In vitro propagation of medicinal and ornamental plant … · 2017-02-21 · V o l u m e 8 2 I s s u e 1 Fe b r u a r y 2 0 1 7 55 Yanni Zhang et al. | In vitro propagation of medicinal

54 E u r o p e a n J o u r n a l o f H o r t i c u l t u r a l S c i e n c e

In vitro propagation of medicinal and ornamental plant Lysimachia davurica Yanni Zhang, Dongling Sun and Shangchun HuCollege of Landscape Architecture, Northeast Forestry University, Harbin 150040, China

Eur. J. Hortic. Sci. 82(1), 54–60 | ISSN 1611-4426 print, 1611-4434 online | https://doi.org/10.17660/eJHS.2017/82.1.6 | © ISHS 2017

Original articleGerman Society for Horticultural Science

IntroductionThe genus Lysimachia (family Primulaceae) contains

more than 180 species of perennial herbs widely distrib-uted across temperate and subtropical regions. Lysimachia davurica Ledeb. is native to temperate Asia, including China and Siberia (U.S. National Plant Germplasm System, 2016). It is a traditional Chinese medicinal plant for treating high blood pressure and insomnia. As a wild perennial flower with long flowering period, L. davurica could also be grown as a bedding, border or potted plant. At present, the research on L. davurica is mainly focused on germination and chemical separation (Tian et al., 2005; Zhang et al., 2013).

Seed germination rate of L. davurica is relatively low (Zhang et al., 2013). To facilitate horticultural production of this promising ornamental species, an efficient propaga-tion and regeneration system is needed. Micropropagation

SummaryLysimachia davurica is an important medicinal

and ornamental plant species. An efficient in vitro regeneration system on excised leaves of L. davurica was developed. The experimental conditions of tissue culture system were investigated based on the effect of different concentrations and combinations of plant growth regulators (PGRs), explant orientations, leaf segments, and concentrations of activated charcoal. Two types of callus induction medium using Murashige and Skoog (MS) medium + 1.0 mg L-1 NAA + 0.5 mg L-1 6-BA and 1.0 mg L-1 NAA + 1.0 mg L-1 6-BA had a callus inducing rate of 95.56%. The shortest time to callus appearance was 7 d. Proximal segments were better for callus induction than middle and distal ones. Placing leaf with abaxial side down on medium was better than adaxial side for inoculation. For the combinations of 6-BA and NAA tested, MS + 0.5 mg L-1 6-BA and 0.1 mg L-1 NAA was best for callus proliferation, while MS + 1.0 mg L-1 6-BA and 0.1 mg L-1 NAA was optimal for callus differentiation and shooting. For the combinations of PGRs tested, the best rooting medium is 1/2 MS + 0.5 mg L-1 IBA + 0 mg L-1 NAA, with 99.89% rooting rate. On plantlets rooting, 1 g L-1 activated charcoal performed better than others (0, 2, 3, and 4 g L-1). The optimal transplanting medium was using vermiculite and humus as substrate in the proportion of 2:1 (v/v), with 97.78% survival rate. This protocol could be an efficient method for the micropropagation of L. davurica.

Keywordsmicropropagation, tissue culture, leaf, shoot regeneration, callus

Significance of this studyWhat is already known on this subject?• Micropropagation of L. davurica had been achieved

using hypocotyl and cotyledon stem section as explants while its efficiency of plant regeneration is relatively low. To date, a tissue culture protocol for regeneration of L. davurica using leaf tissue explants has not been reported.

What are the new findings?• Our study provides a tissue culture protocol

for regeneration of L. davurica using leaf tissue explants, based on the effect of different hormone concentrations and combinations, explant orientations, leaf segments, and concentrations of activated charcoal.

What is the expected impact on horticulture?• The results could help promote rapid production of

L. davurica for growers. The in vitro plantlets may be suitable to set up a transformation system to facilitate the production of transgenic plants with desirable horticultural traits or medicinal contents.

of L. davurica had been achieved via hypocotyl and cotyle-don stem section as explants induced by cytokinins (Qu et al., 1990). However, the efficiency of plant regeneration (shoots per culture) is relatively low. Therefore, this research explored ways to achieve both direct shoot formation and indirect plantlet regeneration through inducing leaves of L. davurica. To date, a tissue culture protocol for regener-ation of L. davurica using leaf tissue explants has not been reported. The main purposes of our study were to enhance multiplication and promote rapid production of L. davurica for growers.

