International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:12 No:06
Shoot Induction from Broccoli Explant
Hypocotyls and Biosynthesis of Sulforaphane
Wenny Tilaar, Sumeru Ashari, Bagyo Yanuwiadi, Jeany Polii-Mandang, Francien H. Tomasowa
Abstract The research aimed to assess combination effect of
naphthalene acetic acid (NAA) and benzylaminopurine (BAP) to
shoot induction from broccoli explants hypocotyl and to evaluate
sulforaphane synthesis. Factorial design arranged in randomized
complete design was used with 0 and 1 ppm concentration of
NAA and 0, 2.5, and 5 ppm of BAP. Each combination of NAA
and BAP was replicated five times. Parameters observed were
time of adventive shoot formation, number of adventive shoot,
fresh shoot weight, shoot height, and sulforaphane content in
broccoli shoot. Data was analyzed using variance analysis. The
result showed that significant difference in the number of shoots,
shoot height, and sulforaphane synthesis on broccoli shoot was
evident in the treatment combination with NAA and BAP. The
highest shoot number and the sulforaphane content were
detected in the treatment combination with 1 ppm NAA and 5
ppm BAP. The highest shoot height was yielded by the
combination of 0 ppm NAA and 0 ppm BAP. The treatment with
NAA revealed significant difference in shoot formation time
while that with BAP showed significant difference in fresh shoot
weight.
Index Term broccoli, substant growth, shoot induction and
sulforaphane
I. INT RODUCT ION
BROCCOLI is a horticulture plant and belongs to the family
of Brassicaceae [1]. The broccoli (Brassica oleracea) is rich
in amount and variety of antioxidant compounds. One of the
antioxidant compounds of broccoli is sulforaphane [2]. In
addition, broccoli is also rich in fat, protein, carbohydrate,
fiber, water, and vitamin [3]. In addition, broccoli is also rich
of fat, protein, carbohydrate, fiber, water, and vitamin [4].
Broccoli is not an indigenous Indonesian plant. Yet, d ue to
the economic value of the plant, the broccoli has been
cultivated in Indonesia since 1970. Since the broccoli
cultivated in Indonesia is a hybrid variety, it is difficult to use
the progeny as it will segregate, leading to the problem of
seedling availability in order to fulfill the demands of industry.
One solution is using in vitro propagation such as tissue
culture. The advantages of using tissue culture in seedling
W. T ilaar is with the Faculty of Agriculture, Sam Ratulangi University, Jl.
Kampus Kleak, Manado 95115, North Sulawesi, Indonesia (corresponding
author, phone +62-81430844760, e-mail: tilaarwenny@yahoo.com).
S. Ashari is with the Faculty of Agriculture, Brawijaya University, Jl.
Veteran 2, Malang 65145, Indonesia.
B. Yanuwiadi is with Department of Biology, Faculty of Mathematics and
Natural Sciences, Brawijaya University, Jl. Veteran 2, Malang 65145,
Indonesia.
J. Polii-Mandang is with the Faculty of Agriculture, Sam Ratulangi
University, Jl. Kampus Kleak, Manado 95115, North Sulawesi, Indonesia.
F.H. T omasowa is with Department of English, Faculty of Culture Studies,
Brawijaya University, Jl. Veteran 2, Malang 65145, Indonesia.
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44
production are free of pests and diseases, short time
consumption, rapid production, and no dependence on season
and climate.
Hormone was categorized as growth regulator which was
synthesized by plant [5]. Currently, growth regulator used in
tissue culture is naphthalene acetic acid (NAA) which is used
as auxin and benzyl amino purine (BAP) used as cytokinin [6].
Interaction between auxin and cytokinin was demonstrated by
Skoog and Miller for organogenesis using tissue culture of
tobacco. If concentrations of auxin lower than cytokine,
explants would induce shoot. On the contrary, cytokinin lower
then auxin would induce root. We previously reported that
broccoli hypocotyls could produce callus on 1 ppm of NAA
and combination of 0.1 ppm NAA and 0.5 ppm of BAP. A
combination of 0.1 ppm NAA and 4 ppm BAP gave the best
formation of shoot from broccoli hypocotyls [7, 8].
Broccoli is also used as medicinal plant. The plant has been
reported containing sulforaphane which function as
antioxidant [9, 10]. Sulforaphane was produced from
glucosinolate hydrolysis [11, 12]. Glucosinolate is a
glucoraphanin that produces sulforaphane with the help from
myrosinase [13]. Broccoli seed is rich of myrosinase and
glucoraphanin [1]. It is, therefore, important to conduct a
research on broccoli using tissue culture to increase
sulforaphane with the application of growth regulator.
