Artificial Seeds and Their Applications PDF

GENERAL I ARTICLE Artificial Seeds and their Applications G V S Saiprasad Plant propagation using artificial or synthetic seeds devel- oped from...

GENERAL I ARTICLE Artificial Seeds and their Applications G V S Saiprasad Plant propagation using artificial or synthetic seeds devel- oped 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 and plant lines with problems in seed propagation. Being clonal in nature G V S Saiprasad is a the technique cuts short laborious selection procedure of scientist in Division of the conventional recombination breeding and can bring the Biotechnology, Indian Institute of Horticulture advancements of biotechnology to the doorsteps of the Science, Bangalore. He is farmer in a cost-effective manner. currently working on molecular events during Need for Artificial or Synthetic Seed Production Techno- ripening in mango and logy banana and on molecular markets. Development of micropropagation techniques will ensure abun- dant supply of the desired plant species. In some crop species seed propagation has not been successful. This is mainly due to heterozygosity of seed, minute seed size, presence of reduced endosperm and the requirement of seed with mycorrhizal fungi association for germination (eg. orchids), 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 vegetative propagation techniques are time consuming and expensive. Development of artificial seed pro- duction technology is currently considered as an effective and efficient alternate method of propagation in several commer- cially important agronomic and horticultural crops. It has been suggested as a powerful tool for mass propagation of elite plant species with high commercial value. Characteristics of clonal propagation systems are discussed in Box 1. Artificial seed technology involves the production of tissue culture derived somatic embryos encased in a protective coat- ing. Artificial seeds have also been often referred to as synthetic ------~~-------­ RESONANCE I May 2001 39  GENERAL I ARTICLE Box 1. Characteristics of Clonal Propagation Systems Micropropagation Low volume, small scale propagation method Maintains genetic uniformity of plants Acclimatisation of plantlets required prior to field planting High cost per plantlet Relatively low multiplication rate Greenhouse cuttings Low volume, small scale propagation method Maintains genetic uniformity of plants Rooting of plantlets required prior to field planting High cost per plantlet Multiplication rate limited by mother plant size Artificial seeds High volume, large scale propagation method Maintains genetic uniformity of plants Direct delivery of propagules to the field, thus eliminating transplants Lower cost per plantlet Rapid multiplication of plants. seeds. However, the term 'synthetic seed' should not be con- fused with commercial seeds of a synthetic cultivar which is defined as an advanced generation of an open pollinated popula- tion composed of a group of selected inbred clones or hybrids. The concept of artificial or synthetic seed is shown in Figure 1. ~--- Artificial seed coat ;''''·i~~~''''''''''''''''- Somatic embryo.~-~~ -.....,.a;iIiIii!o'Il!-ooOi~-- Artificial endosperm Figure 1. Artificial seed con- cept. -40-----------------------------~-------------R-ESO--~-----I-M-a-Y-2-00-1  GENERAL I ARTICLE Box.2. Advantages of Artificial or Synthetic Seeds over Somatic Embryos for Propagation Ease of handling while in storage Easy to transport Has potential for long term storage without losing viability Maintains the clonaJ nature of the resulting plants Serves as a channel for new plant lines produced through biotechnological advances to be delivered directly to the greenhouse or field Allows economical mass propagation of elite plant varieties. These synthetic seeds would 'also be a channel for new plant lines produced through biotechnological advances to be deliv- ered directly to the greenhouse or field. Advantages of artificial! synthetic seeds over somatic embryos for propagation are listed in Box 2. This synthetic seed production technology is a high volume, low-cost production technology. High volume propa- gation 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. What are Somatic Embryos? Somatic embryos are bipolar structures with both apical and basal meristematic regions, 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 vs Zygotic Embryos and their Advantages Since the natural Somatic embryos are structurally similar to zygotic embryos seed develops as found in seeds and possess many of their useful features, includ- a result of a sexual ing the ability to grow into complete plants. 