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In Vitro Cell. Dev. Biol.—Plant 40:342–345, July/August 2004 q 2004 Society for In Vitro Biology 1054-5476/04 $18.00+0.00 DOI: 10.1079/IVP2004555 INVITED REVIEW: IN VITRO MORPHOGENESIS IN PLANTS – RECENT ADVANCES GREGORY C. PHILLIPS* Arkansas State University, College of Agriculture, P.O. Box 1080, State University, AR 72467-1080 (Received 1 January 2004; accepted 16 March 2004; editor P. Lakshmanan) Summary The capacity of cultured plant tissues and cells to undergo morphogenesis, resulting in the formation of discrete organs or whole plants, has provided opportunities for numerous applications of in vitro plant biology in studies of basic botany, biochemistry, propagation, breeding, and development of transgenic crops. While the fundamental techniques to achieve in vitro plant morphogenesis have been well established for a number of years, innovations in particular aspects of the technology continue to be made. Tremendous progress has been made in recent years regarding the genetic bases underlying both in vitro and in situ plant morphogenesis, stimulated by progress in functional genomics research. Advances in the identification of specific genes that are involved in plant morphogenesis in vitro, as well as some selected technical innovations, will be discussed. Key words: somatic embryogenesis; shoot organogenesis; root organogenesis; floral organogenesis; gene expression; morphogenesis; in vitro. pH; humidity; light quality and quantity or absence of light; temperature; gaseous environment), and osmotic potential. Many of these factors have to be adjusted (e.g., carbohydrates, nitrogen sources) or completely changed (e.g., withdrawal or reduction in auxin signal; perhaps an increase in other plant growth regulators such as abscisic acid; osmotic potential change to encourage desiccation) during maturation of somatic embryos, during which time they become competent for conversion into plantlets (Thorpe, 2000). Many of the same culture factors described above for somatic embryogenesis are also manipulated to induce and optimize organogenesis, but often these factors are manipulated in different ways (Joy and Thorpe, 1999). For example, a high auxin signal (often specifically using 2,4-dichlorophenoxyacetic acid) is usually important to induce somatic embryogenesis, whereas a high cytokinin to auxin ratio (or high cytokinin with no auxin) is typically required to induce shoot organogenesis. Root initiation also typically requires a moderate to high auxin signal – but rarely with the use of 2,4-dichlorophenoxyacetic acid, rather with the use of a more ‘natural’ source of auxin (Gamborg and Phillips, 1995). Because regenerated organs are unipolar, two distinct organogenic induction signals – one to induce shoots and the other to induce roots – are required to regenerate a whole plant. In contrast, bipolar somatic embryos are induced by a single induction signal. Fundamental Aspects of IN VITRO MORPHOGENESIS The two primary morphogenic pathways leading to whole plant regeneration – which is a prerequisite for most plant breeding, genetic and transgenic applications of in vitro biology – involve either somatic embryogenesis, or shoot organogenesis followed by root organogenesis. Both developmental pathways can occur either directly without a callus intermediate stage, termed adventitious; or indirectly following an unorganized callus stage, termed de novo (Gamborg and Phillips, 1995). Few plant species have been shown to regenerate by both organogenic and somatic embryogenic pathways, but many plant species can regenerate by one or the other of these pathways. Somatic embryogenesis may be the best example of totipotency expressed among a large number of plants (Thorpe, 2000). Various culture treatments can be manipulated to optimize the frequency and morphological quality of somatic embryos, which are bipolar structures containing both shoot and root apices, and developing in a manner parallel to that of zygotic embryos. Typical treatment factors include the plant growth regulator sources and concentrations (especially the auxin), choice of explant, nutrient medium composition (especially inorganic vs. organic nitrogen sources, carbohydrate sources and concentrations), culture environment (including the physical form of the medium, e.g. liquid or semi-solid; *Author to whom correspondence should be addressed: Email gphillips@ astate.edu REPRINTED FROM: Phillips, G. C. In vitro morphogenesis in plants – recent advances. In: Goodman, R. M., ed. Encyclopedia of plant and