J Plant Biotechnol 2020; 47(3): 235-241
Published online September 30, 2020
https://doi.org/10.5010/JPB.2020.47.3.235
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: loandhhv@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.
This is the first study to establish a complete protocol for micropropagation of Rehmannia glutinosa from root segments. The study involved investigating the effect of plant growth regulators on in vitro shoot regeneration and rooting and identifying substrates supporting survival and growth performance of ex vitro seedlings. A Murashige and Skoog (MS) medium containing 30 g/L sucrose for shoot induction and 0.2 mg/L indole-3-acetic acid (IAA), 1 mg/L 6-benzylaminopurine (BAP), and 1 g/L polyvinylpyrrolidone (PVP) for shoot multiplication resulted in the highest number of shoots per explant and shoot height. Applying a medium containing 0.5 mg/L IAA and 1 g/L PVP yielded optimal rooting of the shoots grown in vitro. Compost enriched with microbial inoculants and perlite enhanced seedling growth better than that with organic biofertilizer-free substrates (soil and sand). We recommend the continuous production of micropropagated R. glutinosa seedlings from root segments under the aforementioned conditions as a possible propagation technique for crops of this species.
Keywords IAA, Multiple shoots, NAA, Rehmannia, PVP, Root induction, Tissue culture
In Vietnam, the market demand for medicinal materials from
Few studies developed micropropagation protocols that produced seedlings from leaf mesophyll protoplast (Xu and Davey 1983), leaves (Sang et al. 2009), shoot tips (Shoyama et al. 1983), auxiliary buds (Piątczak et al. 2014b). A recent study has developed a protocol to produced calli with subsequent regeneration of the plant, followed by genetic analysis of the regenerated plants (Piątczak et al. 2014a). The results demonstrated that the regenerative capability accumulation of bioactive metabolites to be reserved for four years of culture. So far, none of the developed
The sterilized root segments excised from
The shoot multiplication experiment was carried out after the success of the initiation phase. Initially, shoot tip excised from the explants were inoculated in MS medium supplemented with 30 g/L sucrose, 7 g/L agar, pH 5.8, and with various concentrations (0.0, 0.3, 0.5, 1.0, and 1.5 mg/L) of BAP (Bio Basic, Canada). The optimal BAP concentration was then selected to final shoot multiplication experiment in which, 1 g/L PVP (Fisher Chemicals, Germany) and various concentrations (0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/L) of IAA (MERCH, Germany) were added to investigate the effect of auxin on shoot multiplication. After four weeks, shoot multiplication rate and shoot height were recorded.
The rooting experiment was carried out after the success of the shoot multiplication phase. Selected
Four-week-old rooted seedlings were transferred from rooting cultured environment into pots with five different growing substrate compositions, including 50% alluvium soil + 50% sand (ASS); 50% alluvium soil + 50% rice husk (ASRH); a compost including organic biofertilizer: a mixture of soil and sand (3:2) topped with 3~4 cm sand layer (COSS); 100% carbonized rice husk (RH); and 90% compost + 10% perlite (COP). Organic biofertilizer was based on chicken manure, sawdust, and coconut peat, in a ratio of 1: 4: 4 (v/v) with the addition of microbial inoculants including
Observed data were analyzed using Microsoft Excel and Irristart 5.0 software. Means and standard errors were applied to assess the experiment results using the ANOVA test at P < 0.05. Treatments were designed randomly with three replicates.
The results from Table 1 showed that all three types of basal media-induced multiple shoots effectively and were significantly different (P ≤ 0.05). The highest percentage of shooting explants (56%) and the number of shoots per explant (5.05) were observed in MS medium. The lowest results were obtained for VW medium with 24.66 % explant with shoots and 3.69 shoots per explant (Fig. 1A). Some previous
Table 1 Differences in multiple shoot induction in
Medium | Shooting explant | Shooting rate | Average number of shoots per explant |
---|---|---|---|
MS | 28.00a | 56.00% | 5.02a |
Knudson | 15.33b | 30.66% | 4.12a |
VW | 12.33c | 24.66% | 3.69c |
Each basal medium was supplemented with 30 g/L sucrose. Different letters (a, b, c) indicate significant differences in shooting between media (P ≤ 0.05).
