AN EFFICIENT IN VITRO PROPAGATION SYSTEM FOR PURPLE CONEFLOWER ( ECHINACEA PURPUREA L )

Echinacea purpurea L (Asteracae) is a perennial herb that has been used as a medicinal plant for hundreds of year. The main prerequisite for the development of high-quality medicinal products is a consistent source of high-quality plant material (Murch et al. 2004). However, purple coneflower is heterozygous, the content of medicinal compounds might differ significantly among individual plants and the quality of the medicine manufactured from these plants may not be the same. Because of this, techniques for in vitro propagation of seedlings of elite genotype in purple coneflower have high application value. Plant regeneration in coneflower has been reported by culturing leaf and petiole explants (Koroch et al. 2002; Roger et al. 2004; Choffe et al. 2000a; Kristen et al. 2000). In the present paper, we report an efficient in vitro propagation system for this important medicinal plant.


INTRODUCTION
Echinacea purpurea L (Asteracae) is a perennial herb that has been used as a medicinal plant for hundreds of year.The main prerequisite for the development of high-quality medicinal products is a consistent source of high-quality plant material (Murch et al. 2004).However, purple coneflower is heterozygous, the content of medicinal compounds might differ significantly among individual plants and the quality of the medicine manufactured from these plants may not be the same.Because of this, techniques for in vitro propagation of seedlings of elite genotype in purple coneflower have high application value.Plant regeneration in coneflower has been reported by culturing leaf and petiole explants (Koroch et al. 2002;Roger et al. 2004;Choffe et al. 2000a;Kristen et al. 2000).In the present paper, we report an efficient in vitro propagation system for this important medicinal plant.

Plant source
Seeds of purple coneflower were purchased at a supermarket (Supplier Company of Plantation Products, Norton, MA 02766, USA) and cultivated at the Chinese Medicinal Plant Garden in the campus of South China Agricultural University.Seeds were collected from these seed-grown plants and used for the present studies.

Establishment of aseptic cultures
Seeds were surface-sterilized by immersing in 70% ethanol for 1 minute and soaking in a 0.1% mercuric chloride for 10 minutes followed by 1% sodium hypochlorite solution containing one drop of Tween 20 per 50ml for 10 minutes.Sterilized seeds were then rinsed three times in sterile deionized water and inoculated on a medium comprised of halfstrength MS (Murashige and Skoog 1962) salts, 1% sucrose and 500mg/l lactalbumin hydrolysis and the medium was solidified with 0.2% Phytagel prior to autoclaving.The seeds were cultured first under dim-light for 14d and then the seedlings were transferred to a medium containing full-strength MS salts, 1% sucrose and 0.2% Phytagel and kept at 25 0 -27 0 C with 12h photoperiod under cool-white light (50μmol m -2 s -1 ).

Preparation of medium
All the media used were adjusted to a pH value of 6.0 with 1N NaOH or 1N HCl solution, gelled with 0.6% agar (except those for seed germination and growth) prior to autoclaving at 1.4kgcm -2 for 20 minutes.

Regeneration ability of explants from different maturity plantlets
Leaf, petiole and root explants of aseptic plantlets were cultured on MS basal medium supplemented with 0.3mg/l BA (Benzyladenine) and 0.01mg/l NAA (Naphthaleneacetic acid) to investigate the regeneration ability with the age of mother plantlets.In cultures, leaves were cut into sections (0.5cm 2 ) and placed on media with the adaxial surface toward the media, while petioles and roots were cut into about 5 mm and cultured by laying randomly on the media.The optimum plantlet age for shoot initiation was determined by comparing the regeneration ability of roots, petioles and leaves taken from plantlets of one and a half months, 2 months, 2 and a half months, and 3 months old.

Capacity of plantlet production with the established methods
Five healthy plantlets were randomly selected as explant source and 50% of each type (root, petiole and leaf) of explants taken from these plantlets were cultured onto the above mentioned regeneration medium.Regenerated buds were rooted and the resulted plantlets were again divided into explants of different kinds and cultured for regeneration of buds.This cycle was repeated again and again and all the healthy buds and plantlets produced from all explants were counted.

Rooting of adventitious buds
Healthy shoots longer than 1.5cm regenerated from all explants types were isolated and cultured on MS basal medium containing 0.01 mg/l NAA.All cultures were kept in 12-h under cool-white light (50 μ mol m -2 s -1 ) at 25 0 C for 40 days.