Materials and methods

Plant materials and disinfection L. davurica plants were used as the initial explant source.

One-year-old parent plants were grown in a greenhouse at Northeast Forestry University in Harbin, China. In May, leaves from upper part of L. davurica were obtained as explants.

About 100 leaves of L. davurica collected from live plants were washed in detergent solution (Liby Inc., Guangzhou) for 20 min, rinsed for 2–3 h under running water, and then placed in sterile console for further processing. All leaves were sterilized with 75% ethanol for 30 s, washed 2–3 times with sterile water, soaked with 0.1% corrosive sublimate (HgCl2) for 4, 6, 8, 10, and 15 min respectively, washed three

V o l u m e 8 2 | I s s u e 1 | F e b r u a r y 2 0 1 7 55

Yanni Zhang et al. | In vitro propagation of medicinal and ornamental plant Lysimachia davurica

times with sterile distilled water, and then blotted dry on sterile filter paper. The leaves were cut into 0.5 cm2 pieces. All samples were inoculated onto Murashige and Skoog (MS) (1962) medium. Each treatment containing 18 explants was repeated three times.

Callus inductionLeaves were sterilized and processed using the optimum

sterilizing method from the previous experiment. Then leaf cuttings were inoculated onto MS media with plant growth regulators (PGRs) of 0.5, 1.0, 2.0, 2.5 mg L-1 6-BA (6-Benzyl-aminopurine) and 0.05, 0.2, 0.5, 1.0 mg L-1 of NAA (1-Naph-thaleneacetic acid) applied in combinations, resulting in 16 PGR combinations amended media. To test the effect of explant orientation on callus induction, sterile leaves were placed with adaxial or abaxial side down on the most optimal medium (from the PGR experiment mentioned above) and cultivated. To test the effect of different leaf segment on cal-lus induction, each leaf was cut along the vertical direction of the main vein into three sections, resulting in distal, middle, and proximal plant segments.

Each treatment consisted of 18 explants and was re-peated three times. After initial cultivation of 30 d, callus induction rate, differentiation rate, and growth status were recorded.

Callus proliferation and shoot regenerationEach callus in good condition was cut into 0.5 cm3 blocks

which were then inoculated onto MS media consisting of 6-BA (0.5 or 1.0 mg L-1) and NAA (0.03, 0.05 or 0.1 mg L-1) in combinations.

The treatment contained 18 samples and was repeated three times. After 30 d of cultivation on media, the data of proliferation multiples, callus differentiation rate, and shoot-ing index was obtained.

Rooting cultivation and acclimatization When adventitious shoots were developed, the shoots

were cut into 2.0 cm length and then inoculated onto 1/4 MS, 1/2 MS, or MS media. And each medium was treated with two levels of NAA (0, 0.5 mg L-1) and three levels of IBA (In-dole-3-Butyric Acid) (0, 0.5, 1.0 mg L-1), resulting in six PGR combinations per kind of medium. Each PGR treated medium was then amended with different concentrations of activated charcoal (1, 2, 3, and 4 g L-1) to promote root growth.

Each treatment contained 30 shoots and was repeated three times. After 30 d of culturing, rooting rate, mean root length, and mean rooting number per shoot were recorded.

Plantlets with well-developed roots and robust growth were hardened for 3 d. Then, plantlets were rinsed under tap water to remove medium remains. These cleaned plantlets were subsequently transplanted into plastic pots filled with different mixing ratios of vermiculite and humus (0:1, 1:1, 2:1, and 3:1), and placed on metal benches in a greenhouse.

Each treatment contained 30 plantlets and was repeated three times. After three months of plant growth, the survival rate of transplanted plantlets was evaluated.

Culture conditionIf not otherwise specified, all MS media used in this study

consisted of 7.8 g L-1 of agar (Nachuan Inc.) and 30 g L-1 of sucrose, and had PH value of 5.9 ± 1. All media were sterilized in an autoclave at 121°C for 20 min. Explants were cultured in a growth chamber with a constant temperature of 25°C, ambient light of 1,500-2,000 lx light, and a photoperiod of

16 h light/8 h dark.

Statistical analysisAll the data were analyzed using Excel 2007 (Microsoft

Office) software with one-way ANOVA. Data were evaluated by the analysis of variance and significant by Duncan’s multi-ple range test with Statistical Package for the Social Sciences (SPSS) 17.0 Software. A difference was considered statisti-cally significant when p < 0.05.

