GUAVA CALLUS PRODUCTION UNDER DIFFERENT CULTURE MEDIUM AND PLANT GROWTH REGULATOR CONDITIONS

Proceedings 33rd PGRSA Annual Meeting
GUAVA CALLUS PRODUCTION UNDER DIFFERENT CULTURE MEDIUM AND
PLANT GROWTH REGULATOR CONDITIONS
Guochen Yang and Zhongge Lu1
ABSTRACT
This research evaluated culture media and plant growth regulators for their influences on callus
initiation. Guava (Psidium guajava L.) is an important tropical fruit species that is rich in
vitamins, minerals, organic acids, and pectins. Different concentrations of 6-benzyladenine
(BA), kinetin, or 2,4-dichlorophenoxyacetic acid (2,4-D), and naphthaleneacetic acid (NAA)
were added to Murashige and Skoog (MS) and woody plant medium (WPM) and tested for their
influences. There are differences in callus initiation and morphology between MS and WPM,
and among PGR concentration treatments.
Additional index words: Psidium guajava, plant growth regulators, cultivar.
INTRODUCTION
Guava (Psidium guajava L.) is a rich source of vitamins, minerals, organic acids, and pectins
(Chan et al 1971, Rathore 1976, Wilson 1980, Campbell 1984, Menzel 1985, Loh and Rao 1989,
Yadava 1994). Seventy-nine different phytochemicals provide guava with many unique
properties and actions including anti-microbial, astringent, bactericidal, cicatrizant,
emmenagogue, hypoglycemic, laxative, nutritive, and spasmolytic. Singh et al (1992) reported
that regular consumption of guava fruit significantly increases the good cholesterol level (high
density lipoprotein) and significantly decreases serum total cholesterole and blood pressures.
Through pharmacological studies of guava with mice, Olajide et al (1999) demonstrated that
guava leaf extract considerably inhibited paw oedema, reduced pain, exhibited an antipyretic
effect, and prevented diarrhea. Medicinal uses of guava have been reported involving
gastroenteritis, dysentery, wounds, ulcers, rheumatics, and toothache (Rathore 1976). Our own
preliminary research has demonstrated antimicrobial activity of guava juice, fruit, and leaf
extract against E. coli O157:H7 (Yang et al., 2004). Because of its considerable store of
vitamins and vitamin precursors, minerals, organic acids, and pectins, there is a need to fully
explore the possibilities of using this tropical fruit for phytochemical production and
antimicrobial applications. It is our belief that with appropriate biotechnological approaches it is
possible to generate production of specific phytochemicals in guava that can be used to improve
overall health and well-being of the general public. The research objectives were to study
suitable PGR concentration and combination for guava callus production; to investigate cultivar
responses on callus production; and to determine optimum culture medium strength for guava
callus production. By achieving these three objectives we believed we would have advance our
knowledge base that could facilitate phytochemical production and extraction.

1 Department of Natural Resources & Environmental Design, North Carolina A&T State University, Greensboro,
NC 27411

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MATERIALS AND METHODS
MS (Murashige and Skoog, 1962) and WPM (Lloyd and McCown, 1981) were used as the base
media. These media were supplemented with 3% sucrose and 0.7% agar. Medium pH was
adjusted to 5.8 using 1N KOH or 1 N HCl before agar was added. The culture media were
sterilized by autoclaving at 121oC for 20 minutes.
Softwood shoots from greenhouse-grown stock guava plants were used as explant materials,
which were disinfested for 15 minutes in 15% bleach solution (0.78% NaOCl) containing 20
drops Tween-20 per liter, and rinsed immediately with sterile distilled water 4 times. The
softwood shoots was cut into 0.5 cm segments as explants. Each explant was transferred onto 10
ml of a particular treatment medium in 25 x 95 mm shell vials with transparent polypropylene
closures. Each shell vial containing one explant represented an experiment unit.
A completely randomized design (CRD) was used. Each individual explant in a shell vial was a
replication, as there was only one explant per shell vial. Callus fresh weight (mg) per explant
was recorded after 10-12 weeks in culture. Average weights were obtained for replication
groups and compared using the Least Significant Difference procedure in SAS version 8 (SAS
Institute, Cary, NC, USA). Treatment differences were considered significant at the ?=0.05
level.
The cultures were placed under 16 hours of light per day provided by cool white fluorescent
tubes at 37.6±10.1 ?mol m-2s-1 and at 23±1oC. Cultures were transferred onto the same fresh
media every four weeks.
Different concentrations of 2,4-D (0.8, 1.0, 1.2, 1.4, 1.6, or 1.8 mg/l) were added as treatments to
the base MS or WPM. Explants were cultured on each treatment medium. Two sets of
experiments were conducted one using MS base medium, the other using the WPM. Each
concentration treatment was tested with 14 explant replications. One explant was transferred
onto each shell vial as one experiment unit or replication.
The test cultivars include Ruby Supreme, RDF, Pear, Allahabad, RDE, L-49, and Beaumont.
Explants (0.5 cm in length) from each cultivar were cultured on MS or WPM with supplements
of 2,4-D at 0.5 mg/l and BA at 1.0 mg/l, plus the base supplements of 3% of sucrose and 0.7%
agar. The same procedure used for PGR concentration comparison experiments was followed
here. There were 14 replications for each cultivar treatment.
MS base medium strengths (½ x, 1x, or 2x) were used as treatments. Each strength MS base
medium was supplemented with 3% sucrose, 0.7% agar, 2,4-D at 0.5 mg/l and BA at 1.0 mg/l.
‘RDF’ explants (0.5 cm in length) were cultured on each strength MS treatment medium. The
same procedure used for PGR concentration comparison experiments was followed here, except
the strength treatment trials used 18 shell vials resulting in 18 replications.
RESULTS AND DISCUSSION
Concentration levels of 2,4-D significantly affected callus production by Guava ‘Beaumont’
(Fig. 1). Explants cultured on 2,4-D at 1.6 mg/l treatment produced the most amount of callus.
The callus fresh weight difference among 2,4-D concentration treatments was significant at

