J Appl Phycol (2008) 20:609–617DOI 10.1007/s10811-007-9205-4 Seaweed micropropagation techniques and their potentials: an overview C. R. K. Reddy & Bhavanath Jha & Yuji Fujita & Masao Ohno Received: 22 April 2007 / Revised and Accepted: 13 June 2007 / Published online: 16 August 2007 # Springer Science + Business Media B.V. 2007 Abstract The seaweed industry worldwide uses 7.5–8.0 million tonnes of wet seaweeds annually with a majority of it derived from cultivated farms, as the demand for seaweed based-products exceeds the supply of seaweed raw material from natural stocks. The main advantage of cultivation is that it not only obviates overexploitation of natural populations but also facilitates the selection of germplasm with desired traits. To enhance the economic prospects of seaweed cultivation, varied practices, such as simple and cost effective cultivation methods, use of select germplasm as seed stock coupled with good farm management practices, etc., are adopted. Nevertheless, in vitro cell culture techniques have also been employed as they facilitate development and propagation of genotypes of commercial importance. There are more than 85 species of seaweeds for which tissue culture aspects have been reported. Although the initial aim of these techniques focuses mostly on genetic improvement and clonal propagation of seaweeds for mariculture, recently the scope of these techniques has been extended for use in bioprocess technology for production of high value chemicals of C. R. K. Reddy (*) : B. Jha Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Bhavnagar 364 002, India e-mail:
[email protected] Y. Fujita Faculty of Fisheries, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan M. Ohno Usa Marine Biological Institute, Kochi University, Usa-Cho, Tosa, Kochi 781-1164, Japan immense importance in the pharmaceutical and nutraceutical sectors. Recently, there has been a phenomenal interest in intensifying seaweed tissue and cell culture research to maximize the add-on value of seaweed resources. This paper deals with the status of seaweed micropropagation techniques and their applications in the context of the marine biotech industry. Further, it also provides an analysis of the problems to be resolved for removing the barriers that are impeding the true realization of potentials offered by these techniques for sustainable development and utilization of seaweed resources. Keywords Callus . Clonal propagation . Plant growth regulators . Polyamines . Somaclones . Tissue culture Introduction The development of techniques for the culture of isolated plant organs, tissues and cells have led to several exciting opportunities in the area of plant biotechnology, and allowed widespread use of cell cultures for in vitro genetic manipulation, plant propagation and production of commercially useful products (Cocking 1990). Following the success achieved by application of these techniques in higher plants, tissue culture of seaweeds was initiated as early as 1978 (Chen and Taylor 1978) with considerable interest and optimism to further enhance the economic prospects of seaweed resources as a whole. Since then, concerted efforts were made to develop basic background technologies for consistent production and regeneration of calluses from diverse group of seaweeds (see reviews by Polne-Fuller 1988; Butler and Evans 1990; Garcia-Reina et al. 1991; Aguirre-Lipperheide et al. 1995; Rajakrishna 610 Kumar 2002). Several protocols for routine callus induction and regeneration are now available in the literature for a wide range of seaweeds (Polne-Fuller and Gibor 1987; Huang and Fujita 1997; Reddy et al. 2003; Rajakrishna Kumar 2002; Rajakrishna Kumar et al. 2004, 2007). Recently, the scope of these techniques has been extended for use in bioprocess technology for production of high value chemicals of immense commercial value in the pharmaceutical and nutraceutical sectors (Rorrer 2000; Rorrer and Cheney 2004; Munoz et al. 2006). Therefore, in vitro cell culture technology facilitates development of new generation technologies in the area of seaweed cultivation and utilization, that collectively help intensive cultivation of higher yielding strains, coupled with green processing technologies with controlled production of products of potential commercial applications at competitive rates. The earlier articles have dealt at great length about the status, applications, potentials and needs in tissue culture of seaweeds (see reviews of Butler and Evans 1990; GarciaReina et al. 1991; Aguirre-Lipperheide et al. 1995). Therefore, a review of basic aspects of seaweed tissue culture is beyond the scope of this article. Instead, this article provides a brief review of the current status of the micropropagation techniques and their potential applications in overall seaweed biotechnology, including