sativa (Alfalfa)

Somatic Embryo

Morus indica (Mulberry)

Shoot Bud

Musa (Banana cv. Basrai)

Shoot Bud Pistacia vera L. (Pistachio)

Somatic Embryo &

Embryogenic Masses

Psidium guajava (Guava)

Somatic Embryo

Rubus idaeus L. (Raspberry)

Shoot Bud

Rubus (Blackberry cv. Jumbo, Veten)

Shoot Bud Santalum album (Sandalwood)

Somatic Embryo

Solanum melongena (Eggplant)

Somatic Embryo

Vitis vinifera (Grape) Somatic Embryo

Zingiber officinale Rosc. (Ginger)

Shoot Bud

Advantages of Artificial or Synthetic Seeds over Somatic Embryos for Propagation:

1. Ease of handling while in storage2. Easy to transport3. Has potential for long term storage without loosing

viability4. Maintains clonal of the resulting plants5. 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

What are Somatic Embryos?

12

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.

13

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 better protection (than agar) to the encased somatic embryos against mechanical injury.

14

Principles and Conditions for Encapsulation with Alginate Matrix:

Alginate is a straight chain, hydrophilic, colloidal polyuronic acid composed primarily of hydro-β-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.

Addition of Adjuvant to the Matrix:

15

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 plants2. Multiplication of ornamental hybrids, currently

propagated by cuttings3. Propagation of polyploid plants with elite traits; or

plants with meiotically-unstable elite genotypes.4. Propagation of male or female sterile plants for hybrid

seed production5. Cryo-preserved artificial seeds may also be used for

germplasm preservation, particularly in recalcitrant species (such as mango, cocoa and coconut), as these seeds will not undergo desiccation.

6. Transgenic plants which require separate growth facilities to maintain original genotype may also be preserved using artificial seeds. Somatic embryogenesis is a potential tool in the genetic engineering of plants. Potentially, a single gene can be inserted into a somatic cell. In plants that are regenerated by somatic embryos from a single transgenic cell, the progeny will not be chimeric. Multiplication of elite plants selected in plant breeding programs via somatic embryos avoids the genetic

16

recombination, and therefore does not warrant continued selection inherent in conventional plant breeding, saving considerable amount of time and other resources.

7. Artificial seeds produced in tissue culture are free of pathogens; thus, another advantage is the transport of pathogen free propagules across the international borders avoiding bulk transportation of plants, quarantine 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 encapsulation to exploit this potential, but success has been relatively limited except for Medicago sativa.

17

In alfalfa (M. sativa) desiccation-tolerance of somatic embryos was induced by exogenous application of abscisic acid (ABA) by Senaratna, Mckersie and Bowley. Subsequently, the embryos were dried to 10–15% moisture and stored for at least 3 weeks in the dry state. Under appropriate treatment conditions, 65% of these somatic embryos survived and germinated in a manner analogous to a true seed. Desiccation-tolerance has also been induced in alfalfa somatic embryos by exposure to sub-lethal levels of low temperature, water, nutrient or heat stress. However, these pretreatments had deleterious effects on embryo maturation and plantlet vigor.

Onishi, Sakamoto and Hirosawa have demonstrateda 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 conversionof celery and carrot embryos produced in bioreactors, could be raised to 53–80% from 0% by three sequential treatments:

(i) Culturing the embryos for 7 days in a medium of high osmolarity (with 10% mannitol) less than 16 h photoperiod with 300 lux of illumination for promoting embryo development. This treatment increased the size of embryos from 1–3 mm to 8 mm and their chlorophyll content.

(ii) (ii) Dehydration of embryos to reduce their water content from 95–99% to 80–90% by keeping them for 7 days on 2–7 layers of filter paper under a 16 h photoperiod of 14 mE m–2 s–1 irradiance.

18

(iii) 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 20°C 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 Santalum album24,26, Pistaciavera22 and Mangifera indica 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. Axillary shoot buds and apical shoot tips

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 encapsulated buds of banana and mulberry converted into plantlets without specific root induction treatments. In

19

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 3–6 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 axillary microcuttings 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 alginate gel using the method of Redenbaugh et al. and stored at 4°C after treatment with BAP. Onay, Jeffree and

20

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-like bodies

In orchids such as Cymbidium giganteum, endrobiumwardianum, Geodorum densiflorum, 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.

