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

Echinacea purpurea (purple coneflower) is being used for the preparation ofmore 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 900mgL), peptone (0.0, 100, 300, and 900mgL), and yeast (0.0, 100, 300, and 900mgL) to media containing 0.3mgL BA (6-benzyladenine) and 0.01mgL NAA (naphthaleneacetic acidplant 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 12mgL) and silver nitrate (0.0, 0.5, 2.5, and 12.5mgL) supplements and at higher concentrations of the above inhibited plant growth.


Introduction
Large-scale in 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][2][3][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 successful in vitro culturing of cornflower and to regenerate buds and induce roots to enable mass propagation of selected clones.

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.

Establishment of Aseptic Seedlings.
Seeds were surfacesterilized 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 (50  mol m −2 s −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.

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.

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).

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 (Figure 1 and Table 1).Sugar has provided the tissue culture plant with carbon in organic form that is not required for those grown from seeds.

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 (Table 2).

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).

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.

Table 1 :
Weight (g) of plant parts of purple coneflower grown under different sucrose concentrations ( = 8).Means followed by the same lower case letters in each column are not significantly different at 5% level in Duncan's Multiple Range Test.

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.Means followed by the same lower case letters in each column are not significantly different at 5% level in Duncan's Multiple Range Test.

Table 3 :
Shoot regeneration in callus explants with different concentrations of proline in MS basal medium with 0.1 mgL −1 BA.Means followed by the same lower case letters in each column are not significantly different at 5% level in Duncan's Multiple Range Test.