Large Scale in Vitro Propagation of Anoectochilus Roxburghii For

Industrial Crops and Products 70 (2015) 158–162

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Industrial Crops and Products

jo urnal homepage: www.elsevier.com/locate/indcrop

Large scale in vitro propagation of Anoectochilus roxburghii for

commercial application: Pharmaceutically important and

ornamental plant

a a a,∗ a

Ailian Zhang , Hongzhen Wang , Qingsong Shao , Mengjie Xu ,

b c

Wangshu Zhang , Mingyan Li

The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou 311300, China

Modern Agricultural Research Center in Ningbo, Industrial Technology Research Institute of Zhejiang University, Ningbo 315033, China

Rare Herbal Medicine and Engineering Institute of Zhejiang Province, Wuyi 321200, China

a r t i c l e i n f o a b s t r a c t

Article history: Anoectochilus roxburghii (Orchidaceae family) is an important medicinal and ornamental plant which is in

Received 26 November 2014

extinct condition because of the over-collected and the loss of its suitable habitats. Several protocols for A.

Received in revised form 8 March 2015

roxburghii micropropagation have been tested but none has provided an effective method for large-scale

Accepted 12 March 2015

micropropagation. The study investigates an improved and efﬁcient propagation method including shoots

generation and formation, shoot proliferation, inducing root and acclimatization. The nodal segments

Keywords:

cut from ﬁeld-grown plants were adopted as explants. Half-strength Murashige and Skoog (MS) medium

Anoectochilus roxburghii −1

supplemented with 1.5 mg l 6-benzyladenine (BA) was proved to be the optimal medium for shoots

In vitro propagation

generation and induction, on which, the shoot formation rate was 91.67%. Shoots were inoculated on

Medium composition

such four media as half-strength MS, MS, Vacin and Went (VW), and Gamborg (B5), containing various

Plant growth regulator

−1 −1

concentrations and combinations of the cytokinins BA, 0.5–4.0 mg l , kinetin (Kn), 0.1–2.0 mg l , and

−1

the auxin 1-␣-naphthaleneacetic acid (NAA), 0–0.5 mg l , to select the optimal conditions for shoot

growth and proliferation. Among those, the highest proliferation rate was 4.33, which was obtained on

−1 −1 −1

half-strength MS medium fortiﬁed with 3.0 mg l BA, 1.0 mg l Kn, 0.5 mg l NAA and additives. The

−1 −1

half-strength MS medium supplemented with 0.6 mg l NAA, 0.3 mg l indole-3-butyric acid (IBA) and

−1

100 mg l banana mashes turned out to be best medium for in vitro rooting, which has a inducing root

rate of 93.33%. The rooted plantlets were successfully transferred into plastic cups containing a sterile

sand and peat soil mixture in a ratio of 1:2, and the surface was covered with live moss. The survival rate

for A. roxburghii was 90.2%. The described protocol can be efﬁciently used for the large-scale propagation

and conservation of the germplasm of this endangered medicinal and ornamental plant.

1. Introduction such as its effects in clearing heat and cooling blood, eliminat-

ing dampness, and detoxiﬁcation, A. roxburghii has been called

The genus Anoectochilus (Orchidaceae family) consists of “the king of medicines” (Du et al., 2008; Cui et al., 2013; Shao

approximately 40 species distributed from India, the Himalayas, et al., 2014a,b). Recent research has demonstrated that the entire

Southeast Asia, and Indonesia to New Caledonia and Hawaii (Tseng plant possesses medicinal properties, such as antioxidant, anti-

et al., 2006). The species Anoectochilus roxburghii is used for medic- inﬂammatory and antitumor activities (He et al., 2004; Shao et al.,

inal and ornamental purposes in China and other Asian countries 2014a,b). A. roxburghii is also an expensive ornamental plant that

(Ket et al., 2004). Because of its unique medicinal properties, displays a network of colorful venation in its gorgeous leaves.

Although, the ﬂowers are not large, their white labella are quite

prominent and toothed, such that a grouping of three or four ﬂow-

ering plants makes an attractive, unique addition to one’s orchid

Abbreviations: BA, 6-benzyladenine; IBA, indole-3-butyric acid; Kn, kinetin;

collection (Yoon et al., 2007). A. roxburghii is now facing extinction

NAA, ␣-naphthalene acetic acid; MS, Murashige and Skoog medium; VW, Vacin and

because of the destruction of its habitat, the heavy exploitation of

Went medium; B5, Gamborg’s medium; PGRs, plant growth regulators.

