Agriculture and Natural Resources 51 (2017) 478e484
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Agriculture and Natural Resources j ourna l hom e pa ge : ht t p: / / w w w . j ournal s . el s evi e r. c om / a gri c ul t ure - a ndna t ura l - re s ourc e s /
Original Article
In vitro micropropagation and allelopathic effect of lantana (Lantana camara L.) Varaporn Veraplakorn Department of Biotechnology, Faculty of Science, Ramkhamhaeng University, Huamark, Bangkapi, Bangkok, 10240, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 February 2017 Accepted 30 June 2017 Available online 31 March 2018
The invasive plant, lantana (Lantana camara L.), is well known as a traditional medicinal plant and it may become important in the development of modern drugs. Lantana has long been touted as containing potent allelochemicals and in vitro-produced tissues may be appropriate sources for the production and isolation of bioactive compounds. In this research, effective techniques for shoot multiplication and root and callus induction were developed and the allelopathic efficiency of in vitro leaf and callus was examined. The optimized medium for shoot multiplication was Murashige and Skoog (MS) medium supplemented with 12.0e20.0 m M thidiazuron. For rooting, high root numbers were obtained on MS medium containing 5.0 or 10.0 m M 1-naphthalene acetic acid (NAA). In addition, the highest relative growth rate of callus was achieved when lantana leaf was cultured on NB medium (MS medium with 21.5 mM NAA and 22.5 m M N6-benzyladenine). For allelopathic effects, the results suggested high potential activity of lantana leaf and callus that was able to variably inhibit the seed germination and seedling growth of all four test species. Leaf and callus extract had no significant effect on the germination of Brassica campestris var. chinensis. Callus extract s howed superior ability to suppress germination for Ipomoea aquatica Forsk. and Zea mays L. but inferior inhibition ability for Sorghum bicol or L. These results suggested that the extract of lantana in vitro leaf and callus will be an interesting natural source for further study to develop natural herbicides. Copyright © 2018, Kasetsart University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
Keywords: Allelochemical Callus Germination Herbicide Lantana
Introduction Lantana (Lantana camara L.), belongs to the family Verbenaceae and has been introduced to many countries as a flowering ornamental plant; however, in many places where it has been introduced it has become an invasive weed (Reddy, 2013; Mishra, 2015). Lantana has also been used in traditional medicine and recent studies have examined its chemical composition for potential pharmacological activities (Kalita et al., 2012; Reddy, 2013). Most interestingly, whole plant extracts have been reported to exhibit antibacterial, antifungal and allelopathic activity (Hussain et al., 2011; Reddy, 2013; Saxena et al., 2013; Talukdar, 2013; Gantayet et al., 2014; Mishra, 2015). Lantana has been ranked as one of the top-10, worst weeds and one of the 100 most invasive plants in the world (Chopra and Kumar, 1961; Lowe et al., 2000; Choyal and Sharma, 2011; Invasive Species Specialist Group, 2016). Its invasive capacity has
been attributed to its ability to produce allelopathic chemicals that inhibit the germination, growth and metabolism of many species including crops, weeds, bryophytes and vegetables (Mishra, 2015). Allelopathic effects of lantana on seed germination and seedling growth have been reported in many species such as Brassica juncea L., (Ahmed et al., 2007), Oryza sativa L. (Hossain and Alam, 2010), and Phaseolus radiatus (Gantayet et al., 2014). The allelochemicals found in lantana are present in all parts and extracts comprise monoterpenes and sesquiterpenes, flavonoids, iridoid glycoside, furanonaphoquinones, STH steroids, triterpenes and diterpenes (Mishra, 2015). It has been suggested that many plant species cultured in vitro are able to produce bioactive compounds similar to plants grown in the field (Bhojwani and Razdan, 1996). More importantly, in vitro plant tissue and callus of some species have expressed higher levels of active ingredients than those of natural plants; for example, flavonoids from Centella asiatica L. Urban, camptothecin from Nothapodytes foetida, catharanthine from Catharanthus roseus and anthraquinone from Cassia acutifolia (Tan et al., 2010; Vijaya et al., 2010; Hussain et al., 2012). Extracts from lantana, however, have
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[email protected]. https://doi.org/10.1016/j.anres.2018.03.006 2452-316X/Copyright © 2018, Kasetsart University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http:// creativecommons.org/licenses/by-nc-nd/4.0/).
