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: [email protected]).
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 65 145,
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 6 5145, Indonesia.
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 demonstrat ed 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 µM NAA and 10 µM 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. MAT ERIALS AND MET 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 u sing 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
Wenny Tilaar, Sumeru Ashari, Bagyo Yanuwiadi, Jeany Polii-Mandang, Francien H. Tomasowa
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 ANALYSIS
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 1–2 mL methyl chloride and be mashed. The mashed broccoli shoot was then transferred onto flask and added with 25–50 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 trans ferred to a tube and put on heating block with temperature 70–80 °C until producing supernatant. The supernatant was added 5 ml of NaSO4 and heated again with 70–80 °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
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 7–10 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]. Pavlović 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.
Fig. 1. a). Enlargement of hypocotyls, b). T he meristematic cells were forming globulars, and c). T he shoots initiation.
TABLEI
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
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, Pavlović 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
0.107 μM NAA and 17.76 μM BAP which proved to be the best for broccoli regeneration. Whereas, Pavlović et al. [17] used broccoli hypocotyls in tissue culture with basal medium MS supplemented by the combination of 1 mg/L BA with 0, 0.1 and 0.2 mg/L IBA. The hypocotyls showed the best explants in almost all varieties tested with a minimum regeneration potential of 75 % and producing 3.5–7.4 shoots per explant. This implies that treatments with different concentrations of NAA and BAP yield different numbers of shoots.
TABLEII
NAA AND BAP INTERACTION EFFECTS ON NUMBER OF ADVENTIVE SHOOT
FORMATION
T reatment
Average number of shoot BAP
0 ppm
BAP 2.5 ppm
BAP 5.0 ppm
NAA (ppm) NAA0 7.93ab 17.00c 12.20bc
NAA1 5.07a 15.53c 27.93d
LSD 5% 6.05
C. Adventive Shoot Height
There was interaction of NAA and BAP on the height of adventive shoot. The combination of 0 ppm NAA and 0 ppm BAP yielded the highest adventive shoots (Table III, Fig. 3). No significant difference was shown among the combinations of 0 ppm NAA and 2.5 BAP; 0 ppm NAA and 5 ppm BAP; or 1 ppm NAA and 0 ppm BAP. Significant difference was shown among the combinations of 0 ppm NAA and 2.5 ppm BAP; 1 ppm of NAA and 2.5 ppm BAP as well as combination of 1 ppm NAA and 5 ppm BAP (Table III, Fig. 3). The combination of 0 ppm NAA and 0 ppm BAP yielded the highest shoots, the condition of which is expected to be due to the apical dominance phenomenon. Indoleasetic acid (IAA) is a factor of apical dominance and was formatted in the shoot plant tips. IAA inhibits the outgrowth of side shoots [20]. IAA in the tissue culture promotes not only cell 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.
TABLEIII
EFFECT OF NAA AND BAP INTERACTION ON ADVENTIVE SHOOT HEIGHT
T reatment
Average shoot height BAP
0 ppm
BAP 2.5 ppm
BAP 5.0 ppm
NAA (ppm) NAA0 6.50c 1.68a 2.00a
NAA1 1.71a 3.46b 4.22b
LSD 5% 1.37
D. Shoot Fresh Weight
There was no interaction of NAA and BAP and shoot fresh weight. But on single BAP, the treatment was significant. Significant difference was evident between the results of the treatment with 0 ppm BAP and those with 2.5 ppm BAP and 5 ppm BAP. Significant different results were evident between the treatment with 2.5 ppm BAP and that with 5 ppm of BAP (Table IV).
TABLEIV
BAPEFFECT ON FRESH SHOOT WEIGHT
T reatment Average fresh weight (g/day)
BAP 0 0.62a
BAP 2.5 1.64b
BAP 5 2.41c
LSD 5% 0.65
E. Sulforaphane Content
Interaction between NAA and BAP affected the sulforaphane content of broccoli shoots. There was significant difference was only shown by the combination of 1 ppm NAA and 5 ppm BAP to the other treatment (Table V). The highest sulforaphane content of broccoli shoots was detected at the combination of 1 ppm NAA and 5 ppm BAP with an average of 47.76 ng/mL. Whereas, the lowest was found on combination 1 ppm NAA and 2.5 ppm BAP with average 3.65 ng/mL. The combination NAA and kinetin on Solanum aviculare culture resulted in primary steroid such as cholesterol, stigmasterol, campesterol and B-sitosterol which are secondary metabolite [23]. Kinetin and BAP are group of cytokinin. Pandiangan et al. [24] reported that combination tryptophan 50 to 250 mg/L, NAA 2 ppm and kinetin 0.2 ppm on C. roseus culture can reassign to the catharanthine content. So, it can be concluded that the combination of NAA and BAP or that of NAA and kinetin could produce secondary metabolites such as cholesterol, stigmasterol, campesterol, B– sitosterol, catharanthine and sulforaphane. Nevertheless, only concentration NAA and BAP combination in equal situation can reassign to sulforaphane shoot of broccoli in tissue culture. In our research were the best resulted to concentration 1 ppm NAA and 5 ppm BAP combination. This is a same with response on number shoot and the second parameters very important in the research from shoot induction and synthesis from sulforaphane because the parameters used for multiplication of plant and to increase sulforaphane content of plant by tissue culture.
TABLEV
INTERACTION OF NAA AND BAP COMBINATION ON SULFORAPHANE CONTENT
IN ADVENTIVE SHOOT
T reatment
Average sulforaphane (ng/g fresh shoot weight) BAP 0 ppm BAP 2.5 ppm BAP 5.0 ppm
NAA (ppm) NAA0 9.83c 5.99ab 16.37d
NAA1 7.55bc 3.65a 47.76e
LSD 5% 3.23
V. CONCLUSION
Shoot induction from broccoli explant and synthesis of sulforaphane production had been carried out. We found that the time of adventive shoot formation varied from 10 to 21 days. The use of NAA and BAP was of good advantages, where the combination of NAA and BAP determines the number of shoots that can be produced, the yielded height of shoots and the sulforaphane content. The best combination of NAA and BAP we observed to give the best induction on shoot formation was of 1 ppm NAA and 5 ppm BAP. However, the BAP alone was seen to influence the fresh shoot weight.
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