# Effects of Sucrose and Other Additives onIn Vitro Growth and Development of Purple Coneflower (Echinacea purpurea L.)

Authors: Dahanayake Nilanthi; Yue-Sheng Yang
Journal: Advances in Biology
 (2014)

---

## Abstract

Echinacea purpurea (purple coneflower) is being used for the preparation of more than 240 extracts, salves, and tinctures to help cure diseases like rabies, cold, and upper respiratory infections. Hence, efforts were made to develop a culture medium for successful in vitro culturing of cornflower and to regenerate buds and induce roots to enable mass propagation of selected clones. Of the three levels of sucrose tested as a supplement to MS media (Murashige and Skoog’s medium, 1962) 3% showed better rooting of buds and appeared morphologically normal and identical as compared to those grown at higher and lower concentrations (2 and 4%). The additives hydrolyzed lactabumin (0.0, 100, 300, and 900 mgL−1), peptone (0.0, 100, 300, and 900 mgL−1), and yeast (0.0, 100, 300, and 900 mgL−1) to media containing 0.3 mgL−1 BA (6-benzyladenine) and 0.01 mgL−1 NAA (naphthaleneacetic acid-plant growth regulators) has negatively influenced proliferation of shoots. The higher concentrations of the above have delayed the development of plantlets. Shoot multiplication was enhanced by coconut water with 2% being the best among 4 and 8% tested. Shoot organogenesis was not influenced by copper sulphate (0, 1.5, 3, 6, and 12 mgL−1) and silver nitrate (0.0, 0.5, 2.5, and 12.5 mgL−1) supplements and at higher concentrations of the above inhibited plant growth.


---

## Body

## 1. Introduction

Large-scalein vitro propagation medicinal plants have become vital to meet the increasing demand for high-quality pharmaceuticals and for the conservation of valuable elite stock plants [1–4].Echinacea purpurea (purple coneflower) is known to contain carbohydrates, glycosides, alkaloids, alkylamides (alkamides), polyacetylenes, fatty acids, essential oil, and phytosterols and is being used for the preparation of more than 240 extracts, salves, and tinctures to help cure diseases like rabies, cold, upper respiratory infections, and so forth (http://www.bioalma.com/). Hence, efforts were made to develop a culture medium for successfulin vitro culturing of cornflower and to regenerate buds and induce roots to enable mass propagation of selected clones.


## 2. Materials and Methods

### 2.1. Plant Source

Seeds for the present study were harvested from the purple cornflower clone (source: Norton, MA, USA) maintained at the Chinese Medicinal Plant Garden, South China Agricultural University.


### 2.2. Establishment of Aseptic Seedlings

Seeds were surface-sterilized by sequentially immersing in 70% ethanol for 1 minute, 0.1% mercuric chloride for 10 minutes, and 1% sodium hypochlorite (containing Tween 20, one drop per 50 mL) for 10 minutes. Sterilized seeds were then rinsed three times in sterilized-deionized water and inoculated on a sterilized medium composed of half-strength MS salts, 1% sucrose 500 mgL−1 hydrolyzed lactalbumin and 0.2% phytagel (solidifier).After 14 d under dim light, the germinated seeds were transferred to a sterilized medium containing full-strength MS salts, 1% sucrose, 0.2% phytagel, and 0.6% agar. Seedlings cultures were then incubated in a room at 25°–27°C and 12h photoperiod under cool-white light (50μ mol m−2s−1) for 40 days. All media used were adjusted to the pH 6.0 with 1N NaOH or 1N HCl, prior to autoclaving at 1.4 kg cm−2 for 20 minutes.


### 2.3. Effect of Sucrose on Culture Medium on Shoot and Root Growth

Explants of purple coneflower shoots (1 cm) were introduced to the MS basal medium containing 0.3 mgL−1 BA and 0.01 mgL−1 NAA and three concentrations of sucrose (2%, 3%, and 4%) to assess the shoot growth and root development.