∗

Corresponding author. Tel.: +86 571 63740809; fax: +86 571 63740809. its wild resources, its low propagation rate and its slow growth

E-mail address: [email protected] (Q. Shao).

http://dx.doi.org/10.1016/j.indcrop.2015.03.032

A. Zhang et al. / Industrial Crops and Products 70 (2015) 158–162 159

Fig. 1. Various stages of the in vitro propagation of Anoectochilus roxburghii. (a) Nodal segments used as explants. (b) Shoot bud induction on half-strength MS medium

−1 −1 −1 −1

supplemented with 1.5 mg l 6-benzyladenine (BA). (c) Shoot growth and proliferation on half-strength MS medium fortiﬁed with 3.0 mg l BA, 1.0 mg l kinetin, 0.5 mg l

−1 −1

˛-naphthalene acetic acid (NAA) and additives. (d) and (e) In vitro rooting on half-strength MS medium supplemented with 0.6 mg l NAA, 0.3 mg l indole-3-butyric acid

−1

and 100 mg l banana homogenate. (f) Acclimatized Anoectochilus roxburghii plants.

◦

(Wang et al., 2009; Li et al., 2012). Therefore, to meet the growing autoclaved at 121 C for 20 min. Cultures were grown under light

−2 −1

demand of the herbal and pharmaceutical industries, artiﬁcial cul- provided by cool white ﬂuorescent tubes (40 ␮mol m s photo-

◦

tivation is beginning to be investigated and attempted. A. roxburghii synthetic photon ﬂux) at 23 ± 2 C under a 14/10-h light/dark cycle.

is usually propagated sexually by seeds. However, the conventional The percentage of shoot induction, shoot lengths, shoot diameters

method of propagation is very inefﬁcient and time-consuming as and shoot fresh weights were recorded after 30 d of growing.

the seeds lack endosperm and require mycorrhizal association for

germination (Kong, 2001; Shao et al., 2014a,b). In this paper, we

2.3. Shoot growth and proliferation

demonstrate an improved and efﬁcient propagation system, fol-

lowed by shoot bud induction, shoot proliferation, rooting and

The surface-decontaminated explants were inoculated on

acclimatization. Such a protocol would allow for large-scale prop-

four media, half-strength MS, MS, Vacin and Went (VW),

agation to meet commercial demand and conserve this threatened

and Gamborg (B5), containing 0.7% agar, 3% sucrose, 0.1%

orchid species by reducing wild collection. −1 −1

Hyponex (N:P:K = 7:6:19), 3.0 mg l BA, 1.0 mg l kinetin (Kn)

−1

and 0.5 mg l 1-␣-naphthaleneacetic acid (NAA), in order to select

2. Materials and methods the optimal medium for shoot growth and proliferation. The cul-

ture conditions were the same as described in Section 2.2. The

2.1. Plant material and explant preparation proliferation rate, shoot lengths, shoot diameters and shoot fresh

weights were recorded after 60 d of culture. Since half-strength

Plants of A. roxburghii were collected from the Baicaoyuan (lat- MS medium was considered the most effective among the four

◦ ◦

itude 30 15 , longitude 119 43 ), and their identity was conﬁrmed kinds of media tested, this medium was used in all the subsequent

by Professor Runhuai Hu at Zhejiang A & F University. The stems cultures and experiments in this study. To optimize the type and

were rinsed with running tap water to get ride of adherent soil concentration of plant growth regulators (PGRs) for shoot growth

and were cut into 1.5–2.5-cm-long nodal segments having one axil- and proliferation, the shoots were inoculated on half-strength MS

lary bud (Fig. 1a). The stem segments were surface sterilized after medium replenished with different combinations of the cytokinins

−1 −1

immersed in 75% ethanol for 30 s, followed by the treatment of BA, 0.5–4.0 mg l , and Kn, 0.1–2.0 mg l , and the auxin NAA,

−1

0.1% HgCl2 reagent for 8 min and, ﬁnally, washed three times with 0–0.5 mg l . Observations were recorded after 60 d of culture.

sterilized distilled water for 5 min each.

