Trees (2010) 24:112 DOI 10.

1007/s00468-009-0384-2

REVIEW

Biotechnological advances in guava (Psidium guajava L.): recent developments and prospects for further research
Manoj K. Rai Pooja Asthana V. S. Jaiswal U. Jaiswal

Received: 3 January 2009 / Revised: 22 September 2009 / Accepted: 24 September 2009 / Published online: 6 October 2009 Springer-Verlag 2009

Abstracts Guava (Psidium guajava L.), an important fruit crop of several tropical and sub-tropical countries, is facing several agronomic and horticultural problems such as susceptibility to many pathogens, particularly guava wilting caused by Fusarium oxysporium psidii, low fruit growth, short shelf life of fruits, high seed content, and stress sensitivity. Conventional breeding techniques have limited scope in improvement of guava owing to long juvenile period, self incompatibility, and heterozygous nature. Conventional propagation methods, i.e., cutting, grafting or stool layering, for improvement of guava already exist, but the long juvenile period has made them time consuming and cumbersome. Several biotechnological approaches such as genetic transformation may be effective practical solutions for such problems and improvement of guava. The improvement of fruit trees through genetic transformation requires an efcient regeneration system. During the past 23 decades, different approaches have been made for in vitro propagation of guava. An overview on the in vitro regeneration of guava via organogenesis, somatic embryogenesis, and synthetic seeds is presented. Organogenesis in several different genotypes through various explant selection from mature tree and seedling plants has been achieved. Factors affecting somatic embryogenesis in guava have been reviewed.

Production of synthetic seeds using embryogenic propagules, i.e., somatic embryos and non-embryogenic vegetative propagules, i.e., shoot tips and nodal segments have also been achieved. Development of synthetic seed in guava may be applicable for propagation, short-term storage, and germplasm exchange, and distribution. An initial attempt for genetic transformation has also been reported. The purpose of this review is to focus upon the current information on in vitro propagation and biotechnological advances made in guava. Keywords Guava Genetic transformation In vitro propagation Organogenesis Somatic embryogenesis Synthetic seeds

Introduction Guava (Psidium guajava L., family Myrtaceae), poor mans fruit or apple of tropics, is an important fruit crop of tropical and sub-tropical regions of the world. Guava fruit contains 25 times more vitamin C than orange and is also good source of calcium, phosphorus, and iron (Singh 2005). Traditionally, different parts of plants, i.e., fruits, leaves, roots, and bark are used in the treatment of gastroenteritis, diarrheoa, and dysentery (Jaiswal and Amin 1992). High concentration of pectin in guava fruit may play a signicant role in the reduction of cholesterol and thereby decrease the risk of cardiovascular disease (Singh 2005). Despite these advantages, there are a number of problems that affect guava production. Being a cross-pollinated species, substantial variability is available in seedling populations in different guava growing regions (Srivastava 2005). Guava wilting disease caused by Fusarium oxysporium psidii is a serious problem faced by guava growers,

Communicated by J. Carlson. M. K. Rai P. Asthana V. S. Jaiswal U. Jaiswal Laboratory of Morphogenesis, Department of Botany, Banaras Hindu University, Varanasi 221005, UP, India Present Address: M. K. Rai (&) Centre for Plant Biotechnology, CCSHAU Campus, Hisar 125004, Haryana, India e-mail: mkraibhu@gmail.com

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and loss due to this disease is substantial (Mishra 2005). Other important eld diseases of guava are anthracnose (Gloeosporium psidii), and canker (Pestalotia psidii). Guava suffers from different rot pathogens which cause maximum loss (Mishra 2005). Before initiation of any crop improvement program in guava, priority needs to be given to the following: good fruit quality, increasing yields, disease resistance, longer shelf life of fruits, high vitamin C and pectin content, good aroma, attractive skin, esh color, and soft seeds (Dinesh and Iyer 2005). Such an ideal phenotype cannot be met by conventional breeding. Floral structure (epigynous ower, with abundant incurred stamens of various sizes), long juvenile period, self incompatibility, and heterozygous nature limit the scope of breeding programs for improvement of guava (Jaiswal and Amin 1992). Despite these problems, a few successful reports on guava breeding have also appeared (Ribeiro and Pommer 2004; Pommer and Murakami 2008). Guava is conventionally propagated through cutting, grafting, stooling, or air layering, but these methods are time consuming (Chandra et al. 2004). The planting of extensive new orchards of vegetatively propagated clones of some tropical fruits has some times been limited by pathogens (Litz and Jaiswal 1991). In majority of trees, propagation by root cutting is often characterized by a rapid loss of rooting capacity of the cutting with increasing age of parent plant (Thorpe et al. 1991). Clonal propagation using cell, tissue, and organ culture techniques have considerable potential for the improvement of economically important trees within a limited time frame (Giri et al. 2004; Singh et al. 2004). Generation of new and promising variability through somaclonal variant selection, production of androgenic, and gynogenic haploids to achieve homozygosity, freeing plants from disease-causing organisms by shoot tip culture, production of industrial compounds by cell culture, and development of stress-tolerant plants are some well-known applications of plant tissue culture. This review not only highlights the major biotechnological advances made in guava during past years, but also suggests how present technologies in tissue culture and genetic engineering might affect the direction of future research. The attempted and possible biotechnological interventions in guava are presented in Fig. 1.