### 2.4. Effect of Other Additives on Culture Medium on Shoot and Root Growth

Explants of purple coneflower petiole were grown in media containing 0.3 mgL−1 BA and 0.01 mgL−1 NAA (plant growth regulators) with the following additives hydrolyzed lactalbumin (0.0, 100, 300, and 900 mgL−1), peptone (0.0, 100, 300, and 900 mgL−1), yeast (0.0, 100, 300, and 900 mgL−1), coconut water (0, 2, 4, and 8%), copper sulphate (0, 1.5, 3, 6, and 12 mgL−1), silver nitrate (0.0, 0.5, 2.5, and 12.5 mgL−1), and proline (0, 150, 450, and 1000 mgL−1) to evaluate effect of these additives on growth and regeneration of callus explants.


### 2.5. Data Collection and Analysis

All experiments were replicated four times having 4 explants per culture bottle. The influence of different media on explant growth was estimated through weighing the shoots and roots separately. Statistical analysis was carried out using the Student Newman-Keuls Means Separation Test of SAS (SAS Institute, Cary, NC, 1995).


## 2.1. Plant Source

Seeds for the present study were harvested from the purple cornflower clone (source: Norton, MA, USA) maintained at the Chinese Medicinal Plant Garden, South China Agricultural University.


## 2.2. Establishment of Aseptic Seedlings

Seeds were surface-sterilized by sequentially immersing in 70% ethanol for 1 minute, 0.1% mercuric chloride for 10 minutes, and 1% sodium hypochlorite (containing Tween 20, one drop per 50 mL) for 10 minutes. Sterilized seeds were then rinsed three times in sterilized-deionized water and inoculated on a sterilized medium composed of half-strength MS salts, 1% sucrose 500 mgL−1 hydrolyzed lactalbumin and 0.2% phytagel (solidifier).After 14 d under dim light, the germinated seeds were transferred to a sterilized medium containing full-strength MS salts, 1% sucrose, 0.2% phytagel, and 0.6% agar. Seedlings cultures were then incubated in a room at 25°–27°C and 12h photoperiod under cool-white light (50μ mol m−2s−1) for 40 days. All media used were adjusted to the pH 6.0 with 1N NaOH or 1N HCl, prior to autoclaving at 1.4 kg cm−2 for 20 minutes.


## 2.3. Effect of Sucrose on Culture Medium on Shoot and Root Growth

Explants of purple coneflower shoots (1 cm) were introduced to the MS basal medium containing 0.3 mgL−1 BA and 0.01 mgL−1 NAA and three concentrations of sucrose (2%, 3%, and 4%) to assess the shoot growth and root development.


## 2.4. Effect of Other Additives on Culture Medium on Shoot and Root Growth

Explants of purple coneflower petiole were grown in media containing 0.3 mgL−1 BA and 0.01 mgL−1 NAA (plant growth regulators) with the following additives hydrolyzed lactalbumin (0.0, 100, 300, and 900 mgL−1), peptone (0.0, 100, 300, and 900 mgL−1), yeast (0.0, 100, 300, and 900 mgL−1), coconut water (0, 2, 4, and 8%), copper sulphate (0, 1.5, 3, 6, and 12 mgL−1), silver nitrate (0.0, 0.5, 2.5, and 12.5 mgL−1), and proline (0, 150, 450, and 1000 mgL−1) to evaluate effect of these additives on growth and regeneration of callus explants.


## 2.5. Data Collection and Analysis

All experiments were replicated four times having 4 explants per culture bottle. The influence of different media on explant growth was estimated through weighing the shoots and roots separately. Statistical analysis was carried out using the Student Newman-Keuls Means Separation Test of SAS (SAS Institute, Cary, NC, 1995).


