J Plant Biotechnol 2022; 49(2): 124-130
Published online June 30, 2022
https://doi.org/10.5010/JPB.2022.49.2.124
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: camellia@mardi.gov.my
†Contributed equally
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
The application of traditional taro propagation methods for large-scale cultivation would be insufficient to meet the high demand for quality planting materials. Therefore, this study aimed to develop an in vitro micropropagation technique for two local taro cultivars (cv.), Wangi and Putih. Taro cormels were collected from the Malaysian Agricultural Research and Development Institute (MARDI) germplasm (Serdang, Malaysia). Explants were taken from the shoot tip of cormels and initially cultured on Murashige and Skoog (MS) basal media for four weeks. The explants were then transferred to different multiplication media supplemented with different types and concentrations of cytokinins such as 6-benzylaminopurine (BAP ) and Thidiazuron (TDZ). Shoot production was quantified after six weeks of culture. The highest mean number of new shoots was produced by the Wangi cultivar on MS medium supplemented with 2.0 mg/l BAP (2.10 shoots), MS medium supplemented with 0.5 mg/l TDZ (2.18 shoots), and Gamborg B5 medium supplemented with 6.0 mg/l BAP (2.43 shoots). The maximum average number of the Putih cultivar shoots was obtained on MS supplemented with 2.0 mg/l BAP (3.57 shoots). MS basal media was used for root initiation, as it produced an average of 25 roots with an 11-cm length. Various types of substrate mixtures were used during acclimatization. The best acclimatization substrate for the Wangi cultivar was 100% peat soil, whereas the Putih cultivar grew optimally in a combination of peat and perlites at a 1:1 ratio. Taro plantlets require approximately 4 to 6 weeks to acclimatize before they can be transferred to the field.
Keywords Multiplication, Microcorms, Colocasia esculenta, Acclimatization, Keladi
Taro (
Taro also being a significant export commodity in numerous countries, and it is highly priced in the market. Taro has been widely used in rural agroindustrial for raw material product like syrup, gum, edible film for modified atmosphere packaging (MAP) and renewable energy source. In 2010, taro world production is approximately 9,006,116 metric tonnes (Mg). However, only 133,676 Mg of taro from the world production was exported worldwide (Joe 2012). There were demands from countries such as United States of America, Japan, China, and these are some of the world’s top global importers. In 2010, production of taro in the Asia region was approximately 2.1 million Mg and about 23% of global production (Joe 2012). In Malaysia, production of taro hugely decreased from 4,856.81 Mg in 2015 to 4,129.15 Mg in 2016 and 4,634.8 Mg in 2017 respectively (DOA 2015; 2016; 2017). The main constraints for taro production are a scarcity of disease-free planting materials (Mbong et al. 2013). This plant, which belongs to the Araceae family, is commonly propagated by suckers. It has been reported that cultivars that produce large corms produce a small number of suckers (Sivan and Liyanage 1992; Sivan 1984).
Through conventional method, the amount of taro planting materials yielded is low and probably disease-carrying. The crop also susceptible to fungal, viral, and nematode infections (Gadre and Joshi 2003). Taro leaf blight disease, which caused by
The technique provides an economical, mass propagation, space, and labour-saving method for the preservation of germplasm. They can be stored as tissue culture in nutrient medium rather than repeatedly growing germplasm collections in the field. It only needs to be subcultured once in several months (Inno 1999). The method described in this study for
Suckers of taro were obtained from Malaysian Agricultural Research and Development Institute (MARDI) aroids germplasm collection, number 18 (keladi wangi) and 31 (keladi putih). (Ghazali et al. 2016) (Fig. 1 and 2). Experiments were performed in the Cryopreservation laboratories of the MyGeneBank Complex in MARDI, Serdang (2°58’42.3”N 101°41’17.4”E). To obtain initial cultures of taro, suckers were cut and trimmed to about 4cm length shoot and 1.5cm width base with 3 to 4 leaf sheaths (Fig. 2). The explants were then washed with Decon 90 before rinsed with tap water. The explants were then washed under running tap water for 1 hour, soaked in 20ml dettol antiseptic liquid in 500 ml distilled water for 30 second up to 1 min and rinsed with distilled water. Explants were then soaked in 0.5 g/l Kenlate fungicide (50% benomyl) for 2 hours and rinsed with distilled water. Afterward, under the airstream horizontal laminar airflow cabinet (ESCO, USA), explants were then transferred and soaked into 50% commercial bleach Clorox® (5.65% Sodium hypochlorite) with 3 to 5 drops of tween 20 for 17 minutes under laminar airflow chamber followed by rinsing three times with double distilled water. Then, the explants were cut and trimmed into smaller pieces leaving only 1 to 2 leaf sheaths. The explants were further disinfected with 70% ethanol for 30 seconds and 10% Clorox® for 5 minutes followed by rinsing with double distilled water for four times and blotted dry on sterilized paper before cultured to Murashige and Skoog (1962) (MS) media supplemented with 30 g/l sucrose and solidified with 3 g/l GelriteTM.
