Journal of Plant Biotechnology 2016; 43(1): 104-109
Published online March 31, 2016
https://doi.org/10.5010/JPB.2016.43.1.104
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: paekky@cbnu.ac.kr
Keywords Total phenolics, Flavonoids, Acclimatization, Thidiazuron, Multiple shoot
Micropropagation offers the potential to produce a large number of cloned individuals from tissue culture using various plant tissues and organs. Since the first
Plant secondary metabolites have various functions throughout the life cycle of a plant. It is clear that some secondary metabolites play important roles in defense mechanisms and signaling in plants (Dai and Mumper 2010). In the past decade, their importance has increased rapidly, as these molecules also determine important aspects of human food quality and have pharmaceutical effects. Thus, it is important to increase the production of these useful bioactive compounds and to exploit plant materials through the large-scale culture of medicinal plants. During the culture period of a medicinal plant, the content of bioactive compounds changes with the physiological changes in the propagules, such as proliferation and development (Park et al. 2005). As a result, it is important to know the relationship between bioactive compounds and plant development or tissue type, in order to determine the ideal tissue (or explant) type and harvesting time. Secondary metabolites often accumulate in special types of cells or organs, as their biosynthesis is often coupled with certain morphological differentiations (Alfermann and Petersen 1995).
Rugosa rose (
In the present study, culture conditions were optimized for shoot multiplication and bioactive compounds were analized during acclimatization in
Mature seeds of
Around 1.5~2 cm length of node was used as an explant. For inducing shoot multiplication, MS medium supplemented with 2.5 μM IBA, 0~13.5 μM BA or TDZ alone, or combination of 2.5 μM IBA, 4.4 μM BA or 4.5 μM TDZ were used. All media contained 3% sucrose, and were solidified with 0.3% gelrite. The cultures were performed in 100 mL Erlenmeyer flasks containing 30 mL of medium. The experiments were conducted with 5 times replication. The cultures were maintained for 8 weeks. After completing the experiment, shoot proliferation rate, number of shoots per explant, number of nodes per explant, number of leaves, and shoot length were investigated.
For rooting, terminal 3~4 cm long portions from 8-week-old
After 8 weeks, rooted shoots were washed with water to remove the agar and were transferred to plastic pots (5 cm) containing a mixture of autoclaved vermiculite, perlite and garden soil (1:1:1) and maintained at greenhouse conditions. The plants were acclimatized by covering the pots with a polythene bag to maintain high humidity for 6~7 days. After acclimatization, ten plants were sampled every one week for 4 weeks for the analysis of bioactive compounds.
Fresh weight, dry weight, shoot length, stem diameter, root length, and leaf areas were measured at every sampling time, and the number of leaves, shoots, and nodes were counted.
The dried explants (0.1~0.2 g) were refluxed (LS-2050-S10, LS-TECH, Korea) with 20 ml 80% ethanol at 80°C for 1 h and filtered through filter paper (Advantec 110 mm, Toyo Rosihi Kaisha Ltd., Japan). The final volume of the solution was set at 15 ml using 80% ethanol.
Total phenolics were analyzed by the Folin?Ciocalteu colorimetric method (Folin & Ciocalteu 1927). The ethanolic explant extracts (0.1 ml) were mixed with 2.5 ml distilled water, followed by the addition of 0.1 ml (2N) Folin?Ciocalteu reagent. A 0.5 ml solution of 20% Na2CO3 was added after 5 min and mixed well. The color was developed after 30 min in the dark at room temperature and the absorbance was detected at 760 nm on a visible spectrophotometer (UV-1650 PC, Shimadzu, Japan). These measurements were compared to a standard curve for gallic acid (Sigma Chemical Co., St. Louis, MO, USA) and were expressed as mg of gallic acid equivalents per gram of dry explant.
The contents of total flavonoids were determined colorimetrically, using the method described by Wu et al. (2006). The ethanolic explant extracts and standard (0.25 ml) were mixed with 1.475 ml distilled water. Subsequently, 0.075 ml 5% NaNO2 solution was added and the mixture was shaken vigorously. After a 6-min reaction time, 0.15 ml 10% AlCl3 solution was added. After waiting for 5 min, the absorbance was measured immediately at 510 nm using a spectrophotometer. The results were expressed as mg of (+)-catechin (Sigma Chemical Co., St. Louis, MO, USA) equivalents per gram of dry explant.
