Abstract: Background and Objective: The possibilities of using J. curcas seeds oil as bio-fuel in Nigeria requires large scale production of the plant which can be achieved through plant tissue culture technique. This study aimed to determine the effects of varying concentrations of silver nitrate (AgNO3) solution on the regeneration of J. curcas from cotyledonary petiole explants. Materials and Methods: The experimental layout followed completely randomized design with 20 replications for factors such as varying concentrations of silver nitrate, soaking period and orientation of the explants on MS-media. Data were analyzed using analysis of variance and means were separated using Duncan Multiple Range Test at p<0.05. Results: Application of AgNO3 solution at 10 mg L–1 promoted greater percentage shoot buds regeneration and number of shoot buds per explant. Twenty minutes process-time favoured higher shoot buds regeneration and placement of explants in slant position was found to be conducive orientation for regeneration of shoot buds. L-arginine between 7 and 10 mg L–1 promoted elongation of regenerated shoot buds. Rooting of the elongated shoot buds was faster in culture receiving 20 mg L–1 of indole-3-butyric acid (IBA). Regeneration of shoot buds regeneration was better in in vitro than in in vivo. It took 126-135 to achieve regeneration of plantlets. Conclusion: Recovery of plantlets was better when in vitro cotyledonary petiole explants were treated with 10 mg L–1 of AgNO3 solution, soaked for 20 min and placed in a slanted position on MS media and receiving further treatments of 7-10 mg L–1 of L-arginine and 20 mg L–1 of IBA.
INTRODUCTION
Jatropha curcas, is a large drought-resistant multipurpose shrub with several potentials. Special interest has been shown in the cultivation of J. curcas plantation because of its seed oil, which could be easily converted into quality bio-diesel1. More recently, the clear oil expressed from the seed has been suggested for energetic purposes as a substitute for diesel during2 the World War II. The usefulness of this plant most importantly its seed calls for the need to enter into massive production through the technique of tissue culture.
Plant tissue cultures are known to be used to create a quick and efficient way to regenerate many plants from a single piece of a “mother” plant. An explant is a piece of living tissue that is transferred to an artificial growth medium. The explant could come from numerous parts of the plant, including the leaf, stem, root, flower or seed3. There have been many studies using various tissue culture methods on different explants of J. curcas4-6. Workers found out good callusing on media with different combinations of benzene amino purine (BAP) and indolebutyric acid (IBA) (90%) from 3rd position leaves, leading to a protocol for successful indirect regeneration of J. curcas7. They also reported direct adventitious shoot regeneration of J. curcas and used hypocotyl, leaf and petiole explants. In 2005, auxiliary nodes were used for shoot bud proliferation in addition to leaf explants for adventitious shoot regeneration8. A successful protocol for somatic embryogenesis using seven leaves from seven months old J. curcas plants was reported9 in 2007. A year later, indirect regeneration protocol from J. curcas leaf explants was reported by Li et al.10. About the same time, 80% rooting using full strength MS medium with 0.5 mg L–1 IBA was recorded by a researcher and he developed a protocol for direct shoot regeneration from leaf explants10. Recently, direct regeneration from leaf, petiole and cotyledonary petiole explants has been recorded with established protocol11,12.
J. curcas, most importantly its seed has great economic, agricultural and nutritional potential13. In recent times, the plant has attracted attention of various research organizations, governments, public and international developmental agencies and industries in the tropics and subtropics due to its adaptability to semi-arid marginal sites, the possibility of using its oil as a diesel fuel substitute and its role in erosion control13. Massive production of this plant that will promote its plantation within short period of time via the technique of tissue culture has not been given serious consideration most importantly in Nigeria which the current study aimed to address.
MATERIALS AND METHODS
Study site and sample collection: The research was carried out at the National Centre for Genetic Resource and Biotechnology (NACGRAB), Ibadan, Oyo state, Nigeria between October, 2017 and March, 2018. The materials used were supplied by the Tissue Culture Unit of the centre. The reagents used were of analytical grade. Mature seeds of J. curcas were collected from Faculty of Agricultural Unit of University of Ilorin, Ilorin, Nigeria.
Sterilization of glass wares: Glass wares after being thoroughly washed using liquid soap and hypochlorite for an hour were subjected to both dry and wet sterilization in an oven an autoclave, respectively.
Preparation of Murashige and Skoog media (MS): Prepared MS media contained all the growth adjuvants such as macro and micro nutrient, carbon source and vitamins. The pH of the MS medium was adjusted to 5.8 with 0.1 N NaOH and 0.1 N HCl before adding gelling agent of 2 g phytagel (Sigma). The media was then covered with a foil paper and heated until a clear solution was seen. The molten media was distributed into culture bottles and subsequently autoclaved before inoculation.
