Taro (Colocasia esculenta) and tannia (Xanthosoma sagittifolium) are commonly referred to as cocoyam in Nigeria. They are cherished for their rich taste, nutritional and medicinal properties. Traditionally, cocoyams are vegetatively propagated from tuber fragments, a practice that encourages pathogen distribution. For rapid multiplication and production of quality planting materials, tissue culture technology offers promising alternative compared to the traditional production methods. In this study different concentrations of ripped sweet orange (Citrus sinensis) juice were screened for regeneration and multiplication of 2 months old in vitro cocoyam shoot explants. Among the concentrations, maximum numbers of roots (37 ± 5) were observed in Murashige and Skoog (MS) medium supplemented with 10% orange juice after 8 weeks in culture compared to 16 ± 4 (roots) observed in the control medium. On shoot multiplicity, 16 ± 3 shoots were induced in the control medium in contrast to 12 ± 0.8 shoots in the MS medium supplemented with 10% orange juice after 8 weeks of culture. Higher concentrations (25% and 35%) of the orange juice supplemented medium resulted in concomitant inhibition of all the growth parameters. The study successfully established that ripped orange juice could substitute the use of conventional growth hormones cytokinins (BAP) and auxins (NAA) in in vitro regeneration and rapid multiplication of cocoyam shoot explants.
It is 113 years since the concept of isolation and growth of plant cells in artificial culture medium was proposed by Gotlieb Haberlandt. Eighty years later, precisely in 1983, the first transgenic plant was engineered. The two events (1902 and 1983), marked the beginning in our long scientific journey towards today’s achievements in the plant and agricultural biotechnology revolution [
Plant physiological processes are regulated by chemical signals known as plant growth regulators (PGRs). They are normally active at very low concentrations in plants. Indeed for the specific regulation of many plant processes and the differentiation of cells into specific plant parts, a variety of ratios and concentrations of these plant hormones are required rather than a single hormone acting alone [
Production challenges confronting cocoyam cultivation in the last three decades in Nigeria ranges from scarcity of quality planting materials, low multiplication ratio to incidences of pests and diseases [
Ripped oranges (Citrus sinensis) were washed in running tap water, followed by immersion in 70% ethanol for 20 min. Sterilization was carried out in 3.5% sodium hypochlorite solution in combination with 2 drops of Tween 20 for 15 min. This was followed by rinsing three times in sterile distilled water (10 min per each rinse) and allowed to dry at room temperature. The oranges were cut with sterile scalpel and hand-squeezed. The pulps were sieved through sterile cheesecloth and centrifuged for 20 minutes at 2000 rpm.
The control medium was composed of Murashige and Skoog’s basal medium supplemented with vitamins, myo- inositol, L-cysteine, BAP, NAA, gelrite and sucrose. Other media were composed of all the aforementioned components with different concentrations (10%, 15%, 25%, and 35%) of orange juice excluding the two growth hormones (BAP and NAA) (
The results of in vitro regeneration and multiplication of young cocoyam shoot explants are as captured in Tables 2-5. Various concentrations of the orange juice in the culture media induced varied growth responses on
Media description | Components |
---|---|
Murashige and Skooge (MS) (control) | MS media, vitamin mixture, myo-inositol, L-cysteine, BAP, NAA, gelrite + sucrose |
MS + 10% orange juice | MS media, vitamin mixture, myo-inositol, L-cysteine, 10% orange juice, gelrite + sucrose. |
MS + 15% orange juice | MS media, vitamin mixture, myo-inositol, L-cysteine, 15% orange juice, gelrite + sucrose. |
MS + 25% orange juice | MS media, vitamin mixture, myo-inositol, L-cysteine, 25% orange juice, gelrite + sucrose. |
MS + 35% orange juice | MS media, vitamin mixture, myo-inositol, L-cysteine, 35% orange juice, gelrite + sucrose. |
Media description | 2 weeks | 4 weeks | 6 weeks | 8 weeks |
---|---|---|---|---|
MS (control) | 0b | 0b | 8 ± 2c | 16 ± 4c |
MS + 10% orange juice | 8 ± 2a | 17 ± 4a | 28 ± 6a | 37 ± 5a |
MS + 15% orange juice | 0b | 0b | 12 ± 3b | 18 ± 4b |
MS + 25% orange juice | 0b | 0b | 8 ± 1.6c | 14 ± 3d |
MS + 35% orange juice | 0b | 0b | 6 ± 0.8d | 10 ± 0.9e |
Key: Values represents mean ± standard errors for three replications for each treatment. Mean in a column with the same superscript letter do not differ significantly according to DMRT at 5% level of probability.
