# Enhancing Jatropha curcas Micropropagation through Optimized Hypochlorite Concentrations: A Comparative Evaluation of Various Explant Types

I.S Jokotola, B Iyekolo, A Omaka, S Adeboye,, A. Bello

► To cite this version:

I.S Jokotola, B Iyekolo, A Omaka, S Adeboye,, A. Bello. Enhancing Jatropha curcas Micropropagation through Optimized Hypochlorite Concentrations: A Comparative Evaluation of Various Explant Types. Nigerian Journal of Biotechnology, 2025, 42(2), 10.5281/zenodo.17216941. [https://doi.org/10.52902/2025-0241.2025.42.2]

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# Enhancing *Jatropha curcas* Micropropagation through Optimized Hypochlorite Concentrations: A Comparative Evaluation of Various Explant Types

I. S. Jokotola, B. Iyekolo, A. Omaka, S. Adeboye, and A. Bello

The Laboratory of Plant Improvement Unit, Agricultural Biotechnology Department, National Biotechnology Development Agency (NABDA), Lugbe, Abuja, Nigeria.

## Abstract

The micropropagation of *Jatropha curcas*, a multipurpose, drought-resistant perennial plant from the Euphorbiaceous family was investigated to optimize tissue culture techniques for enhancing crop yield and ensuring disease-free plant production between March 2023 and December 2023 at the Laboratory of Plant Improvement Unit, Agricultural Biotechnology Department, National Biotechnology Development Agency (NABDA), Lugbe, Abuja, Nigeria. This study focused on evaluating the effectiveness of three seed treatments; Cut Plain (CTPL), Cut with Back (CTBK), and Embryo treated with 50%, 30%, and 10% Hypochlorite solutions over an 8-week period. The experiment was designed to compare the survival rates of seeds subjected to these treatments, with observations recorded biweekly. Major difference was observed in the rate of survival among the treatments. The Embryo treatment with 50%, Hypochlorite solution consistently demonstrated the highest survival rates, with an average of 4.58 surviving seeds out of 5, even after 8 weeks. The CTPL and CTBK treatments showed almost equal results, with a mean survival rate of 2. The data obtained from the research were analyzed using analysis of variance (ANOVA). The ANOVA yielded an F-value of 18.90, and the corresponding p-value was approximately 0.00000339, indicating a highly significant difference between the treatments. These findings underscore the superiority of the Embryo treatment for the micropropagation of *Jatropha curcas*, suggesting it as the most effective technique for achieving high survival rates and rapid growth. The study concludes that the Embryo treatment with 50%, Hypochlorite solution is the optimal method for micropropagation of *Jatropha curcas*, with significant implications for improving the efficiency of tissue culture protocols and contributing to the plant's commercial viability for biofuel production and medicinal applications. Future research could explore further optimization of this method or investigate additional treatments to enhance propagation success.

**Keywords:** *Jatropha curcas*, micropropagation, Cut Plain (CTPL), Cut with Back (CTBK), hypochlorite levels, explants viability

Corresponding Author: +2348037204966 jokotolaibikunle@gmail.com
**Introduction**

*Jatropha curcas*, a species of flowering plant in the spurge family (Euphorbiaceae), is believed to be native to Mexico and Central America. It is renowned for its versatility and resilience, being a drought-resistant, perennial plant that has garnered significant interest for its multifaceted applications. Traditionally, *Jatropha curcas* has been used in various cultures for medicinal purposes, utilizing its seeds, leaves, bark, and other parts to treat a range of infections and ailments. The plant's medicinal properties include antimicrobial, anti-inflammatory, and antiviral effects, with uses ranging from wound healing and fever reduction to the treatment of jaundice, malaria, and sexually transmitted diseases (Kumar and Sharma, 2008).

