Effects of different types of phytophormones on organogenesis of Jatropha curcas.
Jatropha curcas is a well known oil- bearing shrub that belongs to Euphorbiaceae family. It is originated from South America and has been cultivated in many countries including Malaysia. The plant is able to grow on poor- nutrient lands . Traditionally, J. curcas is grown for various purposes such as making soaps, fencing live animals and reclaiming wasteland . It is also recommended as a potential plant for soil erosion control in many countries . In Malaysia, it is conventionally grown as living hedge and therefore it is known as jarak pagar in Malay.
Biodiesel is a fast developing alternative fuel in many countries in the world and its global production is set to reach 24 billion litres by 2017 . Oil production from non-edible crops such as J. curcas has gained interest from tropical and sub-tropical countries. In addition, biodiesel is a renewable energy with reduced environmental problems and J. curcas is an attractive potential biofuel feed stock . In recent years, J. curcas is cultivated in many countries mainly due to its high oil content seeds. Jatropha seed oil is suitable for the production of low--cost biofuel [2,20]. Furthermore, Jatropha seed oil is non--edible as compared to other biofuel production plants such as oil palm and rapeseed. Thus, competition to be used as food source could be avoided . Conventional propagation through seeds and cuttings are insufficient to meet the large demand from the market. Seed propagation is often influenced by sowing time and soil depth that affect the germination rate of the seeds. While propagating by cuttings faces problems such as shallow root of plants . The plants are also susceptible to pest infection which causes flower falling, fruit abortion and malformation of seeds . Hence, plant tissue culture technique could be a good alternative for mass propagation and improvement of J. curcas" s plant quality via genetic manipulation.
Somatic organogenesis and embryogenesis are two main routes used for plant regeneration in plant tissue culture. Different plant species requires different in vitro cultural conditions to initiate and develop organogenesis and embryogenesis. Besides genotype, the type of explants and phytohormones used affect the occurrence of these processes in plant tissue culture. The addition of additives such as adenine sulphate (ADS) and activated charcoal into the medium is also known to improve morphogenesis. Many studies found that ADS is able to enhance shoot regeneration while activated charcoal is used to promote rooting in some plant species. As for J. curcas, plant regeneration via organogenesis and embryogenesis were reported before using different types of explants such as cotyledons, shoot apices, leaf-disc, shoot tips, axillary nodes and hypocotyls [35,29,36,6,27,37]. Many of the reports used the in vitro germinated seedlings to obtain the explants in order to achieve plant regeneration in their study.
This study was conducted to investigate the feasibility of using the leaf and petiole explants from the field plants for direct organogenesis of plant tissues. It is a big challenge to use the field obtained explants as there is a high risk of contamination and the physiological conditions of the explants used can be varied greatly. It is an important milestone achieved using these explants J. curcas to establish the plant regeneration system as leaf and petiole explants are easily available for mass propagation of elite plant. Besides, the important role of ADS in shoot formation was observed in organogenesis of leaf and petiole explants was highlighted in this study. The plant efficient regeneration system is vital for performing plant transformation studies of J. curcas using leaf, petiotle or the induced shoot buds as the target tissues.
MATERIALS AND METHODS
Plant materials and sterilization:
Petiole and leaf blade explants obtained from the field were surface sterilized using 5 % (v/v) Clorox[R] for 5 min before the explants were rinsed using distilled-water for another 5 min. The above procedures were then repeated using 2.5 % (v/v) Clorox[R]. Sterilized leaf blade and petiole explants were ready to be used for the subsequent study after rinsing with distilled-water to remove the residue of Clorox[R].
Medium and culture conditions:
Murashige and Skoog (MS) medium  was used in this study. MS medium consists of macro and micro salts, vitamins, [Na.sub.2]FeEDTA and 3 % (w/v) sucrose. Approximately 0.28 % (w/v) Gelrite was added into the medium. The pH of the medium was adjusted to 5.8 prior to autoclaving at 121[degrees]C and 15 psi for 15 min. All cultures were kept at 25 [+ or -] 1[degrees]C with 16 h photoperiod (1000 lux) and 8 h darkness.
