REVIEW

Biotechnological advances in guava (Psidium guajava L.): recentdevelopments 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 sus-

ceptibility to many pathogens, particularly guava wilting

caused by Fusarium oxysporium psidii, low fruit growth,

short shelf life of fruits, high seed content, and stress sensi-

tivity. 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 trans-

formation may be effective practical solutions for such

problems and improvement of guava. The improvement of

fruit trees through genetic transformation requires an effi-

cient regeneration system. During the past 2–3 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 propa-

gules, i.e., somatic embryos and non-embryogenic vegeta-

tive 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

man’s fruit’’ or ‘‘apple of tropics’’, is an important fruit

crop of tropical and sub-tropical regions of the world.

Guava fruit contains 2–5 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 significant 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 oxys-

porium 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

123

Trees (2010) 24:1–12

DOI 10.1007/s00468-009-0384-2

and loss due to this disease is substantial (Mishra 2005).

Other important field 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, flesh color, and soft seeds (Dinesh and Iyer

2005). Such an ideal phenotype cannot be met by conven-

tional breeding. Floral structure (epigynous flower, 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, graft-

ing, 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, pro-

duction of industrial compounds by cell culture, and deve-

lopment of stress-tolerant plants are some well-known

applications of plant tissue culture.

This review not only highlights the major biotechno-

logical 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.

Achievements made in guava through tissue culture

The efficient 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

embryogenesis, are distinctly different process (Chris-

tianson 1987; Litz and Gray 1992). Successful regenera-

tion 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 first

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) sug-

gested the pretreatment of explants with antioxidant solu-

tions. Explants were agitated for 30–40 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, 2–3 changes of

medium for the initial 10–15 days were essential for

Fig. 1 Biotechnological interventions in guava

2 Trees (2010) 24:1–12

123

controlling phenolic exudation and establishment of cul-

tures (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.

Table 1 Summary of work on organogenesis in guava

Cultivar Explant Mature/

juvenile

Medium ? PGRs References

Shoot multiplication Rooting

Banaras local Nodal segments M MS ? BAP � MS ? IBA NAA ? AC Amin and Jaiswal (1987)

– Shoot tips M MS ? BAP � MS ? IBA NAA ? AC Jaiswal and Amin (1987)

Chittidar Nodal segments M MS ? BAP � MS ? IBA NAA Amin and Jaiswal (1988)

– Shoot tips, Nodal segments,

Hypocotyl, Leaf segments

(Seedling)

J MS ? BAP MS basal Loh and Rao (1989)

– Shoot tips from seedling J OM ? BAP OM ? NAA ? IBA Papadatau et al. (1990)

Red Indian Nodal segments from seedling J MS ? BAP MS ? IBA or AC Yasseen et al. (1995)

Mara-7 Stem shoot M MS ? BAP, NAA, IBA Fuenmayor and Montero

(1997)

Allahabad

Safeda

Hypocotyl from seedling J MMS ? TDZ ? NAA � MMS ? IBA ? AC Singh et al. (2002)

– Nodal segments from green

house grown plants (GHRP)

and in vitro harvested axillary

buds (IVDS)

M/J MS ? BAP MS ? IBA Ali et al. (2003)

Aneuploid no.

82

Nodal segments M WPM ? BAP MS ? IBA ? NAA Meghwal et al. (2003)

Sardar Apical shoot tips M MS ? BAP ? IBA � MS ? IBA ?NAA Chandra et al. (2005b)

Pant Prabhat Nodal segments M MS ? BAP ? IBA � MS ? IBA ? NAA Mishra et al. (2005)

Safeda Shoot tips M MS ? BAP ? L-

glutamine

MS ? IAA ? IBA Zamir et al. (2007)

Safeda Seedling explants J MS ? Zea ? GA3 � MS ? IBA ? NAA Shah et al. (2008)

Banarasi local Nodal segments J MS ? BAP MS ? IBA 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 Modified 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 References

Induction of somatic embryos Germination of somatic embryos

Sardar Immature and mature

fruit mesocarp

MMS ? L-glutamine

(400 mg l-1) ? ascorbic acid

(100 mg l-1) ? 2, 4-D (2 mg l-1)

MMS ? L-glutamine

(400 mg l-1) ? ascorbic acid

(100 mg l-1) ? 2, 4-D (2 mg l-1)

Chandra et al.