The results shown are the mean values of three independent experiments. One-way analysis of variance (ANOVA) was used to determine if the groups differed significantly. Statistical assessments of the difference between mean values were then assessed by the least significant difference (LSD) test. A P-value of <0.05 was considered to indicate statistical significance and all data were analyzed using the SAS program (SA 9.3; SAS Institute, Inc., Cary, NC, USA).
In
Effect of different concentrations of plant growth regulators on adventitious shoot formation from node cultures of Rosa rugose. A. Shoot proliferation rate, B. No. of shoot, C. Regenera [Con: non-treated control, B: BA, I: IBA, T: thidiazuron]. Bars represent means ± SE (n = 6)
Effect of different concentrations of plant growth regulators (PGRs) on multiple shoot formation from node cultures of Rosa rugosa after 8 weeks of culturing. Fresh weight (A) and dry weight (B) of plants. Con: non-treated control; B: BA; I: IBA; T: thidiazuron. Bars represent means ± SE (n = 6)
BA and TDZ showed a synergistic effect on shoot proliferation in many plants such as rose (Ibrahim and Debergh 2001) and Eastern redbud (Distabanjong and Geneve 1997). Nodal segments produced the highest number of shoots in medium containing a combination of BA and TDZ in Eastern redbud (Distabanjong and Geneve 1997). The highest number of shoots was obtained when explants were treated with a combination of 10~15 ?M BA and 0.5~1.0 ?M TDZ before being transferred to the same medium without TDZ (Distabanjong and Geneve 1997). A rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants, and in the plant TDZ was also more effective than BA in inducing multiple shoots (Siddique and Anis 2007).
After the transfer of shoots to the greenhouse, ten shoots were sampled and were analyzed for growth and bioactive compound production (Table 1). Amongst various growth aspects, the highest increase in fresh weight, shoot length, and leaf number was achieved at 4 weeks of acclimatization (Table 1). Dry matter (%) was the highest at 0 weeks, whereas it was the lowest at 4 weeks, which showed dramatic growth increase. Number of shoots and stem diameter were not significantly changed during the acclimatization period. On the other hand, the number of nodes increased with acclimatization time. Leaf area did not increase at 0~3 weeks after acclimatization, but significantly increased around 2 times at 4 weeks. Number of leaves decreased at the 1st week because of acclimatization stress, and it increased again in 4 weeks of acclimatization. Total phenolics and flavonoids were the highest at 1st week of acclimatization (Fig. 3). The results of total phenolics and flavonoids can be interpreted as
Table 1 Effect of acclimatization periods on plant growth of Rosa rugosa
Time (week) | Fresh weight (mg) | % dry weight | No. of shoots | No. of nodes | No. of leaves | Shoot length (cm) | Root length (cm) | Stem diameter (mm) | Leaf area (cm2) |
0 | 40.7±2.6z | 35.0±2.3 | 1.0±0.0 | 5.0±0.4 | 29.5±3.1 | 3.2±0.1 cy | 0.6±0.1 | 0.8±0.1 | 0.2±0.0b |
1 | 77.2±5.8 | 20.5±0.8 | 1.2±0.2 | 4.8±0.8 | 23.2±3.1 b | 3.9±0.2 c | 1.1±0.1 | 0.9±0.1 | 0.3±0.0b |
2 | 77.5±6.6 | 21.4±1.3 | 1.2±0.2 | 7.0±0.4 | 28.5±2.2 b | 5.1±0.3 b | 1.0±0.1 | 0.8±0.1 | 0.3±0.0b |
3 | 88.0±8.8 | 20.4±1.6 | 1.2±0.2 | 6.8±0.6 | 25.3±3.2 b | 5.5±0.3 b | 1.3±0.2 | 0.8±0.1 | 0.3±0.0b |
4 | 132.7±8.9 | 18.9±0.4 | 1.2±0.0 | 7.8±0.7 | 34.7±1.9 a | 7.5±0.2 a | 1.3±0.1 | 0.8±0.0 | 0.6±0.0a |
zValues represent means ± SE (n = 5)
yWithin each column, different letters indicate mean separation using Duncan’s multiple range test at 5 % level of significance
Changes in bioactive compound content during acclimatization periods of Rosa rugosa. A: Total phenolics, B: Total flavonoids. Bars represent means ± SE (n = 3)
Figure 4 shows that the content of total phenolics and flavonoids depends on the developmental stage and age of