Fig. 1 Biotechnological interventions in guava

embryogenesis, are distinctly different process (Christianson 1987; Litz and Gray 1992). Successful regeneration of plants from tissue culture offers excellent opportunities for the improvement of guava. A report on some morphological and cultural aspects of in vitro grown guava tissue from fruit mesocarp was perhaps the rst attempt to manipulate somatic tissue (Schroeder 1961) of this important fruit crop (Jaiswal and Amin 1992). However, in recent years, several reports have been published on regeneration of guava through organogenesis and somatic embryogenesis (Tables 1, 2). Problems associated with guava micropropagation and their practical solutions Morphogenesis from explants derived from mature trees is of great commercial value because it can be applied in direct cultivar improvement. However, there are several problems associated with in vitro culture of explants obtained from mature trees of guava such as browning or blackening of medium and/or explants due to leaching of phenolics, microbial contamination, and in vitro tissue recalcitrance etc. High phenolic exudation during the excision of plants, explant browning, medium discoloration, and slow growth response have made an ordeal for workers dealing with several woody tree species including guava. Browning of media occurred as a result of oxidation of polyphenols exuded from explants (Rout et al. 2000). In order to reduce phenolic exudation, Amin and Jaiswal (1987, 1988) suggested the pretreatment of explants with antioxidant solutions. Explants were agitated for 3040 min in 0.5% (w/v) solution of polyvinylpolypyrrolidone (PVPP) containing 2% sucrose followed by dip in an antioxidant solution (75 mg citric acid and 50 mg ascorbic acid l-1 water) after surface sterilization of explants. Besides, 23 changes of medium for the initial 1015 days were essential for

Achievements made in guava through tissue culture The efcient regeneration of plants from cell, tissue, and organ culture is recognized as prerequisite for application of most modern genetic and biotechnological approaches to crop improvement (Litz and Gray 1992). Several workers have recognized that the two patterns of in vitro differentiation, i.e., organogenesis and somatic

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Trees (2010) 24:112 Table 1 Summary of work on organogenesis in guava Cultivar Explant Mature/ Medium ? PGRs juvenile Shoot multiplication M M M J MS ? BAP MS ? BAP MS ? BAP MS ? BAP References Rooting

Banaras local Chittidar

Nodal segments Shoot tips Nodal segments Shoot tips, Nodal segments, Hypocotyl, Leaf segments (Seedling) Shoot tips from seedling Stem shoot Hypocotyl from seedling

MS ? IBA NAA ? AC Amin and Jaiswal (1987) MS ? IBA NAA ? AC Jaiswal and Amin (1987) MS ? IBA NAA MS basal Amin and Jaiswal (1988) Loh and Rao (1989)

Red Indian Mara-7 Allahabad Safeda

J M J

OM ? BAP MS ? BAP MS ? BAP, NAA, IBA MMS ? TDZ ? NAA MS ? BAP

OM ? NAA ? IBA MS ? IBA or AC

Papadatau et al. (1990) Yasseen et al. (1995) Fuenmayor and Montero (1997)

Nodal segments from seedling J

MMS ? IBA ? AC MS ? IBA

Singh et al. (2002) Ali et al. (2003)

Aneuploid no. 82 Sardar Pant Prabhat Safeda Safeda Banarasi local

Nodal segments from green M/J house grown plants (GHRP) and in vitro harvested axillary buds (IVDS) Nodal segments M Apical shoot tips Nodal segments Shoot tips Seedling explants Nodal segments M M M J J

WPM ? BAP MS ? BAP ? IBA MS ? BAP ? IBA MS ? BAP ? Lglutamine MS ? Zea ? GA3 MS ? BAP

MS ? IBA ? NAA MS ? IBA ?NAA MS ? IBA ? NAA MS ? IAA ? IBA MS ? IBA ? NAA MS ? IBA

Meghwal et al. (2003) Chandra et al. (2005b) Mishra et al. (2005) Zamir et al. (2007) Shah et al. (2008) Rai et al. (2009b)

AC activated charcoal, BAP 6, benzylaminopurine, IAA indole-3- acetic acid, IBA indole-3- butyric acid, MS Murashige and Skoog (1962) medium, MMS Modied MS medium, NAA a naphthalene acetic acid, OM Rugini Olive medium, TDZ N-phenyl-1, 2, 3-thidiazol-5yl-urea, WPM woody plant medium Table 2 Summary of work on somatic embryogenesis in guava Cultivar Explant Medium ? PGRs Induction of somatic embryos Sardar Immature and mature fruit mesocarp Immature zygotic embryo Immature fruit mesocarp Immature zygotic embryo MMS ? L-glutamine (400 mg l-1) ? ascorbic acid (100 mg l-1) ? 2, 4-D (2 mg l-1) GSEM ? 1.75 lM IAA ? 58.4 lM L-glutamine MS ? 1, 2, 4-1-H Triazol (4 mg l-1) MS ? L-glutamine (400 mg l-1) ? ascorbic acid (100 mg l-1) ? 6% sucrose ? 2, 4-D (1 mg l-1) MS ? 5% sucrose ? 2, 4-D (1 mg l-1) Germination of somatic embryos MMS ? L-glutamine (400 mg l-1) ? ascorbic acid (100 mg l-1) ? 2, 4-D (2 mg l-1) MS medium Liquid MS ? BAP (0.25 mg l-1) ? Biobras-6 (10 lg l-1) MS ? 3% sucrose Chandra et al. (2004) Biswas et al. (2007) Chandra et al. (2005a) Kosky et al. (2005) References