Well-developed plantlets in six weeks culture were removed carefully from the medium, washed with tap water to remove the traces of agar. Plantlets were then planted in germination trays with four different substrates mixture: T1) peat, perlite and vermiculate (1:1:1), T2) peat and vermiculate (1:1), T3) peat and perlite (1:1), and T4) peat. To maintain humidity, the plantlets were covered with transparent plastic sheets and seedling tray was located inside a plastic tray containing 1 cm height of water. Watering was done weekly. The plantlets in the trays were placed in a cool place with 50% shade. After four weeks of acclimatisation, data on the percentage of survival, plant height, root number, root length, number of microcorms, and number of new shoots were all recorded.
All data were analysed by analysis of variance (ANOVA) using Statistical Analysis System Software (SAS) release 9.4. The experiment was set up in a Completely Randomized Design (CRD). The significance of differences among means was done by using Duncan’s Multiple Test Range (DMRT). Statistical significance was considered at P ≤ 0.05.
Direct regeneration of taro from suckers as explants was observed in all concentrations of BAP or TDZ supplemented media. The highest multiplication of shoots for cv. wangi was found on MS media containing 2 mg/l BAP or 0.5 mg/l TDZ with a mean number of shoots of 2.10 ± 0.33 and 2.18 ± 0.36, respectively, while 6 mg/l BAP contained in B5 media with a mean number of shoots of 2.43 ± 0.24. Similarly, Chand et al. (1999) discovered that supplementing 1 mg/l BAP or TDZ as a single hormone resulted in 2 to 2.5 shoots of
Out of nine hormone concentration treatments in MS media and six treatments in B5, only the control treatment did not result in shoot proliferation. Table 1 and 2 showed a significant difference between treatments for all parameters observed in MS and B5 media at P ≤ 0.05. Figure 3 and 4 showed the condition of taro cultured on MS and B5 media. After 2-3 weeks of culture in B5 media, the matured leaves from the plantlets turned brown, and senescence took over, leaving only the young shoots. This incident, however, did not occur in taro cultured on MS media. Bogale (2019) also reported that MS medium is the most suitable medium for in vitro production of C. esculenta cv. Bolosso I. compared to B5 media and Chu (N6) media.
Table 1 Effects of different concentrations of single hormones (BAP and TDZ) on number of shoot and root, shoot height and root length of cv. wangi after 4 weeks of culture in Murashige & Skoog medium
Concentrations of single hormones (BAP/ TDZ) | Number of shoots | Shoot height (cm) | Number of roots | Root length (cm) |
---|---|---|---|---|
Control | 0.32 ± 0.12a | 13.11 ± 0.36f | 21.25 ± 1.10g | 11.25 ± 0.69e |
0.5 mg/l BAP | 0.96 ± 0.23ab | 4.18 ± 0.61a | 12.14 ± 0.93e | 2.34 ± 0.22cd |
1.0 mg/l BAP | 1.32 ± 0.24bc | 6.21 ± 0.48b | 14.32 ± 1.00f | 2.86 ± 0.21d |
2.0mg/l BAP | 2.10 ± 0.33d | 6.54 ± 0.53bc | 3.46 ± 0.58b | 0.54 ± 0.09a |
3.0 mg/l BAP | 2.04 ± 0.27cd | 7.61 ± 0.47cd | 5.96 ± 0.89c | 0.99 ± 0.14ab |
0.5 mg/l TDZ | 2.18 ± 0.36d | 8.68 ± 0.61d | 9.11 ± 0.99d | 2.45 ± 0.17d |
1.0 mg/l TDZ | 2.0 ± 0.20cd | 10.71 ± 0.40e | 2.04 ± 0.29ab | 2.11 ± 0.21cd |
2.0 mg/l TDZ | 1.50 ± 0.36bcd | 5.64 ± 0.39b | 1.04 ± 0.24a | 1.14 ± 0.2ab |
3.0 mg/l TDZ | 1.46 ± 0.30bcd | 5.22 ± 0.48ab | 1.64 ± 0.33ab | 1.64 ± 0.22bc |
Data followed by different letters showed significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 28.
Table 2 Effects of different concentrations of BAP on number of shoot and root, shoot height and root length of cv. wangi after 6 weeks of culture in Gamborg B5 media
BAP concentrations | No. of Shoot | Shoot height (cm) | Root numbers | Root length (cm) |
---|---|---|---|---|
0 mg/l BAP | 0.03 ± 0.03a | 8.1 ± 1.02c | 8.23 ± 0.55c | 10.37 ± 1.20b |
0.5 mg/l BAP | 1.47 ± 0.25b | 4.22 ± 0.16b | 0.93 ± 0.28b | 0.1 ± 0.03a |
2.0mg/l BAP | 2.07 ± 0.41bc | 3.33 ± 0.15ab | 0a | 0a |
4.0 mg/l BAP | 1.73 ± 0.29b | 2.67 ± 0.14a | 0a | 0a |
6.0 mg/l BAP | 2.43 ± 0.24c | 2.97 ± 0.11a | 0a | 0a |
8.0 mg/l BAP | 1.87 ± 0.30bc | 2.93 ± 0.16a | 0a | 0a |
Data followed by different letters showed significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 30.