2.4. Rooting

2.2. Culture media and growth conditions

Small shoots of 2–3 cm in length were transferred onto half-

The surface-decontaminated explants were inoculated on half- strength MS medium supplemented with different concentrations

−1

strength MS medium (Murashige and Skoog, 1962) containing of the auxins NAA, 0.3–1.2 mg l , and indole-3-butyric acid (IBA),

−1 −1 −1

0.7% agar, 3% sucrose, 6-benzyladenine (BA) (0.5–3.0 mg l ) and 0.3–1.2 mg l , and banana homogenate, 0–100 g l , for root

−1 −1 −1

additives (50 mg l ascorbic acid, 25 mg l citric acid, 25 mg l induction. The culture conditions were the same as mentioned

−1

adenine sulfate and 25 mg l l-arginine) for shoot bud induction. above. The percentage of rooting, root numbers and root lengths

The pH of the medium was adjusted to 5.8 with HCl or NaOH and were observed and recorded after 60 d of culture.

160 A. Zhang et al. / Industrial Crops and Products 70 (2015) 158–162

Table 1

Effects of different concentrations of 6-benzyladenine (BA) on shoot bud induction from nodal segments of Anoectochilus roxburghii.

−1

BA (mg l ) Percent of shoot Shoots length (cm) Shoots diameter (mm) Shoots fresh weight

induction (%) (mean ± SD) (mean ± SD) (mg) (mean ± SD)

0.5 66.67 ± 2.38 d 1.18 ± 0.04a 2.28 ± 0.02a 22.10 ± 0.09a

1.0 81.67 ± 1.67b 1.07 ± 0.03b 2.24 ± 0.07a 20.79 ± 0.13b

1.5 91.67 ± 1.04a 1.04 ± 0.05b 2.25 ± 0.03a 20.84 ± 0.11b

2.0 74.67 ± 2.16c 1.02 ± 0.02b 2.06 ± 0.02b 17.95 ± 0.09c

2.5 63.33 ± 0.98 d 0.91 ± 0.01c 1.84 ± 0.06c 14.60 ± 0.07 d

3.0 48.33 ± 1.15e 0.78 ± 0.03 d 1.82 ± 0.08c 11.67 ± 0.10e

Means followed by the different letter within columns are signiﬁcantly different (P < 0.05) using Duncan’s multiple range test.

2.5. Acclimatization of plantlets ascorbic acid and citric acid are sources of antioxidants, adenine

sulfate works as a supplementary cytokinin and L-arginine pro-

Plantlets with well-developed roots were transferred to a green- vides reduced nitrogen. These media additives have been found to

house with 30% natural irradiance for 15 d, and then softly washed be conducive to other plants, such as Eulophia nuda (Panwar et al.,

with running tap water to remove adhering medium before the pot 2012), Leptadenia reticulata (Rathore et al., 2013) and Caralluma

transfer. Subsequently, they were transferred to plastic cups con- edulis (Patel et al., 2014).

taining a sterile sand and peat soil mixture with a ratio of 1:2. The

cultivation medium’s surface was covered with a layer of live moss. 3.2. Effect of media composition on shoot growth and

The planting density of plantlets was 3 cm × 3 cm. The temperature proliferation

◦

of the greenhouse was regulated by a thermostat set at 15 C min-

◦

imum and 30 C maximum, and the humidity was between 80 and Media composition exerted a strong effect on shoot growth

90%. After 30 d, the survival rate was calculated. and proliferation. The present study revealed that half-strength MS

medium was optimal for shoot growth and proliferation of A. rox-

2.6. Statistical analysis burghii in comparison with MS, VW and B5 media. The proliferation

rate turned out to be 4.33 in half-strength MS medium, signiﬁcantly

All experiments were conducted in a completely randomized higher than those obtained in other media (Table 2, Fig. 1c). The pro-

design. The data collected were square root transformed (where liferation rate in VW medium proved to be the lowest (3.67). The

appropriate) and subjected to an analysis of variance (ANOVA). average shoot length (2.46 cm) and shoot fresh weight (262.2 mg)

Where there was a signiﬁcant difference (P 0.05), the mean values obtained by using half-strength MS medium were also signiﬁcantly

were further separated through Duncan’s multiple range test. The greater, as compared with those obtained in VW and B5 media.