(2004)

– Immature zygotic

embryo

GSEM ? 1.75 lM IAA ? 58.4 lM

L-glutamine

MS medium Biswas et al. (2007)

Allahabad Safeda Immature fruit

mesocarp

MS ? 1, 2, 4-1-H Triazol

(4 mg l-1)

– Chandra et al.

(2005a)

Cuban Red Dwarf

EEA

Immature zygotic

embryo

� MS ? L-glutamine

(400 mg l-1) ? ascorbic acid

(100 mg l-1) ? 6% sucrose ? 2,

4-D (1 mg l-1)

Liquid � MS ? BAP

(0.25 mg l-1) ? Biobras-6

(10 lg l-1)

Kosky et al. (2005)

Banarasi local Immature zygotic

embryo

MS ? 5% sucrose ? 2, 4-D

(1 mg l-1)

� MS ? 3% sucrose Rai et al. (2007)

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

Trees (2010) 24:1–12 3

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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 identified several microbial contaminants/

pathogens, i.e., Alternaria, Aspergillus, Cladosporium,

Colletotrichum, Curvularia, Fusarium, Nigrospora, Peni-

cillium, and Trichoderma, during in vitro culture of guava cv.

Enana Roja cubana and all the pathogens, except Nigros-

pora, 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 67–95% seeds of guava were contaminated by

bacteria and fungi using standard tissue culture methods.

They recommended the application of 10% HCl for 24–72 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 specific responsive stage of a mature tree’s

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 (off-

shoots) 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 influenced by several factors

namely kinds of tissue or explants, composition of med-

ium, plant growth regulators (PGRs), media additives,

culture environment etc.

Successful micropropagation, especially for difficult and

recalcitrant tree species, is mainly dependent on the quality

of explants and the response of explants is primarily deter-

mined 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 seg-

ments (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 phe-

nolic 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., greenhouse-

grown 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 opti-

mal 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 multi-

plication 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

4 Trees (2010) 24:1–12

123

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 con-

centrations 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 propaga-

tion (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 significant 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 estab-

lishment 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 med-

ium 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 combi-

nation 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 bio-

chemical 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 differenti-

ation 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 recalci-

trant 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 geno-

type (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-

Trees (2010) 24:1–12 5

123

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 modified 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 cyto-

kinins (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 deve-

lopment 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 embryo-

genesis. Continuous treatment for 60 days allowed differ-

entiation 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

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 4–5 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

6 Trees (2010) 24:1–12

123

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 5–6% 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 (solidified 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 meta-

bolized 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 16–20 days. After 4–5 weeks of culture, entire sur-

face of zygotic embryos were covered with lower-stage

somatic embryos. Synchronization of embryogenic cultures

is difficult 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

Germination of somatic embryos and growth of regene-

rated plants depends on the conditions provided at earlier

stages when somatic embryos mature. In some cases, pre-

cocious germination of somatic embryos takes place

(Arnold et al. 2002; Jimenez 2005). Therefore, in order to

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 med-

ium 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-solidified

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 pro-

pagated crop (Doijode 2001). Therefore, it is a particularly

suitable candidate for synthetic seed technology. Success-

ful 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 combina-

tion 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 short-

term storage and germplasm exchange of elite genotype of

guava (Rai et al. 2008b, c).

In vitro storage of synthetic seeds

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

Trees (2010) 24:1–12 7

123

ABA (1 mg l-1) or 9% sucrose prior to culturing on ger-

mination 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 (4�C) 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 (4�C) 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 quan-

titatively 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 micro-

propagation via shoot tips was carried out by Zamir et al.

