J Plant Biotechnol 2018; 45(2): 140-145
Published online June 30, 2018
https://doi.org/10.5010/JPB.2018.45.2.140
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: feridabdul24@gmail.com
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.
Inducing genetic and morphological variation through conventional method is very difficult. Therefore, mutation induction through
Keywords Keywords Tanduk, LD50, mutation breeding, In vitro
Banana and plantains (
Gamma rays are the most commonly used mutagenic agent in an
However, in the recent studies by Karmarkar et al. (2001), who had made an attempt to induce mutation using
The sword sucker of banana cultivar Tanduk (25~35 cm) in height approximately 3~4 months old were used. Shoot tip culture were established and maintained until fourth (M1V4) sub-culture cycle using MS medium supplemented with 5 mg/l BAP. Individual shoot (1~2 cm in length) were isolated and used as explant (Fig. 1). The roots, necrotic tissue and traces of solidifying agent were removed from the explant and then placed in the sterile Petri dish wetted with distilled water. The individual shoot in the Petri dishes were irradiated with different doses of gamma (γ) rays (10, 20, 30, 40, 50, 60 and 70 Gy) using Cesium-137 source at a dose rate of 5.5 Grey/minute to study the radiosensitivity. The total samples of (140) individual shoot were irradiated and the final treatment were selected after this test. The treated explants were then washed thoroughly with sterilized distilled water prior to culture and then cultured aseptically on MS medium fortified with 2 mg/l activated charcoal to regenerate into complete plantlets. The basal medium contained inorganic salts of MS (Murashige and Skoog, 1962) plus Myo-inositol 100 mg/L, Nicotinic Acid 0.5 mg/L, Pyridoxine HCL 0.5mg/L, Thiamine HCl 0.5 mg/L, Glycine 2.00 mg/L and sucrose 30 g/L. The pH was adjusted to 5.7 prior to adding Agar (7 g/L). All cultures were incubated in the culture room at 25 ± 2°C and 55~65% relative humidity with photoperiod of 16 hours light under fluorescent light (1000 lux) and 8 hours dark. After 4 weeks, well grown rooted plantlets (7~9 cm in length) were carefully removed from the culture vessel and then the roots were washed thoroughly in tap water to remove the traces of nutrients and were transplanted in the greenhouse in polybags containing a ratio 2:1 mixture of peat moss and coco peat.
Individual shoot explant.
The LD50 values were determined based on the percentage of surviving of explants. Post-irradiation rooting responses of
In the present study, the survival rate of irradiated explants was found to decrease linearly with the increasing of the gamma doses (Fig. 2). A remarkable decrease in survival rate was observed at 40, 50, 60 and 70 Gy of gamma dosage. Meanwhile, among the treated explants maximum survival rate of 71% was recorded at 10 Gy, followed by 69.5% at a dose of 20 Gy, 67 % at 30 Gy and minimum of survival rate was observed following the T7 (70 Gy) treatment, where remarkably dropped from 100% to 42% at the first week of culture and 15.5% at the fourth weeks of culture resulting in a total reduction of 74.5%. The non- irradiated explant recorded the highest survival percentage of 100%. The lethal dose (LD50) was found to be 33 Gy based on the survival percentage, the estimation was carried out using linear regression analysis. Karmarkar et al. 2001 had made similar observations in cv. Basrai (AAA) which corroborate our finding.In principle, the most effective doses for mutation induction are thought to be the gamma doses lower than the LD50. As a result of these measurements, a large quantity of individual shoot was finally treated with 10, 20 and 30 Gy doses of gamma radiation and then the explants were transferred on rooting medium.