Disinfection of seeds: Shelled and unshelled seeds of the plant (Fig. 1) were surface sterilized with liquid soap and properly rinsed with several distilled water. It should be noted that disinfections of the seeds were done using absolute sodium hypochlorite before inoculation.
| Fig. 1(a-b): | Jatropha curcas seeds (a) With shells and (b) Without shells |
Inoculation of growth media with J. curcas seeds: This was done in laminar flow hood using atomizer to make the chamber sterile. Forceps and blade holder were again sterilized under the spirit lamp to begin the inoculation process. Shelled seeds and unshelled seeds were inoculated on the growth media and germinations were monitored to generate in vitro cotyledonary petiole explants. While J. curcas seeds were planted in potted soil in screen house to generate in vivo cotyledonary petioles explants.
Regeneration of shoot buds from cotyledonary petiole as influenced by silver nitrate: Twenty-day old cotyledonary petiole explants each of 0.5 cm length grown from in vivo and in vitro propagation were treated with different concentrations of AgNO3 solution (0, 10, 20, 30, 40 mg L–1). The explants were soaked at different time periods (0, 10, 20, 30 and 40 min) in each of the concentrations of AgNO3 solution. The cotyledonary petioles explants generated from in vivo and in vitro propagation were placed in different orientations: horizontal, vertical and slant on hormone-free MS medium for regeneration of explant. The percentage induction of shoot buds and the number of shoot buds per explants was determined after 30 days of culture.
Shoot buds elongation culture: Regenerated shoot buds along with the mother tissues was moved to Jatropha shoot elongation media (JSR) after the formation of calli. JRS contains full strength MS and some other growth adjuvants. Various concentration of L-arginine (0, 5, 7, 10 and 20 mg L–1) was supplemented with the elongation medium and the length of the elongated buds recorded after 15 days of culture.
Rooting culture: Shoots of 10 mm in length was isolated from the mother tissue and transferred to half-strength MS medium containing indole-3-butyric acid (IBA, Sigma-Aldrich Co., St Louis, MO, USA of different concentrations ( 0,0.5, 0.1, 1.5 and 3.0 mg L–1) and number roots recorded after 40 days in culture media.
Acclimatization and transplantation of the regenerated plantlets: The regenerated plantlets were taken from culture bottles, washed properly and gently for purpose of removing residuals of medium and thereafter transplanted to a tray containing sterile polythene bags filled with sterile soil. The tray was covered with a transparent plastic sheet for 2 weeks. The established plants were then transferred to a screen house for acclimatization and further growth.
Statistical analysis: All the experiments were set up in a completely randomized factorial design with 20 replicates per treatment. Data were analyzed used Statistical Package for Social Science (SPSS) 20.0 software and means were separated by Duncan’s multiple range test14 at p<0.05. Figures were used where appropriate.
RESULTS
In this study, the overall Univariate analysis variance of in vivo and in vitro cotyledonary petiole explants that were treated with different concentration of silver nitrate (AgNO3) soaked at different time and placed in MS medium at different orientation on induction of shoot bud is presented in Table 1. All the factors considered were found to be significant p<0.05. The interaction effects were also significant. The significant interactions of all the factors required that the main effect had to be down played and that the induction of the shoot buds depends largely on the factors that were considered (Table 1).
Silver nitrate effect on regeneration percentage and number of shoot buds per explant of J. curcas: Table 2 shows the effect of different concentration of AgNO3 on the regeneration percentage and number of shoot buds per explant. In both in vitro and in vivo as sources of cotyledonary petiole explants, varying concentration AgNO3 showed significantly higher shoot buds regeneration compared with control (Table 2). In in vitro, the concentration of AgNO3 at 10 mg L–1 was found to be most effective with highest percentage of shoot buds induction (76.80%) and followed by 20 (39.40%) and 30 (28.81%) and 40 mg L–1 (22.35%). Significantly lowest shoot buds regeneration was recorded from the control with percentage value of 4.00 % (Table 2). Whereas in vivo, significantly higher percentage shoot buds regeneration was recorded for 20 mg L–1 when compared to other cotyledonary petiole explants. Similar trends of results were recorded for regeneration percentage (Table 2). It should be noted that in both in vitro and in vivo, higher concentration (30-40 mg L–1) of AgNO3 reduced the number of shoot buds regenerated compared to lower concentrations (10-20 mg L–1) (Table 2). Similarly, shoot bud regeneration was greater in vitro than in vivo cotyledonary petiole explants (Table 2).