Changes in contents of total phenolics and flavonoids during the developmental process of Rosa rugosa. SE: somatic embryo, In-1: in vitro 1-yr-old, In-5: in vitro 5-yr-old, Acclimatization: 0-day, Ex-1: ex vitro 1-yr-old, Ex-2: ex vitro 2-yr-old, Ex-3: ex vitro 3-yr old, Ex-4: ex vitro 4-yr-old, Ex-5: ex vitro 5-yr-old, Ex-6: ex vitro 6-yr-old. Bars represent means ± SE (n = 3)
Numerous reports indicate that it is difficult to select an ideal stage of plant source, which would ensure good production of the selected secondary metabolites and simultaneously, fast and continuous growth of propagules that are capable of consistently producing bioactive compounds. One reason is that the primary and secondary metabolism pathways often compete for nutrients and precursors, and are often mutually exclusive (Hagendoorn et al. 1999; Luczkiewicz and Cisowski 2005).
Shoot cultures have been established for many medicinal plants; these can accumulate a higher amount of secondary metabolites than natural plants. Recently, many reports have shown the possibility of shoots acting as a source of industrial product ingredients. For example, shoot cultures were established for
Our study showed that young plants such as
This work was supported by the intramural research grant of Chungbuk National University in 2015.
Journal of Plant Biotechnology 2016; 43(1): 104-109
Published online March 31, 2016 https://doi.org/10.5010/JPB.2016.43.1.104
Copyright © The Korean Society of Plant Biotechnology.
Hae-Rim Jang1, Byung-Jun Park2, Seung-A Park2, Ok-Ja Pee3, So-Young Park1, and Kee-Yoeup Paek1,*
1Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea,
2Korea Kolmar, Deokgogaegil 12-11, Jeoneui-myeon, Sejong 339-851, Republic of Korea,
3Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea; Sejong Agricultural Technology & Extension Center, Sejong, 30048, Republic of Korea
Correspondence to:e-mail: paekky@cbnu.ac.kr
Keywords: Total phenolics, Flavonoids, Acclimatization, Thidiazuron, Multiple shoot
Micropropagation offers the potential to produce a large number of cloned individuals from tissue culture using various plant tissues and organs. Since the first
Plant secondary metabolites have various functions throughout the life cycle of a plant. It is clear that some secondary metabolites play important roles in defense mechanisms and signaling in plants (Dai and Mumper 2010). In the past decade, their importance has increased rapidly, as these molecules also determine important aspects of human food quality and have pharmaceutical effects. Thus, it is important to increase the production of these useful bioactive compounds and to exploit plant materials through the large-scale culture of medicinal plants. During the culture period of a medicinal plant, the content of bioactive compounds changes with the physiological changes in the propagules, such as proliferation and development (Park et al. 2005). As a result, it is important to know the relationship between bioactive compounds and plant development or tissue type, in order to determine the ideal tissue (or explant) type and harvesting time. Secondary metabolites often accumulate in special types of cells or organs, as their biosynthesis is often coupled with certain morphological differentiations (Alfermann and Petersen 1995).
Rugosa rose (
In the present study, culture conditions were optimized for shoot multiplication and bioactive compounds were analized during acclimatization in
Mature seeds of
Around 1.5~2 cm length of node was used as an explant. For inducing shoot multiplication, MS medium supplemented with 2.5 μM IBA, 0~13.5 μM BA or TDZ alone, or combination of 2.5 μM IBA, 4.4 μM BA or 4.5 μM TDZ were used. All media contained 3% sucrose, and were solidified with 0.3% gelrite. The cultures were performed in 100 mL Erlenmeyer flasks containing 30 mL of medium. The experiments were conducted with 5 times replication. The cultures were maintained for 8 weeks. After completing the experiment, shoot proliferation rate, number of shoots per explant, number of nodes per explant, number of leaves, and shoot length were investigated.