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mainly been taken from plant material growing in the field including leaves, stems, roots, flowers and fruits (Ahmed et al., 2007; Choyal and Sharma, 2011; Hussain et al., 2011; Mishra, 2015). There is only one report that has examined in vitro cultured lantana and this exhibited toxicity to the growth of Salvinia molesta Mitchell (Saxena et al., 2013). There are only a few successful reports of the tissue culture of lantana (Waoo et al., 2013; Charan and Kamlesh, 2015; Veraplakorn, 2016). The current research was conducted to develop micro-propagation and callus proliferation techniques for lantana to provide in vitro plant material to examine the allelopathic effects of extracts from these tissues.
Materials and methods Explant preparation Single shoots of L. camara were surface sterilized by soaking in 1.0% NaOCl for 15 min and subsequent rinsing three times in sterile distilled water for 10 min. Shoots were initially cultured on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962). Aseptic shoots were multiplied by culturing on MS medium supplemented with 4.5 mM N6-benzyladenine (BA) for 8 wk. Each 1.0 cm shoot tip was transferred onto MS medium without plant growth regulator for 6 wk to prepare explants for the subsequent experiments. The incubation conditions were a 16.0 h photoperiod 2 (40.0 mmol/m s) at 25 ± 2 C.
Shoot multiplication Single shoots of 1.0 cm length were cultured on MS medium supplemented with thidiazuron (TDZ) at concentrations of 0.0 mM, 2.0 mM, 4.0 m M, 8.0 mM, 12.0 mM, 14.0 m M, 16.0 mM, 20.0 m M or 40.0 mM. The results of shoot number, shoot length and shoot characteristics were recorded after 4 wk.
Root induction Single shoots of 1.0 cm length were cultured on MS medium supplemented with 1-naphthalene acetic acid (NAA) at concentrations of 0.0 mM, 0.5 mM, 2.5 mM, 5.0 mM, 10.0 mM or 20 mM for 4 wk. The numbers of roots and their length and the characteristics of plantlets were recorded.
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Callus induction Callus of leaves of lantana was induced by cutting across the midrib twice and culturing on four media formulae: MS (MS medium without hormone), NB (MS medium with 21.5 mM NAA and 22.5 mM BA), B1 (MS medium with BA 4.5 mM) and B2 (MS medium with BA 22.5 mM). The relative growth rate (RGR ¼ 100 dry weight of treatment/dry weight of control) of callus was recorded after 2 wk. Allelopathic effect of in vitro leaf and callus Extract preparation Lantana leaves were harvested from in vitro shoots cultured on hormone-free MS medium. Callus was proliferated on MS medium supplemented with 21.5 m M NAA and 22.5 mM BA. Shoot and callus were subcultured to fresh medium of the same composition every 4 wk for four passages before use for extraction. Leaf and callus were dried at 60 C for 24 h and subsequently powdered using an electric blender. The powder was measured to 0.3 g, 0.6 g, 1.25 g, 2.5 g, 5.0 g, 7.5 g or 10 g and aqueous extracts were prepared by soaking in 100 mL distilled water at 5 C for 24 h. The extract solution was subsequently filtered through Whatman No.1 filter paper. Germination inhibition Whatman No.1 filter paper was placed in sterile 12 cm Petri dishes. The leaf and callus extracts of each concentration were added to each Petri dish in amounts to keep the seeds moist enough to provide favorable conditions for germination and growth. The control was treated only with distilled water. Twenty seeds of Brassica campestris var. chinensis, Ipomoea aquatica Forsk. and Sorghum bicolor L. and 10 seeds of Zea mays L. were placed in separate Petri dishes with five replicates at each concentration and incubated at room temperature. The number of germinated seeds and the length of the primary root and main shoot were determined after 7 d. The germination inhibition percentage (IP) was calculated following Chung et al. (2003), namely, IP ¼ (CeT)/C 100, where C is the germination percentage of the control and T is the germination percentage of treatment. Statistical analysis A completely randomized design with 10 replicates was performed to determine the effect of TDZ and NAA on shoot multiplication and root induction as well as the effect of the different
Fig. 1. Shoot multiplication of Lantana camara L. on MS medium supplemented with different concentrations of thidiazuron (TDZ) for 4 wk: (A) shoot number and (B) shoot length (Error bars indicate SE; n ¼ 10; different lowercase letters above bars indicate significant differences at p 0.05.).