Without any additional hormones, cv. wangi
Table 3 and Figure 5 revealed a highly significant difference between treatments of different concentrations of BAP for all parameters observed for cv. putih. Only the control treatment does not induce shoot proliferation among the six hormone concentration treatments. The highest multiplication of shoots was found on MS media containing 2 mg/l BAP, as with cv. wangi. Cultivar putih produced more shoots than cv. wangi, according to the results. Cultivar putih produced 3 to 4 (3.57b ± 0.26) shoots. The intensification in BAP concentrations beyond 2 mg/l has not improved the multiplication of shoots and did not affect the shoot length, root numbers and root length. In contrast to Bogale’s (2018) study, taro cv. Bolosso I supplemented with BAP at concentrations ranging from 4 mg/l to 8 mg/l resulted in 3.83 to 6.13 shoots.
Table 3 Effects of different concentrations of BAP on number of shoot and root, shoot height and root length of cv. putih after 6 weeks of culture in Murashige & Skoog medium
Concentrations of BAP | Number of shoots | Shoot height (cm) | Number of roots | Root length (cm) |
---|---|---|---|---|
0 mg/l BAP | 0.03 ± 0.03a | 10.23 ± 0.31d | 12.27 ± 0.40bc | 12.85 ± 0.50d |
0.5 mg/l BAP | 2.27 ± 0.28b | 7.40 ± 0.41c | 12.57 ± 0.62c | 7.90 ± 0.91c |
2.0mg/l BAP | 3.57 ± 0.26d | 5.97 ± 0.34b | 10.67 ± 0.74b | 1.41 ± 0.19b |
4.0 mg/l BAP | 3.10 ± 0.28cd | 4.63 ± 0.32a | 4.43 ± 0.67a | 0.53 ± 0.09ab |
6.0 mg/l BAP | 3.00 ± 0.19cd | 4.43 ± 0.31a | 3.17 ± 0.56a | 0.26 ± 0.05a |
8.0 mg/l BAP | 2.53 ± 0.20bc | 3.90 ± 0.20a | 2.83 ± 0.60a | 0.21 ± 0.04a |
Data followed by different letters showed significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 30.
Root crops culture is difficult to initiate especially if the storage organ such as corm or tuber is being used as starting material. These organs may have contaminated with soil micro flora that become problematic during tissue culture process. In this study, surface sterilization technique does not include mercury (II) chloride that is very toxic to humans. It has been excessively used in explant surface sterilization due to strong sterilization efficiency to kill microbes (soil-borne, epiphytic fungi and bacteria). Hence, the sterilization used in this study gave 0% contamination. The trick behind the success of this sterilization technique is the initial size of explant taken from the corm. The smaller the size of explant, less contamination was found in the culture. If the initial explant size is big around 6-7cm length and 3-4cm width, normally bacterial infection will interfere in the culture. Additional fungicide (benomyl) used in this study also help to reduce fungi contamination.
The survival rate of plantlets transplanted from culture jars to seedling trays was 100% for all substrate media used in this study (Table 4). The difficulties associated with tissue cultured plant survival and growth following transplantation are attributed to the plant’s inability to control water due to the type of media used (Lavanya et al. 2009). Peat was the best substrate media for cv. wangi in this study, with a plant height of 26.34 cm, 12 number of roots with 19.2 cm length. While for cv. putih, a mixture of 1:1 peat and perlite found to be the best with a plant height of 28.76 cm, 11 number of roots with 21.84 cm length (Table 4 & Fig. 6). This response could be attributed to the mixture’s ability to provide adequate moisture and aeration to the plants, resulting in healthy root growth. Perlite is an important component in the potting mixture when mixed with peatmoss. The addition of perlite to peatmoss increases both the amount of oxygen and the amount of water retained by the peatmoss. This obviously improves plant growth conditions (Miller and Donahue 1990). After 4 to 6 weeks planted in the nursery and acclimatized under 50% shades, the plantlets were transferred to the field. Fig. 7 showed the tissue culture plant of cv. wangi and cv. putih that had been successfully planted in the field for eight months.