21

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:

Limited production of viable micropropagules useful in synthetic seed production.

Anomalous and asynchronous development of somatic embryos.

Improper maturation of the somatic embryos that makes them inefficient for germination and conversion into normal plants.

Lack of dormancy and stress tolerance in somatic embryos that limit the storage of synthetic seeds.

Poor conversion of even apparently normally matured somatic embryos and other micropropagules into plantlets that limit the value of the synthetic seeds and ultimately the technology itself.

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. Hence, maturation of somatic embryos, which eventually controls germination and conversion rate, is one of the major

22

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 vitro culture system the somatic embryos show great diversity in their morphology and accordingly in their response which greatly limits the use of synthetic seed technology. Lee and Soh have indicated that continuous ABA treatment increases the formation of somatic embryos with anomalous cotyledons, while in some instances ABA has been found to promote the normal development of both somatic and zygotic embryos in vitro. Cytokinin treatment also increases the number of somatic embryos with multiple cotyledons.

23

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 vitro. 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. These embryos were stored at –20°C for a year and

24

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 non-damaging 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 capsule gel can potentially serve as a reservoir for nutrients

25

(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.Either hydrated calcium alginate-based or desiccated polyoxyethylene glycol-based artificial seeds might be used,

26

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. Akhtar, N., Ph D thesis, Banaras Hindu University, Varanasi, 1997

2. Ammirato, P. V., in Handbook of Plant Cell Culture (eds Evans, D. A., Sharp, W. R., Ammirato, P. V. and Yamada, Y.), Macmillan Publishing Co, New York, 1983, vol. 1, pp. 82–123

3. Ammirato, P. V., Plant Physiol., 1977, 59, 579–5864. Ara, H., Jaiswal, U. and Jaiswal, V. S., Plant Cell Rep.,

1999, 19, 166–1705. Ara, H., Ph D thesis, Banaras Hindu University,

Varanasi, 19986. Arya, K. R., Beg, M. U. and Kukreja, A. K., Indian J. Exp.

Biol., 1998, 36, 1161–11647. Attree, S. M., Moore, D., Sawhney, V. K. and Fowke, L.

C., Ann. Bot., 1991, 68, 519–5258. Attree, S. M., Pomeroy, M. K. and Fowke, L. C., J. Exp.

Bot., 1995, 46, 433–4399. Attree, S. and Fowke, L., Plant Cell Tissue Org. Cult.,

1993, 35, 1–3510. Attree, S. M., Pomeroy, M. K. and Fowke, L. C., Plant

Cell Rep., 1994, 13, 601–60611. Bapat, V. A. and Rao, P. S., Plant Cell Tissue Org. Cult.,

1990, 20, 69–7012. Bapat, V. A. and Rao, P. S., J. Plant Biochem.

Biotechnol., 1992, 1, 109–11313. Bapat, V. A. and Rao, P. S., Plant Cell Rep., 1988, 7,