Effect of duration on the regeneration percentage and number of shoot buds per explants of J. curcas: The results of in vitro cotyledonary petiole explants showed that regeneration of shoot buds and number of buds per explants was significantly influenced by process-time (Table 3).
| Table 1: | Univariate factorial results of the regenerated shoot buds |
| vs: Interaction | |
| Table 2: | Silver nitrate effects on regeneration (%) and number of shoot buds/explants of J. curcas after 30 days |
Data in the same column followed by different letters are significantly different at p<0.05 | |
| Table 3: | Effect of duration on the regeneration (%) and number of shoot buds/explant of J. curcas after 30 days |
Data in the same column followed by different letters are significantly different at p<0.05 |
|
Twenty minutes process-time resulted in greater percentage of shoot buds regeneration (75.13%) and number of shoot buds per explant than other periods. (Table 3). Significantly lowest percentage regeneration and number of shoot buds/explants were recorded from the control (Table 3). The same trend of results was recorded for in vivo cotyledonary petiole explants. Like the effect of concentrations, the means shoot buds regeneration as well as number of shoot buds per explant for process-time was higher in in vitro than in vivo (Table 3).
| Table 4: | Effect of orientation on the regeneration (%) and number of shoot buds/explants of J. curcas after 30 days |
Data in the same column followed by different letters are significantly different at p<0.05 | |
| Table 5: | Effect of L-arginine on the elongation of the regenerated shoot buds from in vitro explants of J. curcas after 15 days |
Data in the same column followed by different letters are significantly different at p<0.05 | |
Effect of orientation on the regeneration percentage and number of shoot buds per explants of J. curcas: Considering the effect of orientation that is the horizontal, slant and vertical placement of explants on the MS medium, significant differences were recorded in both in vitro and in vivo regeneration of cotyledonary petiole explants. (Table 4). In vitro, slant placement showed significantly highest regeneration of shoot buds of 79.10±0.37% and number of shoot buds per explant (10.66±1.56) and followed in decreasing order of magnitude by those of horizontal and vertical placement (Table 4). Whereas, in vivo source of explants, horizontal placement markedly favored higher shoot buds regeneration percentage and number of buds per explant (Table 4). This was closely followed by slant. Significantly lowest shoot buds regeneration percentage and number of buds per explant was recorded from vertical orientation of cotyledonary petiole explants (Table 4).
In Fig. 2a, the in vitro explants treated with 10 mg L–1 of silver nitrate, soaked for 20 min and placed in slant position showed robust shoot buds regeneration when compared to those of in vivo explants receiving 20 mg L–1 of silver nitrate, 20 min soaking period with horizontal placement (Fig. 2b).
Elongation of the regenerated shoot buds from in vitro explant of J. curcas as affected by L-arginine: The elongation of regenerated shoot buds as influenced by varying concentration of L-arginine is shown in Table 5.
| Fig. 2(a-b): | Direct induction of adventitious buds from cotyledonary petiole explants of J. curcas, (a) Treatment of in vitro (with slant orientation) and (b) In vivo (horizontal orientation) petiole explants for 20 min duration AgNO3 treatment |
The most suitable concentration for promoting the elongation of shoot was between 7 and 10 mg L–1. The effect of these concentrations was statistically similar. It was however, significantly higher than those recorded for 5 and 20 mg L–1 and the control. Shoot buds receiving the most effective treatments showed good vigor and healthy shoot buds (Table 5) after 15 days of culture. When L-arginine was used at a higher concentration (20 mg L–1) and concentration as low as 5 mg L–1 the elongation of shoot buds was markedly inhibited (Table 5).
Effect of indole-butyric acid (IBA) on the rooting of regenerated shoots from explant of J. Curcas: The results obtained at day 40 after treatment of elongated J. curcas with indole-butyric acid (IBA) at varying concentration for root initiation is presented in Table 6.
| Fig. 3: | Acclimatization of regenerated plantlets |
| Table 6: | Effects of IBA on the rooting of regenerated shoots from explant of J. Curcas |
Data in the same column followed by different letters are significantly different at p<0.05 | |
It tooks a longer time for rooting to be initiated. However, number of roots per shoot increased with increase in concentration of IBA. It showed that the response of the shoot to this hormone was dose dependent (Table 6). Root initiation was not possible when no IBA was used (control) as well as for those receiving 5 mg L–1 of IBA (Table 6). The resulting plantlets were transferred to the screen house for acclimatization as shown in four weeks after rooting (Fig. 3) It should be noted that in this study, it tooks 126-135 days to obtain regenerated plantlets.