'However, somatic process in cross- embryos differ in that they develop from somatic cells, instead of pollinating species, zygotes (i.e., fusion product of male and female gametes) and it is not genetically thus, potentially can be used to produce duplicates of a single identical to one genotype. Since the natural seed develops as a result of a sexual single parent. process in cross-pollinating species, it is not genetically identi- --------~-------­ RESONANCE I May 2001 41  GENERAL I ARTICLE cal to one single parent. In contrast, somatic embryo develops from somatic cells (non-sexual) and does not involve sexual recombination. This characteristic of somatic embryos allows not only clonal propagation but also specific and directed changes to be introduced into desirable elite individuals by inserting isolated gene sequences into somatic cells. This bypasses ge- netic recombination and selection inherent in conventional breeding technology. If the production efficiency and conve- nience comparable to that of a true seed are achieved, somatic embryos can be potentially used as a clonal propagation system. The lack of synchrony of Basic Requirement for Production of Artificial somatic embryos Seeds is, arguably, the single most Recently, production of synthetic seeds by encapsulating so- important hurdle to matic embryos has been reported in few species. One pre- be overcome requisite for the application of synthetic seed technology in before advances micropropagation is the production of high-quality, vigorous leading to somatic embryos that can produce plants with frequencies com- widespread parable to natural seeds. Inability to recover such embryos is commercialization often a major limitation in the development of synthetic seeds. of synthetic seeds Synthetic seed technology requires the inexpensive production can occur. oflarge numbers of high quality somatic embryos with synchro- Synch ron ized nous maturation. The overall quality of the somatic embryos is embryoid critical for achieving high conversion frequencies. Encapsula- development is tion and coating systems, though important for delivery of required for the somatic embryos, are not the limiting factors for development of efficient production syn thetic seeds. of synthetic seeds. At present, the characteristic lack of developmental synchrony in embryogenic systems stymies multi-step procedures for guid- ing somatic embryos through maturation. The lack of syn- chrony of somatic embryos is, arguably, the single most impor- tant hurdle to be overcome before advances leading to wide- spread commercialization of synthetic seeds can occur. Syn- chronized embryoid development is required for the efficient production of synthetic seeds. -42-------------------------------~~-------------R-ES-O-N-A-N-C-E--I-M--aY--2-0-0-1  GENERAL I ARTICLE Procedure for Production of Artificial Seeds Establish somatic embryogenesis J, Mature somatic embryos J, Synchronize and singulate somatic embryos J, Mass production of somatic embryos ~ Standardization of encapsulation J, Standardization of artificial endosperm Alginate hydrogel ~ is frequently Mass production of synthetic seeds selected as a ~ matrix for synthetic Greenhouse and field planting seed because of its moderate Types of Gelling Agents used for Encapsulation viscosity and low spinnability of Several gels like agar, alginate, pol yco 2133 (Bordon Co.), carboxy solution, low methyl cellulose, carrageenan, gelrite (Kelko. Co.), guargum, toxicity for somatic sodium pee tate, tragacanth gum, etc. were tested for synthetic embryos and quick seed production, out of which alginate encapsulation was found gellation, low cost to be more suitable and practicable for synthetic seed produc- and bio- tion. Alginate hydrogel is frequently selected as a matrix for compatibility synthetic seed because of its moderate viscosity and low characteristics. spinnability 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. Alginate encapsulated somatic embryos of orchids are shown in Figure 2 and the plantlets derived from artificial or synthetic seeds of orchid are shown in Figure 3. --------~-------- RESONANCE I May 2001 43  GENERAL I ARTICLE Figure 2 (left). Artificial or Principle and Conditions for Encapsulation with synthetic seed produced in Alginate Matrix orchids by alginate encap- sulation. Alginate is a straight chain, hydrophilic, colloidal polyuronic Figure 3 (right). Artificial or acid composed primarily of hydro-/3-D-mannuronic acid resi- synthetic seed derived dues with 1-4 linkages. The major principle involved in the plantlets in orchid. alginate encapsulation process is that the sodium alginate drop- lets containing the somatic embryos when dropped into the Somatic embryos CaClz-2H20 solution form round and firm beads due to ion lack seed coat exchange between the Na+ in sodium alginate with Ca2 + in the (testa) and CaCI 2.2H20 solution. The hardness or rigidity of the capsule endosperm that mainly depends upon the number of sodium ions exchanged provide protection with calcium ions. Hence, the concentration of the two gelling and nutrition for agents i.e., sodium alginate and CaCI 2.2H 20, and the complexing zygotic embryos in time should be optimized for the formation of the capsule with developing seeds. optimum bead hardness and rigidity. In general, 3% sodium To augment these alginate upon complexation with 75 mM CaCI2.2H 20 for half an deficiencies, hour gives optimum bead hardness and rigidity for the produc- addition of tion of viable synthetic seeds. nutrients and Artificial Endosperm growth regulators to the Somatic embryos lack seed coat (testa) and endosperm that encapsulation provide protection and nutrition for zygotic embryos in devel- matrix is desired, oping seeds. To augment these deficiencies, addition of nutri- which serves as an ents and growth regulators to the encapsulation matrix is de- artificial sired, which serves as an artificial endosperm. Addition of endosperm. nutrients and growth regulators to the encapsulation matrix --------~------~ 44 RESONANCE I May 2001  GENERAL I ARTICLE results in increase in efficiency of germination and viability of encapsulated somatic embryos. These synthetic seeds can be stored for a longer period of time even up to 6 months without losing