In terms of the use of growth regulator, Morris [14] reported
that callus and cell aggregates of Catharanthus roseus in
culture to medium with additional NAA and P could produce
high ajmalicine and serpentine compared to 2,4_D and kinetin
[14]. Darsini [15] reported that the best callus production on
C. roseus were resulted using combination of 2.5 M NAA
and 10 M BAP. Therefore, it needs to study the effect of
combination of NAA and BAP on shoot induction as well as
induction secondary metabolite such as sulforaphane in
medium Murashige and Skoog.
II. M AT ERIALS AND M ET HOD
A. Sample Preparation
The research was carried out in December 2010 March
2011. The explants used were hypocotyls of broccoli of
cultivar Green Royal. The broccoli seeds were sterilized using
Bayclean. Afterwards, the broccoli seed were soaked in 30%
bleach solution for ten minutes followed by rinsing with sterile
water three times. This process was replicated with
concentration of bleach viz 20% and 10%. Later, the seeds
were sown on Murashige and Skoog (MS) medium for two
weeks to allow production of germination hypocotyls. After
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 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:12 No:06
two weeks, the hypocotyls of broccoli were cut in pieces of
1.5 cm in length. The cuttings were then planted on MS media
supplemented with NAA and BAP with various combinations.
III. EXPERIMENT AND A NALYSIS
The treatments were NAA, BAP and combinations of
NAA and BAP. The NAA treatments were 0 ppm and 1 ppm
added into the MS media. The BAP treatments were 0, 2.5 and
5 ppm added into the MS media. The combination treatments
of NAA and BAP (made five replicates) were:
0 ppm NAA + 0 ppm BAP,
0 ppm NAA + 2.5 BAP,
0 ppm NAA + 5 ppm of BAP,
1 ppm NAA + 0 ppm BAP,
1 ppm NAA + 2.5 ppm BAP, and
1 ppm NAA + 5 ppm BAP.
Sulforaphane extraction was initially with measurement of
broccoli shoot. The broccoli shoots were weighted on digital
balance. Afterwards, the broccoli shoots were put in a mortar
then added with 12 mL methyl chloride and be mashed. The
mashed broccoli shoot was then transferred onto flask and
added with 2550 mL methyl chloride. Moreover, sonification
was performed for 30 minutes to release sulforaphane from
tissue of shoot broccoli. Furthermore, the mashed broccoli
shoot was filtered using Whatman paper and transferred to a
tube and put on heating block with temperature 7080 C until
producing supernatant. The supernatant was added 5 ml of
NaSO4 and heated again with 7080 C for drying the
supernatant. The drying supernatant was added 10 mL
acetonitrile then sonification was again performed for 30
minutes. The drying supernatant was then centrifuged for 15
minutes at 4000 rpm. Afterwards, the drying supernatant was
transferred to vial bottle then put in LC tandem MSMS
quadruple to determine the sulforaphane content.
Parameters observed were time of adventive shoot
formation, number of adventive shoot formation, fresh shoot
a b
Fig. 1. a). Enlargement of hypocotyls, b). T he meristematic cells were forming globulars, and c). T he shoots initiation.
T ABLE I
EFFECT OF NAA CONCENTRATION ON TIME FOR ADVENTIVE SHOOT
FORMATION
T reatment average/day
NAA 0 17.40a
NAA 1 21.53b
LSD 5% 3.57
B. Number of Adventive Shoot Formation
The number of adventive shoot showed that there was
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weight, shoot height, and sulforaphane content in broccoli
shoot. Data was analyzed using variance analysis .
IV. RESULT S AND DISCUSSION
The result showed that shoots forming were generated from
direct organogenesis of explants hypocotyls. Organogenesis of
hypocotyls explants was detected on all treatments. Shoot was
initiated by forming meristematic cells on explants cutting and
wounded explants. Moreover, the meristematic cells form
globules followed by shoot induction (Fig. 1).
A. Time of adventive shoot Formation
No interaction was revealed between NAA and BAP on
the time of adventive shoot formation. This may be due to the
sufficient availability of endogen cytokinin in the explants.
Furthermore, NAA treatment affected on time of the adventive
shoot formation indicated that increasing of NAA
concentration could slow the time of adventive shoot
formation (Table I). The 1 ppm NAA treatment resulted in a
14-to-27-day adventive shoot formation time. The 0 ppm
NAA treatment resulted in a 10-to-21-day adventive shoot
formation time.