For rooting, terminal 3~4 cm long portions from 8-week-old
After 8 weeks, rooted shoots were washed with water to remove the agar and were transferred to plastic pots (5 cm) containing a mixture of autoclaved vermiculite, perlite and garden soil (1:1:1) and maintained at greenhouse conditions. The plants were acclimatized by covering the pots with a polythene bag to maintain high humidity for 6~7 days. After acclimatization, ten plants were sampled every one week for 4 weeks for the analysis of bioactive compounds.
Fresh weight, dry weight, shoot length, stem diameter, root length, and leaf areas were measured at every sampling time, and the number of leaves, shoots, and nodes were counted.
The dried explants (0.1~0.2 g) were refluxed (LS-2050-S10, LS-TECH, Korea) with 20 ml 80% ethanol at 80°C for 1 h and filtered through filter paper (Advantec 110 mm, Toyo Rosihi Kaisha Ltd., Japan). The final volume of the solution was set at 15 ml using 80% ethanol.
Total phenolics were analyzed by the Folin?Ciocalteu colorimetric method (Folin & Ciocalteu 1927). The ethanolic explant extracts (0.1 ml) were mixed with 2.5 ml distilled water, followed by the addition of 0.1 ml (2N) Folin?Ciocalteu reagent. A 0.5 ml solution of 20% Na2CO3 was added after 5 min and mixed well. The color was developed after 30 min in the dark at room temperature and the absorbance was detected at 760 nm on a visible spectrophotometer (UV-1650 PC, Shimadzu, Japan). These measurements were compared to a standard curve for gallic acid (Sigma Chemical Co., St. Louis, MO, USA) and were expressed as mg of gallic acid equivalents per gram of dry explant.
The contents of total flavonoids were determined colorimetrically, using the method described by Wu et al. (2006). The ethanolic explant extracts and standard (0.25 ml) were mixed with 1.475 ml distilled water. Subsequently, 0.075 ml 5% NaNO2 solution was added and the mixture was shaken vigorously. After a 6-min reaction time, 0.15 ml 10% AlCl3 solution was added. After waiting for 5 min, the absorbance was measured immediately at 510 nm using a spectrophotometer. The results were expressed as mg of (+)-catechin (Sigma Chemical Co., St. Louis, MO, USA) equivalents per gram of dry explant.
The results shown are the mean values of three independent experiments. One-way analysis of variance (ANOVA) was used to determine if the groups differed significantly. Statistical assessments of the difference between mean values were then assessed by the least significant difference (LSD) test. A P-value of <0.05 was considered to indicate statistical significance and all data were analyzed using the SAS program (SA 9.3; SAS Institute, Inc., Cary, NC, USA).
In
Effect of different concentrations of plant growth regulators on adventitious shoot formation from node cultures of Rosa rugose. A. Shoot proliferation rate, B. No. of shoot, C. Regenera [Con: non-treated control, B: BA, I: IBA, T: thidiazuron]. Bars represent means ± SE (n = 6)
Effect of different concentrations of plant growth regulators (PGRs) on multiple shoot formation from node cultures of Rosa rugosa after 8 weeks of culturing. Fresh weight (A) and dry weight (B) of plants. Con: non-treated control; B: BA; I: IBA; T: thidiazuron. Bars represent means ± SE (n = 6)
BA and TDZ showed a synergistic effect on shoot proliferation in many plants such as rose (Ibrahim and Debergh 2001) and Eastern redbud (Distabanjong and Geneve 1997). Nodal segments produced the highest number of shoots in medium containing a combination of BA and TDZ in Eastern redbud (Distabanjong and Geneve 1997). The highest number of shoots was obtained when explants were treated with a combination of 10~15 ?M BA and 0.5~1.0 ?M TDZ before being transferred to the same medium without TDZ (Distabanjong and Geneve 1997). A rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants, and in the plant TDZ was also more effective than BA in inducing multiple shoots (Siddique and Anis 2007).