the cultivation, secondary metabolite production and genetic improvement. Further, it also provides an analysis of the problems to be resolved for removing the barriers that are impeding the true realization of potentials offered by these techniques for sustainable development and utilization of seaweed resources. Clonal propagation and selection of strains with superior traits The earlier studies have used clonal propagation as the commonest and simplest approach to select superior strains from wild populations to improve the performance of cultivated crops (Santelices 1992). There are several studies which have exploited the organogenetic potential of seaweeds for isolating superior clones from several economically important seaweeds like Chondrus (Cheney et al. 1981), Gigartina (Sylvester and Waaland 1983), Gracilaria (Patwary and van der Meer 1982, 1983) and Kappaphycus (Doty and Alvarez 1973). Most of the seaweed selection studies have been approached from an empirical perspective for a single trait and a few have been concerned with superior growth potential combined with either sterility or with high polysaccharide yields and gel strength. Although this approach is generally regarded as successful (van der Meer and Patwary 1983: van der Meer 1986), the findings suggest the need for continuous monitoring and isolation of clones with J Appl Phycol (2008) 20:609–617 superior quality for a given trait, and finally the maintenance of the selected strain, eliminating the variants arising spontaneously from the selected clones (Santelices 1992). Most recently, Titlyanov et al. (2006a, b) while working on Gelidium described methods for mass generation of planting material for tank-bubbling and field cultivation using fragments and cell aggregates of apical meristem. Further, freeze-thawing of apical meristem tissues enabled the production of plantlets producing rhizoids that could be used for cultivation in the sea. Both these methods could effectively maximize the number of propagules per donor plant and further facilitate mass production of seed stock required for mariculture round the year. A similar approach has also been successfully demonstrated for producing mass plantlets and tetraspores from fragments and cell aggregates of meristematic and submeristematic tissue of Palmaria palmata (Titlyanov et al. 2006a, b). Simple vegetative propagation of thallus segments (2–3 cm long) in laboratory culture is also utilized to ascertain various culture conditions and media required for successful tissue and protoplast culture in seaweeds. This practice is very useful, especially when one deals with species which are nonresponsive to tissue and protoplast culture. The culture media and conditions generally employed for tissue and cell cultures are similar to those optimized for growing intact plants. Nevertheless, the selection of elite germplasm through clonal propagation is a continuous process and a substantial amount of harvest is utilized as seed material for subsequent cultivation. Further, isolation of useful mutants for cultivation through this process is very cumbersome and less likely to be successful as genetic variant cells in thallus are small in number and are veiled by more common nonvariant cells (Garcia-Reina et al. 1991). This has eventually led to the exploration of the possibilities of developing in vitro cell culture technology for this group of plants. Seaweed tissue and callus culture The seaweed tissue culture is, relatively, a developing area of research with just three decades of development and lags far behind that of higher plants, which got started more than a century ago. The revolution made in plant cell and tissue culture studies has formed the foundation for the genetic engineering of crop plants (Cocking 1990). Micropropagation using tissue culture methods are currently used for the large scale propagation of clones with superior traits in a number of crop species. Furthermore, cell culture offers several advantages for the isolation of mutants over conventional selection procedures using whole plant material. Cell suspensions allow a very large number of cells to be screened simultaneously for a desired trait in a reasonable time frame and reproducible J Appl Phycol (2008) 20:609–617 611 manner. In addition, regeneration of plants from callus can result in the recovery of new genetic variants as a consequence of the well known phenomenon of somaclonal variation (Evans and Sharp 1983). Unlike higher plants, seaweeds show distinct alternation of generations (haploid and diploid) that can be effectively utilized for genetic improvement. Haploid tissues enable easy detection of mutants, while subsequent chromosome doubling produces fertile individuals homozygous at all loci, and these provide pure breeding lines for selection and