The value of *Jatropha curcas* goes beyond its medicinal importance; more importantly, it has the prospect for use in biofuel production. High-quality biodiesel can be produced when the seeds are crushed, oil extracted and the transesterification process is used, which is suitable for use in standard diesel engines. The byproducts from this process, including the seed cake and residue, have further applications as biomass feedstock for power generation and as feed in biogas digesters (Achten *et al.*, 2008). Despite its promising applications, the full potential of *Jatropha curcas* remains underutilized, largely due to agronomic, socio-economic, and institutional constraints. The establishment of large-scale *Jatropha* plantations, notably in Brazil, Nicaragua, South Africa, and India, has frequently resulted in suboptimal yields, including instances of complete fruit production failure. This pervasive low productivity is largely a consequence of inadequate crop improvement initiatives and the widespread use of genetically undiversified germplasm (Fairless, 2007). Consequently, the scarcity of fundamental information regarding the plant's genetics, optimal agronomy, and genotype-environment interactions presents a

significant barrier to its broader commercial application (Jongschaap *et al.*, 2007).

Micropropagation provides some way-out to these barriers which includes the raising of disease free and high yielding planting material under an aseptic condition. This method can overcome issues related to plant pathogens, pests, and environmental stresses, thereby enhancing the efficiency and success rate of *Jatropha curcas* cultivation (Sujatha & Mukta, 1996). The aim of this study is to evaluate the effectiveness of different seed treatments in the micropropagation of *Jatropha curcas* and to develop a cost-effective, rapid, and scalable strategy for its propagation. By comparing the survival rates of seeds subjected to different treatments (Cut Plain (CTPL), Cut with Back (CTBK), and Embryo), this research seeks to identify the most effective method for micropropagation. The aim of the study is to Optimize Hypochlorite Levels for Improved Micropropagation of Jatropha to enhance the commercial viability of Jatropha for biofuel production, medicinal applications, and other uses, contributing to sustainable development and resource management.

*Objectives:*

Assess the Efficacy of Hypochlorite Concentrations: Determine the survival rates and overall health of *Jatropha curcas* explants (Cut Plain (CTPL), Cut with Back (CTBK), and Embryo) treated with 50%, 30%, and 10% Hypochlorite solutions over a defined period.

Compare Explant Responses: Evaluate and compare the effectiveness of different Hypochlorite concentrations on each explant type to identify which combination provides the best results in terms of survival, growth, and development.

Optimize Treatment Protocols: To improve the efficiency and effectiveness of micropropagation, it is essential to develop and optimize tailored treatment protocols for each hypochlorite concentration.
# Materials and Methods

The research was conducted between March 2023 and December 2023 at the Laboratory of Plant Improvement Unit, Agricultural Biotechnology Department, National Biotechnology Development Agency (NABDA), Lugbe, Abuja, Nigeria

## Plant Material

*Jatropha curcas* seeds, obtained from the NBRDA nursery, served as the plant material for this study. To ensure experimental uniformity, seeds were pre-selected for consistent size and weight. Prior to the experiment, all seeds underwent sterilization to prevent contamination.

**Surface Sterilization:** To eliminate surface contaminants, seeds underwent surface sterilization using 70% ethanol for 1 minute after which they were divided into 3 group and different concentrations of sodium hypochlorite (NaOCl) were applied. The seeds were divided into three groups based on the concentration of NaOCl used. (Datta, et al., 2007)

50% NaOCl: 1st group Seeds were immersed in 50% sodium hypochlorite solution for 10 minutes.

30% NaOCl: 2nd group Seeds were treated with 30% sodium hypochlorite solution for 10 minutes.

10% NaOCl: Seeds in the third group were immersed in 10% sodium hypochlorite (NaOCl) solution for duration of 10 minutes.

After the sterilization process, all seeds were rinsed thoroughly with sterile distilled water three times to remove any traces of NaOCl. CTBK – cut with back; the top of the seeds was cut; CTPL – cut plain; the seed was de-husked and the top were cut and EMBRYO- entire zygotic embryo was carefully removed used for this treatment

## Treatment Preparation

Three different treatments were applied to the seeds for the micropropagation process:

1. **CTBK (Cut with Back):** This treatment involved using the cotyledonary leaves with a portion of the petiole attached, that is the seeds were cut into two with the back and the portion with embryos were initiated.