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Fig. 2. In vitro shoot growth of Lantana camara L.: (A) clump of shoots cultured on Murashige and Skoog (MS) medium containing 12.0 mmol/L thidiazuron (TDZ); (B) long single shoot after culturing on MS medium containing 2.0 mmol/L TDZ.
Fig. 3. Root induction of Lantana camara L. on MS medium containing different concentration of NAA for 4 wk; (A) Root number and (B) root length. Error bars indicate SE; n ¼ 10; different lowercase letters above bars indicate significant differences (p 0.05).
media on callus induction and proliferation. For the allelopathic effect experiment, two-way analysis of variance was performed, Equal variances were tested using Levene's method. Where significant differences were found due to treatment, Tukey's B multiple range test was applied. Differences were considered significant at p 0.05. All analyses were performed using the PASW Statistics 18 software (SPSS Inc.; Quarry Bay, Hong Kong). Results Shoot multiplication Single shoots of lantana were induced to multiply on MS medium with 0e40 mM TDZ for 4 wk and multiplication peaked at 12e20 mM (Fig. 1A). The highest shoot number was found on MS medium containing 12.0e20.0 mM TDZ which gave 10.2e14.8 shoots/explant (Fig. 1A). The shoot clump was compact with short internodes and small leaves (Fig. 2A). The highest concentration of 40.0 mM TDZ reduced shoot multiplication to only 3.6 shoots/ explant which was not significantly different from the 3.4 shoots/ explant derived from 8.0 mM TDZ. The media with 2.0 mM TDZ or without TDZ was unable to induce new shoots (Fig. 1A). Shoot length, however, was found to be the greatest (3.3 cm) on the medium supplemented with 2.0 mM TDZ (Figs. 1B and 2B). Shoots cultured on media containing 4.0 mM and 8.0 mM TDZ showed no significant difference in length from those on MS without TDZ. In addition, the medium containing 12.0e40.0 mM TDZ showed the lowest shoot elongation of 0.5e0.9 cm with no significant difference between them (Fig. 1B).
Root induction Lantana shoots were induced to root on MS medium supplemented with 0.0e20.0 mM NAA. High root numbers were found on MS media containing 5.0 mM or 10.0 mM NAA with 9.2 and 6.7 roots being produced, respectively (Fig. 3A). The plantlets appeared healthy with green, expanded leaves and plump roots (Fig. 4A and B). Lantana shoots cultured on 0.5 mM, 2.5 mM or 20.0 mM NAA produced lower root numbers of 4.0, 5.7 and 4.1 roots, respectively. For hormone-free MS medium, the lowest root number of 2.4 roots was derived; however, this was not significantly different from the number of roots produced on MS medium containing 0.5 mM NAA
Fig. 4. Rooting of Lantana camara L. cultured on Murashige and Skoog medium containing 5.0 m M NAA: (A) plantlet with roots; (B) cluster of plump roots.
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Fig. 5. Relative growth rate (RGR) of Lantana camara L. callus cultured on the four media formulae which were MS (Murashige and Skoog without hormone), NB (MS medium with 21.5 mM NAA and 22.5 m M BA), B1 (MS medium with BA 4.5 mM) and B2 (MS medium with BA 22.5 m M) (Error bars indicate SE; n ¼ 10; different lowercase letters above bars indicate significant differences at p 0.05).