The results from Table 2 showed that the highest rate of multiplication and the average height of shoots were observed in explants treated by BAP 1.0 mg/L. However, the quality of shoots generated was under standard and characterized by the abnormal morphological shape of shoots, yellow color of leaves, and several symptoms of mosaic phenomena. This event was probably due to phytotoxins (some phenolic acids) released during the explant excising process (Du et al. 2009). Therefore, PVP was added to the medium to eliminate the toxicity of possible phenolic acids. The results showed a positive effect on the growth and quality of regenerated shoots, as shown in Table 3.
Table 2 Comparison of shooting
Cytokinin (mg/L) | Shoot multiplication | Shoot height (cm) |
---|---|---|
Control (BAP-free) | 2.05g | 2.32f |
BAP 0.3 | 2.37f | 2.84d |
BAP 0.5 | 2.87bc | 3.08c |
BAP 1.0 | 3.09a | 3.50a |
BAP 1.5 | 2.90b | 3.22b |
The MS medium was supplemented with 30 g/L sucrose, 7 g/L agar, and with various concentrations (0.0, 0.3, 0.5, 1.0 and 1.5 mg/L) of BAP and maintained at a pH of 5.8. Different letters (a, b, c) indicate significant differences between media (P ≤ 0.05).
Table 3 Comparison of shooting
Auxin (mg/L) | Shoot multiplication | Shoot height (cm) |
---|---|---|
Control (auxin-free) | 2.86f | 2.94f |
IAA 0.1 | 3.95c | 4.46c |
IAA 0.2 | 4.74a | 5.16a |
IAA 0.3 | 4.39b | 5.03b |
IAA 0.4 | 3.79d | 4.26d |
IAA 0.5 | 3.60e | 3.81e |
The control included MS + 1 g/L PVP + 1mg/L BAP. Different letters (a, b, c) indicate significant differences between media (P ≤ 0.05).
Shoot tips were excised from shooting explants and cultured on MS medium with various concentrations of IAA (0.1, 0.2, 0.3, 0.4, and 0.5 mg/L) in combination with 1.0 mg/L BAP containing 1 g/L PVP to promote regeneration of adventitious shoots. The results showed that the effect of IAA with low concentrations (0.1, 0.2, and 0.3 mg/L) substantially induced multiple shoots. In contrast, the higher concentration of IAA (0.4 and 0.5 mg/L) significantly decreased the shoot multiplication (Fig. 1B). The highest number of shoots (4.74) and shoot height (5.16 cm) was recorded when shoot tip explants were supplemented with 0.2 mg/L IAA, whereas the lowest number of shoots (2.86), as well as shoot height (2.94 cm), were observed in the auxin-free medium. These above variations were statistically different (P ≤ 0.05). The results indicated that plant growth regulators proved their essential role in promoting multiple shoots. The effect of a combination of BAP and IAA (1.0 mg/L and 0.2 mg/L, respectively) was more effective in shoot multiplication in
In rooting experiments, PVP was also supplemented to the media to eliminate browning and abnormal morphological characteristics of plantlets caused by phenolic acids and other toxins from excised explants. Each shoots induced in ½ MS medium with 30 g/L sucrose, 7 g/L agarose, pH 5.8, 1 g/L PVP, and with either NAA or IAA. Root induction was observed after three weeks. The results presented in Table 4 showed that the addition of growth regulators and PVP positively affected to the rooting of
Table 4 Comparison of
Auxin (mg/L) | Number of rooting plantlets | Number of roots per plantlet | Root length (cm) | Plant height (cm) |
---|---|---|---|---|
Control (growth regulator-free) | 43b | 10.37f | 1.36d | 3.52d |
NAA 0.3 | 45a | 22.24e | 1.59c | 3.33e |
NAA 0.5 | 45a | 22.60e | 1.74b | 4.25a |
NAA 1.0 | 45a | 23.33d | 2.09a | 3.60d |
NAA 1.5 | 45a | 22.00e | 2.11a | 3.82c |
IAA 0.3 | 45a | 24.32c | 1.52c | 3.21f |
IAA 0.5 | 45a | 26.78a | 1.76b | 4.05b |
IAA 1.0 | 45a | 25.68b | 1.25e | 3.28ef |
IAA 1.5 | 45a | 24.72c | 1.14f | 3.12f |
The control included half-strength MS + 1 g/L PVP. Different letters (a, b, c) indicated significant differences between media (P ≤ 0.05).