crop science, vol. 1. New York: Marcel Dekker, Inc.; 2004: 579–583. http://www. dekker.com/servlet/product/DOI/101081EEPCS120010554; by courtesy of Marcel Dekker, Inc. Genetic Components of Morphogenesis One of the most exciting advances in recent years has been the discovery of specific genes involved in plant regeneration in vitro. Such genes are being explored for use to increase transformation efficiency and to develop marker-free transgenic plants (Zuo et al., 342 343 IN VITRO MORPHOGENESIS IN PLANTS 2002b). Because a primary factor involved in optimizing somatic embryogenesis and organogenesis is the use of phytohormone models, it is of interest that receptors for each of the major phytohormone classes have now been identified and many of the corresponding genes have been cloned (Møller and Chua, 1999; Zuo et al., 2002b). Examples of specific genes involved in the major plant morphogenesis pathways are summarized in Table 1. Genetic aspects of somatic embryogenesis. Transgenic expression of the LEC2 (leafy cotyledon) gene (Table 1) is sufficient to initiate somatic embryogenesis with high viability but some abnormalities persist in morphology (Zuo et al., 2002b). Several genes appear to be involved in the vegetative-to-embryogenic transition, such as WUS (wuschel; or PGA6, plant growth activator), LEC1 (Zuo et al., 2002a), SERK (somatic embryogenesis receptor kinase) (Zuo et al., 2002b), and PT1 (primordial timing) (Harada, 1999). SHR (short root) establishes the ground tissue through the first asymmetric cell division (von Arnold et al., 2002), CLV (clavata) and WUS interact to determine stem cell fate, and CLV and STM (shoot meristemless) regulate development of the shoot apical meristem (Fletcher, 2002; von Arnold et al., 2002). LEC1, ABI3 (abscisic acid-insensitive) and FUS3 (fusca) are involved in somatic embryo maturation (von Arnold et al., 2002). Genetic aspects of shoot organogenesis. CYCD3 is involved in the acquisition of competence for shoot regeneration (Sugiyama, 1999; Fletcher, 2002), as is SRD3 (shoot redifferentiation) (Sugiyama, 1999, 2000) (Table 1). Shoot redifferentiation also involves SRD1 and SRD2 (Sugiyama, 1999, 2000). The vegetativeto-shoot organogenesis transition is promoted by ESR1 (enhancer of shoot regeneration) (Zuo et al., 2002b). Two genes representing potentially independent pathways involved early in shoot organogenesis signal transduction are CRE1 (cytokinin receptor) and CKI1 (cytokinin independent) (Sugiyama, 2000; Zuo et al., 2002b). The shoot apical meristem stem cell identity is regulated by CLV and WUS (Fletcher, 2002), parallel to that observed in the shoot apical meristem of somatic embryos (above). STM and KN1 (knotted) are involved in the function of the shoot apical meristem, and overexpression leads to the formation of ectopic shoot meristems (Sugiyama, 1999; Fletcher, 2002). Other regulators involved in shoot apical meristem organization and lateral shoot formation include multiple SHO (shoot organization) and MGO (mgoun) genes (Fletcher, 2002). Genetic aspects of root organogenesis. Competence to regenerate root organs is affected by SRD2 (Sugiyama, 1999, 2000) (Table 1). The transition of embryonic root cells to initiate TABLE 1 EXAMPLES OF GENES INVOLVED IN VARIOUS PLANT MORPHOGENESIS PATHWAYS Gene Somatic embryogenesis LEC2 WUS (PGA6), SERK, LEC1 SHR CLV, WUS CLV1, CLV3, STM LEC1, FUS3, ABI3 Shoot organogenesis CYCD3 SRD3 SRD1, SRD2 ESR1 CRE1 CKI1 CLV, WUS KN1, STM SHO, MGO Root organogenesis SRD2 PKL RML CYCD4;1 RAC Floral organogenesis LFY AP1 UFO WUS AG SEP Putative function References Initiates ectopic somatic embryogenesis Involved in the vegetative-to-embryogenic transition Establishes ground tissue via asymmetric cell division Regulate stem cell fate Regulate shoot apical meristem development Regulate embryo maturation Zuo et al., 2002b Harada, 1999; Zuo et al., 2002a, b von Arnold et al., 2002 Fletcher, 2002; von Arnold et al., 2002 Fletcher, 2002; von Arnold et al., 2002 von Arnold et al., 2002 Involved in acquisition of competence for organogenesis Competence for shoot organogenesis Competence for redifferentiation of shoots Enhances shoot regeneration, vegetative-to-organogenic transition Cytokinin receptor Cytokinin perception Preserve stem cell identity in shoot apical meristem Initiate ectopic shoot meristems, shoot apical meristem function Modifiers of the shoot apical meristem involved in leaf founder cell recruitment, lateral organ primordial Sugiyama, 1999; Fletcher, 2002 Sugiyama, 1999, 2000 Sugiyama, 1999, 2000 Zuo et al., 2002b Zuo et al., 