Radiosensitivity test curve illustrating the effect of increasing dose of gamma rays on survival rate of individual
The shoot length reduced prominently with increase in gamma irradiation doses (Table 1). The shoot length was significantly lower at higher dose (30 Gy), however lower gamma doses at 10 and 20 Gy showed no significance difference over the control. The highest shoot length (13.6 cm) was observed at control treatment, followed by 10 Gy (12.7 cm), 20 Gy (11.6 cm), and the shortest shoot length (10.4 cm) was observed at 30 Gy (Fig. 3). As reported by Majeed et al. (2009) and Khalil et al. (1986), reduction of a shoot and root growth at higher doses of gamma rays is due to reduced mitotic activity in meristematic tissues and reduced moisture contents of explants. In agreement with the present investigations, Al-Salhi et al. (2004) observed that decrease in shoot length is in proportion with increasing gamma dose in corn
Table 1 Effect of gamma irradiation on the
Gamma treatment | Growth parameter | ||||||
---|---|---|---|---|---|---|---|
Shoot length | Leaf number | Days to root initiation | Root number | Root length | Plant height | Chlorophyl 1 (SPAD) | |
T0: Control | 13.6±0.6a | 7.01±0.5a | 7.2±0.3a | 20.02±1.4a | 15.1±0.5a | 27.8±1.7a | 44.2±2.1b |
T1: 10 Gy | 12.7±0.1ab | 7.02±0.3a | 11.1±1.1b | 18.3±1.3a | 13.2±0.3b | 25.5±0.5a | 49.8±3.2a |
T2: 20 Gy | 11.6±0.2bc | 5.00±0.6ab | 12.5±1.2b | 12.1±1.3b | 12.5±0.2b | 24.4±0.3a | 42.4±3.2bc |
T3: 30 Gy | 10.4±0.2c | 4.99±0.4c | 12.3±2.5b | 8.2±0.40c | 10.1±0.4c | 21.2±0.4b | 39.6±3.0c |
Means sharing the same letters within a column are not significantly different from each other at
Irradiated and rooted
Significant difference was observed in rooting of explants subjected to the higher dose of γ (30 Gy) (Table 1). It was observed that increase in gamma irradiation dose resulted inhibition of root growth. Non-treated explants had the best rooting ability with the maximum number of root per explant (20) within a short period of time (6 d), with the highest length of a root (15.1 cm). The longer period in rooting (12 d) and lowest number of root per explant (8) with shortest root length (10.1cm) were recorded at 30 Gy treatment. Generally, the non-treated explants exhibited better rooting performance in all root growth parameter. This could be due to perturbations of hormones balance inside the explant and the activity of hormone synthesized and transported from tip to the site of action or exogenous supply of auxin used for rooting. The inhibition of rooting at higher gamma doses have been reported by several researchers (Karmarkar et al. 2001, Wi et al. 2007 and Kiong et al. 2008) who described that rise of radiation dose increases plants sensitivity to gamma rays effect and reduces the amount of endogenous growth regulators, particularly the auxin and cytokines. This would resulted breakdown, or lack of synthesis and or disruption of hormonal balance and enzymatic activities. In contrast, Muthusamy and Jayabalan, (2014) reported that the days to shoot and root initiation was faster when the explants exposed to gamma radiation at a lower dose in cotton seed.
Acclimatizaion of irradiated plantlets
From the data obtained the leaf formation of irradiated plantlets with 10 Gy and 20 Gy was not significantly different over the control but treated explants with 30 Gy showed significant difference with that of control. The maximum number of leaves (7) was recorded at control plants. The lowest leaf number (5) was observed at 30 Gy compared with all treatments. Leaves number decreased in proportion with increasing gamma dose. All explants exposed to gamma dose showed a reducing trend in leaves number and found to be inhibitory. From the data obtained, gamma radiation had no significant impact on leaf number in all treated explant rather it showed reduction trend in leaf number as the gamma dose increases. In contradiction with earlier findings, Yadav, (2016) had observed highly significant impact on leaf number in Cv
The effect of gamma irradiation on chlorophyll content showed 10 Gy showed significant diffence over the control treatment (Table 1). The highest chlorophyll content (49.8) was recorded at 10 Gy treated plantlets, followed by 44.6, 42.4 and 39.6 in treatment 20 Gy, 30 Gy, and control respectively. Our experiments are consistent with recent studies by Sakr et al. (2013) stated that exposure of
In conclusion, the result from the current experiment indicated that γ treatments induced morphological and physiological variations on banana cultivar Pisang Tanduk. Lower dose of γ irradiation induced positive impact on the growth of in vitro plantlets during the multiplication phase and rooting stage, whereas higher γ irradiation had caused a negative impact on the growth, in terms of higher mortality rate and reduction in growth.
Therefore, we suggested that γ radiation can be usefully employed in banana for induction of desirable mutations and further, work on field performance of mutants, in terms of early flowering, yield quantity, quality and other agronomical traits need to be looking next.
The author would like to acknowledge Islamic Development Bank Group (IDBG) for generous funding of this research.
J Plant Biotechnol 2018; 45(2): 140-145
Published online June 30, 2018 https://doi.org/10.5010/JPB.2018.45.2.140
Copyright © The Korean Society of Plant Biotechnology.
Ferid Abdulhafiz, Fatimah Kayat, and Suhana Zakaria
Faculty of Agro Based Industry, University Malaysia Kelantan, 17600, Jeli, Kelantan, Malaysia
Correspondence to:e-mail: feridabdul24@gmail.com
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.