Shoot bud induction and elongation:
The sterilized leaf blade and petiole explants were used to induce shoot buds on three different types of shoot-bud induction media. The media investigated were the MS medium supplemented with single cytokinin at various concentrations, 5, 10, 15 and 20 [micro]M, MS medium supplemented with single cytokinin (5, 10, 15 and 20 [micro]M) in combination with 0.5 [micro]M 3-indolebutyric acid (IBA) and MS medium supplemented with single cytokinin (5, 10, 15 and 20 [micro]M) in combination with 0.5 [micro]M IBA and 50 [micro]M adenine sulphate (ADS). Cytokinins used in this study were 6-benzylaminopurine (BAP), kinetin, thidiazuron (TDZ) and zeatin. The medium without phytohormone was used as the control in this study. Triplicate with ten explants for each replicate were performed and the study was repeated twice.
Rooting of the regenerated shoot buds:
The elongated shoots ([greater than or equal to] 1 cm) were excised and transferred to the half- strength MS medium containing 1 naphthylacetic acid (NAA) to study the rooting. Three different concentrations (0.5, 1 and 2 [micro]M) were investigated. The effect of activated charcoal was also investigated by addition of 1 % (w/v) activated charcoal into the media. Triplicate with each replicate consisted of three excised shoots were used and the study was repeated twice.
Data recorded were analyzed using one-way ANOVA in SPSS software (SPSS Inc. USA). Significant differences between groups were compared using Tukey's HSD test at significance level of 0.05.
Shoot bud induction and elongation:
Three different types of shoot- bud induction media were tested. They were MS medium supplemented with single cytokinin, cytokinin in combination with IBA and cytokinin in combination with IBA and ADS. For all the media supplemented with cytokinin only, callus formation was observed for both the leaf blade and petiole explants except in the control medium. The compact and green callus was observed after two weeks for both the leaf blade and petiole explants. However, shoot bud induction was observed only in the medium supplemented with TDZ after four weeks (Figure 1). The highest shoot bud induction for leaf blade (25.9%) and petiole explants (22.2%) was observed on the MS medium supplemented with 15 [micro]M TDZ (Table 1). Similarly, callus formation was also obtained on the media supplemented with cytokinin and IBA and shoot buds were only observed in the MS medium containing TDZ and IBA for both leaf blade and petiole explants. The highest shoot bud induction from the leaf explants was 33.3% which was obtained on the MS medium containing 15 [micro]M and 20 [micro]M TDZ in combination with IBA (Table 2); while for petiole explants, the highest shoot bud induction, 25.9%, was obtained on the MS medium containing 15 [micro]M TDZ in combination with IBA (Table 2).
As for the media supplemented with a single cytokinin in combination with 0.5 [micro]M IBA and 50 [micro]M ADS, high level of callus formation was again observed for both the leaf blade and petiole explants and shoot bud formation was observed only in the medium containing BAP and TDZ. The highest bud induction, 66.7% and 59.3% for leaf blade and petiole explants, respectively, were obtained on the medium containing 15 [micro]M TDZ and 0.5 [micro]M IBA and 50 [micro]M ADS (Table 3). The results obtained showed that the MS medium supplemented with TDZ in combination with 0.5 [micro]M IBA and 50 [micro]M ADS was more suitable than the MS medium supplemented with TDZ and the MS medium supplemented with TDZ in combination with 0.5 [micro]M IBA for shoot bud induction. Higher frequency of shoot bud induction was obtained from the medium containing 15 [micro]M TDZ regardless of whether TDZ was used in combination with IBA and ADS for both leaf blade and petiole explants. Addition of ADS in the medium obviously promoted the bud induction. Thus, the induced shoot buds were proliferated and multiplied in this medium.
Rooting of the regenerated shoot buds:
Different rooting media were investigated to induce roots from the excised shoots. For all the rooting media studied, callus formation was observed at the basal region of the shoots after one week and the roots were formed at week two (Figure 1E and F). The highest root formation, 55.6 %, was obtained on the half-strength MS medium supplemented with 0.5 [micro]M NAA containing 1 % (w/v) activated charcoal (Table 4). The results showed that higher concentration of NAA would promote callus formation at the basal area of the shoot rather than root formation. From the results obtained, enhancement of root formation was observed on the medium containing both NAA and activated charcoal if compared with the medium supplemented with NAA only. Addition of NAA into the medium was required for root formation in this study.
Plant regeneration via shoot organogenesis had been reported in many plant species. Shoot organogenesis is greatly associated with the explant source and phytohormones used . In most of the studies, phytohormones, particularly cytokinins, are used singly or in combination to induce shoot from various types of explants . Similarly, the media containing various phytohormone combinations were used for induction of shoot organogenesis for J. curcas [35,36,6,23,27].
