With the confirmed non-specific immunostimulant bioactivities, purple coneflower (Echinacea purpurea (L.) Moench) has gained much attention for decades   . Traditionally, people use it as prevention or a treatment of upper respiratory infections in common cold in nations across Europe and North America  . In recent years, five purple coneflower products have been certified as the new veterinary drugs in China (Certification Nos. 2012-23, -24, -25, and 2014-44, -45)  . These promulgations promote the opening of a brand-new market of biological feed additives. It is foreseeable that the scale of commercial cultivation of this plant is going to expand rapidly and steadily. However, this plant is highly cross-pollinated  and the commercial plantations were still growing this medicinal plant with seeds. Consequently, the yield and the quality varied because of segregation of genetic characters. In the aspect of breeding, both transgenic    and ploidy breeding technique    were applied on breeding of new variety. Some kinds of the newly created materials were more valuable than the normal ones, because they grew with higher biomass and contained higher concentration of high-value secondary metabolites  . All these studies were performed by in vitro culture.
To obtain qualified regenerated plants for commercial production and breeding studies, many efforts have been made on in vitro culture in the past fifteen years. Leaf , petiole , and root  were the most frequently used explant for inducing adventitious buds. Various Regular Plant Growth Regulators (PRGs) had been tested, such as 6-benzylaminopurine (BA), naphthylacetic acid (NAA), thidiazuron (TDZ), Kinetin (KT), 3-indolebutyric acid (IBA), and 3-indolylacetic acid (IAA)    .
However, the efficiency of the micropropagation of purple coneflower was still not high enough. As the breeding researches processed, there were many individuals with special genotypes created    . Part of them were found to be sterility or with low fertility, and hard to be reproduced and maintained by performing the present in vitro culture protocols.
The diethyl aminoethyl hexanoate (DA-6), was normally used in field   and was believed to be safe , but uncommon used in tissue culture. Previously, our researches indicated that this PGR was highly effective in in vitro cultures of the purple coneflower . Later, the effects of DA-6 on enhancement of microalgae growth and high-value biocompounds produced were tested, and the results were impressive as well  . It was even been considered an effective PGR to alleviate salinity stress .
Despite of the high efficiency, the effect of DA-6 seems uncontrollable under former propagation strategy. Unlike the BA or NAA  , the effect of DA-6 is insensitive to gene dosage difference. The 0.08 mg・L−1 of DA-6 may increase the regeneration rate of an explant, but strongly decreased the regeneration rate of another explant with same or higher gene dosage; the 0.16 mg・L−1 of DA-6 may promote the growing of a shoot, but severely inhibit the growing of another shoot . The affecting factors may contain the treatment concentration, explant source, or something else, which had not being counted in in those studies. As in the process of tissue culture plantlet production, the medium was usually mass-produced, this fact severely restrain the practical application of the DA-6.
No researches have studied the influence of biomass dosage of explants on the effects of DA-6. To address this problem, we did the following researches. Results showed that the biomass dosage influenced the effects of DA-6 on in vitro culture of the purple coneflower plants. For explants with less biomass, the optimum concentrations of DA-6 to improve the regeneration or growing situations were lower, for tissues and buds with more biomass, higher. This finding communicated in this manuscript may help people applied the DA-6 better in culture of this plant and other creatures.
2. Materials and Methods
2.1. Plant Materials
Diploid E. purpurea were grown up from seeds from the Company of Plantation Products (Norton, MA, USA). Tetraploid was obtained by treating diploid explants with colchicine . We got the triploid plants by crossing diploid and tetraploid plants, and growing the seeds. The ploidy levels plants were confirmed by counting chromosome numbers in at least three root tips in at least five cells . Plantlets of different genotypes were cloned by stimulating axillary bud proliferation  from one individual plant.