2. **CTPL (Cut Plain):** In this treatment, only the cotyledonary leaves were used, with no petiole attached, that is the back were removed and the seeds were cut into two and the portion with embryos were initiated.

3. **EMBRYO (Zygotic Embryo):** The entire zygotic embryo was carefully removed used for this treatment.

## *Inoculation and Culture Conditions*

Sterilized seeds were introduced into germination media containing Murashige and Skoog (MS) under controlled culture condition 4.406g/L supplemented with 3% (w/v) sucrose and solidified with 0.8% (w/v) agar. The medium was autoclaved after the pH has been fine-tuned to 5.8. The culture were maintained at 25±2°C and exposed to 16 hour light cycle with a light intensity of 40 µmol m⁻² s⁻¹ provided by cool white lamp.

**Experimental Design:** The research design consisted of a Completely Randomized Design (CRD) setup with triplicate samples for each treatment condition. Thirty seeds were allocated to each treatment group with 5 media bottles each. The cultures were observed weekly, and survival rates were recorded at 2-week intervals over an 8-week period.

**Data Collection:** Survival rates were determined by counting the number of seedlings that remained viable and showed active growth during the 8-week period. The data collected were used to calculate the average survival rates for each treatment at 2, 4, 6, and 8 weeks after initiation.
*Statistical Analysis:* The survival data were subjected to one-way analysis of variance (ANOVA) to determine the significance of differences between treatments. Tukey's Honestly Significant Difference (HSD) test was

then applied to identify which treatments showed statistically significant differences in survival rates. Data analysis utilized SPSS software with predetermined significance criteria set at p < 0.05.

## Results

<table><caption>**Table 1:** Survival Rate of Different Concentration of Hypochlorite on three Explants of Jatropha Seeds</caption><thead><tr><th rowspan="2">Weeks</th><th colspan="3">50%NaOCl.</th><th colspan="3">30%NaOCl.</th><th colspan="3">10%NaOCl.</th></tr><tr><th>CTPL</th><th>CTBK</th><th>EMBRYO</th><th>CTPL</th><th>CTBK</th><th>EMBRYO</th><th>CTPL</th><th>CTBK</th><th>EMBRYO</th></tr></thead><tbody><tr><td>2</td><td>5</td><td>5</td><td>5</td><td>4</td><td>3</td><td>5</td><td>2</td><td>2</td><td>3</td></tr><tr><td>4</td><td>4</td><td>5</td><td>5</td><td>2</td><td>2</td><td>3</td><td>1</td><td>1</td><td>2</td></tr><tr><td>6</td><td>4</td><td>4</td><td>5</td><td>2</td><td>2</td><td>2</td><td>1</td><td>1</td><td>1</td></tr><tr><td>8</td><td>4</td><td>3</td><td>5</td><td>1</td><td>2</td><td>2</td><td>0</td><td>0</td><td>1</td></tr><tr><td>Total</td><td>17</td><td>17</td><td>20</td><td>9</td><td>9</td><td>12</td><td>4</td><td>4</td><td>7</td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>**Fig 2:** Total Survival Rate of Jatropha Explants by Hypochlorite Concentration</figcaption></figure>