analysis was performed by SPSS software version 19.0. By using the B5 medium, the largest shoot diameter (4.22 mm)

was recorded, but it was not signiﬁcantly different from the val-

3. Results and discussion ues obtained on half-strength MS and MS media. Similarly, the

implication of media composition for shoot growth and prolifer-

3.1. Culture establishment ation has been reported in various plant species, including Vitis

vinifera (Matsumoto and Sakai, 2003), Vitis champinii (Mukherjee

The stem segments from A. roxburghii were used as explants et al., 2010) and Hemsleya zhejiangensis (Lei et al., 2011). That half-

and incubated half-strength MS medium supplemented with dif- strength MS was the most effective compared with MS, VW and

−1

ferent concentrations of BA (0.5–3.0 mg l ) for shoot bud initiation B5 media probably because the MS full-strength medium contains

(Fig. 1b). Among the cytokinin concentrations tested, the high- a comparatively very high amount of nitrogen. Additionally, there

est percentage of shoots formation stood at 91.67%, which was are differences in micronutrient compositions among MS, VW and

−1

observed on half-strength MS medium containing 1.5 mg l BA B5 media that might cause differences in the shoot growth and

(Table 1). However, the shoots formation was suppressed by higher proliferation of A. roxburghii (Lu, 2005; Mukherjee et al., 2010).

concentrations of BA. When the concentration of BA was increased Consequently, in all subsequent experiments only half-strength MS

−1

to 3.0 mg l , the percentage of shoot induction was less than medium was adopted as the base medium.

50%. Furthermore, with the increase in BA concentrations, the

shoot lengths, shoot diameters and shoot fresh weights decreased. 3.3. Effects of PGRs on shoot growth and proliferation

Likewise, the impact of BA on shoot bud initiation from nodal

explants has been demonstrated in various plant species, such as In vitro propagation of shoots was strongly inﬂuenced by the

Hibiscus cannabinus (Ayadi et al., 2011), Stevia rebaudiana (Thiya- types and concentrations of PGRs used. Half-strength MS medium

garajan et al., 2012) and Coleonema album (Fajinmi et al., 2014). supplemented with different combinations of cytokinins and auxin

In present study, half-strength MS medium was fortiﬁed with was investigated in this study. Cytokinins promoted the shoot pro-

−1 −1 −1

50 mg l ascorbic acid, 25 mg l citric acid, 25 mg l adenine liferation at low concentrations. The proliferation was enhanced

−1

sulfate and 25 mg l L-arginine. Among the different additives, when the concentration of cytokinins was increased, to a point.

Table 2

Effects of medium compositions on shoot growth and proliferation in Anoectochilus roxburghii.

Medium compositions Proliferation rate Shoots length (cm) Shoots diameter Shoots fresh weight

(mean ± SD) (mean ± SD) (mm) (mean ± SD) (mg) (mean ± SD)

Half-strength MS 4.33 ± 0.48a 2.46 ± 0.16a 4.11 ± 0.17a 262.2 ± 14.2a

MS 3.75 ± 0.44ab 2.45 ± 0.21a 4.16 ± 0.15a 267.5 ± 13.1a

VW 3.67 ± 0.49b 1.76 ± 0.13b 3.01 ± 0.08b 125.8 ± 15.3c

B5 3.83 ± 0.28ab 1.89 ± 0.10b 4.22 ± 0.18a 215.1 ± 9.6b

Means followed by the different letter within columns are signiﬁcantly different (P < 0.05) using Duncan’s multiple range test.

A. Zhang et al. / Industrial Crops and Products 70 (2015) 158–162 161

Table 3

Effects of plant growth regulators on shoot growth and proliferation in Anoectochilus roxburghii.