Table 4 The effect of different substrate media on survival percentage and growth of cv. wangi and cv. putih attained at week 4 of acclimatization. T1) peat, perlite and vermiculate (1:1:1), T2) peat and vermiculate (1:1), T3) peat and perlite (1:1), and T4) peat
Type of taro | Substrate composition | Survival (%) | Plant height (cm) | Root length (cm) | No. of roots | Plantlets producing new shoots (%) | Plantlets producing micro-corm (%) |
---|---|---|---|---|---|---|---|
cv. wangi | T1 | 100 | 22.38a ± 1.36 | 17.02a ± 1.40 | 9.72a ± 0.66 | 0ns | 28a ± 10.19 |
T2 | 100 | 26.22b ± 1.13 | 18.14ab ± 0.33 | 11.16bc ± 0.48 | 0ns | 56b ± 11.66 | |
T3 | 100 | 26.74b ± 2.27 | 18.18ab ± 1.19 | 10.28ab ± 0.77 | 0ns | 52ab ± 16.25 | |
T4 | 100 | 26.34b ± 1.44 | 19.2b ± 1.08 | 12.08c ± 0.41 | 4ns | 40ab ± 8.94 | |
cv. putih | T1 | 100 | 25.76a ± 2.40 | 21.26a ± 1.48 | 10.68ns | 28ns | 88ns |
T2 | 100 | 25.8a ± 2.24 | 22.88a ± 1.91 | 10.6ns | 20ns | 100ns | |
T3 | 100 | 28.76b ± 1.20 | 21.84a ± 0.82 | 11.84ns | 12ns | 100ns | |
T4 | 100 | 26.04ab ± 1.75 | 21.26a ± 1.43 | 9.64ns | 16ns | 100ns |
Data followed by different letters showed a significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 25.
An effective protocol needs to be established to provide a rapid technique for mass multiplication of these potential crop cultivars. Initiation of cv. wangi and putih through micropropagation begin after 1 week in culture. At multiplication stage, both cultivars multiplied when cultured in MS medium supplemented with selected cytokinin (BAP/ TDZ) during this study. MS medium supplemented with 2 mg/l BAP showed the highest number of shoots produced for both cultivars. At rooting stage, the highest number of roots was achieved on MS medium without hormone. After acclimatisation, 100% of seedlings were survived in green house. A complete cycle of initiating taro tissue culture takes around 4 to 5 months from surface sterilization to acclimatise before field planting.
This study was financially supported by the Malaysian Agricultural Research and Development Institute (MARDI) under the Eleventh Malaysia Plan (RMKe-11), the Ministry of Agriculture and Food Industries (MAFI), and the international project Benefit-sharing Fund (BSF) under The International Treaty.
J Plant Biotechnol 2022; 49(2): 124-130
Published online June 30, 2022 https://doi.org/10.5010/JPB.2022.49.2.124
Copyright © The Korean Society of Plant Biotechnology.
Noor Camellia Noor Alam·Abdul Muhaimin Abdul Kadir
Industrial Crop Research Center, Malaysian Agricultural Research and Development Institute, Serdang, Selangor, Malaysia
Correspondence to:e-mail: camellia@mardi.gov.my
†Contributed equally
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
The application of traditional taro propagation methods for large-scale cultivation would be insufficient to meet the high demand for quality planting materials. Therefore, this study aimed to develop an in vitro micropropagation technique for two local taro cultivars (cv.), Wangi and Putih. Taro cormels were collected from the Malaysian Agricultural Research and Development Institute (MARDI) germplasm (Serdang, Malaysia). Explants were taken from the shoot tip of cormels and initially cultured on Murashige and Skoog (MS) basal media for four weeks. The explants were then transferred to different multiplication media supplemented with different types and concentrations of cytokinins such as 6-benzylaminopurine (BAP ) and Thidiazuron (TDZ). Shoot production was quantified after six weeks of culture. The highest mean number of new shoots was produced by the Wangi cultivar on MS medium supplemented with 2.0 mg/l BAP (2.10 shoots), MS medium supplemented with 0.5 mg/l TDZ (2.18 shoots), and Gamborg B5 medium supplemented with 6.0 mg/l BAP (2.43 shoots). The maximum average number of the Putih cultivar shoots was obtained on MS supplemented with 2.0 mg/l BAP (3.57 shoots). MS basal media was used for root initiation, as it produced an average of 25 roots with an 11-cm length. Various types of substrate mixtures were used during acclimatization. The best acclimatization substrate for the Wangi cultivar was 100% peat soil, whereas the Putih cultivar grew optimally in a combination of peat and perlites at a 1:1 ratio. Taro plantlets require approximately 4 to 6 weeks to acclimatize before they can be transferred to the field.
Keywords: Multiplication, Microcorms, Colocasia esculenta, Acclimatization, Keladi
Taro (
Taro also being a significant export commodity in numerous countries, and it is highly priced in the market. Taro has been widely used in rural agroindustrial for raw material product like syrup, gum, edible film for modified atmosphere packaging (MAP) and renewable energy source. In 2010, taro world production is approximately 9,006,116 metric tonnes (Mg). However, only 133,676 Mg of taro from the world production was exported worldwide (Joe 2012). There were demands from countries such as United States of America, Japan, China, and these are some of the world’s top global importers. In 2010, production of taro in the Asia region was approximately 2.1 million Mg and about 23% of global production (Joe 2012). In Malaysia, production of taro hugely decreased from 4,856.81 Mg in 2015 to 4,129.15 Mg in 2016 and 4,634.8 Mg in 2017 respectively (DOA 2015; 2016; 2017). The main constraints for taro production are a scarcity of disease-free planting materials (Mbong et al. 2013). This plant, which belongs to the Araceae family, is commonly propagated by suckers. It has been reported that cultivars that produce large corms produce a small number of suckers (Sivan and Liyanage 1992; Sivan 1984).