2.2. Preparation of Explants
Leaves, petioles, and roots were isolated from in vitro maintained clones of diploid plants. All these explants were prepared by cutting: leaf explants: 4 × 4, 7 × 7, and 10 × 10 mm2, petiole and root explants: 4, 9, and 25 mm in length. We identified these leaf, petiole, and root explants with different sizes, from small, medium, to large, as the low, medium, and high biomass explants, respectively.
Buds of diploid, triploid, and tetraploid were proliferated on agar-gelled medium containing MS basal elements, 3% sucrose, 0.01 mg・L−1 NAA and 0.5 mg・L−1 BA . Bud explants were isolated from these proliferated shoots and buds. Buds with two to four leaves and 15 mm in height were identified as low biomass bud explants; four to six leaves and 25 mm in height, medium biomass bud explants, six to eight leaves and 40 mm in height, high biomass bud explants. If a bud had grown root(s) when it was isolated for the experiment, we cut off the root(s) before inoculating it onto the medium.
2.3. Preparation of the Medium
Glass jars of about 250 ml inside volume, filled with 40 mL of medium and covered with a polycarbonate screw cap were used. The medium for inducing adventitious bud formation from explants contained Murashige and Skoog (MS) medium  containing 3% sucrose, 0.3 mg・L−1 BA, 0.01 mg・L−1 NAA , and DA-6 of different concentrations. The essential components of the medium for culture of the regenerated buds included MS medium including 3% sucrose, 0.05 mg・L−1 NAA, and DA-6 of different concentrations. All the media used were adjusted to a pH 6.0, gelled with 0.45% agar prior to autoclaving at 1.4 kg・cm−2 for 20 min.
2.4. Maintenance of the Cultures
The cultures temperature is 25˚C. For seed germination, cultures were first kept in darkness for 10 days, then shift to 12-hours photoperiod (about 50 μmol・m−2・s−1). For regeneration of adventitious buds, and rooting of the isolated buds, cultures were kept directly under 12-hours photoperiod (about 50 μmol・m−2・s−1).
2.5. Data Collection and Analysis
Data for adventitious bud formation were recorded 30 days after initiation of the bud regeneration cultures. The regeneration rate was calculated by the number of buds per explant. Buds of at least 1 cm in height, without hyperhydricity  and at least two visual leaves were considered as valid. The values of the shoots per explant, and the roots number, as well as those of height were expressed as means ± SE.
Statistical analysis of the data was carried out by using the Statistical Product and Service Solution (SPSS) 19.0 software. The significant differences among the means were determined by the one-way ANOVA and then Duncan’s multiple range tests for pairwise comparison judgment of more than two data sets, or by the independent sample t-test for two sets of data. The differences were considered significant when 𝑃 < 0.05. Graphs were draw by using Origin 8.5 software. Data-fitting curves were carried out in b-spline style. Photographs were recorded by D90 digital camera (Nikon) with Speedlight SB-26 flash (Nikon), and arranged by the Adobe Photoshop CC software.
3.1. Effects of DA-6 on Bud Regeneration in Explants with Different Biomass Dosage
Leaf, petiole, and root explants with different biomass dosage were inoculated onto medium with various concentrations of DA-6. Effects of DA-6 on the regeneration of adventitious buds were evaluated.
As shown in Figure 1(a) and Figure 2, demonstrated that DA-6 could stimulate the regeneration of adventitious buds in leaf explants when used at suitable concentrations, which varied for biomass dosage. The medium and high biomass explant required higher concentration of DA-6 (0.08 mg・L−1) in order to maximize the number of regenerate buds.
The DA-6 affected the regeneration of petiole explants with different biomass differently. In Figure 1(b) and Figure 3, for small petiole explants, 0.08 mg・L−1 DA-6 significantly inhibited the regeneration. For large petiole explants, 0.08 mg・L−1 of DA-6 increased the regeneration.
As well as to leaf and petiole explants, the DA-6 also affected the regeneration of root explants with different biomass differently. In Figure 1(c) and Figure 3, for small root explants, 0.01 mg・L−1 and higher concentrations of DA-6 significantly inhibited the
Figure 1. Effects of DA-6 on the regeneration of buds from leaf (a), petiole (b), and root (c) explants with different biomass demonstrated by scatter points with standard error (SE) bars and b-spline curve fittings.
regeneration, while for large ones, the same concentration of DA-6 significantly increased the regeneration.