(Fig. 3A). In addition, long roots of 2.5 cm appeared on the medium containing 0.5 mM NAA. Root length, however, from all the concentrations of NAA produced no significant differences (Fig. 3B). Callus induction Leaf explants of lantana were induced to form callus after 2 wk on the media formulae; MS, NB, B1 and B2. Callus appeared along the cut edge of the leaf cultured on all media except MS which was the control treatment (Fig. 6). NB gave the highest RGR being 269.5% of the control (Figs. 5 and 6B). B1 and B2 could produce tiny callus on the leaf surface around the incision (Fig. 6C). The RGR of both were 109.1% and 119.2% of the control, respectively, which were not significantly different from the control treatment (Fig. 5). Allelopathic effect of leaf and callus on seed germination Both leaf and callus extract expressed varied effects on germination and seedling growth of the four species tested (B. campestris var. chinensis, I. aquatica Forsk., Z. mays L. and S. bicolor L). Increasing the extract concentration significantly reduced seed germination. Seedlings of all the test species that were not exposed to the extracts were healthy. The highest concentration of leaf and callus extract at which germination occurred for B. campestris, Z. mays and S. bicolor were 0.6%, 10.0% and 1.25%, respectively; the highest concentration at which I. aquatica germinated was 1.25% for leaf extracts and 0.6% for callus extracts. The treated seedlings of B. campestris were abnormal compared to the controls with plump hypocotyls, stubby roots and brown spots on the cotyledons.
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Seedlings of treated I. aquatica seed showed no abnormal characteristics but had significantly lower growth compared to the control treatment. For Z. mays, the concentration of 10% leaf and callus extract induced some parts of the seed (coleoptile and root) to turn brown. In addition, S. bicolor seed treated with the concentration of 1.25% leaf and callus extract produced translucent white coleoptiles and brown roots. The allelopathic capacity of lantana leaf was different from lantana callus and varied with the test species. Leaf and callus extract showed similar effects on seed germination of B. campestris. The concentrations that achieved 50% germination inhibition of leaf and callus extract were 0.62% and 0.65%, respectively (Figs. 7A and 8A). In addition, callus extract showed higher efficacy to inhibit germination of I. aquatica and Z. mays than leaf extract. For I. aquatica, the concentrations of 50% germination inhibition of leaf and callus extract were 0.94% and 0.45%, respectively (Figs. 7B and 8B). For Z. mays, the concentrations of 50% germination inhibition of leaf and callus extract were 4.39% and 3.05%, respectively. However, at 10% leaf and callus extract, seed of Z. mays was still able to germinate with germination inhibition of 86.1% and 88.2%, respectively (Figs. 7C and 8C). On the contrary, leaf extract revealed higher efficiency on seed germination inhibition of S. bicolor than that of callus extract. The concentrations of 50% germination inhibition of leaf and callus extract were 0.95% and 1.19%, respectively (Figs. 7D and 8D).
Growth inhibition Lantana extract reduced seedling growth of the four species tested. Increasing the extract concentration caused a significant reduction of both shoot and root growth. For all of the test species, both leaf and callus extracts at a concentration of 0.3% significantly inhibited shoot and root growth compared to the control (Tables 1 and 2). For B. campestris, increasing the leaf and callus extract concentration significantly inhibited shoot and root elongation at all extract levels (Table 1). For I. aquatica, leaf and callus extract significantly inhibited shoot and root growth at all concentrations with the exception of roots exposed to 0.3% and 0.6% leaf extract (Tables 1 and 2). For Z. mays, shoot and root length exposed to 0.3% leaf and callus extract were not significantly different from shoot and root length exposed to 0.6% extract. In addition, shoots of Z. mays were not significantly suppressed by 5.0%e10.0% leaf and callus extract. For roots, leaf and callus extract showed no significant suppression from 7.5% to 10.0% and from 2.5% to 10.0%, respectively (Table 2). For S. bicolor, shoot growth was not significantly reduced from 0.6% to 1.25% (Table 1) and root growth was not significantly reduced when exposed to 0.3e1.25% leaf extract but was significantly reduced when exposed to 1.25% callus extract (Table 2).