−1

Plant growth regulators (mg l ) Proliferation rate Shoots length (cm) Shoots diameter (mm) Shoots fresh weight (mg)

± ± ± ±

(mean SD) (mean SD) (mean SD) (mean SD)

BA Kn NAA

0.5 1.0 – 3.38 ± 0.49bcd 2.25 ± 0.10b 4.03 ± 0.16abc 230.6 ± 10.2abcd

1.0 1.0 – 3.71 ± 0.46abcd 2.20 ± 0.12b 4.01 ± 0.15abc 223.2 ± 16.3bcde

2.0 1.0 – 3.75 ± 0.44abcd 2.18 ± 0.09bc 3.98 ± 0.07abc 217.8 ± 11.7cde

3.0 1.0 – 3.96 ± 0.55abc 2.18 ± 0.06bc 3.92 ± 0.23abc 211.3 ± 18.9cde

4.0 1.0 – 3.63 ± 0.49abcd 2.04 ± 0.13 cd 3.81 ± 0.12bcde 186.8 ± 23.4ef

0.5 1.0 0.5 3.63 ± 0.58abcd 2.51 ± 0.08a 4.02 ± 0.06abc 258.8 ± 12.9ab

1.0 1.0 0.5 3.83 ± 0.38abcd 2.46 ± 0.11a 4.13 ± 0.21a 268.6 ± 15.6a

2.0 1.0 0.5 4.08 0.41ab 2.48 ± 0.13a 4.07 ± 0.18ab 259.2 ± 13.3ab

3.0 1.0 0.5 4.33 ± 0.48a 2.46 ± 0.16a 4.11 ± 0.17a 262.2 ± 14.2a

4.0 1.0 0.5 3.92 ± 0.58abc 2.19 ± 0.06b 3.89 ± 0.05abcd 209.1 ± 9.9cde

3.0 0.1 – 3.08 ± 0.28 d 2.43 ± 0.04a 3.82 ± 0.12bcde 223.7 ± 20.1bcde

3.0 0.5 – 3.17 ± 0.48 cd 2.40 ± 0.06a 3.76 ± 0.08cde 211.0 ± 10.7cde

3.0 1.0 – 3.96 ± 0.55abc 2.18 ± 0.06bc 3.92 ± 0.23abc 211.3 ± 18.9cde

3.0 1.5 – 3.63 ± 0.58abcd 2.20 ± 0.05b 3.60 ± 0.18e 194.9 ± 13.9de

3.0 2.0 – 3.46 ± 0.51bcd 1.91 ± 0.14 d 3.63 ± 0.20de 158.8 ± 14.4f

3.0 0.1 0.5 3.17 ± 0.38 cd 2.52 ± 0.06a 4.04 ± 0.09abc 242.2 ± 15.2abc

3.0 0.5 0.5 4.05 ± 0.71ab 2.50 ± 0.04a 3.99 ± 0.12abc 237.4 ± 12.6abc

3.0 1.0 0.5 4.33 ± 0.48a 2.46 ± 0.16a 4.11 ± 0.17a 262.2 ± 14.2a

3.0 1.5 0.5 3.94 ± 0.55abc 2.43 ± 0.10a 3.90 ± 0.18abcd 203.5 ± 13.0cde

3.0 2.0 0.5 3.67 ± 0.56abcd 2.00 ± 0.12 d 3.78 ± 0.08bcde 185.4 ± 10.7ef

Means followed by the different letter within columns are signiﬁcantly different (P < 0.05) using Duncan’s multiple range test.

However, cytokinins inhibited the shoot proliferation at higher con- in other plant species, including Stevia rebaudiana (Thiyagarajan

centrations. BA, alone or with other PGRs, is the most effective and Venkatachalam, 2012), Paphiopedilum hangianum (Zeng et al.,

inducer of explant proliferation in A. roxburghii (He et al., 2013). 2013) and Caralluma edulis (Patel et al., 2014).

The presence of BA, combined with Kn and NAA is indispensable to

explant development. Of the combinations tested, half-strength MS 3.4. Rooting

−1 −1 −1

medium fortiﬁed with 3.0 mg l BA, 1.0 mg l Kn and 0.5 mg l

NAA was found to bring about the greatest levels of shoot prolifer- Of the different additive and PGR combinations tested in

ation and further growth. The highest proliferation rate (4.33) was half-strength MS medium for in vitro root induction, the sup-

−1 −1 −1

produced on this medium, which was better than those produced plementation with 0.6 mg l NAA, 0.3 mg l IBA and 100 mg l

with most of the other treatments (Table 3, Fig. 1c). The greatest banana homogenate was found to be the best (Table 4). On this

average shoot diameter (4.13 mm) and average shoot fresh weight medium, the maximum percentage of rooting, 93.33%, with the

−1 −1

(268.6 mg) were obtained using 1.0 mg l BA, 1.0 mg l Kn and greatest root length, 2.20 cm, was observed (Fig. 1d and e). The high-