Allahabad Safeda Cuban Red Dwarf EEA

Banarasi local

Immature zygotic embryo

Rai et al. (2007)

BAP 6, benzylaminopurine, 2, 4-D 2, 4-dichlorophenoxyacetic acid, GSEM guava somatic embryogenesis medium, IAA indole-3-acetic acid, MS Murashige and Skoog (1962) medium, MMS Modied MS medium

controlling phenolic exudation and establishment of cultures (Amin and Jaiswal 1987, 1988; Chandra et al. 2005b). Mishra et al. (2005) also recommended the

supplementation of 500 mg citric acid in the MS media and initial incubation of cultures in complete dark for 24 h to reduce phenolic browning of media and explants.

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Papadatau et al. (1990) explored the morphogenic potential of shoot tip explants excised from seedlings grown in a growth chamber. Blackening of medium and necrosis of explants were not observed by Papadatau et al. (1990). This is probably because the young seedlings do not synthesize higher quantities of phenolics when grown in a growth chamber (Chandra et al. 2005b). Microbial contamination is one of the most important limiting factors for culture initiation using vegetative plant parts (Krishna and Singh 2007). Microbial contamination can result in the death of cultures, growth retardation, necrosis, and altered morphogenic potential such as reduced rates of multiplication and rooting (George 1993). Acosta et al. (2002) have identied several microbial contaminants/ pathogens, i.e., Alternaria, Aspergillus, Cladosporium, Colletotrichum, Curvularia, Fusarium, Nigrospora, Penicillium, and Trichoderma, during in vitro culture of guava cv. Enana Roja cubana and all the pathogens, except Nigrospora, have been detected during the in vitro establishment of nodal segments. They noticed that approximately 50% of the pathogens were eliminated by 10-min exposure to 3% hypochlorite or use of 0.05 and 0.1% HgCl2. Ali et al. (2007) observed that 6795% seeds of guava were contaminated by bacteria and fungi using standard tissue culture methods. They recommended the application of 10% HCl for 2472 h followed by a 30-min treatment with 10% bleach (NaOCl) to guava seeds to reduce contamination load. Recalcitrance of tissue is another major problem for the culture initiation using mature plant parts (Krishna and Singh 2007). In the case of guava, the donor plants may be limited and successful regeneration was observed mostly from seedling explants (Loh and Rao 1989; Papadatau et al. 1990; Yasseen et al. 1995; Fuenmayor and Montero 1997; Singh et al. 2002; Shah et al. 2008). The selection of explants at a specic responsive stage of a mature trees life cycle is of great importance to overcome recalcitrance (Benson 2000; Krishna and Singh 2007). New vegetative growth that occurs from the base of the main stem (offshoots) serves as a reliable source of shoot tip and nodal segments for guava tissue culture (Amin and Jaiswal 1987; Jaiswal and Amin 1987; Singh et al. 2004). Organogenesis Organogenesis involves adventitious and axillary shoot production. Organogenesis comprises the formation of unipolar structure (either shoot or root meristems) from callus or directly from organized tissues (Singh et al. 2004). In guava, organogenesis has been induced in vitro both from mature tree explants (Amin and Jaiswal 1987, 1988; Jaiswal and Amin 1987) and seedling explants (Loh and Rao 1989; Papadatau et al. 1990; Yasseen et al. 1995; Fuenmayor and Montero 1997; Singh et al. 2002; Shah et al. 2008).

Factors controlling organogenesis in guava Success of in vitro regeneration depends on the control of morphogenesis, which is inuenced by several factors namely kinds of tissue or explants, composition of medium, plant growth regulators (PGRs), media additives, culture environment etc. Successful micropropagation, especially for difcult and recalcitrant tree species, is mainly dependent on the quality of explants and the response of explants is primarily determined by genotype, physiological state of the tissue, and time of the year when the explants are collected and cultured (Giri et al. 2004). Amin and Jaiswal (1987, 1988) have compared the responses of nodal segments of mature tree and nodal segments taken from in vitro proliferated shoots. The performance of nodal segment taken from in vitro proliferated shoots was better in comparison to that obtained from mature tree. The probable reason for better response of in vitro nodal segments as suggested by Amin and Jaiswal (1987) is the absence of lag period between explanting and adaptation of explants to in vitro conditions. Survival and growth response of shoot tips were not as satisfactory as those of nodal segments (Amin and Jaiswal 1987). The greater responsiveness of nodal segments over the shoot tips can be attributed to the absence of apical dominance and presence of axillary buds at a more advanced stage of development (Amin and Jaiswal 1987). Numerous studies have addressed the effect of season on culture establishment in guava (Amin and Jaiswal 1987; Mishra et al. 2005; Singh et al. 2005). The minimum phenolic exudation and culture contamination, and maximum survival and shoot proliferation were obtained from explants harvested between April and June (Amin and Jaiswal 1987). Ali et al. (2003) devised a protocol for regeneration of guava using two different sources of explant, i.e., greenhousegrown plants (GHRP) and in vitro harvested axillary buds (IVDS). The largest number of shoots and comparatively better shoot growth was observed with IVDS. In plant tissue culture, nutritional requirement for optimal growth of a tissue in vitro may vary with species (Bhojwani and Razdan 1996). Hence, media compositions play a key role in morphogenesis and responses of explants. In the case of guava, a number of media have been used for initiation of culture during organogenesis. But mostly MS (Murashige and Skoog 1962) medium was used for shoot multiplication (Table 1). In a separate study, Singh et al. (2002) reported the use of MS medium with major salts reduced to one-half strength for shoot multiplication in cv. Allahabad safeda. In a few studies, other media have also been used for optimum morphogenesis (Papadatau et al. 1990; Meghwal et al. 2003). Before exploiting plant tissue culture for commercial purposes, detailed information regarding the requirement of PGRs is necessary, and it has become a necessity to