Nutrients are the most important factor when transplanting plantlets from
Table 5 Comparison of survival rate and growth of plantlets by the acclimatization medium used
Acclimatization medium | Survival rate (%) | Average number of leaves per plant | Leaf length (cm) | Plant height (cm) |
---|---|---|---|---|
50% alluvium soil + 50% sand | 51.66e | 4.95e | 2.18e | 6.81e |
50% alluvium soil + 50% rice husk | 54.33d | 5.17d | 2.32d | 7.25d |
Compost : soil + sand (3:2)* | 74.67b | 5.61c | 2.65c | 7.56c |
100% rice husk | 65.00c | 6.27b | 3.08b | 8.00b |
90% Compost + 10% perlite | 88.00a | 7.00a | 3.32a | 8.26a |
*Mixture of organic biofertilizer (compost): a mixture of soil and sand (3:2), topped with a 3~4 cm layer of sand. The organic biofertilizer was based on chicken manure, sawdust, and coconut peat in a ratio of 1:4:4 (v/v) with the addition of microbial inoculants (CP, Thailand). Different letters (a, b, c) indicate significant differences between media (P ≤ 0.05).
The present study assessed for the first time, the effect of principal factors involved in a complete process of
The authors are grateful to the Institute of Applied Research and Development, Hung Vuong University, Vietnam and the scientific and technological project “Study and develop
J Plant Biotechnol 2020; 47(3): 235-241
Published online September 30, 2020 https://doi.org/10.5010/JPB.2020.47.3.235
Copyright © The Korean Society of Plant Biotechnology.
Thanh Loan Pham ・Van Huy Nguyen ・Thi Le Thu Hoang ・Thi Tam Tien Ha ・Trung Kien Tran ・ Xuan Duong Vu ・Phi Bang Cao ・Quang Trung Nguyen
Institute of Applied Research and Development, Hung Vuong University, Phu Tho, Vietnam
Faculty of Natural Sciences, Hung Vuong University, Phu Tho, Vietnam
Faculty of Agriculture, Hung Vuong University, Phu Tho, Vietnam
Correspondence to:e-mail: loandhhv@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.
This is the first study to establish a complete protocol for micropropagation of Rehmannia glutinosa from root segments. The study involved investigating the effect of plant growth regulators on in vitro shoot regeneration and rooting and identifying substrates supporting survival and growth performance of ex vitro seedlings. A Murashige and Skoog (MS) medium containing 30 g/L sucrose for shoot induction and 0.2 mg/L indole-3-acetic acid (IAA), 1 mg/L 6-benzylaminopurine (BAP), and 1 g/L polyvinylpyrrolidone (PVP) for shoot multiplication resulted in the highest number of shoots per explant and shoot height. Applying a medium containing 0.5 mg/L IAA and 1 g/L PVP yielded optimal rooting of the shoots grown in vitro. Compost enriched with microbial inoculants and perlite enhanced seedling growth better than that with organic biofertilizer-free substrates (soil and sand). We recommend the continuous production of micropropagated R. glutinosa seedlings from root segments under the aforementioned conditions as a possible propagation technique for crops of this species.
