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Documenti di Professioni
Documenti di Cultura
ISSN 0031-7454
Key Words: callus culture, dedifferentiation, elicitor, flavonoid, Jatropha curcas, ultraviolet-B radiation
Abbreviations: CHS chalcone synthase, HPLC high performance liquid chromatography, MS Murashige and
Skoog, NAA naphthalene-acetic acid, PAL phenylalanine ammonia lyase, PCR polymerase chain reaction,
RAPD Random Amplified Polymorphic DNA, UV-B ultraviolet-B
INTRODUCTION
Flavonoids comprise a large group of natural phenolic
compounds found in vascular plants as products of
secondary metabolism. Their therapeutic potential as
antioxidants and their promising role in anti-cancer
therapy, along with their manifold applications in the
cosmetic and pharmaceutical industry, have aroused
interest in developing alternative systems for increased
production. Biotechnological approaches such as cell or
tissue cultures, which allow optimization of growth
conditions and genetic manipulation to enhance flavonoid
production, offer an opportunity to circumvent problems
regarding insufficient yield. Of the various tissue culture
methods, callus induction is frequently used for flavonoid
production
since
extraction
procedures
from
undifferentiated tissues are easier to perform (Jedinak et
The Philippine Agricultural Scientist Vol. 95 No. 4 (December 2012)
336
Kin
0
10
20
30
40
50
0
10
20
30
40
50
RESULTS
Callus Induction and UV Irradiation
In the two trials performed, all the tested culture media
were able to induce callus formation in more than 60% of
the leaf explants, except for the medium without plant
growth regulators (Table 2). Calli were first observed 67
d after inoculation and were found predominantly on the
cut portions of the midrib and secondary veins. Most of
the calli formed were green in color and exhibited a
slightly compact structure. Callus growth rate varied
among the different media but all cultures exhibited
about 3- to 5- fold increases in a 5-wk period based on
fresh weight (Fig. 1). By the end of the fifth week, the
calli grown in medium containing 10 M NAA + 10 M
kinetin were found to have significantly lower fresh
weights than those cultured in other growth media (Fig.
2) and reached fresh weights averaging 4 g.
Among the growth media that were able to produce
at least 5 g of calli on the fifth week of culture, the
medium supplemented with the lowest levels of plant
growth regulators (20 M NAA + 20 M kinetin) was
chosen to initiate and maintain the callus cultures that
were used in the irradiation experiments in order to
minimize the probable effects of high hormone levels on
the degree of genetic variation in the cultures. After 89
d from the start of culture, 8083% of the explants
cultured in the two trials had formed callus. By the fifth
week, the calli reached an average weight of 5.02 0.432
g and were then subjected to the UV treatments. UV-B
irradiation had no noticeable effect on the growth and
morphology of the treated calli.
Genetic Variation
In the two trials performed, sizes of the products
amplified using 10 random primers varied from 150 to
4,000 bp (Table 3). The number of bands for each primer
varied from 8 (OPAL 8) to 17 (OPAB 5), with an average
of 12.44.94 bands per primer. The RAPD analyses of
DNA obtained from the leaf (mother plant), along with
the control and UV-B irradiated calli generated a total of
890 bands, all of which exhibited monomorphic patterns.
Examples of the monomorphic bands and their intensities
are shown in Figure 3. These identical banding patterns
generated by the 10 primers were observed in the two
independent amplifications performed. The dendrograms
338
DISCUSSION
Before embarking on any genetic transformation or
elicitation experiment using in vitro grown plant tissues,
the feasibility of establishing tissue cultures must first be
determined. The experiments conducted in the present
study showed that MS medium supplemented with equal
concentrations of NAA and kinetin, ranging from 10 to
50 M, can induce callus growth on leaf explants of J.
curcas. The resulting calli were green and nonembryogenic which is important since chloroplastcontaining cells are known to possess flavonoid
biosynthetic potential and non-embryogenic callus
cultures
containing
homogenous
clumps
of
dedifferentiated cells are often used for experiments on
secondary metabolite production (Havsteen 2002;
Jedinak et al. 2004).
Kin
Total No.
of
Explants
0
10
20
30
40
50
0
10
20
30
40
50
30
30
30
30
30
30
% Callus
Induction
3.354.74
63.54.95
80.08.90
83.54.95
76.54.95
77.014.1
Callus Characteristics
Nature
Color
Dry, compact
Dry, compact
Slightly compact
Slightly compact
Slightly compact
Slightly compact
Light green
Light green
Green
Green
Green
Light green
Growth Rate**
Very slow
Slow
Moderate
Moderate
Fast
Fast
MS basal medium + 2% sucrose + different combinations of naphthalene acetic acid (NAA) and kinetin (Kin). Data were recorded after 20 d of culture.
Each treatment was repeated twice and each replicate consisted of 15 explants. Callus induction data are means SD.
**
Callus growth rate: very slow (callus first appeared after 1315 d), slow (callus first appeared after 1012 d), moderate (callus first appeared after 79
d), fast (callus first appeared after 46 d).
10 NAA :
20 NAA :
30 NAA :
40 NAA :
50 NAA :
10 Kin
20 Kin
30 Kin
40 Kin
50 Kin
OPAK 14
OPD14
OPAW 7
OPAB 5
OPF 11
OPF 2
OPA 4
OPAD 11
OPAL 8
OPAB 6
0
0
14
21
28
35
CTGTCATGCC
CTTCCCCAAG
AGCCCCCAAG
CCCGAAGCGA
TTGGTACCCC
CCTGATCACC
AATCGGGCTG
CAATCGGGTC
GTCGCCCTCA
GTGGCTTGGA
14
12
11
17
13
12
13
14
8
10
Total: 124
200-3000
300-1500
500-3000
150-2000
250-3000
250-1000
500-4000
400-2500
400-1500
250-1500
8
b
a
4
2
in
:5
0
in
50
N
A
:4
0
in
A
N
A
40
30
N
A
:3
0
in
K
:2
0
A
N
A
20
10
N
A
:1
0
in
Hormone Combinations
A. Vitexin
1200
1000
800
600
400
200
lo
w
VB
U
VB
(+
)
(+
)
(-)
Le
af
B. Isovitexin
a
1200
1000
600
bc
400
200
lo
w
VB
U
VB
(+
)
(+
)
(-)
Le
af
hi
gh
C. Apigenin
1200
1000
800
ab
600
400
a
200
hi
gh
lo
w
VB
(+
)
VB
VB
U
(+
)
(-)
Le
af
g/g
g-1 Dry Weight
g
1400
340
ac
800
VB
1400
hi
gh
VB
1400
ACKNOWLEDGMENTS
We would like to thank the Institute of Biology, College
of Science, University of the Philippines Diliman for
providing us with financial and logistical support. Special
thanks also to the Philippine Council for Health Research
Development (PCHRD) - Department of Science and
Technology (DOST) and to Congressman Michael John
Duavit for the grants awarded to E. M. Alvero-Bascos.
We would also like to express our gratitude to Dr. Ian
Fontanilla, Dr. Jonas Quilang, Dr. Windell Rivera, Dr.
Sonia Jacinto, Dr. Ernelea Cao, Dr. Juliana Janet Puzon,
Edric Adao and Chris Mendoza for all the help that they
have extended to us.
341
REFERENCES CITED
342
343