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standardize when dealing with tree species. The levels and kinds of PGRs included in the culture medium largely determine the success of tissue culture work. Root and shoot initiations are closely regulated by the relative concentrations of auxin and cytokinin in the medium (Rout et al. 2000). Cytokinin levels were shown to be the most critical for multiplication of many tropical fruit tress. BAP was the most common cytokinin used for guava propagation (Amin and Jaiswal 1987, 1988; Loh and Rao 1989; Papadatau et al. 1990; Yasseen et al. 1995; Ali et al. 2003). Superiority of BAP for shoot induction may be attributed to the ability of plant tissues to metabolize BAP more readily than other synthetic growth regulators or to the ability of BAP to induce production of natural hormones such as zeatin within the tissue (Malik et al. 2005). Singh et al. (2002) were able to prompt shoot multiplication in cv. Allahabad safeda after treatment with TDZ and NAA. Other growth-enhancing medium additives including sucrose (Amin and Jaiswal 1989a) and adenine sulfate (Singh et al. 2002) had also signicant effect on shoot multiplication and elongation. Agar is most frequently used as a gelling agent because of its desirable characteristics such as clarity, stability, and its inertness (Pati et al. 2006). Agar minimizes the water loss and allows good nutrient diffusion (Amin and Jaiswal 1989a). For any micropropagation protocol, successful rooting of microshoots is a pre-requisite to facilitate their establishment in soil (Pati et al. 2006). Root initiation has been encouraged in guava by incorporating either IBA alone or with combination of NAA (Table 1), although Loh and Rao (1989) were able to stimulate rooting only on MS basal medium. Relatively low salt concentrations in medium are known to enhance rooting of microshoots. Several studies on guava indicate that half-strength MS medium was adequate for root induction. Activated charcoal, often used in plant tissue culture to improve cell growth and differentiation, alone or sequentially after an auxin induced rooting step of micropropagated shoots (Thomas 2008). Rooting of shoots of guava has also been promoted by the addition of activated charcoal (Amin and Jaiswal 1987; Yasseen et al. 1995; Singh et al. 2002). Phloroglucinol was found to inhibit in vitro rooting of shoots of guava and this inhibition was more striking when it was used in combination with auxins (Amin and Jaiswal 1989b). Somatic embryogenesis Somatic embryogenesis is the process by which somatic cells, under inductive conditions, generate embryogenic cells, which undergo a series of morphological and biochemical changes resulting in the formation of somatic embryos (Zimmerman 1993; Komamine et al. 2005). Somatic embryogenesis plays an important role in clonal

propagation. When integrated with conventional breeding programs and molecular and cell biological techniques, somatic embryogenesis provides a valuable tool to enhance the pace of genetic improvement of commercial crop species (Stasolla and Yeung 2003). As compared to organogenesis, somatic embryogenesis provides an ideal experimental process for investigation of plant differentiation as well as a mechanism for expression of totipotency in plant cells (Litz and Gray 1992). Many workers have emphasized somatic embryogenesis as a preferred method for genetic improvement and multiplication of valuable germplasm of a number of woody perennials (Gupta and Durzan 1987; Raj Bhansali 1990; Litz and Gray 1992). In guava, considerable efforts have been made for in vitro regeneration via somatic embryogenesis (Akhtar 1997; Akhtar et al. 2000; Chandra et al. 2004, 2005a; Jaiswal and Jaiswal 2005; Biswas et al. 2007; Kosky et al. 2005; Rai et al. 2007). Immature zygotic embryos have been utilized as the primary explants for the induction of somatic embryogenesis by most of workers. Other explants such as leaf, node, internode, petal, and mesocarp have also been tested but failed to induce somatic embryos except mesocarp with some success (Biswas et al. 2005; Chandra et al. 2004, 2005a). Immature zygotic embryos have proved to be an effective regenerable tissue for the many recalcitrant tropical fruit species. Zygotic embryos are made up of PEDCs (preembryogenic determined cells), in which, cells have the embryogenic competence and can be easily induced to follow the embryogenic pathways (Sharp et al. 1980). Development of different stages of somatic embryos on zygotic embryo and germination of induced somatic embryos of guava are presented in Fig. 2. Induction of somatic embryogenesis Induction of somatic embryogenesis in guava was affected by nature of explants, physiological age of explants, duration of treatment of 2, 4-dichlorophenoxyacetic acid (2, 4-D) to explant, an interactive effect of 2, 4-D and sucrose, different PGRs and their combination, and genotype (Akhtar et al. 2000; Jaiswal et al. 2005; Rai et al. 2007). Requirement of auxin or other PGRs for the initiation of somatic embryogenesis is largely determined by the developmental stage of the explant tissue. The initiation of the embryogenic pathway is restricted only to certain responsive cells in the primary explant which have the potential to activate those genes involved in the generation of embryogenic cells. The competence for embryogenic induction may be the result of varying auxin sensitivity to these cells (Dudits et al. 1995; Arnold et al. 2002). Rai et al. (2007) noted the zygotic embryos obtained from 10-