Data collection and analysis
Experiment was arranged according to the Randomized Complete Block Design.Regeneration cultures were evaluated 40 days after initiation and regeneration percentage of adventitious buds was calculated on the ratio of number of explants regenerated buds to the number of explants cultured.All experiments had four replicates, each with eight explants per bottle.Statistical analysis was carried out using the Student Newman-Kuells Means Separation Test of SAS (SAS Institute, Cary, NC, 1995).

Regeneration ability of explants
Differences in maturity of the material plantlets greatly influenced the regeneration of shoot buds from the explants (Table1).Number of shoot buds

No of buds from 1 g of leaf explants
No of buds from 1 g of petiole explants regenerated from all root, petiole and leaf explants increased with increasing plantlet maturity up to two and a half months.Petiole and root explants of different maturity exhibited much higher regeneration ability than leaf explants with no exception, while vitrification of the regenerated buds was generally slight (Figure 1).

Capacity of plantlet production
It took 40 days to regenerate buds of 1.5cm height, and two and a half months was necessary for a bud to grow to a plantlet of suitable maturity for providing explant material with high regeneration potential.It means, to complete a propagation culture cycles required about 4 months.In that case, it is possible to get 3 propagation culture cycles within one year duration.
According to the data shown in Table 2, explants of leaf, petiole and root taken from a 75 days old plantlet could in average regenerate 104 buds in 40 days, and after subsequent 75 days of rooting culture of these buds, these buds would become plantlets explants taken from which had high adventitious bud regeneration potential and would regenerate 10816 (104 times 104) buds in another 40 days.As culture continuing, more number of plantlets would be propagated.Details in the estimated production capacity of buds and plantlets are summarized in Table 3. From Table 3 it is clear that within one year more than one million plantlets ready for transplanting could be propagated by the established culture methods.

DISCUSSION
Previous work in our laboratory has demonstrated that purple coneflower root explants easily regenerate shoots under in vitro culture conditions (Dahanayake 2009).Root tissue has been used successfully for regeneration in a range of plant species (Bhat et al. 1992;Knoll et al. 1997;Sankhla et al. 1995;Vinocur et al. 2000;Chaudhuri et al. 2004).It is obvious that making full use of all the organs including the root as explant source can effectively increase the number of buds from a certain plantlet.
Efficient plant regeneration systems are required in this species to propagate unique lines and to improve the quality based on somatic cell genetics and recombinant DNA technology.Age of plantlets greatly influenced the regeneration ability of explants excised from garlic root tips, which reached the maximum value (95%) in 15 days old plantlets (Muhammad et al. 1997).In addition the highest number of shoots per explant was obtained from 30 days old seedlings in Aeschynomene sensitiva (Claudine et al. 1996).Up to the date the effect of planting material with plantlets age on shoot regeneration has not been reported in E. purpurea.In the present experiment, it was clear that explant age had a positive effect on shoot induction and  also it seemed to be a factor interfering with the level of regeneration.These results agreed with the previous findings in Brassica (Sharma and Thorpe 1989), garlic (Muhammad et al. 1997), legume (Claudine et al. 1996) and Tylophora indica (Chaudhuri et al. 2004).
The present study clearly demonstrates that all parts of a plant can be used as explants for regeneration of adventitious shoots and two and a half months old plantlets were most suitable for providing explant materials.By culturing the explants on MS basal medium with 0.3mg/l BA and 0.01mg/l NAA, large number of buds could be regenerated in 40 days.Such a prolific rate of multiplication cannot be expected by any of the in vivo methods of clonal propagation.Because it was difficul to maintain a so large number buds, then we had to randomly select 5 plantlets to continue the study.All of the hardened plants transferred to the field survived, and no phenotypic variations were observed.
A novel shoot regeneration methodology from root, petiole and leaf explants has been developed for E. purpurea.This method is promising for rapid multiplication of purple coneflower.

Figure 1
Figure 1 Regeneration of buds from explants of two and a half months old plantlets.Photos were taken after 40 days of culture.a. leaf explants; b. petiole explants; c. root explants.

Table 1 Effects of the age of the mother plantlet on regeneration of buds
*Means fallowed by the same lower case letters in each column are not significantly different at 5% level in Duncan's Multiple Range Test.