Through conventional method, the amount of taro planting materials yielded is low and probably disease-carrying. The crop also susceptible to fungal, viral, and nematode infections (Gadre and Joshi 2003). Taro leaf blight disease, which caused by
The technique provides an economical, mass propagation, space, and labour-saving method for the preservation of germplasm. They can be stored as tissue culture in nutrient medium rather than repeatedly growing germplasm collections in the field. It only needs to be subcultured once in several months (Inno 1999). The method described in this study for
Suckers of taro were obtained from Malaysian Agricultural Research and Development Institute (MARDI) aroids germplasm collection, number 18 (keladi wangi) and 31 (keladi putih). (Ghazali et al. 2016) (Fig. 1 and 2). Experiments were performed in the Cryopreservation laboratories of the MyGeneBank Complex in MARDI, Serdang (2°58’42.3”N 101°41’17.4”E). To obtain initial cultures of taro, suckers were cut and trimmed to about 4cm length shoot and 1.5cm width base with 3 to 4 leaf sheaths (Fig. 2). The explants were then washed with Decon 90 before rinsed with tap water. The explants were then washed under running tap water for 1 hour, soaked in 20ml dettol antiseptic liquid in 500 ml distilled water for 30 second up to 1 min and rinsed with distilled water. Explants were then soaked in 0.5 g/l Kenlate fungicide (50% benomyl) for 2 hours and rinsed with distilled water. Afterward, under the airstream horizontal laminar airflow cabinet (ESCO, USA), explants were then transferred and soaked into 50% commercial bleach Clorox® (5.65% Sodium hypochlorite) with 3 to 5 drops of tween 20 for 17 minutes under laminar airflow chamber followed by rinsing three times with double distilled water. Then, the explants were cut and trimmed into smaller pieces leaving only 1 to 2 leaf sheaths. The explants were further disinfected with 70% ethanol for 30 seconds and 10% Clorox® for 5 minutes followed by rinsing with double distilled water for four times and blotted dry on sterilized paper before cultured to Murashige and Skoog (1962) (MS) media supplemented with 30 g/l sucrose and solidified with 3 g/l GelriteTM.
Well-developed plantlets in six weeks culture were removed carefully from the medium, washed with tap water to remove the traces of agar. Plantlets were then planted in germination trays with four different substrates mixture: T1) peat, perlite and vermiculate (1:1:1), T2) peat and vermiculate (1:1), T3) peat and perlite (1:1), and T4) peat. To maintain humidity, the plantlets were covered with transparent plastic sheets and seedling tray was located inside a plastic tray containing 1 cm height of water. Watering was done weekly. The plantlets in the trays were placed in a cool place with 50% shade. After four weeks of acclimatisation, data on the percentage of survival, plant height, root number, root length, number of microcorms, and number of new shoots were all recorded.
All data were analysed by analysis of variance (ANOVA) using Statistical Analysis System Software (SAS) release 9.4. The experiment was set up in a Completely Randomized Design (CRD). The significance of differences among means was done by using Duncan’s Multiple Test Range (DMRT). Statistical significance was considered at P ≤ 0.05.
Direct regeneration of taro from suckers as explants was observed in all concentrations of BAP or TDZ supplemented media. The highest multiplication of shoots for cv. wangi was found on MS media containing 2 mg/l BAP or 0.5 mg/l TDZ with a mean number of shoots of 2.10 ± 0.33 and 2.18 ± 0.36, respectively, while 6 mg/l BAP contained in B5 media with a mean number of shoots of 2.43 ± 0.24. Similarly, Chand et al. (1999) discovered that supplementing 1 mg/l BAP or TDZ as a single hormone resulted in 2 to 2.5 shoots of
Out of nine hormone concentration treatments in MS media and six treatments in B5, only the control treatment did not result in shoot proliferation. Table 1 and 2 showed a significant difference between treatments for all parameters observed in MS and B5 media at P ≤ 0.05. Figure 3 and 4 showed the condition of taro cultured on MS and B5 media. After 2-3 weeks of culture in B5 media, the matured leaves from the plantlets turned brown, and senescence took over, leaving only the young shoots. This incident, however, did not occur in taro cultured on MS media. Bogale (2019) also reported that MS medium is the most suitable medium for in vitro production of C. esculenta cv. Bolosso I. compared to B5 media and Chu (N6) media.
Table 1 . Effects of different concentrations of single hormones (BAP and TDZ) on number of shoot and root, shoot height and root length of cv. wangi after 4 weeks of culture in Murashige & Skoog medium.