## 3. Results and Discussion

### 3.1. Effect of Sucrose on Culture Medium on Shoot and Root Growth

MS media supplemented with 3% sucrose showed better rooting of buds and appeared morphologically normal roots as compared to those grown at higher and lower concentrations (Figure1 and Table 1). Sugar has provided the tissue culture plant with carbon in organic form that is not required for those grown from seeds.Table 1
Weight (g) of plant parts of purple coneflower grown under different sucrose concentrations (n=8).
Sucrose conc. %
Whole plant
Leaves
Petiole
Roots
Residues
2
1.26
ab
*
0.42a
0.30a
0.38ab
0.10ab
3
1.69a
0.56a
0.36a
0.61a
0.12a
4
0.77b
0.35a
0.19a
0.14b
0.05b
*Means followed by the same lower case letters in each column are not significantly different at 5% level in Duncan’s Multiple Range Test.Figure 1
Root system of purple coneflower grown under different sucrose concentrations: (a) 4%; (b) 3%; (c) 2%.


### 3.2. Effect of Other Additives on Culture Medium on Shoot and Root Growth

Nitrogenous additives, hydrolyzed lactalbumin, peptone, and yeast in the medium have negatively influenced proliferation of shoots. The higher concentrations of the above have delayed the development of plantlets. Shoot multiplication has been enhanced by coconut water with 2% being the best. Shoot organogenesis was not influenced by copper sulphate and silver nitrate supplements and at higher concentrations of the above inhibited plant growth (Table2).Table 2
Potential of shoot regeneration in petiole explants with different concentrations of additives in MS basal medium with 0.3 mgL−1 BA and 0.01 mgL−1 NAA.
Treatment mgL−1
Number of buds per explant
Lactalbumin hydrolysis
0
2.12a
100
0.95b
300
0.20c
900
0.06d
Peptone
0
1.85a
100
0.80b
300
0.31c
900
0.08d
Yeast
0
1.95a
100
0.91b
300
0.28c
900
0.05d
Coconut water
0%
2.05a
2%
2.58b
4%
2.03a
8%
1.91a
Copper sulphate
0
2.00a
1.5
0.10b
3
0.05bc
6
0.01bc
12
0.00c
Silver nitrate
0
1.89a
0.5
0.08b
2.5
0.02b
12.5
0.00b
*Means followed by the same lower case letters in each column are not significantly different at 5% level in Duncan’s Multiple Range Test.


### 3.3. Growth of Callus Derived from Petiole Explants

Callus taken from cell suspension treated with BA and transferred to the MS medium supplemented with 0.6% agar + 3% sucrose with 0.1 mgL−1 BA + 0.0 mgL−1 proline displayed soft texture and no significant growth. Callus on medium containing BA and 150 mgL−1 proline showed brownish nodular callus after 9 weeks of initiation. Greenish well growing nodular callus was observed on the medium supplemented with BA and proline at higher concentrations such as 450 mgL−1 and 1000 mgL−1 (Table 3 and Figure 2).Table 3
Shoot regeneration in callus explants with different concentrations of proline in MS basal medium with 0.1 mgL−1 BA.
Proline  mgL−1
Number of buds per explant
0
0.00a
150
5.09b
450
11.9c
1000
16.7d
*Means followed by the same lower case letters in each column are not significantly different at 5% level in Duncan’s Multiple Range Test.Growth of callus on agar media containing MS basal elements, 3% sucrose, and other additives: (a) 0.1 mgL−1 BA + 0.0 mgL−1 proline; (b) 0.1 mgL−1 BA + 150 mgL−1 proline; (c) 0.1 mgL−1 BA + 450 mgL−1 proline; (d) 0.1 mgL−1 BA + 1000 mgL−1 proline. All green buds develop to shoots within 6 weeks.
(a)
(b)
(c)
(d)