(2003). Shoot tips were irradiated with gamma rays at

15–90 Gy using 60Co gamma cell source and cultured in

MS medium containing 3.0% sucrose, BAP and L-gluta-

mine. 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 identifications are traditionally based on various

morphological characters; however, morphological char-

acters 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 environ-

mental alterations can create problems in correct identifi-

cation 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 undesir-

able somaclonal variations. In guava, recently, a few reports

have been made on assessment of genetic diversity using

Random Amplified 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 tech-

nology faces severe problems due to the presence of

inhibitors such as polysaccharides, which inhibit the enzy-

matic DNA processing or phenolics as inhibitors of PCR

reactions (Prakash et al. 2002). The well-established mod-

ified CTAB protocol (Porebski et al. 1997) yielded excel-

lent DNA templates for PCR amplification 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 gene-

tically 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 deter-

mine phylogenetic relationship in 18 cultivars of Taiwan.

Apart from characterization and assessment of chemical

diversity, Feria-Romero et al. (2009) used RAPD amplifi-

cation 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). Microsatel-

lites 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

amplified 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 pro-

vides the means for modifying single horticultural traits

8 Trees (2010) 24:1–12

123

without significantly altering other aspects of the pheno-

type (Singh et al. 2004; Krishna and Singh 2007). The

main target of gene transfer techniques is to produce

improved varieties through the incorporation of horticul-

turally important genes into existing cultivars (Singh et al.

2004). Fruit trees are considered to be recalcitrant mate-

rial 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, efficient 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 mark-

ers (nptII and GUS) with CaMV 35S promoter gene) has

been used for transformation of guava (Biswas et al.

2007). Recently, preliminary work on genetic transfor-

mation 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 identified as

a particularly rich source of hydroperoxide lyase (HPL)

activity. HPL catalyzes the cleavage of 13- and 9-hydro-

peroxides 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 flavor in

the food industry, and are potentially important in plant

defense against pathogens (Croft et al. 1993). The HPL

enzyme purified from guava fruits was cloned by poly-

merase chain reaction with 30 and 50 rapid amplification of

cDNA ends (Tijet et al. 2000). The sequence shows

approximately 60–70% identity to known 13-hydroperox-

ide lyases. The cDNA was expressed in Escherichia coli.

Concluding remarks and future prospects

In the past 2–3 decades, encouraging progress has been

made regarding in vitro propagation of guava via

Table 3 Achievements made in

guava through molecular

approaches

Biotechnological tool Achievement References

DNA markers

RAPD Estimation of molecular diversity of 41

genotype of guava

Prakash et al. (2002)

Determination of genetic relationship in

13 north Indian guava cultivars

Dahiya et al. (2002)

Molecular identification of 18 guava

cultivars of Taiwan.

Chen et al. (2007)

Assessment of genetic relationship among

four Mexican guava cultivars to

estimate chemical (quercetin) diversity

Feria-Romero et al. (2009)

SSR Construction of (GA)n and (GT)nmicrosatellite-enriched library and

characterization of 23 nuclear simple

sequence repeat (SSR) loci in three

guava species for cultivars identification

and linkage mapping

Risterucci et al. (2005)

AFLP Genetic characterization of Mexican

native 48 guava cultivars

Hernandez-Delgado et al.

(2007)

Gene cloning Purification, molecular cloning, and

expression of the gene encoding 13-

hydroperoxide lyase from guava fruit

Tijet et al. (2000)

Genetic transformation Agrobacterium tumefaciens mediated

genetic transformation of guava,

resultants plants showed kanamycin

resistance

Biswas et al. (2007)

Introduction of cold tolerance genes

(CBF1, CBF2 and CBF3) to

organogenic and embryogenic explants

Biswas et al. (2007)

Trees (2010) 24:1–12 9

123

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 exploita-

tion 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 efficiencies 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 efficient

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 beneficial 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

Scientific & 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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