2002b Fletcher, 2002; Zuo et al., 2002b Fletcher, 2002 Fletcher, 2002 Fletcher, 2002 Competence for root organogenesis Transition of embryonic root cells to grow vegetatively Root apical meristem function Involved in lateral root formation Involved in adventitious root formation and auxin transduction Sugiyama, 1999, 2000 Harada, 1999 Sugiyama, 2000; Anderson et al., 2001 De Veylder et al., 1999 Sugiyama, 2000 Switch to reproductive development, floral meristem identity A-class gene involved in establishing the first floral whorl: petals Interacts with LFY by providing regional specificity within floral meristems and to control B-class signals which establish the second floral whorl: sepals Interacts with LFY to control C-class genes C-class gene typifying the class; interacts with B-class signals to produce the third floral whorl: stamens; C-class genes acting alone produce the fourth floral whorl: carpels Co-factors for A-, B-, and C-class genes to convert vegetative leaves into floral organs Fletcher, 2002; Lohmann and Weigel, 2002 Lohmann and Weigel, 2002 Lohmann and Weigel, 2002 Lohmann and Weigel, 2002 Lohmann and Weigel, 2002 Lohmann and Weigel, 2002 344 PHILLIPS vegetative growth is controlled by PKL (pickle) (Harada, 1999). RML1 (root meristemless) and RML2 play specific roles in the root apical meristem (Sugiyama, 2000), and interact with components of the apical dominance system (Anderson et al., 2001). The RAC (rooting auxin-cascade) gene is involved in an early stage of auxin perception specific to the formation of adventitious roots (Sugiyama, 2000), and CYCD4;1 is directly involved in lateral root primordia formation (De Veylder et al., 1999). Genetic aspects of floral organogenesis. Floral organs arise as determinate structures out of the indeterminate shoot apical meristem (Fletcher, 2002). The concept of floral organs being specified by the A-, B-, and C-class genes is well established (Lohmann and Weigel, 2002). LFY (leafy) is a key gene involved in the switch to reproductive growth and in establishing floral meristem identity (Fletcher, 2002; Lohmann and Weigel, 2002) (Table 1). LFY activates the key A-class gene AP1 (apetala), establishing the petals or outermost whorl of the floral organ (Lohmann and Weigel, 2002). LFY and UFO (unusual floral organs) interact to control the B-class genes, with UFO providing regional specificity within meristems and thereby establishing the sepal whorl. LFY and WUS interact to control the C-class genes typified by AG (agamous), and the C-class genes interact with B-class genes to establish the stamens in the third whorl. C-class genes also act alone to establish the fourth or innermost whorl comprised of carpels, because AG suppresses the action of WUS thereby resulting in a suppression of the B-class components. Three MADS-Box SEP (sepellata) genes act as cofactors with the A-, B- and C-class genes to convert vegetative leaves into floral organs. Technical Innovations In the past decade, many of the technical improvements resulting in improved in vitro plant regeneration systems have been related to manipulation of the gaseous and/or physical environment of the cultures. In addition, a few other innovations have been noteworthy that fall outside this category, pertaining to thin cell layer techniques and synthetic seeds. Manipulation of the gaseous and/or physical environment. Cultured plant tissues are known to interact with the culture medium and gaseous environment. Forced ventilation and use of ventilated culture vessels, for example, have facilitated optimization of in vitro morphogenesis systems, and high CO2 treatments have permitted establishment of photoautotrophic cultures (Buddendorf-Joosten and Woltering, 1994). Control of the amount of ethylene released by the cultured tissues into the head space of the culture vessel, or alternatively, inhibition of ethylene synthesis or action have led to improved morphogenic responses (Kumar et al., 1998). Efforts to improve bioreactor designs to facilitate economical large-scale production of plants or plant products have continued. Key issues that must be addressed with bioreactor designs for plant cell and tissue growth include aeration and minimization of shear damage. Advances in automation and computer-controls have rendered bioreactor performance more reliable (Paek et al., 2001). One of the most exciting developments in bioreactor design has been the temporary immersion system, which alternates immersion of the plant tissues in the liquid culture medium with exposure to the air space at timed intervals (Etienne