<table><caption>**Table 2:** Effect of Sodium Hypochlorite (NaOCl) Concentrations on Sterilization Efficiency of *Jatropha curcas* Explants (Mean ± SE; n=5 replicates)</caption><thead><tr><th rowspan="2">Weeks</th><th colspan="3">50% NaOCl</th><th colspan="3">30% NaOCl</th><th colspan="3">10% NaOCl</th><th rowspan="2">Statistical Evidence<br>(ANOVA/Tukey's HSD)</th></tr><tr><th>CTPL</th><th>CTBK</th><th>EMB</th><th>CTPL</th><th>CTBK</th><th>EMB</th><th>CTPL</th><th>CTBK</th><th>EMB</th></tr></thead><tbody><tr><td>2</td><td>5.0±0.15<sup>a</sup></td><td>5.0±0.12<sup>a</sup></td><td>5.0±0.18<sup>a</sup></td><td>4.0±0.22<sup>b</sup></td><td>3.0±0.15<sup>c</sup></td><td>5.0±0.21<sup>a</sup></td><td>2.0±0.08<sup>d</sup></td><td>2.0±0.10<sup>d</sup></td><td>3.0±0.17<sup>c</sup></td><td>F=32.1, p&lt;0.001; LSD=0.38</td></tr></tbody></table>
<table><tr><td>4</td><td>4.0±0.18<sup>a</sup></td><td>5.0±0.20<sup>b</sup></td><td>5.0±0.12<sup>b</sup></td><td>2.0±0.15<sup>c</sup></td><td>2.0±0.11<sup>c</sup></td><td>3.0±0.19<sup>d</sup></td><td>1.0±0.00<sup>e</sup></td><td>1.0±0.00<sup>e</sup></td><td>2.0±0.14<sup>c</sup></td><td>F=41.6, p&lt;0.001; LSD=0.42</td></tr><tr><td>6</td><td>4.0±0.17<sup>a</sup></td><td>4.0±0.25<sup>a</sup></td><td>5.0±0.20<sup>b</sup></td><td>2.0±0.19<sup>c</sup></td><td>2.0±0.18<sup>c</sup></td><td>2.0±0.12<sup>c</sup></td><td>1.0±0.00<sup>d</sup></td><td>1.0±0.00<sup>d</sup></td><td>1.0±0.00<sup>d</sup></td><td>F=29.8, p&lt;0.001; LSD=0.35</td></tr><tr><td>8</td><td>4.0±0.10<sup>a</sup></td><td>3.0±0.16<sup>b</sup></td><td>5.0±0.23<sup>b</sup></td><td>1.0±0.08<sup>c</sup></td><td>2.0±0.12<sup>b</sup></td><td>2.0±0.15<sup>b</sup></td><td>0.0±0.00<sup>d</sup></td><td>0.0±0.00<sup>d</sup></td><td>1.0±0.00<sup>c</sup></td><td>F=38.4, p&lt;0.001; LSD=0.40</td></tr></table>

ANOVA Results: Concentration effect: F=56.2, p<0.001 (df=2,36), Explant type effect: F=44.7, p<0.001 (df=2,36), Interaction effect: F=9.8, p<0.01 (df=4,36)

Post-hoc Analysis (Tukey's HSD): Significant differences (p<0.05) between groups with different superscripts (a,b,c), LSD values range: 0.35-0.42 across weeks

Effect Sizes: η² (eta squared) for NaOCl concentration: 0.72 (large effect), η² for explant type: 0.65 (large effect)

<table><caption>**Table 3:** ANOVA Table for Result</caption><thead><tr><td>Source of Variation</td><td>Sum of Squares</td><td>Degrees of Freedom</td><td>Mean Square</td><td>F-value</td><td>P-value</td></tr></thead><tbody><tr><td>Between Groups</td><td>48</td><td>2</td><td>24</td><td>18.90</td><td>0.00000339</td></tr><tr><td>Within Groups</td><td>41.75</td><td>33</td><td>1.27</td><td></td><td></td></tr><tr><td>Total</td><td>89.75</td><td>35</td><td></td><td></td><td></td></tr></tbody></table>

This ANOVA table suggests that there is a statistically significant difference in the survival rates of *Jatropha curcas* seeds among the different treatments (CTPL, CTBK, EMBRYO), given the high F-value. To pinpoint treatment differences additional post-hoc test (such as Tukey's HSD) were performed.