hybridization. The methods employed in seaweed tissue culture are essentially the same as described for higher plant tissue culture. It generally involves preparation of axenic explants and their culture on solid agar medium enriched with a range of macro- and micro-nutrients, vitamins, sugars (as carbon source), and plant growth regulators (auxins and cytokinins). The initial studies carried out from 1980 to 1990 have mostly dealt with fundamental aspects of seaweed tissue culture (Aguirre-Lipperheide et al. 1995). These studies have largely concentrated on the development of basic techniques for routine tissue culture of different seaweeds, with a few attempting to enhance the callus induction rate and growth by using different plant growth regulators and carbon sources in explant culture medium (Lawlor et al. 1989; Kaczyna and Megnet 1993; Yokoya and Handro 1996, 2002; Yokoya et al. 2004; Rajakrishna Kumar et al. 2004). A list of seaweed species in which plants are reported to have been regenerated from tissue or callus culture is provided in Table 1. The benefits that were expected as a result of application of these techniques to Table 1 Seaweed species from which tissue culture have been accomplished Species Chlorophyta Enteromorpha sp. Enteromorpha intestinalis Ulva angusta Ulva sp. Rhodophyta Agardhiella subulata Ahnfeltiopsis flabelliformis Ceramium kondoi Chondrus crispus Carpopeltis affinis C. prolifera Chondrcanthus tenellus Eucheuma alvarezii E. denticulatum E. uncinatum Furcellaria fastigata Gelidiella acerosa G. robustum Gelidium sp. G. vagum Gigartina exasperata Gloiopeltis tenax Gracillaria acuminata G. chilensis G. corticata G. papenfussii G. perplexa G. tenuistipitata G. tenuifrons G. textori G. verrucosa Grateloupia doryphora G. dichotoma G. filiformis G. filicina Status of success Reference – CI CI – Polne-Fuller Polne-Fuller Polne-Fuller Polne-Fuller PR PR CI CI CI & CI & CI & PR SE CI PR PR CI PR PR PR CI PR CI CI CI CI & PR CI CI PR CI CI & BF CI CI & CI Cheney et al. 1987; Bradley and Cheney 1990; Huang et al. 1998 Huang and Fujita 1997 Gusev et al. 1987 Chen and Taylor 1978; Chen 1982 Huang and Fujita 1997 Huang and Fujita 1997 Huang and Fujita 1997 Polne-Fuller and Gibor 1987; Dawes and Koch 1991; Dawes et al. 1993 Reddy et al. 2003 Munoz et al. 2006 Dawes and Koch 1991; Dawes et al. 1993 Polne-Fuller and Gibor 1987 Gusev et al. 1987 Rajakrishna Kumar et al. 2004 Polne-Fuller and Gibor 1987 Titlyanov and Titlyanova 2006 Gusev et al. 1987 Polne-Fuller and Gibor 1987 Huang and Fujita 1997 Huang and Fujita 1997 Collantes et al. 2004 Rajakrishna Kumar et al. 2007 Polne-Fuller and Gibor 1987 Yokoya et al. 2004 Yokoya et al. 2004 Yokoya 2000 Huang and Fujita 1997 Gusev et al. 1987; Kaczyna and Megnet 1993 Robaina et al. 1990 Yokoya and Handro 1996 Yokoya et al. 1993 Huang and Fujita 1997 PR PR PR PR PR PR and and and and Gibor Gibor Gibor Gibor 1986 1987 1987 1986 612 J Appl Phycol (2008) 20:609–617 Table 1 (continued) Species G. turutura G. imbricata Hypnea musciformis Laurencia sp. Meritotheca papulosa Ochtodes secundriramea Phyllophora nervosa Pionitis crispata Porphyra perforata P. lenceolata P. nereocystis P. umbilicalis P. yezoensis Pterocladia capillacea Ptilophora subcostata Smithora naiadum Soleria filiformis Phaeophyta Cystoseira expensa C. osmundacea C. retorta C. retroflexa C. siliquosa Dictyosiphon foeniculaceus Ecklonia cava E. radiata E. stolonifera Eisenia bicyclis Egregia menziesis Fucus sp. Laminaria angustata L. digitata L. hyperborea L. japonica L. saccharina L. setchellii L. sinclairii Macrocystis pyrifera Prionitis lanceolata Pelvetia fastigiat Sargassum confusm S. flutians S. heterophyllum S. muticum S. polycystum Sargassum sp. S. tenerrimum Stylopodium sp. Turbinaria conoides Undaria pinnatifida U. undarioides Status of success Reference CI CI CI PR SD CI CI PR CI & PR CI PR PR PR CI PR CI CI & BS CI PR CI Huang and Fujita 1997 Huang and Fujita 1997 Rajakrishna Kumar et al. 2007 Garcia-Reina et al. 1988 Robaina et al. 1992 Huang and Fujita 1997 Huang and Fujita 1997 Malaikal et al. 2001; Rorrer and Cheney 2004 Gusev et al. 1987 Huang and Fujita 1997 Polne-Fuller and Gibor 1987 Polne-Fuller and Gibor 1987 Polne-Fuller and Gibor 1987 Liu and Kloareg 1992 Yamazaki et al. 1998; Hafting 1999 Liu and Gordon 1987 Liu et al. 1990 Huang and Fujita 1997 Polne-Fuller and Gibor 1987 Robeldo and Garcia-Reina 1993 SD CI SD SD SD CI Lawlor et al. 1990 Polne-Fuller and Gibor 1987 Lawlor et al. 1990 Lawlor et al. 1990 Lawlor et al. 1990 Saga et al. 1982 Notoya and Aruga 1989, 1992a; Kawashima and Tokuda 1990 Notoya 1988; Lawlor et al. 1989 Notoya 1988 Notoya and Aruga 1990 Polne-Fuller and Gibor 1987 Fries 1985 Saga et al. 1978; Saga and Sakai 1983 Fries 1980; Asensi et al. 2001 Fries 1980 Yan 1984 Lee 1985 Qi et al. 1995 Polne-Fuller and