After the transfer of shoots to the greenhouse, ten shoots were sampled and were analyzed for growth and bioactive compound production (Table 1). Amongst various growth aspects, the highest increase in fresh weight, shoot length, and leaf number was achieved at 4 weeks of acclimatization (Table 1). Dry matter (%) was the highest at 0 weeks, whereas it was the lowest at 4 weeks, which showed dramatic growth increase. Number of shoots and stem diameter were not significantly changed during the acclimatization period. On the other hand, the number of nodes increased with acclimatization time. Leaf area did not increase at 0~3 weeks after acclimatization, but significantly increased around 2 times at 4 weeks. Number of leaves decreased at the 1st week because of acclimatization stress, and it increased again in 4 weeks of acclimatization. Total phenolics and flavonoids were the highest at 1st week of acclimatization (Fig. 3). The results of total phenolics and flavonoids can be interpreted as
Table 1 . Effect of acclimatization periods on plant growth of Rosa rugosa.
Time (week) | Fresh weight (mg) | % dry weight | No. of shoots | No. of nodes | No. of leaves | Shoot length (cm) | Root length (cm) | Stem diameter (mm) | Leaf area (cm2) |
0 | 40.7±2.6z | 35.0±2.3 | 1.0±0.0 | 5.0±0.4 | 29.5±3.1 | 3.2±0.1 cy | 0.6±0.1 | 0.8±0.1 | 0.2±0.0b |
1 | 77.2±5.8 | 20.5±0.8 | 1.2±0.2 | 4.8±0.8 | 23.2±3.1 b | 3.9±0.2 c | 1.1±0.1 | 0.9±0.1 | 0.3±0.0b |
2 | 77.5±6.6 | 21.4±1.3 | 1.2±0.2 | 7.0±0.4 | 28.5±2.2 b | 5.1±0.3 b | 1.0±0.1 | 0.8±0.1 | 0.3±0.0b |
3 | 88.0±8.8 | 20.4±1.6 | 1.2±0.2 | 6.8±0.6 | 25.3±3.2 b | 5.5±0.3 b | 1.3±0.2 | 0.8±0.1 | 0.3±0.0b |
4 | 132.7±8.9 | 18.9±0.4 | 1.2±0.0 | 7.8±0.7 | 34.7±1.9 a | 7.5±0.2 a | 1.3±0.1 | 0.8±0.0 | 0.6±0.0a |
zValues represent means ± SE (n = 5)
yWithin each column, different letters indicate mean separation using Duncan’s multiple range test at 5 % level of significance
Changes in bioactive compound content during acclimatization periods of Rosa rugosa. A: Total phenolics, B: Total flavonoids. Bars represent means ± SE (n = 3)
Figure 4 shows that the content of total phenolics and flavonoids depends on the developmental stage and age of
Changes in contents of total phenolics and flavonoids during the developmental process of Rosa rugosa. SE: somatic embryo, In-1: in vitro 1-yr-old, In-5: in vitro 5-yr-old, Acclimatization: 0-day, Ex-1: ex vitro 1-yr-old, Ex-2: ex vitro 2-yr-old, Ex-3: ex vitro 3-yr old, Ex-4: ex vitro 4-yr-old, Ex-5: ex vitro 5-yr-old, Ex-6: ex vitro 6-yr-old. Bars represent means ± SE (n = 3)
Numerous reports indicate that it is difficult to select an ideal stage of plant source, which would ensure good production of the selected secondary metabolites and simultaneously, fast and continuous growth of propagules that are capable of consistently producing bioactive compounds. One reason is that the primary and secondary metabolism pathways often compete for nutrients and precursors, and are often mutually exclusive (Hagendoorn et al. 1999; Luczkiewicz and Cisowski 2005).
Shoot cultures have been established for many medicinal plants; these can accumulate a higher amount of secondary metabolites than natural plants. Recently, many reports have shown the possibility of shoots acting as a source of industrial product ingredients. For example, shoot cultures were established for
Our study showed that young plants such as
This work was supported by the intramural research grant of Chungbuk National University in 2015.