Media description | 2 weeks | 4 weeks | 6 weeks | 8 weeks |
---|---|---|---|---|
MS (control) | 4 ± 0.3b | 9 ± 2b | 16 ± 4b | 24 ± 5b |
MS + 10% orange juice | 3 ± 0.1c | 7 ± 1.4c | 10 ± 2d | 12 ± 3e |
MS + 15% orange juice | 8 ± 2a | 19 ± 4a | 26 ± 7a | 32 ± 6a |
MS + 25% orange juice | 2 ± 0.2d | 6 ± 0.4d | 11 ± 2.5c | 19 ± 3.8c |
MS + 35% orange juice | 2 ± 0.1d | 6 ± 0.7d | 9 ± 2e | 18 ± 4d |
Key: Values represents mean ± standard errors for three replications for each treatment. Mean in a column with the same superscript letter do not differ significantly according to DMRT at 5% level of probability.
Media description | 2 weeks | 4 weeks | 6 weeks | 8 weeks |
---|---|---|---|---|
MS (control) | 1.4 ± 0.1a | 2.15 ± 0.3a | 3.6 ± 0.04a | 5.15 ± 0.2a |
MS + 10% orange juice | 1.2 ± 0.3a | 2.13 ± 0.1a | 3.8 ± 0.2a | 5.23 ± 0.03a |
MS + 15% orange juice | 1.2 ± 0.04a | 2.13 ± 0.01a | 3.1 ± 0.4b | 5.09 ± 0.1a |
MS + 25% orange juice | 0.87 ± 0.02a | 1.62 ± 0.03a | 3.2 ± 0.05b | 4.77 ± 0.06a |
MS + 35% orange juice | 0.64 ± 0.03a | 1.52 ± 0.02a | 2.8 ± 0.1b | 4.33 ± 0.6ab |
Key: Values represents mean ± standard errors for three replications for each treatment. Mean in a column with the same superscript letter do not differ significantly according to DMRT at 5% level of probability.
Media description | 2 weeks | 4 weeks | 6 weeks | 8 weeks |
---|---|---|---|---|
MS (control) | 2 ± 0.1b | 5 ± 0.2c | 8 ± 0.4b | 16 ± 3a |
MS + 10% orange juice | 3 ± 0.2a | 6 ± 0.1b | 9 ± 0.3a | 12 ± 0.8b |
MS + 15% orange juice | 3 ± 0.03a | 7 ± 0.4a | 9 ± 0.6a | 11 ± 2c |
MS + 25% orange juice | 1 ± 0.02c | 3 ± 0.01e | 8 ± 0.2b | 11 ± 3.2c |
MS + 35% orange juice | 1 ± 0.04c | 4 ± 0.3d | 8 ± 0.5b | 11 ± 2.4c |
Key: Values represents mean ± standard errors for three replications for each treatment. Mean in a column with the same superscript letter(s) do not differ significantly according to DMRT at 5% level of probability.
the in vitro culture of the cocoyam shoot explants. Regeneration and proliferation efficiency of the explants were higher in the MS media supplemented with 10% and 15% orange juice compared to the other treatments (Tables 2-5). All the culture media except those supplemented with 25% and 35% orange juice concentrations induced optimal responses on regeneration and propagation of the explants. In 10% and 15% orange juice supplemented media, there were significant improvement in roots and leaf growth compared to the control medium (
The superiority of the orange juice supplemented medium over the control medium was further demonstrated on leaf induction and growth after 2, 4, 6, & 8 weeks in culture (
The effects of the orange juice supplemented media were observed to be insignificant on shoot height (
Orange juice, a complex organic extract used in this study contained carbohydrates, protein, several vitamins, lower levels of some amino acids and organic acids [
At 10% and 15% orange juice concentration, the supplemented media produced the best result, thus recommending its application for in vitro regeneration and multiplication of cocoyam plantlets. We observed that higher concentration of the orange juice (25% & 35%) in the MS medium resulted in the concomitant decrease in the growth responses of roots, leaves, shoot height and shoot multiplicity. Based on our results, we therefore concluded that orange juice at 10% and 15% concentrations in the MS media is a potential alternative organic
additive to synthetic growth hormones in the culture medium for in vitro regeneration and multiplication of cocoyam. Impressively, one cocoyam shoot explant could develop multiples of 9 new shoots in culture within 4 weeks. This cheap and straight forward approach may be economically feasible for large scale production of disease-free cocoyam planting materials through tissue culture technology.
AlfredO. Ubalua,AhamefulaI. Ikpeama,OnyinyechiD. Okeagu, (2015) Effect of Different Concentrations of Orange Juice for in Vitro Regeneration and Multiplication of Cocoyam (Taro). American Journal of Plant Sciences,06,2569-2575. doi: 10.4236/ajps.2015.616259