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6 Fig. 2 Somatic embryogenesis in guava. a Induction of somatic embryogenesis on zygotic embryo. b Development of lower stages somatic embryos on whole surface of zygotic embryo after 45 weeks of culture. c Development of torpedo stages somatic embryos. d Germination of somatic embryos. e Development of a well-developed plantlet from germination of somatic embryo

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week-old fruits have the maximum ability to induce somatic embryos in guava cv. Banarasi local. This is probably due to their favorable physiological make-up at that stage of development. Similarly, mesocarp explant obtained from immature fruits has comparatively better capacity to induce somatic embryos than those obtained from mature fruits in cv. Sardar (Chandra et al. 2004). In order to optimize the somatic embryo induction in guava, a number of media are being used. However, the most of successful cases recommended the use of MS medium or derivative thereof (Chandra et al. 2004; Kosky et al. 2005; Rai et al. 2007). Contrarily, Biswas et al. (2005) did not obtain somatic embryos on MS, B5 or N6 culture media; thus they used a modied GSEM (guava somatic embryogenesis medium) for the induction of somatic embryogenesis. Moreover, all the above-said media have high levels of ammonia and nitrate salts, which played a key role in the induction of somatic embryogenesis (Akhtar et al. 2000). Nitrogen in the form of amino acid such as L-glutamine and organic compounds such as ascorbic acid has been effective in induction of somatic embryogenesis in guava (Chandra et al. 2004; Kosky et al. 2005; Biswas et al. 2005). The importance of reduced nitrogen, certain amino acids, organic, and inorganic nutrients has been reviewed in other tropical fruit species

by Akhtar et al. (2000). PGRs play an important role in the induction of either unorganized callus growth or polarized growth leading to somatic embryogenesis (Arnold et al. 2002; Rai et al. 2007). Auxin, particularly 2, 4-D is required for the induction of somatic embryogenesis in guava (Chandra et al. 2004; Kosky et al. 2005; Rai et al. 2007). Different auxins (IAA, IBA and NAA) and cytokinins (BAP, Kin and TDZ) either alone or in combination with 2, 4-D have been shown to be less effective than 2, 4-D alone for induction of somatic embryogenesis (Akhtar et al. 2000; Jaiswal et al. 2005). Contrarily, Biswas et al. (2005) obtained somatic embryos on IAA-containing medium. The duration of explant exposure to a growth regulator is an important factor for induction and development of somatic embryos. In most cases, auxin is required only for induction of somatic embryogenesis and is subsequently inhibitory for development of somatic embryos. Rai et al. (2007) examined the treatment of zygotic embryo for 8 days with 2, 4-D and observed that this was effective for the induction of somatic embryogenesis. Continuous treatment for 60 days allowed differentiation of somatic embryos only up to lower stage. Various carbon sources were tested on zygotic embryo explants for somatic embryo induction (Akhtar et al. 2000). The rationale behind those experiments lay in the fact that

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the plant cannot use or metabolize all carbon sources effectively, and thus those could be a limiting factor to induction and development of somatic embryos. Sucrose was shown to support somatic embryo induction and addition of 56% sucrose in medium was found best for induction of somatic embryogenesis (Kosky et al. 2005; Rai et al. 2007). Somatic embryo induction in guava is severely hampered by the presence of glucose, maltose, lactose, fructose, sorbitol, and mannitol in medium (Akhtar et al. 2000). Physical state (semisolid or liquid) and strength of medium is often important for induction and maintenance of somatic embryos in several tropical fruit species including mango (Ara et al. 2000, 2004). In case of guava, full-strength semisolid (solidied by 0.8% (w/v) agar) medium was adequate for embryogenic response, while in liquid medium much reduced level of induction was obtained (Akhtar et al. 2000). Development and maturation of somatic embryos Synthetic auxins, particularly 2, 4-D, which are effective for induction of somatic embryos, are usually less metabolized by the cells than other auxins. Therefore, in order to obtain development of somatic embryos it is necessary to transfer the embryogenic cultures to medium lacking auxin (Arnold et al. 2002). In guava, zygotic embryos treated with 2, 4-D for 8 days were transferred to 2, 4-D free medium for the development of somatic embryos (Rai et al. 2007). Initial stage somatic embryos were formed after 1620 days. After 45 weeks of culture, entire surface of zygotic embryos were covered with lower-stage somatic embryos. Synchronization of embryogenic cultures is difcult to achieve on development medium; it may be due to establishment of polarity within culture relative to the accessibility of 2, 4-D (Krishna and Singh 2007). In order to achieve mature somatic embryos, lower-stage somatic embryos induced on zygotic embryos transferred to medium containing different concentrations of sucrose, abscisic acid (ABA), two selected amino acids L-glutamine, and L-proline or PEG (Rai et al. 2008a, 2009a). Among the different concentrations of sucrose tried, a concentration of 5% was most effective for maturation of somatic embryos. Rai et al. (2008a, 2009a) also suggested addition of ABA, L-proline, or PEG to growth regulator free medium to improve maturation of somatic embryos. Germination of somatic embryos and plantlet development