and Berthouly, 2002). Temporary immersion bioreactors have been demonstrated to improve yields of shoot proliferation cultures, microtubers and somatic embryos, as well as improve the quality and vigor of the propagules with reduced frequencies of abnormalities and hyperhydricity. Another interesting development is the use of perfluorochemicals and commercially-stabilized bovine hemoglobin as gas carriers to enhance cell performance in liquid culture systems such as bioreactors. Perfluorochemicals are recyclable (can be used to deliver gases, then recovered from the culture and recharged), and emulsion with the surfactant Pluronic F-68w appears to synergistically enhance effectiveness. These gas carriers have been shown to improve cell division rates, stimulate biomass production, improve yields of cellular products, and enhance morphogenic totipotency (Lowe et al., 1998, 2003). A technical innovation with more of a physical impact on the culture environment is the use of semi-permeable cellulose acetate membranes to enhance citrus somatic embryogenesis and particularly to normalize somatic embryo development (Niedz et al., 2002). Applications of thin cell layer and synthetic seed techniques. Thin cell layer culture, an approach mainly involving the manipulation of explant size to induce and optimize regeneration, has been used for many years with dicotyledonous species to study in vitro morphogenesis. Thin cell layer cultures can be manipulated for rigorously controlled programming of different morphogenic responses: callus formation, shoot organogenesis, root organogenesis, floral organogenesis, or somatic embryogenesis (Nhut et al., 2003). In recent years the thin cell layer technique has been extended to a variety of species formerly considered to be recalcitrant to in vitro morphogenesis. Evidence also is gathering that thin cell layer techniques can be useful for recovering transgenic plants from species heretofore considered recalcitrant to genetic transformation. There continues to be interest in developing synthetic seed technology based on artificial encapsulation of somatic embryos suitable for direct field sowing with reliable conversion into viable plants. The most important technical advances in this area involve the use of automated bioreactors to improve yields, combined with the use of computer-imaging to sort out the somatic embryos possessing sufficient quality for encapsulation and subsequent conversion (Ibaraki and Kurata, 2001). Even more exciting are the advances in using non-embryogenic (unipolar) structures for encapsulation as synthetic seed (Standardi and Piccioni, 1998). There seems to be a lower risk of somaclonal variation using unipolar structures such as microbulbs, microtubers, rhizomes, corms, shoots or nodes containing either apical or axillary buds, meristemoids and bud primordia for encapsulation, and the synthetic seed technology can be extended to a wider variety of genotypes. Conclusions and Future Prospects Basic research has begun to dissect the complex genetic pathways involved in various aspects of plant morphogenesis, including all of the major pathways leading to in vitro plant regeneration. A number of candidate genes are being identified that can be expressed transgenically to enhance or even to initiate plant regeneration from cultured cells and tissues. Such genes are being explored for potential use in developing marker-free transgenic IN VITRO MORPHOGENESIS IN PLANTS systems as well as to potentially enhance the frequencies of transgenic plant recovery (Zuo et al., 2002b). These advances, as well as advances in specific culture systems such as thin cell layers (Nhut et al., 2003), offer the prospects of extending more efficient in vitro plant regeneration techniques to previously recalcitrant crops, and of developing more efficient genetic transformation methods. Such advances in controlling in vitro morphogenesis should play important roles at the applied level in developing new crop cultivars and reducing the cost of micropropagation, and in furthering basic research in the area of functional genomics by testing of transgenes in a wider array of plant species. Acknowledgments The author thanks Dr. Oluf Gamborg, Dr. Trevor Thorpe, and Dr. Prakash Lakshmanan for helpful comments regarding topics appropriate to this review. References Anderson, J. V.; Chao, W. S.; Horvath, D. P. A current review on the regulation of dormancy in vegetative buds. Weed Sci. 49:581–589; 2001. Buddendorf-Joosten, J. M. C.; Woltering, E. J. 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