Concentrations of single hormones (BAP/ TDZ) | Number of shoots | Shoot height (cm) | Number of roots | Root length (cm) |
---|---|---|---|---|
Control | 0.32 ± 0.12a | 13.11 ± 0.36f | 21.25 ± 1.10g | 11.25 ± 0.69e |
0.5 mg/l BAP | 0.96 ± 0.23ab | 4.18 ± 0.61a | 12.14 ± 0.93e | 2.34 ± 0.22cd |
1.0 mg/l BAP | 1.32 ± 0.24bc | 6.21 ± 0.48b | 14.32 ± 1.00f | 2.86 ± 0.21d |
2.0mg/l BAP | 2.10 ± 0.33d | 6.54 ± 0.53bc | 3.46 ± 0.58b | 0.54 ± 0.09a |
3.0 mg/l BAP | 2.04 ± 0.27cd | 7.61 ± 0.47cd | 5.96 ± 0.89c | 0.99 ± 0.14ab |
0.5 mg/l TDZ | 2.18 ± 0.36d | 8.68 ± 0.61d | 9.11 ± 0.99d | 2.45 ± 0.17d |
1.0 mg/l TDZ | 2.0 ± 0.20cd | 10.71 ± 0.40e | 2.04 ± 0.29ab | 2.11 ± 0.21cd |
2.0 mg/l TDZ | 1.50 ± 0.36bcd | 5.64 ± 0.39b | 1.04 ± 0.24a | 1.14 ± 0.2ab |
3.0 mg/l TDZ | 1.46 ± 0.30bcd | 5.22 ± 0.48ab | 1.64 ± 0.33ab | 1.64 ± 0.22bc |
Data followed by different letters showed significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 28..
Table 2 . Effects of different concentrations of BAP on number of shoot and root, shoot height and root length of cv. wangi after 6 weeks of culture in Gamborg B5 media.
BAP concentrations | No. of Shoot | Shoot height (cm) | Root numbers | Root length (cm) |
---|---|---|---|---|
0 mg/l BAP | 0.03 ± 0.03a | 8.1 ± 1.02c | 8.23 ± 0.55c | 10.37 ± 1.20b |
0.5 mg/l BAP | 1.47 ± 0.25b | 4.22 ± 0.16b | 0.93 ± 0.28b | 0.1 ± 0.03a |
2.0mg/l BAP | 2.07 ± 0.41bc | 3.33 ± 0.15ab | 0a | 0a |
4.0 mg/l BAP | 1.73 ± 0.29b | 2.67 ± 0.14a | 0a | 0a |
6.0 mg/l BAP | 2.43 ± 0.24c | 2.97 ± 0.11a | 0a | 0a |
8.0 mg/l BAP | 1.87 ± 0.30bc | 2.93 ± 0.16a | 0a | 0a |
Data followed by different letters showed significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 30..
Without any additional hormones, cv. wangi
Table 3 and Figure 5 revealed a highly significant difference between treatments of different concentrations of BAP for all parameters observed for cv. putih. Only the control treatment does not induce shoot proliferation among the six hormone concentration treatments. The highest multiplication of shoots was found on MS media containing 2 mg/l BAP, as with cv. wangi. Cultivar putih produced more shoots than cv. wangi, according to the results. Cultivar putih produced 3 to 4 (3.57b ± 0.26) shoots. The intensification in BAP concentrations beyond 2 mg/l has not improved the multiplication of shoots and did not affect the shoot length, root numbers and root length. In contrast to Bogale’s (2018) study, taro cv. Bolosso I supplemented with BAP at concentrations ranging from 4 mg/l to 8 mg/l resulted in 3.83 to 6.13 shoots.
Table 3 . Effects of different concentrations of BAP on number of shoot and root, shoot height and root length of cv. putih after 6 weeks of culture in Murashige & Skoog medium.
Concentrations of BAP | Number of shoots | Shoot height (cm) | Number of roots | Root length (cm) |
---|---|---|---|---|
0 mg/l BAP | 0.03 ± 0.03a | 10.23 ± 0.31d | 12.27 ± 0.40bc | 12.85 ± 0.50d |
0.5 mg/l BAP | 2.27 ± 0.28b | 7.40 ± 0.41c | 12.57 ± 0.62c | 7.90 ± 0.91c |
2.0mg/l BAP | 3.57 ± 0.26d | 5.97 ± 0.34b | 10.67 ± 0.74b | 1.41 ± 0.19b |
4.0 mg/l BAP | 3.10 ± 0.28cd | 4.63 ± 0.32a | 4.43 ± 0.67a | 0.53 ± 0.09ab |
6.0 mg/l BAP | 3.00 ± 0.19cd | 4.43 ± 0.31a | 3.17 ± 0.56a | 0.26 ± 0.05a |
8.0 mg/l BAP | 2.53 ± 0.20bc | 3.90 ± 0.20a | 2.83 ± 0.60a | 0.21 ± 0.04a |
Data followed by different letters showed significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 30..
Root crops culture is difficult to initiate especially if the storage organ such as corm or tuber is being used as starting material. These organs may have contaminated with soil micro flora that become problematic during tissue culture process. In this study, surface sterilization technique does not include mercury (II) chloride that is very toxic to humans. It has been excessively used in explant surface sterilization due to strong sterilization efficiency to kill microbes (soil-borne, epiphytic fungi and bacteria). Hence, the sterilization used in this study gave 0% contamination. The trick behind the success of this sterilization technique is the initial size of explant taken from the corm. The smaller the size of explant, less contamination was found in the culture. If the initial explant size is big around 6-7cm length and 3-4cm width, normally bacterial infection will interfere in the culture. Additional fungicide (benomyl) used in this study also help to reduce fungi contamination.