Time of adventive shoot formation on Brassica oleracea
generated from peduncle explants was 710 days [16]. In our
previous study we found that the time of adventive shoot
formation generated from explants hypocotyls broccoli was
ranging from 14 to 20 days [8]. Pavlovi et al. [17] reported
that adventive shoot formation from hypocotyls, cotyledon
and root explants broccoli was four week. Ravantar et al. [18]
reported that time of adventive shoot formation in broccoli
generated from cotyledon explants was eight weeks. Hence, it
could be concluded that the time of adventive shoot formation
of broccoli is determined by broccoli explants and cultivar.
c
interaction between NAA and BAP. The highest adventive
shoot formation (27.93 shoots) was detected on the treatment
combination of 1 ppm NAA and 5 ppm BAP (Table II, Fig. 2).
This result differs from Huang et al. [19] in that the mean
number was 6.4 shoots per explant. Furthermore, Pavlovi et
al. [17] revealed that the number of shoot per explants ranged
from 3.5 to 7.4 shoots per explants. This implies that the
number of shoot varied due to the different concentrations of
NAA and BAP. Huang et al. [19] used the concentrations of
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0.107 M NAA and 17.76 M BAP which proved to be the C. Adventive Shoot Height
best for broccoli regeneration. Whereas, Pavlovi et al. [17] There was interaction of NAA and BAP on the height of
used broccoli hypocotyls in tissue culture with basal medium adventive shoot. The combination of 0 ppm NAA and 0 ppm
MS supplemented by the combination of 1 mg/L BA with 0, BAP yielded the highest adventive shoots (Table III, Fig. 3).
0.1 and 0.2 mg/L IBA. The hypocotyls showed the best No significant difference was shown among the combinations
explants in almost all varieties tested with a minimum of 0 ppm NAA and 2.5 BAP; 0 ppm NAA and 5 ppm BAP; or
regeneration potential of 75 % and producing 3.57.4 shoots 1 ppm NAA and 0 ppm BAP. Significant difference was
per explant. This implies that treatments with different shown among the combinations of 0 ppm NAA and 2.5 ppm
concentrations of NAA and BAP yield different numbers of BAP; 1 ppm of NAA and 2.5 ppm BAP as well as
shoots. combination of 1 ppm NAA and 5 ppm BAP (Table III, Fig.
T ABLE II 3). The combination of 0 ppm NAA and 0 ppm BAP yielded
NAA AND BAP INTERACTION EFFECTS ON NUMBER OF ADVENTIVE SHOOT
FORMATION
the highest shoots, the condition of which is expected to be
Average number of shoot due to the apical dominance phenomenon. Indoleasetic acid
T reatment BAP BAP BAP (IAA) is a factor of apical dominance and was formatted in the
0 ppm 2.5 ppm 5.0 ppm shoot plant tips. IAA inhibits the outgrowth of side shoots
NAA0 7.93ab 17.00c 12.20bc [20]. IAA in the tissue culture promotes not only cell
NAA (ppm)
NAA1 5.07a 15.53c 27.93d
LSD 5% 6.05 elongation but also cell division until the elongation of shoots.

Fig. 2. Adventive shoots formation from broccoli hypocotyls from difference combination of NAA and BAP.

Fig. 3. Height of the adventive shoot s from N0B0 and N0B2.5 treatment combinations.
T ABLE III
EFFECT OF NAA AND BAP INTERACTION ON ADVENTIVE SHOOT HEIGHT D. Shoot Fresh Weight
Average shoot height BAP is a cytokinin growth regulatory compound.
T reatment BAP BAP BAP Cytokinins are often used to stimulate plant growth and
0 ppm 2.5 ppm 5.0 ppm
development [20, 21]. While growth is often designated as an
NAA0 6.50c 1.68a 2.00a
NAA (ppm) irreversible increase in volume [22]. So, BAP is necessary to
NAA1 1.71a 3.46b 4.22b
LSD 5% 1.37 support the shoot fresh weight.

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There was no interaction of NAA and BAP and shoot fresh V. CONCLUSION
weight. But on single BAP, the treatment was significant. Shoot induction from broccoli explant and synthesis of
Significant difference was evident between the results of the sulforaphane production had been carried out. We found that
treatment with 0 ppm BAP and those with 2.5 ppm BAP and 5 the time of adventive shoot formation varied from 10 to 21
ppm BAP. Significant different results were evident between days. The use of NAA and BAP was of good advantages,
the treatment with 2.5 ppm BAP and that with 5 ppm of BAP where the combination of NAA and BAP determines the
(Table IV). number of shoots that can be produced, the yielded height of
shoots and the sulforaphane content. The best combination of
T ABLE IV NAA and BAP we observed to give the best induction on
BAP EFFECT ON FRESH SHOOT WEIGHT shoot formation was of 1 ppm NAA and 5 ppm BAP.
T reatment Average fresh weight (g/day) However, the BAP alone was seen to influence the fresh shoot
BAP 0 0.62a weight.
BAP 2.5 1.64b
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