Calculation formula listContamination rate = (the number of contaminations/

the number of inoculations) × 100%.Mortality rate = (the number of dead/ the number of in-

oculations) × 100%.Callus induction rate = (the number of callus formed/ the

number of inoculations) × 100%.Differentiation rate = (the number of callus differentiated

to adventitious shoots/ the number of inoculations) × 100%.Shooting index = (the number of adventitious shoots/ the

number of inoculated calluses) × 100%.Rooting rate = (the number of rooted adventitious

shoots/ the number of inoculations) × 100%.Mean root length = the sum of each root length/ the num-

ber of total roots.Mean rooting number per shoot = the number of roots/

the number of rooting shoots.Survival rate = (the number of survivals/ the number of

transplanting individuals) × 100%.

Results

Effect of sterilizing time on explantsThe contamination rate of L. davurica leaves decreased

with the increase in sterilizing time using 0.1% HgCl2, and dropped to zero when sterilizing time was more than 8 min. On the contrary, the mortality rate increased as sterilizing time increased. Based on the consideration of both contam-ination and mortality rates, the sterilizing effect of 75% eth-anol for 30 s plus 0.1% HgCl2 for 4 min (with contamination rate 23.46 ± 2.14% and mortality rate 1.23 ± 2.14%) were determined to be the most appropriate combination for ex-plant sterilization.

Effect of explant orientation and leaf segment on callus induction

Explant orientation influenced the efficacy of callus in-duction. It took seven days for callus induction when ex-plants were abaxially cultured on medium. However, when explants were adaxially grown on medium, the extended time of 13 d was required for callus induction. In addition, the higher callus induction rate was observed from the ex-plants that were cultured abaxially (100%) when compared to the other explants grown adaxially (74.07%) (Table 1). Hence, abaxial side was more efficient for L. davurica tissue culture than adaxial side.

When different leaf segments were cultured, the callus in-duction rate significantly dropped from proximal (94.44%), middle (64.81%) to distal segment (37.04%). Also, explant sources of leaf proximal and middle segments displayed fast-er callus development (7 d) if compared to distal segments (14 d). These results indicated that proximal segments are more suitable than middle and distal segment in terms of callus induction.



Table 1. Effect of explant orientation and leaf segment on callus induction.

Treatments Number of segments

The earliest callus induction time (d)

Callus induction rate (%)

Explant orientation Abaxial side down on medium 18 7 100.00 ± 0.00Adaxial side down on medium 18 13 74.07 ± 3.21

Segment Proximal 18 7 94.44 ± 5.56 cMiddle 18 7 64.81 ± 6.42 bDistal 18 11 37.04 ± 8.49 a

The values are the means (± SE) of three replications with 18 explants per replication. Means within a column followed by the same letter are not significantly different at p<0.05.

Table 2. Effect of different concentrations and combinations of 6-BA and NAA on leaf explants.

Concentration of 6-BA (mg L-1)

Concentration of NAA (mg L-1)

Callus induction rate (%)

Callus differentiation rate (%)

0.5 0.05 51.1 ± 3.9 b 8.9 ± 7.8 ab0.5 0.2 84.4 ± 3.7 fg 71.2 ± 8.4 e0.5 0.5 64.4 ± 3.8 bcd 13.7 ± 5.5 ab0.5 1.0 95.6 ± 3.5 g 2.4 ± 4.1 a1.0 0.05 17.8 ± 3.4 a 72.2 ± 25.5 e1.0 0.2 57.8 ± 3.9 bc 53.7 ± 3.2 cde1.0 0.5 75.6 ± 10.2 def 39.0 ± 9. 9 bcd1.0 1.0 95.6 ± 7.7 g 4.8 ± 4.2 a2.0 0.05 17.8 ± 3.1 a 27.8 ± 25.5 abc2.0 0.2 68.9 ± 7.7 cde 9.8 ± 9.2 ab2.0 0.5 68.9 ± 13.9 cde 8.3 ± 4.4 ab2.0 1.0 82.2 ± 13.9 efg 2.4 ± 4.1 a2.5 0.05 22.2 ± 10.18 a 56.7 ± 40.4 cde2.5 0.2 17.8 ± 3.9 a 61.1 ± 9.6 de2.5 0.5 53.3 ± 6.7 b 17.3 ± 18.9 ab2.5 1.0 75.6 ± 10.2 def 14.2 ± 13.7 ab

The values are the means (± SE) of three replications with 18 explants per replication. Means within a column followed by the same letter are not significantly different at p<0.05.