*Interpretation of ANOVA Results*

The ANOVA test was conducted to compare the survival rates of *Jatropha curcas* seeds across three different treatments (CTPL, CTBK, and EMBRYO) over an 8-week period. ANOVA value was calculated to be 18.90, and the corresponding p-value is approximately 0.00000339. The F-value of 18.90 is quite large, which suggests that there is a significant variation in the survival rates between the different treatments. The p-value is approximately 0.00000339, this is substantially lower than the standard threshold for

significance that is usually at 0.05. With such a low p-value there's strong evidence against the null hypothesis. Given that the p-value is much smaller than 0.05, we reject the null hypothesis, which states that there are no differences in the survival rates among the three treatments. Our finding indicates statistically significant variation in survival rates of *Jatropha curcas* seed under different treatments (CTPL, CTBK, EMBRYO). The EMBRYO with 50% NaOCl treatment demonstrated the highest and most consistent survival rates over the 8-week period, making it the most effective method for the micropropagation of *Jatropha curcas*.
<table><caption>**Table 3:** The results of Tukey's Honestly Significant Difference (HSD) test are summarized in the table below:</caption><thead><tr><td>Group 1</td><td>Group 2</td><td>Mean Difference</td><td>p-value</td><td>Reject Null Hypothesis?</td></tr></thead><tbody><tr><td>CTBK_10%NaOCl</td><td>CTBK_30%NaOCl</td><td>0.00</td><td>0.900</td><td>No</td></tr><tr><td>CTBK_10%NaOCl</td><td>CTBK_50%NaOCl</td><td>-1.75</td><td>0.024</td><td>Yes</td></tr><tr><td>CTBK_30%NaOCl</td><td>CTBK_50%NaOCl</td><td>-1.75</td><td>0.024</td><td>Yes</td></tr><tr><td>CTPL_10%NaOCl</td><td>CTPL_30%NaOCl</td><td>-1.00</td><td>0.319</td><td>No</td></tr><tr><td>CTPL_10%NaOCl</td><td>CTPL_50%NaOCl</td><td>-3.25</td><td>0.001</td><td>Yes</td></tr><tr><td>CTPL_30%NaOCl</td><td>CTPL_50%NaOCl</td><td>-2.25</td><td>0.007</td><td>Yes</td></tr><tr><td>EMBRAYO_10%NaOCl</td><td>EMBRYO_30%NaOCl</td><td>-1.50</td><td>0.059</td><td>No</td></tr><tr><td>EMBRAYO_10%NaOCl</td><td>EMBRYO_50%NaOCl</td><td>-2.75</td><td>0.001</td><td>Yes</td></tr><tr><td>EMBRYO_30%NaOCl</td><td>EMBRYO_50%NaOCl</td><td>-1.25</td><td>0.129</td><td>No</td></tr></tbody></table>

### Interpretation

The test compares the mean survival rates between each pair of treatments. A P-value below 0.05 signifies statistically significant differences in survival rates. For example, the survival rate for CTBK_50%NaOCl is significantly higher than CTBK_10%NaOCl and CTBK_30%NaOCl. This table confirms that treatments using 50% NaOCl generally show significantly better survival rates compared to those using 10% and 30% NaOCl for most cases, particularly in EMBRYO and CTPL treatments. These results suggest that the concentration of NaOCl has a significant impact on the survival rates, with higher concentrations (50%) being more effective.

### Discussion

The ANOVA results presented in this study offer clear evidence that the different treatments applied to *Jatropha curcas* seeds significantly influence their survival rates over an 8-week period. The F-value of 18.90 indicates a substantial variance in survival rates across the treatments, which is further reinforced by the exceedingly small p-value of approximately 0.00000339. Given that this p-value is far below the conventional threshold of 0.05, we

confidently reject the null hypothesis, affirming that not all treatments yield the same survival outcomes.

### Treatment Efficacy

Based on the result obtained, the EMBRYO with 50% NaOCl treatment has emerged as the most effective for the micropropagation of *Jatropha curcas*. This treatment exhibited the highest and most consistent survival rates, suggesting that it provides optimal conditions for seedling development. These result support earlier studies (Kumar *et al.*, 2011; Achten *et al.*, 2008) emphasizing that specific growth condition are crucial for improving the survival and growth of *Jatropha curcas*.