In this study, shoot induction was investigated using the leaf blade and petiole explants cultured on media containing different combinations of phytohormones. This study showed that shoot bud induction was mainly influenced by the type of cytokinin supplemented in the induction medium. No shoot bud was induced from both the leaf blade and petiole explants cultured, except those cultured in the medium containing TDZ. Cytokinins play an important role in promoting shoot regeneration through either organogenesis or embryogenesis [10,21]. The effectiveness of cytokinins in shoot regeneration via organogenesis has been reported and TDZ is usually used for inducing shoot organogenesis primarily for woody plants . TDZ was also used for shoot induction from shoot and leaf explants of Ochna integerrima , leaf explants of Cichorium intybus L.  and Paulownia tomentosa . Thus, TDZ might be more effective to induce shoot bud formation from the non--meristematic tissues such as leaf and petiole explants for J. curcas. The choice and effectiveness of cytokinin used in promoting the plant regeneration is also explants dependent. For example, Purkayastha et al.  used BAP to induce shoot from shoot apices of J. curcas while TDZ showed enhancing shoot bud formation effect for the leaf and petiole explants in this study. Similarly Kumar et al.  reported TDZ was most efficient in inducing shoot buds from petiole explants of J. curcas. However, in another study using young cotyledons of J. curcas, Khemkladngoen et al.  found that the MS medium supplemented with BAP and IBA was more effective for plant regeneration. Kumar and Reddy  concluded in their study that the genotypes and explants sources influenced plant regeneration of J. curcas.
This study also showed the importance of auxin in combination with cytokinin in inducing shoot buds from leaf and petiole explants. Roy and Banerjee  and Yancheva et al.  also reported the combining effects of auxin with cytokinin had exerted greater impacts than single cytokinin on induction of shoot organogenesis. In this study, TDZ in combination with IBA was found to improve the shoot induction from leaf and petiole explants. This combination (TDZ and IBA) had been reported to exert the same effects on shoot induction from leaf explants of Fragaria species  and Carthamus tinctorius L. . In studying the level of gene expression, Torelli et al.  reported a sharp increase of LESK1 expression leading to a strong increase in shoot induction when cytokinin and auxin were used in combinations. Similar to the results were obtained from a recent study using leaf explants of J. curcas . They also reported ten times higher concentration of copper sulphate than that of in MS medium was able to enhance the shoot bud induction.
On the other hand, addition of ADS in combination with TDZ and IBA was found to further enhance the shoot induction from both the leaf and petiole explants in our study. Similar observations had been reported in which shoot organogenesis was improved by adding ADS into shoot induction media for many plant species such as Azadirachta indica  and Melia azedarach L. . Siwach and Gill  reported the addition of ADS into the medium enhanced the shoot regeneration of Ficus religiosa originated from the nodal explants. In a plant regeneration study of Plumbago rosea, high concentration of ADS (370 [micro]M) was also recommended for the shoot induction . Datta et al.  also reported that higher concentration of ADS (55 [micro]M) in combination with cytokinin (BAP, kinetin, TDZ or 2ip) was effective to induce shoot multiplication of nodal explants. Similar observation was obtained in our study that the use of ADS in combination with TDZ could further promote shoot induction from leaf and petiole explants of J. curcas. The enhancement on shoot proliferation by ADS was also recorded in shoot induction study for Sida cordifolia L .
For rooting study, the induced shoots were cultured on the phytohormone--free MS medium as reported by Sujatha and Mukta . However, contradict result was obtained in this study as no root was induced in the Phytohormone--free medium except on the medium containing NAA. Interestingly, Kumar et al.  induced rooting of J. curcas elongated shoot using a complex medium as compared to this study. They used half-strength MS medium supplemented with IBA, NAA, IAA and activated charcoal with 2% sucrose. This could be due to different genotype. NAA had been reported to be effective on root induction  other than IAA and IBA [4,22]. Similar to this study, half- strength MS medium was also applied to induce root from regenerated J. curcas shoots by Purkayastha et al.  and Khurana-Kaul et al. . The high concentration of salts present in full-strength MS medium might affect the rooting. Besides, the addition of charcoal to the root induction medium improved the root formation from the shoots which was also observed by Kumar et al. . The addition of charcoal might absorb inhibitors or toxic compounds that affect rooting besides providing the dark condition to promote the root formation .