## 3.1. Effect of Sucrose on Culture Medium on Shoot and Root Growth

MS media supplemented with 3% sucrose showed better rooting of buds and appeared morphologically normal roots as compared to those grown at higher and lower concentrations (Figure1 and Table 1). Sugar has provided the tissue culture plant with carbon in organic form that is not required for those grown from seeds.Table 1
Weight (g) of plant parts of purple coneflower grown under different sucrose concentrations (n=8).
Sucrose conc. %
Whole plant
Leaves
Petiole
Roots
Residues
2
1.26
ab
*
0.42a
0.30a
0.38ab
0.10ab
3
1.69a
0.56a
0.36a
0.61a
0.12a
4
0.77b
0.35a
0.19a
0.14b
0.05b
*Means followed by the same lower case letters in each column are not significantly different at 5% level in Duncan’s Multiple Range Test.Figure 1
Root system of purple coneflower grown under different sucrose concentrations: (a) 4%; (b) 3%; (c) 2%.


## 3.2. Effect of Other Additives on Culture Medium on Shoot and Root Growth

Nitrogenous additives, hydrolyzed lactalbumin, peptone, and yeast in the medium have negatively influenced proliferation of shoots. The higher concentrations of the above have delayed the development of plantlets. Shoot multiplication has been enhanced by coconut water with 2% being the best. Shoot organogenesis was not influenced by copper sulphate and silver nitrate supplements and at higher concentrations of the above inhibited plant growth (Table2).Table 2
Potential of shoot regeneration in petiole explants with different concentrations of additives in MS basal medium with 0.3 mgL−1 BA and 0.01 mgL−1 NAA.
Treatment mgL−1
Number of buds per explant
Lactalbumin hydrolysis
0
2.12a
100
0.95b
300
0.20c
900
0.06d
Peptone
0
1.85a
100
0.80b
300
0.31c
900
0.08d
Yeast
0
1.95a
100
0.91b
300
0.28c
900
0.05d
Coconut water
0%
2.05a
2%
2.58b
4%
2.03a
8%
1.91a
Copper sulphate
0
2.00a
1.5
0.10b
3
0.05bc
6
0.01bc
12
0.00c
Silver nitrate
0
1.89a
0.5
0.08b
2.5
0.02b
12.5
0.00b
*Means followed by the same lower case letters in each column are not significantly different at 5% level in Duncan’s Multiple Range Test.


## 3.3. Growth of Callus Derived from Petiole Explants

Callus taken from cell suspension treated with BA and transferred to the MS medium supplemented with 0.6% agar + 3% sucrose with 0.1 mgL−1 BA + 0.0 mgL−1 proline displayed soft texture and no significant growth. Callus on medium containing BA and 150 mgL−1 proline showed brownish nodular callus after 9 weeks of initiation. Greenish well growing nodular callus was observed on the medium supplemented with BA and proline at higher concentrations such as 450 mgL−1 and 1000 mgL−1 (Table 3 and Figure 2).Table 3
Shoot regeneration in callus explants with different concentrations of proline in MS basal medium with 0.1 mgL−1 BA.
Proline  mgL−1
Number of buds per explant
0
0.00a
150
5.09b
450
11.9c
1000
16.7d
*Means followed by the same lower case letters in each column are not significantly different at 5% level in Duncan’s Multiple Range Test.Growth of callus on agar media containing MS basal elements, 3% sucrose, and other additives: (a) 0.1 mgL−1 BA + 0.0 mgL−1 proline; (b) 0.1 mgL−1 BA + 150 mgL−1 proline; (c) 0.1 mgL−1 BA + 450 mgL−1 proline; (d) 0.1 mgL−1 BA + 1000 mgL−1 proline. All green buds develop to shoots within 6 weeks.
(a)
(b)
(c)
(d)


## 4. Conclusion

Optimum sucrose concentration for shoot growth and root production determined to be 3% as compared to 4% and 2%. Greenish well growing nodular callus was observed on the medium supplemented with BA and proline at concentration as high as 450 mgL−1 and 1000 mgL−1. Coconut water (2%) gave higher shoot regeneration as compared to higher concentrations. Nitrogenous additives, hydrolyzed lactalbumin, peptone, and yeast reduced the proliferation rate of shoots.



---

*Source: 402309-2014-06-16.xml*