develop normal plants from somatic embryos, a dissection of critical factors that might contribute to germination of somatic embryos is required. In some cases, somatic embryos develop into plants on culture medium without PGRs, whereas there are some other cases where auxin or cytokinin stimulates germination or an altered basal medium was necessary (Jimenez 2005). In guava, lowering the medium strength and sucrose concentration was necessary for germination of somatic embryos. Germination of mature somatic embryos was achieved on agar-solidied half-strength MS medium containing 3% sucrose (Rai et al. 2007). Kosky et al. (2005) advocated the use of liquid medium and addition of BAP, Biobras-6 (brassinosteroid analog), and 2% sucrose in medium for germination of somatic embryos. Production of synthetic seeds Encapsulation of somatic embryos or non-embryogenic vegetative propagules to produce synthetic seeds could possibly be utilized as means for germplasm storage and transportation of elite germplasm (Krishna and Singh 2007). Guava is a cross-pollinated and vegetatively propagated crop (Doijode 2001). Therefore, it is a particularly suitable candidate for synthetic seed technology. Successful plantlet regeneration from encapsulated somatic embryos of guava was reported by Akhtar (1997), Biswas et al. (2007), Rai and Jaiswal (2008), and Rai et al. (2008a). Torpedo stage somatic embryos were encapsulated in 2% sodium alginate and 100 mM calcium chloride and cultured on appropriate medium for plant regeneration (Akhtar 1997). Maximum plantlet conversion from encapsulated somatic embryos was obtained on growth regulator free full-strength MS medium. Recently, Rai et al. (2008b, c) have also employed the encapsulation of vegetative propagules (shoot tips and nodal segments) for the development of synthetic seeds in guava. A combination of 3% sodium alginate and 100 mM calcium chloride was most suitable for formation of ideal synthetic seeds. Maximum plantlet conversion from encapsulated shoot tips was achieved on liquid MS medium (Rai et al. 2008b). Plantlet conversion was also affected by medium strength and sucrose concentrations in medium. These encapsulated vegetative propagules could be potentially used in shortterm storage and germplasm exchange of elite genotype of guava (Rai et al. 2008b, c). In vitro storage of synthetic seeds

Germination of somatic embryos and growth of regenerated plants depends on the conditions provided at earlier stages when somatic embryos mature. In some cases, precocious germination of somatic embryos takes place (Arnold et al. 2002; Jimenez 2005). Therefore, in order to

Two different approaches have been applied for storage of synthetic seed of guava using slow-growth procedure (Rai et al. 2008a, b). (1) Transferring the encapsulated somatic embryos onto the full-strength MS medium containing

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ABA (1 mg l-1) or 9% sucrose prior to culturing on germination medium (growth regulator free full-strength MS medium ? 3% sucrose) resulted in extended storage of up to 60 days (Rai et al. 2008a). The temporary suppression of germination in encapsulated somatic embryos by ABA or high sucrose offers a possibility of conservation of elite genotype of guava for short period. (2) Encapsulated shoot tips could be stored at low temperature (4C) or room temperature under minimal growth medium (sucrose lacking medium) for different days (Rai et al. 2008b). Results revealed that storage of encapsulated shoot tips under minimal growth medium was better than storage at low temperature (4C) for conservation of guava. In vitro selection Development of improved variants obtained through in vitro selection pressure technique is recommended for increasing genetic diversity, both qualitatively and quantitatively inherited characters such as biotic and abiotic stress tolerance, fruit quality, and yield etc. In vitro culture of plant cells, tissues or organs on medium containing selective agent offers the opportunity to regenerate and select plants with desirable characteristics. The technique has been effectively utilized to induce tolerance which includes the use of some selective agents that permit the preferential survival and growth of desired phenotypes (Purohit et al. 1998). To create genetic variability for selecting early bearing, short statured and less seeded guava mutants, in vitro mutagenesis followed by micropropagation via shoot tips was carried out by Zamir et al. (2003). Shoot tips were irradiated with gamma rays at 1590 Gy using 60Co gamma cell source and cultured in MS medium containing 3.0% sucrose, BAP and L-glutamine. Sensitivity to radiation was evaluated by determining the percentage shoot tip survival and shoot proliferation. However, recovery of desired variants is still lacking.

Achievements made in guava through molecular approaches Molecular approaches are useful for characterizing the genetic diversity among different cultivars or species, for identifying genes of commercial interest and improvement through genetic transformation technology. Some of the important achievements made in guava through molecular approaches are presented in Table 3. Clonal identications are traditionally based on various morphological characters; however, morphological characters may not be reliable to discriminate between closely related guava genotypes (Chandra et al. 2005b). Most of the cultivars grown on a commercial scale are seedling