Gibor 1987 Polne-Fuller and Gibor 1987 Polne-Fuller and Gibor 1987 Polne-Fuller and Gibor 1987 Kirihara et al. 1997 Polne-Fuller and Gibor 1987 Mooney and Staden 1984 Polne-Fuller and Gibor 1987 Rao and Rao 1996 Yoshida et al. 1999 Rajakrishna Kumar et al. 2007 Polne-Fuller and Gibor 1987 Rajakrishna Kumar et al. 2007 Zhang 1982; Yan 1984; Kawashima and Tokuda 1993; Notoya and Aruga 1992b Kimura and Notoya 1997 PR PR PR CI PR PR PR PR PR PR CI CI CI CI CI PR PR PR PR PR AE CI CI CI PR PR BF, bud formation; CI, callus induction; PR, plant regeneration; SE, somatic embryogenesis; AE, adventitve embryogenesis; BS, biochemical study; SD, shoot development J Appl Phycol (2008) 20:609–617 seaweeds are minuscule and incomparable with that of the higher plants. This is presumably due to many factors. Seaweed calli, compared to those of higher plants, are generally slow growing and small in size. Furthermore, occurrence of calli in some seaweeds is sporadic, and the percentages obtained are often very low (e.g. 4% in Gracilaria verrucosa: Gusev et al. 1987; 1% in Sargassum muticum: Polne-Fuller and Gibor 1986; 15% in Gelidium vagum: Gusev et al. 1987). In addition, the role of plant growth regulators and carbon sources on callus formation in seaweed explants is variable, and hence the occurrence of callus is suggested to be due to internal factors inherent to the explant rather than to the culture conditions employed (Aguirre-Lipperheide et al. 1995). Plant growth regulators (PGRs) and callus induction The inconsistency in responses to PGRs by seaweeds could be mostly due to lack of understanding of the physiological role of these substances in their growth and differentiation. Tarakhovskaya et al. (2007) reviewed phytohormones in algae in general and reported the occurrence of essentially all known phytohormones in concentrations comparable with that of higher plants, indicating their function in various complex metabolisms. Recently, Stirk et al. (2005) identified 19 endogenous cytokinins, including 1 new aromatic cytokinin (benzyladenine and toplin derivatives), in 31 seaweed species. This work reveals some interesting differences in the cytokinin composition in seaweeds compared to higher plants. These differences included an abundance of iP (isopentenyladenine) and cZ (cis-zeatin) conjugates in the seaweed samples and no DHZ (dihydrozeatin). The latter group of cytokinins is more commonly occurring in higher plants than in the former groups (Stirk et al. 2005). The role of plant growth regulators on callus formation in seaweed explants is debatable and showed no definite trend. Nevertheless, indole-3-acetic acid (IAA), 2,4dichlorophenoxyacetic acid (2,4-D) and kinetin had stimulatory role in callus formation, growth and regeneration both in intercalary and apical explants of Gracilaria tenuistipitata, G. perplexa and Grateloupia dichotoma (Yokoya and Handro 1996; Yokoya et al. 2004). Kaczyna and Megnet (1993) also succeeded in induction of callus in Gracilaria verrucosa using a combination of auxins and cytokinins. The effect of PGRs on callus formation also varied with the seaweed and photon irradiance used during explant culture. The brown color morph of Hypnea musciformis had the highest rate of callus formation in high photon irradiance with low concentration of IAA, while green color morph produced calli with 2,4-D irrespective of concentration studied and photon flux densities used. A water soluble extract from Laurencia sp. increased callus formation on 613 explants of the same species (Robaina et al. 1992). However, the type of substances involved in these responses and their mode of action are unknown. In the light of these conflicting findings, it would be an interesting aspect to investigate the use of some PGR conjugates, such as iP and cZ, which have recently been detected in several seaweeds for their abilities to induce callus formation and multiplication, rather than extensively examining the higher plant phytohormones. Production of micropropagules from callus culture as a means of seed stock A number of studies have shown regeneration of micropropagules directly from callus and sometimes from explants of some red seaweeds (Dawes and Koch 1991; Reddy et al. 2003; Rorrer and Cheney 2004; Rajakrishna Kumar et al. 2004, 2007). Dawes and Koch (1991), Reddy et al. (2003) and Rajakrishna Kumar et al. (2004) have used this phenomenon as a means of maintenance and clonal propagation of seed stock for mariculture of economically important seaweeds. Rorrer and Cheney (2004) employed micropropagules in bioreactor cultivation for production of low volume high value products from a variety of seaweeds. Considering their utility in wide range of applications, some studies attempted to enhance the quantity of micropropagules production by developing innovative in vitro