Keywords: IAA, Multiple shoots, NAA, Rehmannia, PVP, Root induction, Tissue culture
In Vietnam, the market demand for medicinal materials from
Few studies developed micropropagation protocols that produced seedlings from leaf mesophyll protoplast (Xu and Davey 1983), leaves (Sang et al. 2009), shoot tips (Shoyama et al. 1983), auxiliary buds (Piątczak et al. 2014b). A recent study has developed a protocol to produced calli with subsequent regeneration of the plant, followed by genetic analysis of the regenerated plants (Piątczak et al. 2014a). The results demonstrated that the regenerative capability accumulation of bioactive metabolites to be reserved for four years of culture. So far, none of the developed
The sterilized root segments excised from
The shoot multiplication experiment was carried out after the success of the initiation phase. Initially, shoot tip excised from the explants were inoculated in MS medium supplemented with 30 g/L sucrose, 7 g/L agar, pH 5.8, and with various concentrations (0.0, 0.3, 0.5, 1.0, and 1.5 mg/L) of BAP (Bio Basic, Canada). The optimal BAP concentration was then selected to final shoot multiplication experiment in which, 1 g/L PVP (Fisher Chemicals, Germany) and various concentrations (0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/L) of IAA (MERCH, Germany) were added to investigate the effect of auxin on shoot multiplication. After four weeks, shoot multiplication rate and shoot height were recorded.
The rooting experiment was carried out after the success of the shoot multiplication phase. Selected
Four-week-old rooted seedlings were transferred from rooting cultured environment into pots with five different growing substrate compositions, including 50% alluvium soil + 50% sand (ASS); 50% alluvium soil + 50% rice husk (ASRH); a compost including organic biofertilizer: a mixture of soil and sand (3:2) topped with 3~4 cm sand layer (COSS); 100% carbonized rice husk (RH); and 90% compost + 10% perlite (COP). Organic biofertilizer was based on chicken manure, sawdust, and coconut peat, in a ratio of 1: 4: 4 (v/v) with the addition of microbial inoculants including
Observed data were analyzed using Microsoft Excel and Irristart 5.0 software. Means and standard errors were applied to assess the experiment results using the ANOVA test at P < 0.05. Treatments were designed randomly with three replicates.
The results from Table 1 showed that all three types of basal media-induced multiple shoots effectively and were significantly different (P ≤ 0.05). The highest percentage of shooting explants (56%) and the number of shoots per explant (5.05) were observed in MS medium. The lowest results were obtained for VW medium with 24.66 % explant with shoots and 3.69 shoots per explant (Fig. 1A). Some previous
Table 1 . Differences in multiple shoot induction in
Medium | Shooting explant | Shooting rate | Average number of shoots per explant |
---|---|---|---|
MS | 28.00a | 56.00% | 5.02a |
Knudson | 15.33b | 30.66% | 4.12a |
VW | 12.33c | 24.66% | 3.69c |
Each basal medium was supplemented with 30 g/L sucrose. Different letters (a, b, c) indicate significant differences in shooting between media (P ≤ 0.05)..
The results from Table 2 showed that the highest rate of multiplication and the average height of shoots were observed in explants treated by BAP 1.0 mg/L. However, the quality of shoots generated was under standard and characterized by the abnormal morphological shape of shoots, yellow color of leaves, and several symptoms of mosaic phenomena. This event was probably due to phytotoxins (some phenolic acids) released during the explant excising process (Du et al. 2009). Therefore, PVP was added to the medium to eliminate the toxicity of possible phenolic acids. The results showed a positive effect on the growth and quality of regenerated shoots, as shown in Table 3.
Table 2 . Comparison of shooting
Cytokinin (mg/L) | Shoot multiplication | Shoot height (cm) |
---|---|---|
Control (BAP-free) | 2.05g | 2.32f |
BAP 0.3 | 2.37f | 2.84d |
BAP 0.5 | 2.87bc | 3.08c |
BAP 1.0 | 3.09a | 3.50a |
BAP 1.5 | 2.90b | 3.22b |
The MS medium was supplemented with 30 g/L sucrose, 7 g/L agar, and with various concentrations (0.0, 0.3, 0.5, 1.0 and 1.5 mg/L) of BAP and maintained at a pH of 5.8. Different letters (a, b, c) indicate significant differences between media (P ≤ 0.05)..