[FIGURE 1 OMITTED]
In brief, this study reported a plant regeneration system of J. curcas via organogenesis. For shoot organogenesis, the medium contaning TDZ, IBA and ADS greatly improved shoot--bud induction and proliferation of shoots while the medium containing NAA and activated charcoal was suitable for rooting. The established plant regeneration system is suitable for both micropropagation and genetic manipulation studies.
Authors are grateful to Universiti Tunku Abdul Rahman (UTAR) for the research fund.
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(1) Siow Then Soong, (2) Tee Chong Siang and (2) Adeline Ting Su Yien
(1) Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Perak, Malaysia.
(2) School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, 46150, Selangor Darul Ehsan, Malaysia. Address For Correspondence:
Tee Chong Siang, Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Perak, Malaysia.
Phone: +605-4688888, Fax: +605-4661676; E-mail: email@example.com
This work is licensed under the Creative Commons Attribution International License (CC BY).
Received 22 March 2016; Accepted 28 May 2016; Available online 12 June 2016
Table 1: Effects of different cytokinins on shoot induction using leaf blade and petiole explants. Cytokinin Leaf blade explants ([micro]M) Callus Shoot bud formation (%) (1) induction (%) (1) BAP 0 0.0 (c) 0.0 (b) 5 40.7 (ab) 0.0 (b) 10 48.2 (ab) 0.0 (b) 15 55.6 (a) 0.0 (b) 20 48.2 (ab) 0.0 (b) kinetin 0 0.0 (c) 0.0 (b) 5 18.5 (b) 0.0 (b) 10 25.9 (b) 0.0 (b) 15 29.6 (b) 0.0 (b) 20 25.9 (b) 0.0 (b) TDZ 0 0.0 (c) 0.0 (b) 5 70.4 (a) 7.4 (ab) 10 63.0 (a) 22.2 (a) 15 51.9 (a) 25.9 (a) 20 40.7 (ab) 25.9 (a) 0 0.0 (c) 0.0 (b) 5 55.6 (a) 0.0 (b) zeatin 10 66.7 (a) 0.0 (b) 15 74.1 (a) 0.0 (b) 20 77.8 (a) Cytokinin Petiole explants ([micro]M) Callus Shoot bud formation (%) (1) induction (%) (1) BAP 0 0.0 (c) 0.0 (b) 5 63.0 (b) 0.0 (b) 10 59.3 (b) 0.0 (b) 15 70.4 (b) 0.0 (b) 20 77.8 (b) 0.0 (b) kinetin 0 0.0 (c) 0.0 (b) 5 55.6 (b) 0.0 (b) 10 63.0 (b) 0.0 (b) 15 74.1 (b) 0.0 (b) 20 66.7 (b) 0.0 (b) TDZ 0 0.0 (c) 0.0 (b) 5 81.5 (a) 7.4 (ab) 10 88.9 (a) 22.2 (a) 15 88.9 (a) 22.2 (a) 20 96.3 (a) 18.5 (a) 0 0.0 (c) 0.0 (b) 5 70.4 (b) 0.0 (b) zeatin 10 70.4 (b) 0.0 (b) 15 88.9 (a) 0.0 (b) 20 92.6 (a) (1) Mean values ([+ or -] SE) in each column of the same letter are not significantly different according to Tukey's HSD test at P = 0.05. Table 2: Effects of combining single cytokinin with 0.5 [micro]M IBA on shoot induction using leaf blade and petiole explants. Leaf blade explants Cytokinin Callus Shoot bud ([micro]M1) formation (%) (2) induction (%) (2) BAP 0 0.0 (c) 0.0 (b) 5 74.1 (ab) 0.0 (b) 10 77.8 (ab) 0.0 (b) 15 81.5 (a) 0.0 (b) 20 88.9 (a) 0.0 (b) kinetin 0 0.0 (c) 0.0 (b) 5 48.2 (b) 0.0 (b) 10 59.3 (b) 0.0 (b) 15 70.4 (b) 0.0 (b) 20 55.6 (b) 0.0 (b) TDZ 0 0.0 (c) 0.0 (b) 5 92.6 (a) 18.5 (a) 