Table 3. Effect of different concentrations and combinations of 6-BA and NAA on callus proliferation and differentiation.

6-BA concentration(mg L-1)

NAA concentration(mg L-1)

Differentiation rate (%)

Shooting index

Callus growth

0.5 0.03 3.7 ± 3.2 a 2.00 ± 1.73 a +0.5 0.05 40.7 ± 3.2 c 1.46 ± 0.10 a ++0.5 0.1 22.2 ± 5.6 b 2.26 ± 0.07 a +++1.0 0.03 37.0 ± 3.2 c 2.44 ± 0.73 a +1.0 0.05 51.9 ± 3.2 d 2.17 ± 0.17 a ++1.0 0.1 74.1 ± 3.2 f 4.56 ± 0.25 b ++

The values are the means (± SE) of three replications with 18 explants per replication. Means within a column followed by the same letter are not significantly different at p<0.05. Percentage of explant surface area that produced callus: + (<25%), ++ (25% to 75%), +++ (>75%).



Effect of PGR combinations on callus induction and differentiation

The regeneration of leaf explants display varying when different concentrations of NAA were used (Table 2). It was shown that leaf edge incisions were thickened and leaves gradually became dark green after 3 d of culturing. After 7 d, leaf edge incisions began to form callus that were then grad-ually enlarged (Figure 1A). Callus induction rate increased as NAA concentration increases. When the NAA concentration was 1.0 mg L-1, the callus induction rate increased to over 95%. The increase in 6-BA concentration accelerated callus differentiation, but decreased callus growth which could produce small-sized shoots. Among all treatments tested, the optimal medium for induction of leaf callus consists of MS + 0.5 mg L-1 6-BA + 1.0 mg L-1 NAA or MS + 1.0 mg L-1 6-BA + 1.0 mg L-1 NAA for highest callus induction rate of over 95%.

Effect of PGR concentration on callus proliferation and shoot regeneration

All combinations of 6-BA and NAA could promote callus growth. After 7 d of cultivation, tiny, dense, ivory, spheroidal dots appeared on callus surface and finally differentiated ad-ventitious shoots (Figure 1B and 1C). When the concentra-tion of 6-BA was constant, 0.1 mg L-1 NAA performed best for shoot regeneration considering both shoot regeneration rate and callus growth status. When the concentration of NAA was constant, the callus proliferation and shoot regeneration increased as the concentration of 6-BA increased. Therefore, the combination of 0.5 mg L-1 6-BA and 0.1 mg L-1 NAA was more suitable for callus proliferation, while 1.0 mg L-1 6-BA and 0.1 mg L-1 NAA was optimal for callus differentiation and shooting (Table 3).

Plantlets rooting and acclimatizationThe plantlets rooting performed varying in different con-

centration of macro-elements in growth media. The rooting rate (84.45%) and highest rooting number in 1/2 MS was significantly higher in 1/4 MS and MS (Table 4). The plant-lets rooting performed poorly in MS medium with the obser-vation of shortest root length and lowest rooting rates. Root lengths in 1/4 MS and 1/2 MS mediums had no significant difference. Plantlets in three media without macro-elements amended all had thin stems, wispy root hair, tawny leaves and few callus in the base (Figure 1D). This indicated using medium only was not enough for efficient rooting induction of L. davurica.

To evaluate the effect of PGRs in plant regeneration, different concentrations of IBA and NAA were added into the basic medium of 1/2 MS. The addition of PGRs resulted in more robust growth observed in both plantlets and root callus (Figure 1E). When only IBA was added, the rooting rate, rooting number and root length were higher at IBA concentration of 0.5 mg L-1 than 0 or 1.0 mg L-1 (Table 4). This indicated that a proper concentration of IBA may promote the plantlets rooting and improve the callus induction, while excess IBA may inhibit root induction and growth, and quality of developed roots. When the 0.5 mg L-1 NAA was added into IBA treated media, rooting number, rooting rate and root length all decreased. Therefore, the optimal medium for rooting was 1/2 MS + 0.5 mg L-1 IBA + 0 mg L-1 NAA (Table 4).

When different concentrations (1, 2, 3, and 4 g L-1) of activated charcoal (AC) were added into the basic medium containing 1/2 MS + 0.5 mg L-1 IBA. Results showed that the rooting rate significantly decreased with the increase of AC concentration (Table 5). The rooting number and root length was highest at the concentration of 1 g L-1 (Figure 1F).

