Preface Nowadays, artificial seed technology is one of the most important tools to breeders and scientists of plant tissue culture. It has offered powerful advantages for large scale mass propagation of elite plant species and conservation of plants. The artificial seed technology is anrousing and fast growing area of research in plant tissue culture. This technology is currently considered as an efficient alternative technique for plant propagation of commercially important plant materials. This technology also facilitates the way of handling cells and tissues, protecting them from external gradients, short-term and long-term storage under low temperature and ultra-low temperature, respectively and as an efficient system of delivery. The synthetic seed technology provides many other advantages which have been discussed in detail in different chapters in this endeavour. The information in the areas of synthetic seed preparation technology, its implications, achievements and limitations are lying unorganized in different articles of journals and edited books. Those information was presented in this monograph in organized way with up-to-date citations, which will provide comprehensive literatures of recent advances.To be an expert in artificial seeds, one should have enough practical hand in plant tissue culture. So, in Chapter 2, Fundamentals of Plant Tissue Culture has been elaborated in simple approach. The base materials for production of synthetic seeds are somatic embryos or tissue culture derived materials, viz. shoot tips, axillary buds, meristem etc. To produce large number of synthetic seeds, a continuous supply of source material is very essential. The different sources of encapsulating plant materials have been detailed in this book as a separate chapter. A separate chapter also has been included on hardening of artificial seed derived plantlets or plant tissue cultured derived plantlets to make the in vitro developed plantlets suitable for planting in field condition. The main aim of artificial seeds is to obtain true-to-type plants; consequently thedifferent methods of testing clonal fidelity have been described in Chapter 9. It is my pleasure to acknowledge the help of Ms. Priyanka Sharma and Ms. Pallabi Saha for their precarious review to improve the manuscript. I also like to acknowledge the service received from my students Mr. Gadge Susant Sundarrao and Mr. Monish Roy during preparation of the manuscript. I am greatly obliged to Prof. Asit Kumar Basu (BCKV, Mohanpur, Nadia, West Bengal, India) and Dr. Asit B. Mandal under whose guidance I learnt the production technology of synthetic seed. I also acknowledge the encouragement and assistance received from the staff of the Department of Seed Science and Technology (UBKV, Pundibari, Cooch Behar, West Bengal) to write this book. I am indebted to the Vice-Chancellor of Uttar Banga Krishi Viswavidyalaya for allowing me in preparation of this book. I am especially indebted to my beloved son KINJAL for his many small pieces of assistance during the preparation of the manuscripts.

Introduction Progress in plant biotechnological research has opened many avenues for basic and applied research in the field of crop plants. Plant tissue culture is an important component of biotechnology that involves in the improvement of crops. Besides this, plant tissue culture provides a good system for many basic studies in plant breeding, plant physiology, genetics and cell biology. Further, manipulation of cells through the modern technique of genetic engineering may lead to an introduction of new gene(s) to a host plant and the techniques of genetic engineering rely on plant tissue culture techniques. The regeneration of plants through plant tissue culture and their subsequent acclimatization and delivery to the field possesses many problems to make tissue culture technology a viable alternative proposition. The successful development of encapsulation technique for preparation of plant propagules in a nutrient gel has initiated a new line of research on artificial seeds. Application of this technology has been well recognized in several agronomically important crops and forest trees. Artificial seeds make a promising technique for propagation of transgenic plants, non-seeds producing plants, polyploids with elite traits and plant lines with problems in seed propagation.

2.1. LABORATORY SETUP The setting up of a tissue culture laboratory needs proper planning. It depends on the nature of research works to be under taken and/or commercialization of tissue culture technology and the availability of space. The ideal tissue culture laboratory should have the following components: Store facility for chemicals Media preparation room Autoclave room Storage facilities for autoclaved laboratory-ware and media Washing facilit Culture room Inoculation room Instrumentation room Hardening facility

To mimic the natural seeds, the embryos from cultures are encapsulated in a nutrient gel containing essential organic/inorganic salts, carbon sources, plant hormones and antimicrobial agents and coated completely to protect the embryos from mechanical damage during handling and to allow the development and germination without any undesirable variation. When looking at the history of synthetic seed technology, a wide number of encapsulating agents have been tested in time for their capacity to produce beads like, potassium alginate, sodium alginate, carregeenan, agar, agarose, guargum, polyox, gelrite, sodium pectate, carboxymethyl cellulose, nitrocellulose, ethylocellulose, polyethyleneamine (Kersulec et al., 1993), chitosane (Tay et al., 1993), tracanth gum etc. have been tested as hydrogels (Table 3.1), out of which alginate emasculation was found to be more suitable and practical for synthetic seed production. Some plants exudates of arabic (Acacia senegal), karaya (Stereculsia sp.), or seed gums of gaur (Cyamopsis tetragonoloba), tamarind (Tamarindus indica) or microbial products like dextran, xantham are also being used for encapsulation.