culture methods. Subculture of thin slices of pigmented callus as embedded culture in 0.4% solidified Provasoli enriched seawater (PES) medium enabled mass production of micropropagules from Kappaphycus alvarezii which is commercially cultivated world over for κ-carrageenan. Development of somaclones from callus culture Successful regeneration of fresh plants from several seaweed species has been reported with a few dealing with inadvertent selection resulting from somaclonal variation. Yan (1984) obtained tissue culture progeny from calli of Laminaria and Undaria with rapid growth and high temperature tolerance for longer periods than the normal sporophytes. The in vitro culture of Laminaria digitata stipe medullary explants produced cell-filament suspension cultures which gave rise to normal sporophytic plants along with other forms. The genetic identity of these plants was confirmed with cell-filament suspension using nine polymorphic micro-satellite markers, although flow cytometric analysis of nuclei of cell-filament and regenerated plants displayed 2C and 4C level (Asensi et al. 2001). GarciaReina et al. (1988) described two types of callus of Laurencia obtained from the same plant with identical 614 appearance in terms of pigmentation and texture. These differed markedly in regeneration potential and growth rate of the regenerated plants. The tissue culture progeny obtained from pigmented callus of Kappaphycus also showed enhanced growth rate as high as 1.5–1.8 times the rate of farmed plants propagated through vegetative means (Reddy et al. 2003). The ploidy and RAPD analysis of tissue culture progeny did not show any evidence for genetic variability (Reddy, unpublished data). The tissue culture of Gelidiella acerosa, Gracilaria corticata and Turbinaria conoides also produced calli which showed regeneration patterns quite similar to that of Kappaphycus, but the amount of propagules produced is comparatively less (Rajakrishna Kumar et al. 2004, 2007). Polyamines as growth promoters in seaweeds Like hormones, another group of PGRs, namely polyamines (PAs), are implicated in many biological processes in higher plants, including cell division, root and floral initiation, fruit development, senescence and abiotic stress responses. PAs, spermidine, spermins and their diamine obligate precursor putrescine belong to a class of aliphatic amines that are commonly present in all living organisms and are labeled as a new class of growth substances or hormonal messengers. PAs are present in plants in amounts varying from micromolar to millimolar and thus their levels are significantly higher than those of plant hormones (Kakkar and Sawhney 2002). Recently, occurrence of PAs has been reported in several intertidal marine macroalgae subjected to lethal hypo-saline stress (Lee 1998). Generally, putrescine and spermidine are the most abundant while spermine only occurs in trace levels. Changes in environmental conditions influence the biosynthesis and accumulation of PAs in algae. This is an important consideration as many marine macroalgae grow in intertidal habitats where there are many environmental fluctuations that they need to tolerate in order to grow. For example, at a low salinity, six green macroalgae accumulated higher levels of putrescine (Lee 1998). As in higher plants, PAs play an important role in cell growth and development of seaweeds, with an increase in endogenous concentrations just prior to cell division (Lee 1998). These few examples suggest that PGRs (both hormones and polyamines) can potentially play a similar role in seaweed growth and development as in higher plants. Cell suspension cultures from seaweed callus Callus development in multicellular marine macroalgae has been related to the anatomical structure and cellular J Appl Phycol (2008) 20:609–617 organization of the thallus (Aguirre-Lipperheide et al. 1995). The earlier studies have often used the term “callus-like formations” to distinguish the callus of macroalgae (Garcia-Reina et al. 1991; Yokoya et al. 1993) from that of higher plants where the callus (disorganized growth of cells) develops from differentiated tissues as a result of wounding (Yeoman 1987). Despite the differences in cellular organization among the red (pseudo-parenchymatous type tissue) and brown (parenchymatous type tissue) seaweeds, the callus obtained from both groups showed more or less similar morphology, with uniseriate, pigmented and branched filamentous outgrowths from both cortical and medullary tissues of explants. The callus in higher plants is friable type and forms numerous cell aggregates when transferred to agitated liquid cultures. In contrast to this, the filamentous callus observed in seaweeds is rigid (non-friable) and regenerates rapidly into full plants when transferred to agitated liquid cultures and