−1 −1

0.5 mg l NAA in half-strength MS medium, but the values were est average root number of 3.50 was obtained by using 0.6 mg l

−1 −1 −1

not signiﬁcantly different from those obtained by using 3.0 mg l NAA, 0.6 mg l IBA and 100 mg l banana homogenate in half-

−1 −1

BA, 1.0 mg l Kn and 0.5 mg l NAA in half-strength MS medium. strength MS medium, but it was not signiﬁcantly different than the

−1 −1 −1

Many developmental processes in plants, such as cell growth, cell value obtained with 0.6 mg l NAA, 0.3 mg l IBA and 100 mg l

division and differentiation, as well as organogenesis in tissue and banana homogenate in half-strength MS medium. The supple-

organ cultures, are controlled by interactions between cytokinins mentation of the medium with banana homogenate resulted in a

and auxins (Gaspar et al., 1996; Coenen and Lomax, 1997; Amoo greater percent of successful rootings, with greater root numbers

and Van Staden, 2013). The ﬁndings in the present study indi- and root lengths. Banana is a complicated additive that contains

cate a synergistic effect of auxins with cytokinins on shoot growth abundant nutritional and hormonal substances. The homogenate

and proliferation in A. roxburghii. The proliferation rates obtained of the banana contains amino acids, organic acids, nucleic acids,

with combinations of cytokinins and auxins were higher than those vitamins and carbohydrates (Zeng et al., 2013). The addition of

with cytokinins alone. Similar observations have been reported banana homogenate to media is appropriate for in vitro rooting

Table 4

Effects of auxins and banana homogenate on the rooting of Anoectochilus roxburghii.

−1 −1

NAA(mg l ) IBA Banana homogenate (g l ) Percent of Root numbers Root length (cm)

−1

(mg l ) rooting (%) (mean ± SD) (mean ± SD)

0.3 0.3 – 63.33 ± 0.05f 2.60 ± 0.50def 1.65 ± 0.07e

0.6 0.3 – 81.67 ± 0.04bcd 3.13 ± 0.35abc 2.03 ± 0.06b

1.2 0.3 – 71.67 ± 0.04e 3.00 ± 0.53bcd 1.90 ± 0.02c

0.3 0.3 100 80.00 ± 0.06 cd 2.80 ± 0.41cde 1.77 ± 0.06 d

0.6 0.3 100 93.33 ± 0.05a 3.47 ± 0.51a 2.20 ± 0.03a

1.2 0.3 100 85.00 ± 0.05bc 3.20 ± 0.41ab 1.98 ± 0.04bc

0.6 0.3 – 81.67 ± 0.04bcd 3.13 ± 0.35abc 2.03 ± 0.06b

0.6 0.6 – 78.33 ± 0.04 d 2.80 ± 0.41cde 1.78 ± 0.09 d

0.6 1.2 – 68.33 ± 0.04ef 2.33 ± 0.48f 1.48 ± 0.06f

0.6 0.3 100 93.33 ± 0.05a 3.47 ± 0.51a 2.20 ± 0.11a

0.6 0.6 100 86.67 ± 0.05b 3.50 ± 0.50a 2.05 ± 0.05b

0.6 1.2 100 78.33 ± 0.04 d 3.07 ± 0.25bcd 1.95 ± 0.05bc

Means followed by the different letter within columns are signiﬁcantly different (P < 0.05) using Duncan’s multiple range test.

162 A. Zhang et al. / Industrial Crops and Products 70 (2015) 158–162

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2012). It is interesting to note that at low concentrations the aux-

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ins stimulated rooting. The percentage of rooting was enhanced

Antihyperglycemic and antioxidant activity of water extract from Anoectochilus

when the concentration of auxins was increased, to a point. How- roxburghii in experimental diabetes. Exp. Toxicol. Pathol. 65, 485–488.