The artificial seed production technique was first used in clonal propagation to cultivate somatic embryos placed into an artificial endosperm and constrained by an artificial seed coat. Today artificial seeds represent capsules with a gel envelope, which contains not only somatic embryos but also axillary and apical buds or stem and root segments (Vdovitchenko and Kuzovkina, 2011). The prime requirements for preparation of artificial seeds are high frequency somatic embryogeniccalli, synchronous embryo development and maximum conversion of embryos into plants. Success in production of synthetic seeds mainly depends on how best callus development and plantlet regeneration are achieved. However, over time the morphogenetic competence of differentiated cultures declines (Lynch and Benson, 1991). Therefore, the primary goal of synthetic seed production is to produce somatic embryos that resemble more closely to the true seed embryo in storage and handling characteristics so that they can be utilized as a unit for clonal propagation and germplasm conservation. Synthetic seeds may be hydrated or dehydrated and may be quiescent or not. Encapsulation of micropropagules enables to satisfy the requirements of synthetic seed development. The gelling agents used for encapsulation for production of synthetic seeds act as protective cover. The encapsulated synthetic seeds also contain growth nutrients, plant growth promoting microorganisms (mycorrhizah, Rhizobium, etc.), and/or other biochemical constituents necessary for optimal embryo-to-plant development (Fig. 4.1). Fungicides may also be used in the liquid gelling medium to protect them during storage and germination in vitro and in vivo.

As stated by Murashige’s definition, artificial seed (or synthetic/synseed) technology was initially restricted to species in which somatic embryogenesis was possible. Later, Bapat and co-workers (1987) proposed to broaden the technology to the encapsulation of various in vitro-derived propagules, and they used axillary buds of Morus indica as a first example of this new application. The new concept paved the way for the encapsulation of explants other than somatic embryos, and to the formulation of a new definition of artificial seeds (Aitken-Christie et al. 1995) as “artificially encapsulated somatic embryos, shoots, or other tissues which can be used for sowing under in vitro or ex vitro conditions”. In other words, the “new” artificial seed, lost the original bond of containing an embryo (zygotic or somatic), can now contain any kind of explant from tissue culture (such as axillary buds, shoot tips, nodal segments, bulblets, protocorms, callus samples, cells) which, following ‘germination’, will evolve into a plantlet or a shoot (artificial seed conversion) or will produce new cell proliferation (artificial seed regrowth). Realization of synthetic seed technology requires manipulation of in vitro culture system for large-scale production of viable materials for preparation of synthetic seed, subsequently conversion into complete plants under in vitro as well as in vivo conditions. Somatic and gametic embryogenesis that enhanced axillary bud proliferation are the efficient techniques for rapid and large-scale multiplication of desirable plant species. Plant tissues such as somatic embryos, apical shoot tips, axillary shoot buds, embryogenic calli, and protocom-like bodies are potential micropropagules that have been considered for creating synthetic seeds. Encapsulations of somatic embryos, apical and axillary shoot buds, and regeneration of whole plants from them have been reported for a number of plant species (Redenbaug et al., 1986; Mathur et al., 1989; Ganapathi et al., 1992; Lulsdorf et al., 1993).

In fact, as the concept of synthetic seed involves the use of small propagules and enables the direct sowing of this material in vitro or in vivo, the technology could provide great flexibility to the breeders by not only reducing the costs when large quantities of propagules are required for handling, shipping and planting, but also eliminating the acclimation step when direct sowing in vivo is applied (Onishi et al. 1994). The artificial seed technology has been applied to a number of plant species belonging to angiosperms. The exact application of artificial seeds will vary from species to species. In self-pollinated crops that currently have good seed production systems, synthetic seeds will not have any practical applications, but in cross pollinating species, especially those where seed production is difficult and expensive, synthetic seeds offer many advantages and opportunities. The importance of encapsulation technique as an aid to propagation in various crop plants has been discussed by many workers. By combining the benefits of vegetative propagation system with the capability of long-term storage and with clonal multiplication, synthetic seeds have many diverse applications in agriculture (Gray and Purohit, 1991; Redenbaugh et al., 1991; Redenbaugh, 1993). In some species seed propagation is not successful. The reason behind this may be due to heterozygosity of seed, small sized seed, presence of reduced or insufficient endosperm, and seedless varieties of crop species like grapes, watermelon, mango, guava etc. Sometimes association of mycorrhizal fungi is essential for seed germination, for example orchids. The germination of these seeds under natural conditions or under control environment is not possible. Propagating of these species through synthetic seed is the added advantage. The possible implications of synthetic seed technology have been detailed hereunder.