The survival rate of plantlets transplanted from culture jars to seedling trays was 100% for all substrate media used in this study (Table 4). The difficulties associated with tissue cultured plant survival and growth following transplantation are attributed to the plant’s inability to control water due to the type of media used (Lavanya et al. 2009). Peat was the best substrate media for cv. wangi in this study, with a plant height of 26.34 cm, 12 number of roots with 19.2 cm length. While for cv. putih, a mixture of 1:1 peat and perlite found to be the best with a plant height of 28.76 cm, 11 number of roots with 21.84 cm length (Table 4 & Fig. 6). This response could be attributed to the mixture’s ability to provide adequate moisture and aeration to the plants, resulting in healthy root growth. Perlite is an important component in the potting mixture when mixed with peatmoss. The addition of perlite to peatmoss increases both the amount of oxygen and the amount of water retained by the peatmoss. This obviously improves plant growth conditions (Miller and Donahue 1990). After 4 to 6 weeks planted in the nursery and acclimatized under 50% shades, the plantlets were transferred to the field. Fig. 7 showed the tissue culture plant of cv. wangi and cv. putih that had been successfully planted in the field for eight months.
Table 4 . The effect of different substrate media on survival percentage and growth of cv. wangi and cv. putih attained at week 4 of acclimatization. T1) peat, perlite and vermiculate (1:1:1), T2) peat and vermiculate (1:1), T3) peat and perlite (1:1), and T4) peat.
Type of taro | Substrate composition | Survival (%) | Plant height (cm) | Root length (cm) | No. of roots | Plantlets producing new shoots (%) | Plantlets producing micro-corm (%) |
---|---|---|---|---|---|---|---|
cv. wangi | T1 | 100 | 22.38a ± 1.36 | 17.02a ± 1.40 | 9.72a ± 0.66 | 0ns | 28a ± 10.19 |
T2 | 100 | 26.22b ± 1.13 | 18.14ab ± 0.33 | 11.16bc ± 0.48 | 0ns | 56b ± 11.66 | |
T3 | 100 | 26.74b ± 2.27 | 18.18ab ± 1.19 | 10.28ab ± 0.77 | 0ns | 52ab ± 16.25 | |
T4 | 100 | 26.34b ± 1.44 | 19.2b ± 1.08 | 12.08c ± 0.41 | 4ns | 40ab ± 8.94 | |
cv. putih | T1 | 100 | 25.76a ± 2.40 | 21.26a ± 1.48 | 10.68ns | 28ns | 88ns |
T2 | 100 | 25.8a ± 2.24 | 22.88a ± 1.91 | 10.6ns | 20ns | 100ns | |
T3 | 100 | 28.76b ± 1.20 | 21.84a ± 0.82 | 11.84ns | 12ns | 100ns | |
T4 | 100 | 26.04ab ± 1.75 | 21.26a ± 1.43 | 9.64ns | 16ns | 100ns |
Data followed by different letters showed a significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 25..
An effective protocol needs to be established to provide a rapid technique for mass multiplication of these potential crop cultivars. Initiation of cv. wangi and putih through micropropagation begin after 1 week in culture. At multiplication stage, both cultivars multiplied when cultured in MS medium supplemented with selected cytokinin (BAP/ TDZ) during this study. MS medium supplemented with 2 mg/l BAP showed the highest number of shoots produced for both cultivars. At rooting stage, the highest number of roots was achieved on MS medium without hormone. After acclimatisation, 100% of seedlings were survived in green house. A complete cycle of initiating taro tissue culture takes around 4 to 5 months from surface sterilization to acclimatise before field planting.
This study was financially supported by the Malaysian Agricultural Research and Development Institute (MARDI) under the Eleventh Malaysia Plan (RMKe-11), the Ministry of Agriculture and Food Industries (MAFI), and the international project Benefit-sharing Fund (BSF) under The International Treaty.
Table 1 . Effects of different concentrations of single hormones (BAP and TDZ) on number of shoot and root, shoot height and root length of cv. wangi after 4 weeks of culture in Murashige & Skoog medium.
Concentrations of single hormones (BAP/ TDZ) | Number of shoots | Shoot height (cm) | Number of roots | Root length (cm) |
---|---|---|---|---|
Control | 0.32 ± 0.12a | 13.11 ± 0.36f | 21.25 ± 1.10g | 11.25 ± 0.69e |
0.5 mg/l BAP | 0.96 ± 0.23ab | 4.18 ± 0.61a | 12.14 ± 0.93e | 2.34 ± 0.22cd |
1.0 mg/l BAP | 1.32 ± 0.24bc | 6.21 ± 0.48b | 14.32 ± 1.00f | 2.86 ± 0.21d |
2.0mg/l BAP | 2.10 ± 0.33d | 6.54 ± 0.53bc | 3.46 ± 0.58b | 0.54 ± 0.09a |
3.0 mg/l BAP | 2.04 ± 0.27cd | 7.61 ± 0.47cd | 5.96 ± 0.89c | 0.99 ± 0.14ab |
0.5 mg/l TDZ | 2.18 ± 0.36d | 8.68 ± 0.61d | 9.11 ± 0.99d | 2.45 ± 0.17d |
1.0 mg/l TDZ | 2.0 ± 0.20cd | 10.71 ± 0.40e | 2.04 ± 0.29ab | 2.11 ± 0.21cd |
2.0 mg/l TDZ | 1.50 ± 0.36bcd | 5.64 ± 0.39b | 1.04 ± 0.24a | 1.14 ± 0.2ab |
3.0 mg/l TDZ | 1.46 ± 0.30bcd | 5.22 ± 0.48ab | 1.64 ± 0.33ab | 1.64 ± 0.22bc |
Data followed by different letters showed significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 28..