thereby limiting the scope of applications offered by cell suspension cultures. However, the subcultured callus tufts excised from Turbinaria conoides explants produced tiny somatic embryo-like colonies on pigmented filamentous cells with occasional friable callus clumps when cultured in soft agar (0.4% agar). This finding perhaps highlights the need of innovative culture techniques for obtaining all categories of in vitro cell technology for seaweeds (Rajakrishna Kumar et al. 2007) Bioprocess engineering of cell and tissue culture of seaweeds Bioprocess engineering of seaweeds is one of the most recent and exciting developments of seaweed biotechnology. This development has opened up new opportunities to produce and recover seaweed products directly from cells and cell aggregates in photo-bioreactors (Huang and Rorrer 2002; Rorrer and Cheney 2004; Munoz et al. 2006) which dispense with the use of whole plants. The main advantage of photo-bioreactor cultivation of cell and tissue cultures of macroalgae is the enablement of continuous, steady and defined production of high yields of quality product, thereby circumventing the barriers of seasonality. The optimization of culture conditions for each cell system can also be undertaken with ease and precision for deriving maximum benefits from photo-bioreactor grown cell cultures (Rorrer and Cheney 2004). Further, the downstream process used for recovery of products from cell culture is more environmentally friendly as compared to the conventional process that utilizes the whole plants as a source of raw material for extraction. The bioprocess technology consists of three main components: cell and tissue culture development, photo-bioreactor design, and identification of J Appl Phycol (2008) 20:609–617 strategies for eliciting secondary metabolite biosynthesis. The former two components have been investigated using selective species known for biosynthesis of novel oxylipins and halogenated terpenoids (Rorrer and Cheney 2004). Rorrer and Cheney (2004) described a variety of methods to develop phototropic suspension culture suitable for in vitro cultivation in photo-bioreactor systems and assessed the factors limiting their process cultivation performance using different photo-bioreactor configurations. The photobioreactor cultivation of cell and tissue culture will no doubt form a potential technology platform for the controlled production of low volume high value products from macroalgal resources, including pharmaceuticals and nutraceuticals. Consequently, the macroalgal culture development providing a controlled growth environment suitable for secondary metabolite biosynthesis may outweigh the need for optimization of growth rate or minimize the inputs required for biomass production, thereby enhancing the efficiency of performance of photo-bioreactor cultivation. Conclusion Development of in vitro cell culture technology is of fundamental importance if seaweed biotechnology is to play a central role in the growth of global seaweed industry in future. Considering the present status of progress made in tissue culture of seaweeds, it would be realistic to believe that these techniques can help to the extent of generating genetic mutants of commercial importance. The techniques which have been so far described for propagation of seaweeds through tissue culture have been tested at laboratory scale and have not been validated for their suitability in commercial scale production. The following aspects have to be critically studied if the economic prospects associated with in vitro cell culture technology is to be realized: & & & & & Methods for producing viable axenic material with greater consistency are required. New tissue and callus culture methods need to be developed for obtaining callus from both pigmented and non-pigmented parts of thallus. Physiology of plant growth regulators in plant cell division and development needs to be studied if high callus induction rate and growth are required. Genotyping and selection of tissue culture progeny with desired stable traits must be studied. Reports describing truly cell suspension cultures from seaweeds are rare and limited to the genus Porphyra. Techniques for developing cell suspension culture have to be carried out if high value secondary metabolites to be produced in vitro. 615 & It is imperative to establish homozygous lines of economically important seaweeds for growth and phycocolloid yields. The benefits of in vitro cell manipulations techniques can be more effective and realized if such select genetic lines are used for improving the traits. Acknowledgments The first two authors (C.R.K. and B.J.) are grateful to Dr P.K. Ghosh, Director, CSMCRI for facilities and encouragement. We would also like to thank Mr. Manoj K. 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