Figure 1. L. davurica tissue culture. (A) Callus induction with 6-BA and NAA combinations, callus were yellow-green dense granular and sprouted small adventitious shoots after a period of continuous culture. (B) Callus proliferation after cultivated for 30 d. (C) Adventitious shoots differentiating from callus after cultivated for 30 d. (D) Rooting in 1/2 MS, plantlets had thin stems, wispy root hair, tawny leaves and few callus in the base. (E) Rooting in IBA. (F) When the medium was added 1 g L-1 AC, plant root was fair and regular, root hair was tiny, dense and even. (G) Plantlets in transplanting substrate. (H) Regenerated L. davurica in open field.

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FIGURE 1. L.davurica tissue culture. (A) Callus induction with 6-BA and NAA combinations, callus were yellow-green dense granular and sprouted small adventitious shoots after a period of continuous culture (B) Callus proliferation after cultivated for 30 d. (C) Adventitious shoots differentiating from callus after cultivated for 30 d. (D) Rooting in 1/2MS, plantlets had thin stems, wispy root hair, tawny leaves and few callus in the base. (E) Rooting in IBA. (F) When the medium was added 1 g L-1 AC, plant root was fair and regular, root hair was tiny, dense and even. (G) Plantlets in transplanting substrate. (H) Regenerated L.davuricain open field.

E F G H

A B C D



Therefore, for the concentrations we tested, the addition of 1 g L-1 AC was more appropriate for plantlets rooting.

Plantlets with robust growth and thick roots were chosen to harden, and recovered to normal growth after transplanting into the substrate mixing vermiculite and humus for 2 weeks. Plantlets survival rate responded varying for different mixing ratios of vermiculite and humus (Table 6). The survival rate was highest for the 2:1 (v/v) mixing vermiculite and humus, but it had no significant difference with their 1:1 mixing. The survival rate was lowest for the 0:1 mixing vermiculite and humus. The results showed that the 2:1 mixing vermiculite and humus performed better than others (0:1, 1:1, and 3:1) for transplanting L. davurica plantlets.

DiscussionAn in vitro regenerated protocol is a method for attaining

a large number of regenerated plants. The selection of effective explants is the first prerequisite for establishment of plant regeneration protocol (Phulwaria et al., 2013).

Different explants, such as axillary buds, shoot tips and nodal stems, are commonly used for micropropagation and germplasm conservation. Leaves have been commonly used as sources for plant tissue culture as they can be easily obtained. In this study, we selected L. davurica leaves as explants to induce callus. The optimal induction medium was MS + 0.5 mg L-1 6-BA + 1.0 mg L-1 NAA, and MS + 1.0 mg L-1 6-BA + 1.0 mg L-1 NAA. The induction rate of 95.56% was high. The result agreed with previous studies which observed that an auxin-cytokine combination provided maximum shoot induction response from callus (Karami et al., 2009; Ahmad et al., 2010; Murthy et al., 2010; Cheruvathur et al., 2013; Thomas and Hoshino, 2015). Zheng et al. (2009) found that MS + 1.0–3.0 mg L-1 BAP + 0.1 mg L-1 NAA was the optimum induction medium for two Lysimachia species: L. christinae and L. nummularia ‘Aurea’. Compared with our results on L. davurica, different species might prefer different optimum induction media.

Recently, the effects of explant orientation on callus induction and adventitious shoots regeneration are discussed in several tissue culture experiments. Putting adaxial side of leaf segments down on medium was more appropriate for obtaining regenerated plantlets of Anthurium andreanum (Cardoso and Habermann, 2014), Ficus carica (Kim et al., 2007), and Vaccinium angustifolium (Debnath, 2009). This is because the stomata in adaxial side of leaf facilitate oxygen exchange, and palisade parenchyma on the adaxial side are better at transferring growth regulators and nutrients from medium to explants (Kim et al., 2007). However, in Jatropha curca (Mazumdar et al., 2010) and Pyrus ussuriensis (Liu et al., 2008), the highest regeneration efficiency was obtained by putting leaf abaxial side in direct contact with the culture medium. Our results concurred with Mazumdar et al. (2010) that leaf edge often wrap upward when adaxial side touches medium, resulting in decreased shoot number per explant since the incision surface cannot touch the medium.

Table 4. Effect of different concentrations and combinations of IBA and NAA on root induction and growth.