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?=0.05 level. There were different cultivars responses in callus production to the MS and WPM
base culture media. General observation of callus fresh weight data indicated that there was the
largest amount of ‘Beaumont’ callus growth occurred when cultured on WPM than explants
cultured on MS (Fig. 1). By contrast, visual assessment of the ‘RDF’ explants produced more
callus when cultured on MS in comparison with explants cultured on WPM (Fig. 2). There were
not enough data on this to conduct statistical analysis on these observations.
Our preliminary research has indicated that the combination of 2,4-D at 0.5 mg/l and BA at 1.0
mg/l was the best for callus initiation. This combination was selected for the cultivar comparison
tests. Significant differences were observed among the cultivars (Fig. 3). ‘Ruby Supreme’,
‘RDF’, ‘L-49’, or ‘Beaumont’ produced much more callus than ‘Pear’, ‘Allahabad’, or ‘RDE’ on
MS. In contrast, ‘Ruby Supreme’, ‘RDF’, ‘RDE’, or ‘Beaumont’ produced more callus than
‘Pear’, ‘Allahabad’, or ‘L-49’ on WPM. ‘Ruby Supreme’, ‘RDF’, or ‘Beaumont’ produced
favorable results in callus production, regardless of the base culture medium.
‘RDF’ explants were cultured on different strength MS plus 2,4-D at 0.5 mg/l and BA at 1.0
mg/l. Significant difference in callus fresh weight were observed among MS strength treatments.
Both visual assessment (Fig. 4) and callus fresh weight data (Fig. 5) indicated that 2x MS
treatment enhanced callus production. Callus also displayed good morphological quality.
However, about 40% of calli produced from this treatment tended to become brown, and
eventually die (data not shown). No similar observation was noticed from ½ or 1x MS
treatment.
LITERATURE CITED
Campbell, C.W. 1984. Guava: Tropical fruits and nuts. In FW Martin (ed.), CRC handbook of
tropical food crops, p254-256, CRC Press.
Chan, H.T. Brekke, J.E. and Chan, T. 1971. Nonvolatile organic acids in guava Journal of Food
Science 36:237-239.
Lloyd, G. and McCown, B.H. 1981. Commercially feasible micropropagation of mountain laurel
(Kalmia latifolia) by use of shoot tip culture. Comb. Proc. Intern. Plant Prop. Soc. 30:421-427.
Loh, C.S. and Rao, A.N. 1989. Clonal propagation of guava (Psidium guajava L.) from seedlings
and grafted plants and adventitious shoot formation in vitro. Scientia Horticulturae 39:31-39.
Menzel, C.M. 1985. Guava: an exotic fruit with potential in Queensland. Queensland Ag. J.
111(2):93-98.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassay with
tobacco tissue cultures. Physiol. Plant. 15:473-497.
Olajide, O.A. Awe, S.O. and Makinde, J.M. 1999. Pharmacological studies on the leaf of
Psidium guajava. Fitoterapia Vol. 70(1), p25-31.
Rathore, D.S. 1976. Effect of season on the growth and chemical composition of guava (Psidium
guajava L.) fruits. J. Hort Sci 51:41-47.

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Singh, R.M. Rastogi, S.S. Singh, S. Ghosh, S. and Niaz, M.A. 1992. Effects of guava intake on
serum total and high-density lipoprotein cholesterol levels and on systemic blood pressure. The
American Journal of Cardiology Vol. 70:1287-1291.
Wilson, C.W. 1980. Guava. In Nagy & Shaw (eds.) Tropical and sub-tropical fruits:
composition, properties, and uses. AVI, Westport, CT. p279-299.
Yadava, U.L. 1994. Physicochemical properties of guava produced in Georgia. HortScience
29:536-537.
Yang, G. Ibrahim, S.A. and Niedziela, C. 2004. Antimicrobial effect of guava products against
foodborne pathogens HortScience, Vol. 39(4) p778, abstract #299.





450
400
350
300
250
MS
200
WPM
150
100
50
0 0.8 1 1.2 1.4 1.6 1.8


Figure 1. Guava (var. Beaumont) callus fresh weight (mg) from MS (left) or WPM (right) plus 2,4-D at 0.8,
1.0, 1.2, 1.4, 1.6 or 1.8 mg/l.



Figure 2. Guava (var. RDF) callus initiation on MS (left) and WPM (right) media.




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1000
800
600
MS
400
WPM
200
0 V 1 V 2 V 3 V 4 V 5 V 6 V 7


Figure 3. Guava callus fresh weight (mg) from seven cultivars (V1-Ruby Supreme, V2-RDF, V3-Pear, V4-
Allahabad, V5-RDE, V6-L49, V7-Beaumont) cultured on MS (left) or WPM (right) basic medium plus 2,4-
D at 0.5 mg/l and BA at 1.0 mg/l.



Figure 4. Guava (var. RDF) callus initiation on different strengths of basic MS medium.


800
700
600
500
400
MS
300
200
100
0
1/2x
1x
2x MS


Figure 5. Guava (var. RDF) callus fresh weight (mg) from ½, 1, or 2x MS plus 2,4-D at 0.5 mg/l and BA at
1.0 mg/l.


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