Inducing genetic and morphological variation through conventional method is very difficult. Therefore, mutation induction through
Keywords: Keywords Tanduk, LD50, mutation breeding, In vitro
Banana and plantains (
Gamma rays are the most commonly used mutagenic agent in an
However, in the recent studies by Karmarkar et al. (2001), who had made an attempt to induce mutation using
The sword sucker of banana cultivar Tanduk (25~35 cm) in height approximately 3~4 months old were used. Shoot tip culture were established and maintained until fourth (M1V4) sub-culture cycle using MS medium supplemented with 5 mg/l BAP. Individual shoot (1~2 cm in length) were isolated and used as explant (Fig. 1). The roots, necrotic tissue and traces of solidifying agent were removed from the explant and then placed in the sterile Petri dish wetted with distilled water. The individual shoot in the Petri dishes were irradiated with different doses of gamma (γ) rays (10, 20, 30, 40, 50, 60 and 70 Gy) using Cesium-137 source at a dose rate of 5.5 Grey/minute to study the radiosensitivity. The total samples of (140) individual shoot were irradiated and the final treatment were selected after this test. The treated explants were then washed thoroughly with sterilized distilled water prior to culture and then cultured aseptically on MS medium fortified with 2 mg/l activated charcoal to regenerate into complete plantlets. The basal medium contained inorganic salts of MS (Murashige and Skoog, 1962) plus Myo-inositol 100 mg/L, Nicotinic Acid 0.5 mg/L, Pyridoxine HCL 0.5mg/L, Thiamine HCl 0.5 mg/L, Glycine 2.00 mg/L and sucrose 30 g/L. The pH was adjusted to 5.7 prior to adding Agar (7 g/L). All cultures were incubated in the culture room at 25 ± 2°C and 55~65% relative humidity with photoperiod of 16 hours light under fluorescent light (1000 lux) and 8 hours dark. After 4 weeks, well grown rooted plantlets (7~9 cm in length) were carefully removed from the culture vessel and then the roots were washed thoroughly in tap water to remove the traces of nutrients and were transplanted in the greenhouse in polybags containing a ratio 2:1 mixture of peat moss and coco peat.
Individual shoot explant.
The LD50 values were determined based on the percentage of surviving of explants. Post-irradiation rooting responses of
In the present study, the survival rate of irradiated explants was found to decrease linearly with the increasing of the gamma doses (Fig. 2). A remarkable decrease in survival rate was observed at 40, 50, 60 and 70 Gy of gamma dosage. Meanwhile, among the treated explants maximum survival rate of 71% was recorded at 10 Gy, followed by 69.5% at a dose of 20 Gy, 67 % at 30 Gy and minimum of survival rate was observed following the T7 (70 Gy) treatment, where remarkably dropped from 100% to 42% at the first week of culture and 15.5% at the fourth weeks of culture resulting in a total reduction of 74.5%. The non- irradiated explant recorded the highest survival percentage of 100%. The lethal dose (LD50) was found to be 33 Gy based on the survival percentage, the estimation was carried out using linear regression analysis. Karmarkar et al. 2001 had made similar observations in cv. Basrai (AAA) which corroborate our finding.In principle, the most effective doses for mutation induction are thought to be the gamma doses lower than the LD50. As a result of these measurements, a large quantity of individual shoot was finally treated with 10, 20 and 30 Gy doses of gamma radiation and then the explants were transferred on rooting medium.
Radiosensitivity test curve illustrating the effect of increasing dose of gamma rays on survival rate of individual
The shoot length reduced prominently with increase in gamma irradiation doses (Table 1). The shoot length was significantly lower at higher dose (30 Gy), however lower gamma doses at 10 and 20 Gy showed no significance difference over the control. The highest shoot length (13.6 cm) was observed at control treatment, followed by 10 Gy (12.7 cm), 20 Gy (11.6 cm), and the shortest shoot length (10.4 cm) was observed at 30 Gy (Fig. 3). As reported by Majeed et al. (2009) and Khalil et al. (1986), reduction of a shoot and root growth at higher doses of gamma rays is due to reduced mitotic activity in meristematic tissues and reduced moisture contents of explants. In agreement with the present investigations, Al-Salhi et al. (2004) observed that decrease in shoot length is in proportion with increasing gamma dose in corn
Table 1 . Effect of gamma irradiation on the
Gamma treatment | Growth parameter | ||||||
---|---|---|---|---|---|---|---|
Shoot length | Leaf number | Days to root initiation | Root number | Root length | Plant height | Chlorophyl 1 (SPAD) | |
T0: Control | 13.6±0.6a | 7.01±0.5a | 7.2±0.3a | 20.02±1.4a | 15.1±0.5a | 27.8±1.7a | 44.2±2.1b |
T1: 10 Gy | 12.7±0.1ab | 7.02±0.3a | 11.1±1.1b | 18.3±1.3a | 13.2±0.3b | 25.5±0.5a | 49.8±3.2a |
T2: 20 Gy | 11.6±0.2bc | 5.00±0.6ab | 12.5±1.2b | 12.1±1.3b | 12.5±0.2b | 24.4±0.3a | 42.4±3.2bc |
T3: 30 Gy | 10.4±0.2c | 4.99±0.4c | 12.3±2.5b | 8.2±0.40c | 10.1±0.4c | 21.2±0.4b | 39.6±3.0c |
Means sharing the same letters within a column are not significantly different from each other at
Irradiated and rooted
Significant difference was observed in rooting of explants subjected to the higher dose of γ (30 Gy) (Table 1). It was observed that increase in gamma irradiation dose resulted inhibition of root growth. Non-treated explants had the best rooting ability with the maximum number of root per explant (20) within a short period of time (6 d), with the highest length of a root (15.1 cm). The longer period in rooting (12 d) and lowest number of root per explant (8) with shortest root length (10.1cm) were recorded at 30 Gy treatment. Generally, the non-treated explants exhibited better rooting performance in all root growth parameter. This could be due to perturbations of hormones balance inside the explant and the activity of hormone synthesized and transported from tip to the site of action or exogenous supply of auxin used for rooting. The inhibition of rooting at higher gamma doses have been reported by several researchers (Karmarkar et al. 2001, Wi et al. 2007 and Kiong et al. 2008) who described that rise of radiation dose increases plants sensitivity to gamma rays effect and reduces the amount of endogenous growth regulators, particularly the auxin and cytokines. This would resulted breakdown, or lack of synthesis and or disruption of hormonal balance and enzymatic activities. In contrast, Muthusamy and Jayabalan, (2014) reported that the days to shoot and root initiation was faster when the explants exposed to gamma radiation at a lower dose in cotton seed.