selections from the well-known parent cultivars (Jaiswal and Amin 1992). A close genetic relationship among cultivars, somatic mutations, and changes due to environmental alterations can create problems in correct identication of germplasm. In recent years, different molecular markers (RAPD, RFLP, AFLP, SSRs, ISSR, VNTRS) have been employed for the investigations of cultivar origins and taxonomic relationships of several plant species. Detection of genetic variation is also important for micropropagation and in vitro germplasm conservation to eliminate undesirable somaclonal variations. In guava, recently, a few reports have been made on assessment of genetic diversity using Random Amplied Polymorphic DNA (RAPD) markers (Dahiya et al. 2002; Prakash et al. 2002; Chen et al. 2007; Feria-Romero et al. 2009). Isolation of adequate quality of genomic DNA for use in PCR-based DNA marker technology faces severe problems due to the presence of inhibitors such as polysaccharides, which inhibit the enzymatic DNA processing or phenolics as inhibitors of PCR reactions (Prakash et al. 2002). The well-established modied CTAB protocol (Porebski et al. 1997) yielded excellent DNA templates for PCR amplication for guava (Prakash et al. 2002). Prakash et al. (2002) analyzed molecular diversity of 41 different genotypes of guava collected from different parts of India by using RAPD markers. The authors suggested that the genetic base of Indian guava can be rated as low to moderate diversity and various triploid seedless cultivars of guava are not genetically identical and have independent origins. Dahiya et al. (2002) also tried to determine genetic relationship in 13 north Indian cultivars of guava using RAPD markers. Chen et al. (2007) using RAPD markers also attempted to determine phylogenetic relationship in 18 cultivars of Taiwan. Apart from characterization and assessment of chemical diversity, Feria-Romero et al. (2009) used RAPD amplication method to identify molecular markers associated with high quercetin accumulation in the leaves of guava trees, selected from four different Mexican agronomic regions. Simple sequence repeats (SSRs), also known as microsatellites markers have been widely utilized in plant genomic studies, and are reported to be more variable than RFLPs and RAPDs (Krishna and Singh 2007). Microsatellites markers to study genetic diversity in guava were developed using a genomic library enriched for (GA)n and (GT)n dinucleotide repeats and 23 nuclear SSR loci were chosen to assess diversity in three guava species (Risterucci et al. 2005). Hernandez-Delgado et al. (2007) studied the amplied fragment length polymorphism (AFLP) analysis of genetic relationship among 48 guava cultivars grown in different parts of Mexico. Genetic transformation opens the opportunity for genetic manipulation of plants at cellular level and provides the means for modifying single horticultural traits

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Trees (2010) 24:112 Table 3 Achievements made in guava through molecular approaches

Biotechnological tool DNA markers RAPD

Achievement

References

Estimation of molecular diversity of 41 genotype of guava Determination of genetic relationship in 13 north Indian guava cultivars Molecular identication of 18 guava cultivars of Taiwan. Assessment of genetic relationship among four Mexican guava cultivars to estimate chemical (quercetin) diversity

Prakash et al. (2002) Dahiya et al. (2002) Chen et al. (2007) Feria-Romero et al. (2009)

SSR

Construction of (GA)n and (GT)n microsatellite-enriched library and characterization of 23 nuclear simple sequence repeat (SSR) loci in three guava species for cultivars identication and linkage mapping Genetic characterization of Mexican native 48 guava cultivars Purication, molecular cloning, and expression of the gene encoding 13hydroperoxide lyase from guava fruit Agrobacterium tumefaciens mediated genetic transformation of guava, resultants plants showed kanamycin resistance Introduction of cold tolerance genes (CBF1, CBF2 and CBF3) to organogenic and embryogenic explants

Risterucci et al. (2005)

AFLP Gene cloning

Hernandez-Delgado et al. (2007) Tijet et al. (2000)

Genetic transformation

Biswas et al. (2007)

Biswas et al. (2007)

without signicantly altering other aspects of the phenotype (Singh et al. 2004; Krishna and Singh 2007). The main target of gene transfer techniques is to produce improved varieties through the incorporation of horticulturally important genes into existing cultivars (Singh et al. 2004). Fruit trees are considered to be recalcitrant material for genetic transformation studies and the main impediment for genetic transformation is the regeneration of transformed plantlets. Choice of explants having competence for transformation and regeneration is a crucial factor. Hence, efcient tissue culture techniques become the base for genetic transformation studies (Giri et al. 2004). The successful regeneration of genetically transformed plants has been achieved in several tropical fruit plant species (Gomez-Lim and Litz 2004). An engineered Agrobacterium tumefaciens strain LBA 4404 (harboring binary vector pBI121 having selectable markers (nptII and GUS) with CaMV 35S promoter gene) has been used for transformation of guava (Biswas et al. 2007). Recently, preliminary work on genetic transformation of guava with cold hardiness genes (CBF1, CBF2 and CBF3) also demonstrated by Biswas et al. (2005, 2007), however, complete regeneration of transformed plants could not be achieved.

The development of recombinant DNA technology has not only extremely impacted on our understanding of gene structures, functions, and regulations, but also greatly facilitated gene cloning, characterization, and their expression into target species. Guava fruit was identied as a particularly rich source of hydroperoxide lyase (HPL) activity. HPL catalyzes the cleavage of 13- and 9-hydroperoxides of linoleic and linolenic acid into volatile C6- or C9-aldehydes and C12- or C9-oxoacids, respectively (Kim and Grosch 1981). The C6 and C9 volatile compounds have a commercial value in the production of natural avor in the food industry, and are potentially important in plant defense against pathogens (Croft et al. 1993). The HPL enzyme puried from guava fruits was cloned by polymerase chain reaction with 30 and 50 rapid amplication of cDNA ends (Tijet et al. 2000). The sequence shows approximately 6070% identity to known 13-hydroperoxide lyases. The cDNA was expressed in Escherichia coli.