DISCUSSION
J. curcas is a multipurpose, drought resistant, perennial plant. The plant is currently gaining a lot of attention being a good source of bio-fuel. In this case, attention should therefore be shifted to develop a relatively efficient protocol for regeneration of plantlets with minimal contamination via the technique of plant tissue culture. In this study, the effect of varying concentration of silver nitrate were tried on the regeneration of J. curcas plantlets and the results showed that several factors such concentration of pre-treated cotyledonary petiole explants with silver nitrate, duration of process-time, orientation of the explant on the MS media and source of explants most importantly in vitro propagation should be taken into consideration in order to regenerate shoot buds development. It had been earlier reported15 that factors such as growth regulators, orientation of the explant on the media have great effect on the response of the explants. It is worth of mentioning that, application of silver nitrate solution at 10 mg L–1 is considered optimum for promoting the regeneration of shoot buds in those cotyledonary petiole explants derived from in vitro source. The results were not in line with the findings of Liu et al.16 where growth media supplemented with 20 mg L–1 of Thidiazuron (TDZ) significantly increased regeneration percentage and number of shoot buds per plant when in vitro cotyledonary petiole explants were used. The variation could be ascribed to growth adjuvant used. Also, the present investigation differed slightly as to the number of days used to regenerate the plantlets which took the authors 105 days as against the 135 days recorded in this study. Other workers have reported7,18,15. better regeneration of adventitious shoot in in vitro multiplication of Jatropha curcas.
With respect to process-time, cotyledonary petioles explant soaked for 20 min in silver nitrate solution gave the best result in terms regeneration of shoot buds and number of shoot buds/explant. All other durations markedly inhibited shoot bud regeneration but were better than the control. The present results indicated that silver nitrate as ethylene modulator is only required for short period of time during induction of shoot buds formation. The results are closely consistent with previous studies in J. curcas and glycine max16,17.
In all the orientations investigated, the present study revealed that slant placement of the explants on the media was confirmed to be much more conducive orientation for regeneration of shoot buds. The percentage of induction of shoot buds and the number of induced shoot buds recorded in this study differed significantly under different orientations and the source of explants and this was at variance with observation reported in earlier researches by Kondamudi et al.15 and Liu et al.16. Available information have established that poor quality of the regenerated shoot buds has been a major problem that hinders further development of the regenerated buds in J. curcas15. In the present study, the problem was partially taken care of by using low concentration of silver nitrate and short treatment duration. Visual observation showed that there was an improvement of the quality of the regenerated shoot buds and shoot bud elongation was further enhanced when the explants were exposed to 5-7.5 mg L–1 of L-arginine L-arginine an amino acid that was scarcely used in plant tissue cultures and it has been reported to have beneficial effects in tissue culture J. curcas and apple18,19. In this study, concentration as low as 5 mg L–1 and as high as 7.5 mg L–1 are considered optimum for the elongation of shoot buds. Like GA3, L-arginine may have positive effect on facilitating the elongation of regenerated buds6,18.
It has been established that for the purpose of obtaining intact plants, auxin, is frequently supplemented into the rooting medium19. The rooting of shoot could indeed begin starting from 20 days of culture and because of slow root development, there was need to extend the time to 40 days for purpose of obtaining satisfying rooting efficiency. The result is suggestive of the fact that 40 days was suitable for rooting. Optimum concentration to achieve better root development was the highest concentration of the IBA (20 mg L–1). The present study slightly agreed to the report of Liu et al.16, who reported 30 days for rooting efficiency in J. curcas treated with TDZ. The variation in number of days for rooting efficiency could be attributed to the growth adjuvant used. This implies that the response of cotyledonary petiole explants of J. curcas will vary with different growth adjuvant as pre-treatment chemicals. Finally, regenerated plantlets were successfully acclimatized in potted soil in the screen house and no abnormal development were observed up to this point.
CONCLUSION
This study has shown that successful regeneration of shoot buds was best achieved if in vitro cotyledonary petiole explants that were treated with 10 mg L–1 silver nitrate solution, soaked for 20 min and placed in slant position. Further still, shoot bud elongation and rooting for regeneration of plantlets were favored upon applications of L-arginine and IBA at 7-10 mg L–1 and 20 mg L–1 respectively.
SIGNIFICANCE STATEMENT
This study discovers that using silver nitrate at 10 mg L–1 as growth adjuvant is beneficial for large production of J. curcas plantlets most importantly when the cotyledonary explants were soaked for 20 min and placed in slant orientation on MS-media. This study will help researcher to uncover the critical role of silver nitrate when used optimally in growth modulation of J. curcas that many other researchers were not able to explore. Thus, a new theory on this compound when utilized appropriately or in combination with other concentrations of compounds such as L-arginine and IBA may be arrived at.