Table 3 . Comparison of shooting
Auxin (mg/L) | Shoot multiplication | Shoot height (cm) |
---|---|---|
Control (auxin-free) | 2.86f | 2.94f |
IAA 0.1 | 3.95c | 4.46c |
IAA 0.2 | 4.74a | 5.16a |
IAA 0.3 | 4.39b | 5.03b |
IAA 0.4 | 3.79d | 4.26d |
IAA 0.5 | 3.60e | 3.81e |
The control included MS + 1 g/L PVP + 1mg/L BAP. Different letters (a, b, c) indicate significant differences between media (P ≤ 0.05)..
Shoot tips were excised from shooting explants and cultured on MS medium with various concentrations of IAA (0.1, 0.2, 0.3, 0.4, and 0.5 mg/L) in combination with 1.0 mg/L BAP containing 1 g/L PVP to promote regeneration of adventitious shoots. The results showed that the effect of IAA with low concentrations (0.1, 0.2, and 0.3 mg/L) substantially induced multiple shoots. In contrast, the higher concentration of IAA (0.4 and 0.5 mg/L) significantly decreased the shoot multiplication (Fig. 1B). The highest number of shoots (4.74) and shoot height (5.16 cm) was recorded when shoot tip explants were supplemented with 0.2 mg/L IAA, whereas the lowest number of shoots (2.86), as well as shoot height (2.94 cm), were observed in the auxin-free medium. These above variations were statistically different (P ≤ 0.05). The results indicated that plant growth regulators proved their essential role in promoting multiple shoots. The effect of a combination of BAP and IAA (1.0 mg/L and 0.2 mg/L, respectively) was more effective in shoot multiplication in
In rooting experiments, PVP was also supplemented to the media to eliminate browning and abnormal morphological characteristics of plantlets caused by phenolic acids and other toxins from excised explants. Each shoots induced in ½ MS medium with 30 g/L sucrose, 7 g/L agarose, pH 5.8, 1 g/L PVP, and with either NAA or IAA. Root induction was observed after three weeks. The results presented in Table 4 showed that the addition of growth regulators and PVP positively affected to the rooting of
Table 4 . Comparison of
Auxin (mg/L) | Number of rooting plantlets | Number of roots per plantlet | Root length (cm) | Plant height (cm) |
---|---|---|---|---|
Control (growth regulator-free) | 43b | 10.37f | 1.36d | 3.52d |
NAA 0.3 | 45a | 22.24e | 1.59c | 3.33e |
NAA 0.5 | 45a | 22.60e | 1.74b | 4.25a |
NAA 1.0 | 45a | 23.33d | 2.09a | 3.60d |
NAA 1.5 | 45a | 22.00e | 2.11a | 3.82c |
IAA 0.3 | 45a | 24.32c | 1.52c | 3.21f |
IAA 0.5 | 45a | 26.78a | 1.76b | 4.05b |
IAA 1.0 | 45a | 25.68b | 1.25e | 3.28ef |
IAA 1.5 | 45a | 24.72c | 1.14f | 3.12f |
The control included half-strength MS + 1 g/L PVP. Different letters (a, b, c) indicated significant differences between media (P ≤ 0.05)..
Nutrients are the most important factor when transplanting plantlets from
Table 5 . Comparison of survival rate and growth of plantlets by the acclimatization medium used.
Acclimatization medium | Survival rate (%) | Average number of leaves per plant | Leaf length (cm) | Plant height (cm) |
---|---|---|---|---|
50% alluvium soil + 50% sand | 51.66e | 4.95e | 2.18e | 6.81e |
50% alluvium soil + 50% rice husk | 54.33d | 5.17d | 2.32d | 7.25d |
Compost : soil + sand (3:2)* | 74.67b | 5.61c | 2.65c | 7.56c |
100% rice husk | 65.00c | 6.27b | 3.08b | 8.00b |
90% Compost + 10% perlite | 88.00a | 7.00a | 3.32a | 8.26a |
*Mixture of organic biofertilizer (compost): a mixture of soil and sand (3:2), topped with a 3~4 cm layer of sand. The organic biofertilizer was based on chicken manure, sawdust, and coconut peat in a ratio of 1:4:4 (v/v) with the addition of microbial inoculants (CP, Thailand). Different letters (a, b, c) indicate significant differences between media (P ≤ 0.05)..