Acclimatizaion of irradiated plantlets
From the data obtained the leaf formation of irradiated plantlets with 10 Gy and 20 Gy was not significantly different over the control but treated explants with 30 Gy showed significant difference with that of control. The maximum number of leaves (7) was recorded at control plants. The lowest leaf number (5) was observed at 30 Gy compared with all treatments. Leaves number decreased in proportion with increasing gamma dose. All explants exposed to gamma dose showed a reducing trend in leaves number and found to be inhibitory. From the data obtained, gamma radiation had no significant impact on leaf number in all treated explant rather it showed reduction trend in leaf number as the gamma dose increases. In contradiction with earlier findings, Yadav, (2016) had observed highly significant impact on leaf number in Cv
The effect of gamma irradiation on chlorophyll content showed 10 Gy showed significant diffence over the control treatment (Table 1). The highest chlorophyll content (49.8) was recorded at 10 Gy treated plantlets, followed by 44.6, 42.4 and 39.6 in treatment 20 Gy, 30 Gy, and control respectively. Our experiments are consistent with recent studies by Sakr et al. (2013) stated that exposure of
In conclusion, the result from the current experiment indicated that γ treatments induced morphological and physiological variations on banana cultivar Pisang Tanduk. Lower dose of γ irradiation induced positive impact on the growth of in vitro plantlets during the multiplication phase and rooting stage, whereas higher γ irradiation had caused a negative impact on the growth, in terms of higher mortality rate and reduction in growth.
Therefore, we suggested that γ radiation can be usefully employed in banana for induction of desirable mutations and further, work on field performance of mutants, in terms of early flowering, yield quantity, quality and other agronomical traits need to be looking next.
The author would like to acknowledge Islamic Development Bank Group (IDBG) for generous funding of this research.
Individual shoot explant.
Radiosensitivity test curve illustrating the effect of increasing dose of gamma rays on survival rate of individual
Irradiated and rooted
Acclimatizaion of irradiated plantlets
Table 1 . Effect of gamma irradiation on the
Gamma treatment | Growth parameter | ||||||
---|---|---|---|---|---|---|---|
Shoot length | Leaf number | Days to root initiation | Root number | Root length | Plant height | Chlorophyl 1 (SPAD) | |
T0: Control | 13.6±0.6a | 7.01±0.5a | 7.2±0.3a | 20.02±1.4a | 15.1±0.5a | 27.8±1.7a | 44.2±2.1b |
T1: 10 Gy | 12.7±0.1ab | 7.02±0.3a | 11.1±1.1b | 18.3±1.3a | 13.2±0.3b | 25.5±0.5a | 49.8±3.2a |
T2: 20 Gy | 11.6±0.2bc | 5.00±0.6ab | 12.5±1.2b | 12.1±1.3b | 12.5±0.2b | 24.4±0.3a | 42.4±3.2bc |
T3: 30 Gy | 10.4±0.2c | 4.99±0.4c | 12.3±2.5b | 8.2±0.40c | 10.1±0.4c | 21.2±0.4b | 39.6±3.0c |
Means sharing the same letters within a column are not significantly different from each other at
Journal of
Plant BiotechnologyIndividual shoot explant.
|@|~(^,^)~|@|Radiosensitivity test curve illustrating the effect of increasing dose of gamma rays on survival rate of individual
Irradiated and rooted
Acclimatizaion of irradiated plantlets