10 100.0 (a) 33.3 (a) 15 100.0 (a) 33.3 (a) 20 100.0 (a) 25.9 (a) zeatin 0 0.0 (c) 0.0 (b) 5 81.5 (a) 0.0 (b) 10 88.9 (a) 0.0 (b) 15 92.6 (a) 0.0 (b) 20 100.0 (a) 0.0 (b) Petiole explants Cytokinin Callus Shoot bud ([micro]M1) formation (%) (2) induction (%) (2) BAP 0 0.0 (c) 0.0 (b) 5 59.3 (ab) 0.0 (b) 10 70.4 (ab) 0.0 (b) 15 74.1 (ab) 0.0 (b) 20 74.1 (ab) 0.0 (b) kinetin 0 0.0 (c) 0.0 (b) 5 29.6 (b) 0.0 (b) 10 48.2 (b) 0.0 (b) 15 37.0 (b) 20 33.3 (b) 0.0 (b) TDZ 0 0.0 (c) 0.0 (b) 5 81.5 (a) 7.4 (ab) 10 92.6 (a) 18.5 (a) 15 96.3 (a) 25.9 (a) 20 100.0 (a) 22.2 [+ or -] 11.1 (a) zeatin 0 0.0 (c) 0.0 (b) 5 81.5 (a) 0.0 (b) 10 85.2 (a) 0.0 (b) 15 92.6 (a) 0.0 (b) 20 100.0 (a) 0.0 (b) (1) A cytokinin (15 gM) in combination with 0.5 gM IBA. (2) Mean values ([+ or -] SE) in each column of the same letter are not significantly different according to Tukey's HSD test at P = 0.05. Table 3: Effects of combinations of single cytokinin with IBA and ADS on shoot induction for leaf blade and petiole explants. Leaf blade explants Cytokinin Callus Shoot bud ([micro]M) (1) formation (%) (2) induction (%) (2) BAP 0 0.0 (b) 0.0 (b) 5 96.3 (a) 14.8 (b) 10 96.3 (a) 11.1 (b) 15 96.3 (a) 0.0 (b) 20 100.0 (a) 0.0 (b) kinetin 0 0.0 (b) 0.0 (b) 5 77.8 (a) 0.0 (b) 10 85.2 (a) 0.0 (b) 15 96.3 (a) 0.0 (b) 20 96.3 (a) 0.0 (b) TDZ 0 0.0 (b) 0.0 (b) 5 100.0 (a) 44.4 (a) 10 100.0 (a) 48.1 (a) 15 100.0 (a) 66.7 (a) 20 100.0 (a) 51.9 (a) zeatin 0 0.0 (b) 0.0 (b) 5 100.0 (a) 0.0 (b) 10 100.0 (a) 0.0 (b) 15 100.0 (a) 0.0 (b) 20 100.0 (a) 0.0 (b) Petiole explants Cytokinin Callus Shoot bud ([micro]M) (1) formation (%) (2) induction (%) (2) BAP 0 0.0 (c) 0.0 (b) 5 92.6 (a) 0.0 (b) 10 92.6 (a) 0.0 (b) 15 100.0 (a) 0.0 (b) 20 100.0 (a) 0.0 (b) kinetin 0 0.0 (c) 0.0 (b) 5 85.2 (a) 0.0 (b) 10 85.2 (a) 0.0 (b) 15 88.9 (a) 0.0 (b) 20 92.6 (a) 0.0 (b) TDZ 0 0.0 (c) 0.0 (b) 5 96.3 (a) 29.6 (a) 10 96.3 (a) 40.7 (a) 15 96.3 (a) 59.3 (a) 20 100.0 (a) 48.1 (a) zeatin 0 0.0 (c) 0.0 (b) 5 100.0 (a) 0.0 (b) 10 100.0 (a) 0.0 (b) 15 100.0 (a) 0.0 (b) 20 100.0 (a) 0.0 (b) (1) A cytokinin (15 [micro]M) in combination with 0.5 [micro]M IBA and 50 [micro]M ADS. (2) Mean values ([+ or -] SE) in each column of the same letter are not significantly different according to Tukey's HSD test at P = 0.05. Table 4: Effects of different concentrations of NAA and 1%(w/v) charcoal on root induction after two weeks. Root formation (%) (1) NAA With charcoal Without charcoal ([micro]M) 0 0.5 55.6 (a) 11.1 (b) 1 44.4 (a) 33.3 (ab) 2 33.3 (ab) 22.2 (ab) (1) Mean values with the same letter are not significantly different according to Tukey's HSD test at P = 0.05.
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|Author:||Soong, Siow Then; Siang, Tee Chong; Yien, Adeline Ting Su|
|Publication:||Advances in Environmental Biology|
|Date:||May 1, 2016|
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