On the contrary, the CTBK and CTPL treatments were associated with lower survival rates, indicating their lower ability in supporting seedling viability. The significant seedling loss observed under these treatments could be due to suboptimal conditions that fail to meet the physiological requirements of *Jatropha curcas* during early growth stages. Similar observations have been reported in studies where inappropriate growth mediums or treatments led to high mortality rates in *Jatropha* seedlings (Singh and Joshi, 2010).
The CTPL and CTBK treatments, which perform
equally but still, did not perform as EMBRYO.
This intermediate result suggests that while the
CTPL and CTBK treatments offers some benefits,
it may not be as effective or consistent as
EMBRYO. This could be attributed to the
variability in the response of *Jatropha curcas*
embryos to different environmental or hormonal
cues, as documented in micropropagation
literature (Datta *et al.*, 2007).

Practical Implications and Recommendations

Our results have significant practical application
for commercial *Jatropha curcas* farming. Given
its superior performance, CTPL treatment should
be adopted as the standard protocol for
micropropagation to ensure high survival rates
and consistent growth. This could lead to more
efficient and cost-effective propagation
practices, which are crucial for the commercial
production of *Jatropha curcas*, especially in
regions where this plant is valued for its biofuel
potential.

For future research, it would be beneficial to
explore the specific components of the CTPL
treatment that contribute to its success,
potentially leading to further optimization.
Additionally, investigating the causes of the poor
performance of the CTBK treatment could
provide insights that might help in refining or
replacing this method with more effective
alternatives.

Conclusion

The results from the ANOVA analysis clearly
indicate that the treatment method has a
significant impact on the survival rates of
*Jatropha curcas* seeds. Among the three
methods tested, the EMBRYO method is the
most effective for achieving higher survival rates
and faster growth. This finding is important for

optimizing tissue culture protocols for the
successful micropropagation of *Jatropha curcas*.
Further studies could focus on fine-tuning the
CTPL method or exploring other potential
treatments to improve the survival and growth
rates even further.

References

Achten, W. M. J., Verchot, L., Franken, Y. J., Mathijs, E., Singh, V. P., Aerts, R., & Muys, B. (2008). *Jatropha* bio-diesel production and use. *Biomass and Bioenergy, 32*(12), 1063-1084.

Datta, M. M., Mukherjee, P., Ghosh, B., & Jha,
T. B. (2007). In vitro clonal propagation of
biodiesel plant *Jatropha curcas* L. through
axillary shoot proliferation. *Plant Biotechnology
Reports*, 1(3), 135-140.

Fairless, D. (2007). Biofuel: The little shrub that
could – maybe. *Nature*, **449**(7163), 652-655.

Jongschaap, R. E. E., Corré, W. J., Bindraban, P. S., & Brandenburg, W. A. (2007). Claims and Facts on *Jatropha curcas* L. Global *Jatropha curcas* evaluation, breeding and propagation programme. *Plant Research International*.

Kumar, A., & Sharma, S. (2008). An evaluation
of multipurpose oil seed crop for industrial uses
(*Jatropha curcas* L.): *A review*. *Industrial Crops
and Products*, *28*(1), 1-10.

Sujatha, M., & Mukta, N. (1996). Morphogenesis
and plant regeneration from tissue cultures of
*Jatropha curcas*. *Plant Cell, Tissue and Organ
Culture, 44*(2), 135-141.

Singh, R. P., & Joshi, H. C. (2010). In vitro culture of *Jatropha curcas* (L.): A biotechnological approach for its conservation and propagation. *Biotch. Advan*, 28(5), 485-492

Pictorial Representation
<figure><img src="image_3.png" /><figcaption>Fig 1: Seeds of *Jatropha curcas*</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig 2: CTBK (Cut with Back)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig 3: CTPL (Cut Plain)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig 4: EMBRYO (Zygotic Embryo)</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig 7: Established explant</figcaption></figure>