viability, especially when stored at 4°C. Addition of Adjuvants to the Matrix In addition to preventing the embryo from desiccation and mechanical injury, a number of useful materials such as nutri- ents, fungicides, pesticides, antibiotics and microorganisms (eg. rhizobia) may be incorporated into the encapsulation ma- trix. Incorporation of activated charcoal improves the conver- sion and vigour of the encapsulated somatic embryos. It has been suggested that charcoal breaks up the alginate and thus increases respiration of somatic embryos (which otherwise lose vigour within a short period of storage). In addition, charcoal retains nutrients within the hydrogel capsule and slowly re- leases them to the growing embryo. Utilization of Artificial Seeds The artificial seeds can be used for specific purposes, notably In addition to multiplication of non-seed producing plants, ornamental hy- preventing the brids (currently propagated by cuttings) or the propagation of embryo from polyploid plants with elite traits. The artificial seed system can desiccation and also be employed in the propagation of male or female sterile mechanical injury, plants for hybrid seed production. Cryo-preserved artificial a number of useful seeds may also be used for germplasm preservation, particularly materials such as in recalcitrant species (such as mango, cocoa and coconut), as nutrients, these seeds will not undergo desiccation. Furthermore, fungicides, transgenic plants, which require separate growth facilities to pesticides, maintain original genotypes may also be preserved using so- antibiotics and matic embryos. Somatic embryogenesis is a potential tool in the microorganisms genetic engineering of plants. Potentially, a single gene can be (eg. rhizobia) may inserted into a somatic cell. In plants that are regenerated by be incorporated somatic embryos from a single transgenic cell, the progeny will into the not be chimeric. Multiplication of elite plants selected in plant encapsulation breeding programs via somatic embryos avoids the genetic re- matrix. -R-ES-O-N-A-N-C-E--I-M-a-Y---2-00-1--------------~~-----------------------------45-  GENERAL I ARTICLE Artificial seeds combination, and therefore does not warrant continued selec- produced in tissue tion inherent in conventional plant breeding, saving consider- culture are free of able amount of time and other resources. Artificial seeds pro- pathogens. Thus, duced in tissue culture are free of pathogens. Thus, another another advantage advantage is the transport of pathogen free propagules across the is the transport of international borders avoiding bulk transportation of plants, pathogen free quarantine and spread of diseases. propagules across the international Potential Uses of Artificial Seeds borders avoiding Delivery systems: bulk transportation of plants, Reduced costs of transplants quarantine and Direct greenhouse and field delivery of: spread of diseases. elite, select genotypes hand-pollinated hybrids genetically engineered plants sterile and unstable genotypes Large-scale mono cultures Mixed-genotype plantations Carrier for adjuvants such as microorganisms, plant growth regulators pesticides, fungicides, nutrients and antibiotics Protection of meiotically-unstable, elite genotypes Can be conceivably handled as seed using conventional plant- ing equipment. Analytical tools: Comparative aid for zygotic embryogeny Production of large numbers of identical embryos Determination of role of endosperm in embryo development and germination Study of seed coat formation Study of somaclonal variation. -------------~~----------­ 46 RESONANCE I May 2001  GENERAL I ARTICLE Applicability and Feasibility of Artificial Seed Production Technology In order to be useful, synthetic seed must either reduce produc- tion costs or increase crop value. The relative benefits gained, when weighed against development costs, will determine whether its use is justified for a given crop species. Considering a combination of factors, including improvement of the existing embryogenic systems, relative cost of seed as well as specific application for synthetic seed allows judgement of relative need for a given crop. For example, synthetic seed of seedless water- melon would actually cost less than conventional seed, provid- ing a benefit at the outset of crop production. Although embryo- genic systems for this crop do not exist, the benefit that could be conferred by use of synthetic seed would be very great. Value- added aspects that would increase crop worth are numerous and include cloning of elite genotypes, such as genetically engi- neered varieties, that cannot produce true seed. Suggested Reading K Redenbaugh, (ed.), Synthetic seeds, CRC Press, Boca Raton, 1993. J A Fujii, D T Slade, K Redenbaugh, and K A Walker, Artificial seeds Address for Correspondence for plant propagation, Trends in Biotechnology, 5, 335-339,1987. G V 5 Saiprasad I Kinoshita, The production and use of artificial seed, Research Journal Division of Biotechnology of Food and Agriculture, 15 (3), 6-11, 1992. IIHR, Hessarghatta Lake Post T Senaratna, Artificial seeds, Biotechnology Adf)Qnces-, 10, 379-392, Bangalore 560 089. India. 1992. ~. There are grounds for cautious opti- mism that we may now be near the end of the search for the ultimate laws of I " nature. Stephen W Hawking A brief history of time: from the Big Bang to Black Holes -R-ES-O-N-A-N-C-E--I-M-a-Y---2-00-1------------~~-------------------------------4-7