3.2. Effects of DA-6 on Rooting of Bud Explants with Different Biomass
The bud explants with the height of 15 and 25 mm were defined as the low, and medium biomass bud explants. The former grew two to four leaves, and the later, four to six. As well as to regeneration of leaf, petiole, and root explants, the DA-6 affected the growth of bud explants with different biomass differently. In Table 1 and Figure 5, for small bud explants, 0.16 mg・L−1 DA-6 significantly inhibited the formation of the adventitious roots, while for large ones, the same concentration of DA-6 significantly increased the rooting.
3.3. Effects of DA-6 on Growing of Bud Explants with Different Gene Dosage
The diploid, triploid, and hexaploid bud explants with the height of 40 mm were tested. The selected buds grew six to eight leaves. In Table 2 and Figure 6, the optimal concentrations of DA-6 to improve the height of diploid, triploid, and hexaploid bud explants were 0.32, 0.32, and 0.64 mg・L−1. The optimal concentration of DA-6 to improve the rooting of the explants was 0.64 mg・L−1. When the concentrations of DA-6 were rose to be higher than the optimal one, the promotional effect decreased. Even so, when
Figure 2. The effect of DA-6 on regeneration of leaf explants with different biomass Leaf explants from left to right were low-, medium-, and high-biomass; concentrations of DA-6 from top to bottom were 0, 0.01, 0.08, 0.16, and 0.32 mg・L−1.
the concentration of DA-6 was three times higher (1.28 mg・L−1) than the optimal one (0.32 mg・L−1), the situations of the plants were still better than the plants grown in the medium with no DA-6.
4. Discussion and Conclusion
Previous studies have proved that certain concentrations of DA-6 improved the regeneration and the growth situation of adventitious buds in purple coneflower. However,
Figure 3. The effect of DA-6 on regeneration of petiole explants with different biomass. Petiole explants from left to right were low-, medium-, and high-biomass; concentrations of DA-6 from top to bottom were 0, 0.01, 0.08, 0.16, and 0.32 mg・L−1.
the effects varied. For example, the optimal concentrations for the diploid and triploid leaf explants to achieve the maximal regeneration rate were higher than that for the tetraploid, and hexaploid ones . In another words, the genedosage may not be the critical factor influenced the effects of DA-6.
In this study, we investigated the effects of DA-6 on in vitro culture of the purple coneflower leaf, petiole, root, and bud explants with different biomass dosages. The results showed that explants with lower biomass were more sensitive to DA-6. On one
Table 1. Effects of DA-6 on rooting of bud explants (initial height 15 and 25 mm).
*The values of the root numbers are expressed as the mean ± SE. The data in the same column followed by different letters are significantly different, as determined by the one-way ANOVA and then the Duncan’s multiple range test at P < 0.05 level.
Table 2. Effect of DA-6 on growth of diploid, triploid, and tetraploid shoots with larger biomass (initial height 40 mm).
*The values of the height and root tip numbers are expressed as the mean ± SE. The data in the same column followed by different letters are significantly different, as determined by the one-way ANOVA and then the Duncan’s multiple range test at P < 0.05 level.
hand, they needed very low concentration of DA-6 to reach their best regeneration rates or growth situation. For example, the leaf and petiole explant with low biomass needed only 0.01 mg・L−1 of DA-6 to increase their regeneration rates (Figure 1(a) and Figure 2). On the other hand, though no studies have demonstrated the application of DA-6 may inhibit the plant growth, the regeneration of adventitious buds was easily inhibited by DA-6 when the concentrations were higher than the optimal ones. Sometimes, a high concentration of DA-6 can be fatal for a low-biomass explant. Such as 0.08 mg・L−1 of DA-6, it was fatal for low-biomass petiole and root explants (Figure 1(b), Figure 1(c), Figure 2 and Figure 3). As a contrast, the explants with high biomass needed higher concentrations of DA-6 to obtain the best regeneration or growth states. Meanwhile, the explants with high biomass were more tolerant to high concentration of DA-6. For instance, regeneration rates of the high-biomass leaf, petiole, and root explants decreased slower than the low-biomass ones as the concentrations of DA-6 increased over the optimal ones (Figures 1-4).