IBA concentration (mg L-1)

NAA concentration (mg L-1)

Rooting rate (%)

Root number

Root length (cm)

0 0 83.7 ± 2.2 d 6.1 ± 0.23 c 3.55 ± 0.06 d0 0.5 81.2 ± 0.6 d 8.7 ± 0.27 e 2.63 ± 0.04 c0.5 0 99.9 ± 0.1 e 9.4 ± 0.04 f 5.35 ± 0.07 e0.5 0.5 61.1 ± 2.8 b 3.1 ± 0.02 a 1.09 ± 0.03 a1.0 0 72.0 ± 1.0 c 6.9 ± 0.03 d 2.23 ± 0.05 b1.0 0.5 21.6 ± 4.2 a 3.6 ± 0.04 b 1.12 ± 0.04 a

The values are the means (± SE) of three replications with 30 plantlets per replication. Means within a column followed by the same letter are not significantly different at p<0.05.

Table 5. Effect of different concentrations of activated charcoal (AC) on rooting.

Activated charcoal (g L-1)

Rooting rate (%)

Root number

Root length (cm)

Plant length (cm)

0 99.89 ± 0.11 d 9.64 ± 0.04 c 5.37 ± 0.07 b 6.93 ± 0.80 b1 99.50 ± 0.50 d 10.33 ± 0.33 c 7.41 ± 0.04 c 11.15 ± 0.43 d2 93.80 ± 0.47 c 9.67 ± 0.33 c 6.67 ± 0.06 b 11.28 ± 0.35 d3 85.22 ± 0.91 b 7.33 ± 0.88 b 6.55 ± 0.21 b 8.85 ± 0.40 c4 75.16 ± 1.83 a 3.67 ± 0.67 a 3.54 ± 0.23 a 5.11 ± 0.31 a

The values are the means (±SE) of three replications with 30 plantlets per replication. Means within a column followed by the same letter are not significantly different at p<0.05.

Table 6. Effect of different mixing ratios of vermiculite and humus on survival rates of plantlets transplanting.

Mixing ratio of substrates(vermiculite: humus)

Survival rate (%)

0:1 37.78 ± 2.22 a1:1 91.11 ± 4.44 c2:1 97.78 ± 2.22 c3:1 55.55 ± 2.22 b

The values are the means (± SE) of three replications with 30 plantlets per replication. Means within a column followed by the same letter are not significantly different at p<0.05.



Several previous studies indicated that 1/2 MS medium could significantly promote plantlets rooting for Cannabis sativa (Cheng et al., 2016) and Orostachys malacophyllus (Zhang and Han, 2012). This is confirmed by our results. In addition, the supplement of exogenous hormones can also promote the rooting of tissue culture seedlings, for Citrus (Tallón et al., 2012) and Passiflora edulis (Prammanee et al., 2011). Higher auxin to cytokinin ratio tend to facilitate early rooting, while root elongation could be inhibited in excessively high amounts of auxin in growth media, for Arabidopsis (Kakani et al., 2009), Arachis paraguariensis (Aina et al., 2015), Caladium × hortulanum (Cao et al., 2016), and Citrus sinensis (Mendes et al., 2011). Therefore, choosing optimal type and amount of auxin is critical for plantlets rooting and root elongation. The quality and size of plantlets root system is one of the most important factors determining the future transplanting survival rate. Turker and Guner (2013) noted that 0.5 mg L-1 IBA was the optimal concentration for rooting of L. vulgaris, which was consistent with our findings in L. davurica.

The best root system of L. davurica plantlets was ob-tained by amending 1 g L-1 AC into basic medium. The posi-tive effects of activated charcoal might due to its adsorption of inhibitory compounds, resulting in decreasing toxic me-tabolites in media (Thomas, 2008).

The regenerated L. davurica have been grown in the open field of our university nursery for three years. No evidence of morphological variation was observed. This protocol could be an efficient method for micropropagation of L. davurica. In addition, the in vitro plantlets may be suitable to set up a transformation system to facilitate the production of trans-genic plants with desirable horticultural traits or medicinal contents.

AcknowledgmentsThis work was supported by the National Natural Science

Foundation of China (No. 31600576), the Fundamental Research Funds for the Central Universities (Grant No. 2572015BX06 and 2572015EY03), and Scientific Research Foundation of North-east Forestry University (No. QG2015-03).

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Received: Oct. 10, 2016Accepted: Jan. 12, 2017

Address of authors: Yanni Zhang, Dongling Sun and Shangchun Hu*College of Landscape Architecture, Northeast Forestry University, 26 Hexing Road, Harbin, Heilongjiang 150040, China* Corresponding author;

E-mail: [email protected]; Tel.: +86 0451 82191564

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