Concluding remarks and future prospects In the past 23 decades, encouraging progress has been made regarding in vitro propagation of guava via

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Trees (2010) 24:112 Amin MN, Jaiswal VS (1989b) Effects of phloroglucinol, sucrose pH and temperature in vitro rooting of guava (Psidium guajava L.) microcutting. Bangladesh J Bot 18:129139 Ara H, Jaiswal U, Jaiswal VS (2000) Plant regeneration from protoplasts of mango (Mangifera indica L.) through somatic embryogenesis. Plant Cell Rep 19:622627 Ara H, Jaiswal U, Jaiswal VS (2004) An improved method of proliferation of proembryogenic calli of Mangifera indica L. var. Amrapali for scale-up of somatic embryo production. Indian J Biotechnol 3:229234 Arnold SV, Sabala I, Bozhkov P, Dyachok J, Filonova L (2002) Developmental pathways of somatic embryogenesis. Plant Cell Tissue Organ Cult 69:233249 Benson EE (2000) In vitro recalcitrance: an introduction. In Vitro Cell Dev Biol Plant 36:141148 Bhojwani SS, Razdan MK (1996) Plant tissue culture: theory and practice. Elsevier, Amsterdam Biswas BK, Yadav A, Joshee N, Yadav AK (2005) In vitro plant regeneration and genetic transformation to enhance cold hardiness in guava: a nutraceutical fruit. In: Proceeding of 1st international guava symposium. CISH, Lucknow, India, (abst.) pp 3132 Biswas BK, Joshee N, Yadav A, Yadav AK (2007) Development and application of biotechnology in guava: a nutraceutical fruit. In: Desjardins Y (ed) ISHS Acta Horticulturae 744: Ist international symposium on human health effects of fruits and vegetables, pp 267276 Chandra R, Bajpai A, Gupta S, Tiwari RK (2004) Embryogenesis and plant regeneration from mesocarp of Psidium guajava L. (guava). Indian J Biotech 3:246248 Chandra R, Mishra M, Abida M, Singh DB (2005a) Paclobutrazol mediated somatic embryogenesis in guava. In: Abstract of 1st international guava symposium. CISH, Lucknow, India, p 34 Chandra R, Mishra M, Bajpai A (2005b) Biotechnological interventions for improvement of guava (Psidium guajava L.). In: Kishun R, Mishra AK, Singh G, Chandra R (eds) Proceeding of 1st international guava symposium. CISH, Lucknow, India, pp 1925 Chen TW, Chang-Chai NG, CY Wang, Shyu YT (2007) Molecular identication and analysis of Psidium guajava L. from indigenous tribes of Taiwan. J Food Drug Anal 15:8288 Christianson ML (1987) Causal events in morphogenesis. In: Green CE, Somers DA, Hackett WP, Biesboer DD (eds) Plant tissue and cell culture. A.R. Liss., New York, pp 4455 Croft KPC, Juttner F, Slusarenko AJ (1993) Volatile products of the lipoxygenase pathway evolved from Phaseolus vulgaris (L.) leaves inoculated with Pseudomonas syringae pv. phaseolicola. Plant Physiol 101:1324 Dahiya KK, Archak S, Karihaloo JL (2002) DNA ngerprinting of guava (Psidium guajava L.) cultivars using RAPD markers. Indian J Plant Genet Resour 15:112115 Dinesh MR, Iyer CPA (2005) Signicant research achievement in guavaimprovement and future needs. In: Kishun R, Mishra AK, Singh G, Chandra R (eds) Proceeding of 1st international guava symposium. CISH, Lucknow, India, pp 716 Doijode SD (2001) Guava: Psidium guajava L. In: Doijode SD (ed) Seed storage of horticultural crops. Haworth Press, New York, pp 6567 Dudits D, Gyorgyey J, Bogre L, Bako L (1995) Molecular biology of somatic embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer Academic Publisher, Dordrecht, pp 267308 Feria-Romero IA, Astudillo-Dela HV, Chavez-soto MA, Rivera-arce E, Lopez M, Serrano H, Lozoya X (2009) RAPD markers associated with quercetin accumulation in Psidium guajava. Bio Plant 53:125128 Fuenmayor MED, Montero NJM (1997) In vitro clonal propagation of guava (Psidium guajava L.) from stem shoot of cv. Mara-7. Acta Hort 452:4751

organogenesis and somatic embryogenesis by manipulation of growth media and culture conditions as well as by testing a variety of explant sources. However, some of the long-standing problems such as guava wilt disease, short shelf life of fruits, and abiotic stress sensitivity requires urgent attention of researchers. There is need to exploitation of modern tools of biotechnology in improvement of guava. An increase in genetic transformation studies aimed at improving visual and growth characteristics of the plants has been hindered by low transformation efciencies and genotype dependence of protocols. As a result, guava regeneration studies have once again emerged as an essential complement of transformation studies. Since genetic transformation system for guava is not yet well developed, efforts need to be made to develop an efcient transformation system for guava. For instance, insertion of genes controlling ethylene biosynthesis could be helpful in increasing shelf life of fruits of guava. Transformation of genes encoding hydrolytic enzymes such as chitinase and glucanase (which can degrade fungal cell wall) could also be benecial in development of wilt resistant plant of guava (Chandra et al. 2005b). Such efforts will ultimately provide the most rapid advances in guava.
Acknowledgments Financial assistance provided by Council of Scientic & Industrial Research (CSIR), New Delhi, to the authors (MKR and PA) is gratefully acknowledged. Suggestions by the anonymous reviewers for improving the manuscript are also very much appreciated.

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