Table 2 . Effects of different concentrations of BAP on number of shoot and root, shoot height and root length of cv. wangi after 6 weeks of culture in Gamborg B5 media.
BAP concentrations | No. of Shoot | Shoot height (cm) | Root numbers | Root length (cm) |
---|---|---|---|---|
0 mg/l BAP | 0.03 ± 0.03a | 8.1 ± 1.02c | 8.23 ± 0.55c | 10.37 ± 1.20b |
0.5 mg/l BAP | 1.47 ± 0.25b | 4.22 ± 0.16b | 0.93 ± 0.28b | 0.1 ± 0.03a |
2.0mg/l BAP | 2.07 ± 0.41bc | 3.33 ± 0.15ab | 0a | 0a |
4.0 mg/l BAP | 1.73 ± 0.29b | 2.67 ± 0.14a | 0a | 0a |
6.0 mg/l BAP | 2.43 ± 0.24c | 2.97 ± 0.11a | 0a | 0a |
8.0 mg/l BAP | 1.87 ± 0.30bc | 2.93 ± 0.16a | 0a | 0a |
Data followed by different letters showed significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 30..
Table 3 . Effects of different concentrations of BAP on number of shoot and root, shoot height and root length of cv. putih after 6 weeks of culture in Murashige & Skoog medium.
Concentrations of BAP | Number of shoots | Shoot height (cm) | Number of roots | Root length (cm) |
---|---|---|---|---|
0 mg/l BAP | 0.03 ± 0.03a | 10.23 ± 0.31d | 12.27 ± 0.40bc | 12.85 ± 0.50d |
0.5 mg/l BAP | 2.27 ± 0.28b | 7.40 ± 0.41c | 12.57 ± 0.62c | 7.90 ± 0.91c |
2.0mg/l BAP | 3.57 ± 0.26d | 5.97 ± 0.34b | 10.67 ± 0.74b | 1.41 ± 0.19b |
4.0 mg/l BAP | 3.10 ± 0.28cd | 4.63 ± 0.32a | 4.43 ± 0.67a | 0.53 ± 0.09ab |
6.0 mg/l BAP | 3.00 ± 0.19cd | 4.43 ± 0.31a | 3.17 ± 0.56a | 0.26 ± 0.05a |
8.0 mg/l BAP | 2.53 ± 0.20bc | 3.90 ± 0.20a | 2.83 ± 0.60a | 0.21 ± 0.04a |
Data followed by different letters showed significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 30..
Table 4 . The effect of different substrate media on survival percentage and growth of cv. wangi and cv. putih attained at week 4 of acclimatization. T1) peat, perlite and vermiculate (1:1:1), T2) peat and vermiculate (1:1), T3) peat and perlite (1:1), and T4) peat.
Type of taro | Substrate composition | Survival (%) | Plant height (cm) | Root length (cm) | No. of roots | Plantlets producing new shoots (%) | Plantlets producing micro-corm (%) |
---|---|---|---|---|---|---|---|
cv. wangi | T1 | 100 | 22.38a ± 1.36 | 17.02a ± 1.40 | 9.72a ± 0.66 | 0ns | 28a ± 10.19 |
T2 | 100 | 26.22b ± 1.13 | 18.14ab ± 0.33 | 11.16bc ± 0.48 | 0ns | 56b ± 11.66 | |
T3 | 100 | 26.74b ± 2.27 | 18.18ab ± 1.19 | 10.28ab ± 0.77 | 0ns | 52ab ± 16.25 | |
T4 | 100 | 26.34b ± 1.44 | 19.2b ± 1.08 | 12.08c ± 0.41 | 4ns | 40ab ± 8.94 | |
cv. putih | T1 | 100 | 25.76a ± 2.40 | 21.26a ± 1.48 | 10.68ns | 28ns | 88ns |
T2 | 100 | 25.8a ± 2.24 | 22.88a ± 1.91 | 10.6ns | 20ns | 100ns | |
T3 | 100 | 28.76b ± 1.20 | 21.84a ± 0.82 | 11.84ns | 12ns | 100ns | |
T4 | 100 | 26.04ab ± 1.75 | 21.26a ± 1.43 | 9.64ns | 16ns | 100ns |
Data followed by different letters showed a significant difference at P ≤ 0.05. Data represent mean ± standard error, n = 25..
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