434–43614. Bapat, V. A., in Synseeds (ed. Redenbaugh, K.), CRC

Press, Boca Raton, 1993, pp. 381–407

27

15. Burns, J. A. and Wetzstein, H. Y., Plant Cell Tissue Org. Cult., 1997, 48, 93–102

16. Capuano, G., Piccioni, E. and Standardi, A., J. Hortic. Sci. Biotechnol., 1998, 73, 299–305

17. Capuano, M. and Debergh, P. C., Plant Cell Tissue Org. Cult., 1997, 48, 23–29

18. Choi, Y. E., Kim, H. S., Soh, W. Y. and Yang, D. C., Plant Cell Rep., 1997, 16, 738–744

19. Compton, M. E., Benton, C. M., Gray, D. J. and Songstad, D. D., In vitro Cell Dev. Biol., 1992, 28, 197–201

20. Corrie, S. and Tandon, P., Indian J. Exp. Biol., 1993, 31, 61–64

21. Crouch, M. L. and Sussex, I. M., Planta, 1981, 153, 64–74

22. Datta, K. B., Kanjilal, B. and Sarker, D., Curr. Sci., 1999, 76, 1142–1145

23. Fowke, L. and Attree, S., Plant Tissue Cult. Biotechnol., 1996, 2, 124–130

24. Ganapathi, T. R., Suprasanna, P., Bapat, V. A. and Rao, P. S., Plant Cell Rep., 1992, 11, 571–575

25. George, L. and Eapen, S., Indian J. Exp. Biol., 1995, 33, 291–293

26. Ghosh, B. and Sen, S., Plant Cell Rep., 1994, 13, 381–385

27. Gray, D. J. and Purohit, A., Crit. Rev. Plant Sci., 1991, 10, 33–61

28. Gray, D. J., HortSci., 1987, 22, 810–81429. Hammatt, N. and Davey, M. R., J. Plant Physiol., 1987,

128, 219–22630. Gupta, P. K., Shaw, D. and Durzan, D. J., in Cell and

Tissue Culture in Forestry (eds Bonga, J. M. and Durzan, D. J.), Martinus Nijhoff Publishers, Dordrecht, 1987, pp. 101–108

31. 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. Kinoshita, The Production and use of artificial seeds, Research Journal of Food and Agriculture, 15(3), 6-11, 1993.

33. J. A. Fujii, D. T. Slade, K. Redenbaugh and K. A. Walker; Artificial Seeds for Plant Propagation, Trends in Biotechnology, 5, 335-339, 1987.

34. Jain, S. M., Gupta, P. K. and Netwon, R. J., Somatic Embryogenesis in Woody Plants, Kluwer Academic Publishers, Dordrecht, 1995

35. Janeiro, L. V., Ballester, A. and Vieitez, A. M., Plant Cell Tissue Org. Cult., 1997, 51, 119–125

36. Janick, J., Kim, Y.-H., Kitto, S. and Saranga, Y., in Synseeds (ed. Redenbaugh, K.), CRC Press, Boca Raton, 1993, pp. 12–34

37. Jeong, R. B., Fujiwara, K. and Kozai, T., in Horticultural Reviews (ed. Janick, J.), Wiley, New York, 1995, vol. 17, pp. 125–172

38. Kitto, S. K. and Janick, J., HortSci., 1982, 17, 48839. Kitto, S. and Janick, J., J. Am. Soc. Hortic. Sci., 1985,

110, 277, 28340. K. Redenbaugh; Synthetic Seeds, CRC Press, Boca

Raton, 1993.41. Lakshmana Rao, P. V. and Singh, B., Plant Cell Rep.,

1991, 10, 7–1142. Lee, K. S. and Soh, W. Y., Korean J. Plant Tissue Cult.,

1994, 21, 287–29143. Liu, C.-M., Xu, Z.-H. and Chua, N.-H., Plant Cell, 1993,

5, 621–63044. Lulsdorf, M. M., Tautorus, T. E., Kikcio, S. I., Bethune, T.

D. and Dunstan, D. I., Plant Cell Rep., 1993, 12, 385–389

45. Malemngaba, H., Roy, B. K. Bhattacharya, S. and Deka, P. C., Indian J. Exp. Biol., 1996, 34, 801–805

46. Marsolais, A. A., Wilson, D. P. M., Tsujita, M. J. and Senaratna, T., Can. J. Bot., 1991, 69, 1188–1193

47. Mckersie, B. D. and Bowley, S. R., in Synseeds (ed. Redenbaugh, K.), CRC Press, Boca Raton, 1993, pp. 231–255

29

48. Michler, C. H. and Bauer, E. O., Plant Sci., 1991, 77, 111–118

49. 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. 257–270

50. 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. 101–108

51. Norstog, K., Am. J. Bot., 1965, 52, 538–54652. Onay, A., Jeffree, C. E. and Yeoman, M. M., Plant Cell

Rep., 1996, 15, 723–72653. Onishi, N., Sakamoto, Y. and Hirosawa, T., Plant Cell

Tissue Org. Cult., 1994, 39, 137–14554. Padmaja, G., Reddy, L. R. and Reddy, G. M., Indian J.