Effect of different concentrations of plant growth regulators on adventitious shoot formation from node cultures of Rosa rugose. A. Shoot proliferation rate, B. No. of shoot, C. Regenera [Con: non-treated control, B: BA, I: IBA, T: thidiazuron]. Bars represent means ± SE (n = 6)
Effect of different concentrations of plant growth regulators (PGRs) on multiple shoot formation from node cultures of Rosa rugosa after 8 weeks of culturing. Fresh weight (A) and dry weight (B) of plants. Con: non-treated control; B: BA; I: IBA; T: thidiazuron. Bars represent means ± SE (n = 6)
Changes in bioactive compound content during acclimatization periods of Rosa rugosa. A: Total phenolics, B: Total flavonoids. Bars represent means ± SE (n = 3)
Changes in contents of total phenolics and flavonoids during the developmental process of Rosa rugosa. SE: somatic embryo, In-1: in vitro 1-yr-old, In-5: in vitro 5-yr-old, Acclimatization: 0-day, Ex-1: ex vitro 1-yr-old, Ex-2: ex vitro 2-yr-old, Ex-3: ex vitro 3-yr old, Ex-4: ex vitro 4-yr-old, Ex-5: ex vitro 5-yr-old, Ex-6: ex vitro 6-yr-old. Bars represent means ± SE (n = 3)
Table 1 . Effect of acclimatization periods on plant growth of Rosa rugosa.
Time (week) | Fresh weight (mg) | % dry weight | No. of shoots | No. of nodes | No. of leaves | Shoot length (cm) | Root length (cm) | Stem diameter (mm) | Leaf area (cm2) |
0 | 40.7±2.6z | 35.0±2.3 | 1.0±0.0 | 5.0±0.4 | 29.5±3.1 | 3.2±0.1 cy | 0.6±0.1 | 0.8±0.1 | 0.2±0.0b |
1 | 77.2±5.8 | 20.5±0.8 | 1.2±0.2 | 4.8±0.8 | 23.2±3.1 b | 3.9±0.2 c | 1.1±0.1 | 0.9±0.1 | 0.3±0.0b |
2 | 77.5±6.6 | 21.4±1.3 | 1.2±0.2 | 7.0±0.4 | 28.5±2.2 b | 5.1±0.3 b | 1.0±0.1 | 0.8±0.1 | 0.3±0.0b |
3 | 88.0±8.8 | 20.4±1.6 | 1.2±0.2 | 6.8±0.6 | 25.3±3.2 b | 5.5±0.3 b | 1.3±0.2 | 0.8±0.1 | 0.3±0.0b |
4 | 132.7±8.9 | 18.9±0.4 | 1.2±0.0 | 7.8±0.7 | 34.7±1.9 a | 7.5±0.2 a | 1.3±0.1 | 0.8±0.0 | 0.6±0.0a |
zValues represent means ± SE (n = 5)
yWithin each column, different letters indicate mean separation using Duncan’s multiple range test at 5 % level of significance
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Plant BiotechnologyEffect of different concentrations of plant growth regulators on adventitious shoot formation from node cultures of Rosa rugose. A. Shoot proliferation rate, B. No. of shoot, C. Regenera [Con: non-treated control, B: BA, I: IBA, T: thidiazuron]. Bars represent means ± SE (n = 6)
|@|~(^,^)~|@|Effect of different concentrations of plant growth regulators (PGRs) on multiple shoot formation from node cultures of Rosa rugosa after 8 weeks of culturing. Fresh weight (A) and dry weight (B) of plants. Con: non-treated control; B: BA; I: IBA; T: thidiazuron. Bars represent means ± SE (n = 6)
|@|~(^,^)~|@|Changes in bioactive compound content during acclimatization periods of Rosa rugosa. A: Total phenolics, B: Total flavonoids. Bars represent means ± SE (n = 3)
|@|~(^,^)~|@|Changes in contents of total phenolics and flavonoids during the developmental process of Rosa rugosa. SE: somatic embryo, In-1: in vitro 1-yr-old, In-5: in vitro 5-yr-old, Acclimatization: 0-day, Ex-1: ex vitro 1-yr-old, Ex-2: ex vitro 2-yr-old, Ex-3: ex vitro 3-yr old, Ex-4: ex vitro 4-yr-old, Ex-5: ex vitro 5-yr-old, Ex-6: ex vitro 6-yr-old. Bars represent means ± SE (n = 3)