The present study assessed for the first time, the effect of principal factors involved in a complete process of
The authors are grateful to the Institute of Applied Research and Development, Hung Vuong University, Vietnam and the scientific and technological project “Study and develop
Table 1 . Differences in multiple shoot induction in
Medium | Shooting explant | Shooting rate | Average number of shoots per explant |
---|---|---|---|
MS | 28.00a | 56.00% | 5.02a |
Knudson | 15.33b | 30.66% | 4.12a |
VW | 12.33c | 24.66% | 3.69c |
Each basal medium was supplemented with 30 g/L sucrose. Different letters (a, b, c) indicate significant differences in shooting between media (P ≤ 0.05)..
Table 2 . Comparison of shooting
Cytokinin (mg/L) | Shoot multiplication | Shoot height (cm) |
---|---|---|
Control (BAP-free) | 2.05g | 2.32f |
BAP 0.3 | 2.37f | 2.84d |
BAP 0.5 | 2.87bc | 3.08c |
BAP 1.0 | 3.09a | 3.50a |
BAP 1.5 | 2.90b | 3.22b |
The MS medium was supplemented with 30 g/L sucrose, 7 g/L agar, and with various concentrations (0.0, 0.3, 0.5, 1.0 and 1.5 mg/L) of BAP and maintained at a pH of 5.8. Different letters (a, b, c) indicate significant differences between media (P ≤ 0.05)..
Table 3 . Comparison of shooting
Auxin (mg/L) | Shoot multiplication | Shoot height (cm) |
---|---|---|
Control (auxin-free) | 2.86f | 2.94f |
IAA 0.1 | 3.95c | 4.46c |
IAA 0.2 | 4.74a | 5.16a |
IAA 0.3 | 4.39b | 5.03b |
IAA 0.4 | 3.79d | 4.26d |
IAA 0.5 | 3.60e | 3.81e |
The control included MS + 1 g/L PVP + 1mg/L BAP. Different letters (a, b, c) indicate significant differences between media (P ≤ 0.05)..
Table 4 . Comparison of
Auxin (mg/L) | Number of rooting plantlets | Number of roots per plantlet | Root length (cm) | Plant height (cm) |
---|---|---|---|---|
Control (growth regulator-free) | 43b | 10.37f | 1.36d | 3.52d |
NAA 0.3 | 45a | 22.24e | 1.59c | 3.33e |
NAA 0.5 | 45a | 22.60e | 1.74b | 4.25a |
NAA 1.0 | 45a | 23.33d | 2.09a | 3.60d |
NAA 1.5 | 45a | 22.00e | 2.11a | 3.82c |
IAA 0.3 | 45a | 24.32c | 1.52c | 3.21f |
IAA 0.5 | 45a | 26.78a | 1.76b | 4.05b |
IAA 1.0 | 45a | 25.68b | 1.25e | 3.28ef |
IAA 1.5 | 45a | 24.72c | 1.14f | 3.12f |
The control included half-strength MS + 1 g/L PVP. Different letters (a, b, c) indicated significant differences between media (P ≤ 0.05)..
Table 5 . Comparison of survival rate and growth of plantlets by the acclimatization medium used.
Acclimatization medium | Survival rate (%) | Average number of leaves per plant | Leaf length (cm) | Plant height (cm) |
---|---|---|---|---|
50% alluvium soil + 50% sand | 51.66e | 4.95e | 2.18e | 6.81e |
50% alluvium soil + 50% rice husk | 54.33d | 5.17d | 2.32d | 7.25d |
Compost : soil + sand (3:2)* | 74.67b | 5.61c | 2.65c | 7.56c |
100% rice husk | 65.00c | 6.27b | 3.08b | 8.00b |
90% Compost + 10% perlite | 88.00a | 7.00a | 3.32a | 8.26a |
*Mixture of organic biofertilizer (compost): a mixture of soil and sand (3:2), topped with a 3~4 cm layer of sand. The organic biofertilizer was based on chicken manure, sawdust, and coconut peat in a ratio of 1:4:4 (v/v) with the addition of microbial inoculants (CP, Thailand). Different letters (a, b, c) indicate significant differences between media (P ≤ 0.05)..
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