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Matsumoto, T., Sakai, A., 2003. Cryopreservation of axillary shoot tips of

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In the present investigation, we report an improved and efﬁcient

299–304.

in vitro propagation procedure, followed by shoot bud induction,

Mukherjee, P., Husain, N., Misra, S.C., Rao, V.S., 2010. In vitro propagation of a grape

shoot proliferation, rooting and acclimatization. This high mul- rootstock, deGrasset (Vitis champinii Planch.): effects of medium compositions

and plant growth regulators. Sci. Hortic. 126, 13–19.

tiplication rate was achieved by using micropropagation instead

Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays

of the conventional propagation method. The regenerated plants

with tobacco tissue culture. Physiol. Plant 15, 473–497.

showed normal growth and displayed no apparent morphological Panwar, D., Ram, K., Shekhawat, N.S.H., 2012. In vitro propagation of Eulophia nuda

Lindl., an endangered orchid. Sci. Hortic. 139, 46–52.

differences when compared with the donor plant. Such a proto-

Patel, A.K., Phulwaria, M., Rai, M.K., Gupta, A.K., Shekhawat, S., Shekhawat, N.S.,

col could be employed for large-scale multiplication, propagation

2014. In vitro propagation and ex vitro rooting of Caralluma edulis (Edgew.)

and conservation of the germplasm of A. roxburghii, an endangered Benth. & Hook. f.: an endemic and endangered edible plant species of the Thar

Desert. Sci. Hortic. 165, 175–180.

medicinal and ornamental plant species.

Rathore, M.S., Rathore, M.S., Shekhawat, N.S., 2013. Ex vivo implications of

phytohormones on various in vitro responses in Leptadenia reticulata (Retz.)

Conﬂict of interest Wight & Arn. – an endangered plant. Environ. Exp. Bot. 86, 86–93.

Shao, Q.S., Deng, Y.M., Liu, H.B., Zhang, A.L., Huang, Y.Q., Xu, G.Z., Li, M.Y., 2014a.

Essential oils extraction from Anoectochilus roxburghii using supercritical

The authors declare that there are no conﬂicts of interest.

carbon dioxide and their antioxidant activity. Ind. Crops Prod. 60, 104–111.

Shao, Q.S., Zhou, A.C., Hu, R.H., Zhang, Y.Y., Liu, T.M., Li, M.Y., 2014b. Inﬂuence of

Acknowledgments seedling grade on plant growth, yield and quality of Anoectochilus roxburghii.

China J. Chin. Mater. Med. 39, 785–789.

Thiyagarajan, M., Venkatachalam, P., 2012. Large scale in vitro propagation of Stevia

This work was supported by the National Natural Sci- rebaudiana (Bert.) for commercial application: pharmaceutically important

and antidiabetic medicinal herb. Ind. Crops Prod. 37, 111–117.

ence Foundation of China (Grant No. 81303167), the Zhejiang

Tseng, C.C., Shang, H.F., Wang, L.F., Su, B., Hsu, C.C., Kao, H.Y., Cheng, K.T., 2006.

Provincial Natural Science Foundation (Grant No. LY13H280010,

Antitumor and immunostimulating effects of Anoectochilus formosanus Hayata.

LY14H280008), Zhejiang Province Public Technology Applied Phytomedicine 13, 366–370.

Research Projects (Grant No. 2014C32017), Science and Technol- Wada, S., Tanimoto, E., Masuda, Y., 1968. Cell elongation and metabolic turnover of

the cell wall as affected by auxin and cell wall degrading enzymes. Plant Cell

ogy Innovation Program for Students of Zhejiang (Xinmiao Talent

Physiol. 9, 369–376.

Scheme) (Grant No. 2014R412004) and Ningbo Agricultural Sci-

Wang, Y.J., Meng, Z.X., Yu, X.M., Wang, C.L., Guo, S.X., 2009. Screening of

ence and Technology Research Projects (Grant No. 2014C10013). endophytic fungi promoting growth and development of Anoectochilus

roxburghii. Chin. Pharm. J. 44, 976–979.

We would also like to extent our appreciation to the anonymous

Wu, K.L., Zeng, S.J., Da Silva, J.A.T., Chen, Z.L., Zhang, J.X., Yang, Y.S., Duan, J., 2012.

reviewers for carefully reviewing our manuscript and putting for-

Efﬁcient regeneration of Renanthera Tom Thumb ‘Qilin’ from leaf explants. Sci.

ward many valuable suggestions. Hortic. 135, 194–201.

Yoon, Y.J., Murthy, H.N., Hahn, E.J., Paek, K.Y., 2007. Biomass production of

Anoectochilus formosanus Hayata in a bioreactor system. J. Plant Biol. 50,

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