During the growth of regenerated buds, the suitable concentration for bud explants with 4 cm in height and 6 - 8 leaves was 0.32 or 0.64 mg・L−1. Either of these was higher than the optimal concentrations for the low and the medium biomass bud explants (0.01, 0.08 mg・L−1 respectively) (Table 1 and Table 2, Figure 5 and Figure 6).
Figure 4. The effect of DA-6 on regeneration of root explants with different biomass. Root explants from left to right were low-, medium-, and high-biomass; concentrations of DA-6 from top to bottom were 0, 0.01, 0.08, 0.16, and 0.32 mg・L−1.
Figure 5. The effect of DA-6 on rooting of bud explants with different biomass. Initial biomass of buds were small (upper), and large (lower); concentrations of DA-6 from left to right were 0, 0.01, 0.08, 0.16, 0.32, and 0.64 mg・L−1.
Figure 6. The effect of DA-6 on growth of shoots of the diploid, triploid, and tetraploid plants with larger biomass. From left to right: concentrations of DA-6 were 0, 0.16, 032, 0.64, and 1.28 mg・L−1; from top to bottom: diploid, triploid, and tetraploid.
The present results evidenced that the effects of DA-6 on micropropagation of this medicinal plant were related with the initial biomass dosages of the explants, which had not being considered as an influence factor in earlier studies. It is understandable because this novo PGR has a very short history of being applied on plant micropropagation , and/or soon later on microalgae . As it had been practically used in field   and had already been proved to be safe , more studies of this PGR on plants and other creatures were worth exploring.
BA and NAA are commonly used plant growth regulators in in vitro culture for the induction of adventitious buds formation. Many researchers used them and obtained high regeneration rates on purple coneflowers  . In the present experiments, we did not obtain high regeneration rates compared with those reported by Choffe et al. and Koroch et al. Even so, the effects of novo PGRs are still worth study, because we found significant variations in adventitious bud regeneration abilities genotypes, and for some genotypes, the effect of BA was limited . Present results proved the application of DA-6 at certain concentration as a supplement to the culture medium was able to enhance the stimulating effects BA on bud regeneration.
As we known, the genedosage influenced the effects of BA   and NAA  on micropropagation of purple coneflower significantly. The way that DA-6 worked on the explants seems to be different from either BA or NAA. The biomass dosage influenced the effects of DA-6 on the micropropagation of purple coneflower. The working mechanism of this novo PGR is still unclear. As the synthetic technology of DA-6 has being well studied and applied    , the application of DA-6 on purple coneflower may help who produce and grow purple coneflower clonal plants improve their productivity with low cost.
To make good use of DA-6, the explants inoculated on the medium with DA-6 should be cut into standard size. For example, prepare the medium contains 0.08 mg・L−1 for leaf explants at least 7 × 7 mm2 in areas, petiole and root explants at least 9 mm at length, and bud explants at least 25 mm in height and with four to six leaves. For leaf explants smaller than 4 × 4 mm2 in areas, petiole and root explants shorter than 4 mm, and bud explants smaller than 15 mm in height and with less than four leaves, no DA-6 should be added into the medium in case of the culture be strongly restrained.
This study was funded by the Natural Science Foundation of Hainan Province (#20153074) and the Science and technology cooperation projects of Hainan Province, China (#KJHZ2015-15).
Conflicts of Interest
The authors declare no conflict of interest.
DA-6: Diethyl Aminoethyl Hexanoate
MS: Murashige and Skoog (1962)
NAA: Naphthalene acetic acid