Exp. Biol., 1995, 33, 967–97155. Parrott, W. A., Dryden, G., Vogt, S., Hildebrand, D. F.,

Collins, G. B. and Williams, E. G., In Vitro Cell Dev. Biol., 1988, 24, 817–820

56. Piccioni, E., Plant Cell Tissue Org. Cult., 1997, 47, 255–260

57. Piccioni, E. and Standardi, A., Plant Cell Tissue Org. Cult., 1995, 42, 221–226

58. Redenbaugh, K., Fujii, J. A. and Slade, in Synseeds (ed. Redenbaugh, K.), CRC Press, Boca Raton, 1993, pp. 38–46

59. Redenbaugh, K., HortSci., 1990, 25, 251–25560. Redenbaugh, K. and Walker, K., in Plant Tissue Culture:

Applications and Limitations (ed. Bhojwani, S.), Elsevier, Amsterdam, 1990, pp. 102–135

61. Redenbaugh, K., Nichol, J., Kossler, M. E. and Paasch, B., In vitro, 1984, 20, 256–257

62. Redenbaugh, K. (ed.), Synseeds, CRC Press, Boca Raton, 1993

63. Redenbaugh, K., Slade, D., Viss, P. and Fujii, J., HortSci., 1987, 22, 803–809

64. Refouvelet, E., LeNours, S., Tallor, C. and Daguin, F., Sci. Hortic., 1998, 74, 233–241

30

65. Saiprasad G. V. S; Resonance, May 2001, page 39-4766. Sharma, A., Tandon, P. and Kumar, A., Indian J. Exp.

Biol., 1992, 30, 747–74867. Sharma, T. R., Singh, B. M. and Chauhan, R. S., Plant

Cell Rep., 1994, 13, 300–30268. Senaratna, T., Mckersie, B. D. and Bowley, S. R., Plant

Sci., 1989, 65, 253–25969. Senaratna, T., Saxena, P. K. Rao, M. V. and Afele, J.,

Plant Cell Rep., 1995, 14, 375–37970. Singh, F., Lindleyana, 1991, 6, 61–6471. T. Senaratna, Artificial Seeds, Biotechnology Advances,

10, 379-392, 1992.72. Wetzstein, H. Y. and Baker, C. M., Plant Sci., 1993, 92,

81–89

Artificial seed technology: Development of a protocol in Geodorum densiflorum (Lam) Schltr. – An endangered orchid

K. B. Datta*, B. Kanjilal 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 University College, Raiganj 733 134, India (Received 10 August 1998; revised accepted 29 January 1999)

The research communication reports the production of artificial seeds through encapsulation of protocorm-like bodies (PLBs) of Geodorum densiflorum (Lam) Schltr. – an endangered orchid taxon of Terai Hills, 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 l–1), 6-benzyl-aminopurine (2 mg l–1), and a -napthaleneacetic acid (1 mg l–1). 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

31

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 densiflorum (Lam) Schltr., as well as by those of a few others7–9, suggests the that most suitable encapsulating agent for orchid protocorm-like bodies (PLBs) is sodium alginate, due to its solubility atroom 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 vitro, 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 l–1 of 6-benzylaminopurine (BAP), 1 mg l–1 a -napthaleneacetic acid (NAA), 2 gl–1 peptone and 15% coconut milk (CM). The pH of the medium was adjusted to 5.6–5.8. 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-head-like 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 15–20 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.

33

Fungicide bavastin was incorporated into the nutrient gel at a constant concentration of 4 mg l–1, 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 l–1. 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 l–1), BAP (2 mg l–1) and NAA (1 mg l–1), 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 6–8 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 l–1 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 vitro 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 6–8 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 non-sterile 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-like bodies (PLBs), encapsulated PLBs and their regeneration under in vitro and natural condition. The bar represents 10 mm for all 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. densiflorum suggests that the lengthy and empirical process of hardening could be avoided for transplantation of in vitro-grown plantlets from laboratory condition to natural conditions (Table 2).

36

Moreover, the development of the protocol for the endangered orchid G. densiflorum may be an useful addition to the in 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, 277–288.

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, 495–497. 6. Morel, G. M., Am. Orchid Soc. Bull., 1964, 31, 473–477. 7. Sharma, A., Tandon, P. and Kumar, A., Indian J. Exp. Biol., 1992,

30, 744–748. 8. Malemngaba, H., Roy, B. K., Bhattacharya, S. and Deka, P. C.,

Indian J. Exp. Biol., 1996, 34, 801–805. 9. Singh, F., Lindleyana, 1991, 6, 61–64. 10. Knudson, L., Am. Orchid Soc. Bull., 1946, 15, 214–217. 11. Wadhwa, M. K., Verma, K. L. and Singh, R., Seed Sci.

Technol., 1989, 17, 99–105. 12. Fernendez, P. C., Bapat, V. A. and Rao, P. C. Indian J. Exp.

Biol., 1992, 30, 839–841.

37

38