Plant tissue culture is mainly referred to as mass-multiplication of in vitro derived plantlets grown under controlled environment. Subsequently, the production of artificial seeds from in vitro grown plant propagules is also a method of mass-production of true-to-type plantlets. Micropropagation allows rapid and mass production of high quality, disease free and uniform planting material irrespective of the season and weather. Micro-shoots on being transferred to ex vitro conditions are exposed to abiotic (altered temperature, light intensity and humidity conditions) and biotic stress conditions i.e. soil micro-flora, so there is need for acclimatization for successful establishment and survival of plantlets (Deb and Imchen, 2010). In order to increase growth and reduce mortality in plantlets at the acclimatisation stage, research has been focused on the control of the environmental conditions (both physical and chemical) and to acclimatize the plants to compete with soil microflora (Mathur et al., 2008). In this chapter methods of acclimatization of plantlets derived from simple plant tissue culture or from synthetic seeds are discussed.

Many researchers successfully prepared synthetic seeds of crop plants. The major success of this technology is being elaborated in this chapter. 8.1. FIELD CROPS Rice is the world’s most important food crop and a primary food source for more than one third of the world’s population. This crop has received considerable attention in biotechnology research progammes. Research in artificial seeds in rice is not scanty and this technology through somatic embryogenesis would offer a great scope for large-scale propagation of superior elite hybrids (Brar and Khush, 1994). However, success on artificial seeds research in field crops, especially cereals is still in infancy. However, many workers successfully encapsulated somatic and androgenic embryos of cereal crops (Datta and Potrykus, 1989; Suprasanna et al., 1996; Roy and Mandal, 2004 & 2008). Brar et al. (1994) emphasized the need for research on artificial seeds in rice through embryogenesis and outlined its impact on mass propagation of true-breeding hybrids. Somatic embryogenesis can also be used in the regeneration of genetically transformed plants (Vicient and Martinez, 1998). Subsequently those somatic embryos could be economically and successfully propagated though synthetic seed technology.

Artificial seeds have been widely used for micro-propagation of many plant species. Establishment of gene banks for ex situ conservation of plant germplasm in the form of field gene banks, seed gene banks, in vitro collections, and cryogenically preserved tissues is a common practice (Withers 1983; Rao, 2004; Borner, 2006). Alginate encapsulation provides a viable approach for in vitro germplasm conservation as it combines the advantages of clonal multiplication with those of seed propagation and storage (Standardi and Piccioni, 1998; Ara et al., 2000). The main aim of artificial seeds is to obtain true to type plants to maintain the germplasm, but, however, during tissue culture, there is a chance of genetic aberration which is commonly known as somaclonal variations. Larkin and Scowcroft (1981) for the first time used the word ‘somaclonal variation’ to describe these variations as displayed among the plants derived from tissue culture. It is clearly understood that genomes modify themselves when exposed to unfamiliar conditions, and these modifications are best described as programmed loss of cellular control (Phillips et al., 1994).

Results of intensive researches in the field of synthetic seed technology seem promising for propagation of crop plants. However, as described in literature, the major stumbling block in establishing artificial seed production as a viable technology is a lack of understanding of the somatic embryo process and an inability to consistently produce high-quality propagules that can germinate in a soil environment with an acceptably high success rate7. Several aspects of the techniques are still underdeveloped and hinder its commercial application. 10.1. LACK OF STANDARD PROTOCOLS The synthetic seed technology application in a commercial scale is still hampered by the low efficiency of the protocols and logistic behind it. Till today standard protocol for synthetic preparation is available for a limited number of plant species.

Micropropagation through artificial seeds may be commercially exploited on a large scale, generating millions of plantlets within a few days, and this may become a profitable multibillion rupees industry in near future. This technology would be feasible and even competitive economically with the other seed production methods. In the near future, the synthetic seed technology could be the key to overcome these problems, e.g., through automated procedures of artificial seed production (Aitken-Christie et al. 1995) followed by autotrophic micropropagation (Jeong et al. 1995). The synthetic seed systems coupled with artificial intelligence and microcomputer systems like the most advanced robots, which can mimic the motions, and functions of living beings (i.e. automated encapsulations). It would tremendously increase the efficiency of encapsulation and production of artificial seeds, and revolutionized the plant propagation method in the years ahead. This technology is gradually moving towards the commercial propagation of high value crops. However, there is a great need for refinement of this technology by tackling of certain technical problems such as the need to produce high quality and high fidelity somatic embryos and to avoid the genetic instability and variability of tissue culture derived plants. The somatic variation can be overcome if the process and regulation of somatic embryogenesis and origin of somaclonal variation are well understood. The understanding about the storage, transport, handling growth habit and harvest index of artificial seeds is essential. The efforts are also needed to increase the output of embryos or plants per gram of callus tissue and conversion frequency of artificial seeds are also needed.

Subject Index β-Lactams 37 β-Naphthoxyacetic acid 49 (NH4)2SO4 50 2,4,5-T 41, 43, 51, 93 2,4-D 41, 43, 49, 51, 75, 93, 94, 102 2-ip 50 3,6-dichloro-2-methoxybenzoic acid 41 4-amino-3,5,6-trichloropicolinic acid 41 6-benzyladenine 41, 43 6-benzylaminopurine 41, 43, 98