Fig. 6. Callus induction of Lantana camara L.: (A) growing callus on leaf explant cultured on NB medium (Murashige and Skoog medium with 21.5 m M NAA and 22.5 mM BA); (B) leaf explant with tiny callus on B1 medium (MS medium with BA 4.5 mM); (C) leaf explant with tiny callus on B2 medium (MS medium with BA 22.5 m M).
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Fig. 7. Allelopathic effect of leaf extract on seed germination inhibition: (A) Brassica campestris var. chinensis; (B) Ipomoea aquatica Forsk.; (C) Zea mays L.; (D) Sorghum bicolor L. where R 2 is the coefficient of determination.
Fig. 8. Allelopathic effect of callus extract on seed germination inhibition: (A) Brassica campestris var. chinensis; (B) Ipomoea aquatica Forsk.; (C) Zea mays L.; (D) Sorghum bicolor L.
Discussion Lantana shoot multiplication has been reported using MS medium supplemented with cytokinins such as BA, kinetin, zeatin and TDZ (Affonso et al., 2007; Waoo et al., 2013; Samani et al., 2014; Veraplakorn, 2016). In the current research, media supplemented
with TDZ performed effectively for shoot multiplication of lantana. Concentrations of 12.0e20.0 m M TDZ were able to induce high shoot production of 10.2e14.8 shoots/explant. This was better than using 16.0 mM BA which produced 4.9 shoots/explant (Veraplakorn, 2016). However, concentrations of TDZ lower than 12.0 mM, did not promote lantana shoot multiplication. This was similar to reports
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Table 1 Allelopathic effect of leaf and callus extract on shoot elongation of Brassica campestris var. chinensis, Ipomoea aquatica Forsk., Zea mays L. and Sorghum bicolor L. Extract concentration (%)
Shoot length (cm) a Leaf extract
0.0 0.3 0.6 1.25 2.5 5.0 7.5 10.0 a
Callus extract
B. campestris
I. aquatica
Z. mays
S. bicolor
B. campestris
3.3 ± 0.2 a 2.6 ± 0.2 b 1.3 ± 0.1 c e e e e e
5.2 ± 0.4 a 2.2 ± 0.1 b 1.4 ± 0.1 c 0.6 ± 0.1 d e e e e
18.5 ± 0.3 a 4.1 ± 0.6 b 3.3 ± 0.5 bc 2.4 ± 0.2 cd 2.3 ± 0.2 cd 1.5 ± 0.2 de 0.9 ± 0.2 e 0.3 ± 0.2 e
17.7 ± 0.4 a 2.5 ± 0.1 b 1.4 ± 0.2 c 1.2 ± 0.1 c e e e e
3.2 ± 0.2 a 2.3 ± 0.1 b 1.3 ± 0.1 c e e e e e
I. aquatica
Z. mays
5.2 ± 0.3 a 2.2 ± 0.1 b 1.3 ± 0.1 c e e e e e
S. bicolor
18.1 ± 0.4 a 4.4 ± 0.2 b 4.1 ± 0.4 b 3.9 ± 0.5 b 2.6 ± 0.2 c 1.7 ± 0.0 cd 0.9 ± 0.4 d 0.9 ± 0.4 d
17.4 ± 0.4 a 2.3 ± 0.2 b 1.7 ± 0.2 bc 1.0 ± 0.1 c e e e e
Mean values (±SE) with different lowercase, superscript letters within each column denote significant (p 0.05) differences between groups.
Table 2 Allelopathic effect of leaf and callus extract on root elongation of Brassica campestris var. chinensis, Ipomoea aquatica Forsk., Zea mays L. and Sorghum bicolor L. Extract concentration (%)
Root length (cm)
a
Leaf extract B. campestris 0.0 0.3 0.6 1.25 2.5 5.0 7.5 10.0 a
a
4.9 ± 0.2 1.9 ± 0.2 b 0.1 ± 0.0 c e e e e e
Callus extract I. aquatica a
3.4 ± 0.2 0.6 ± 0.1b 0.3 ± 0.1bc 0.1 ± 0.0 c e e e e
Z. mays
S. bicolor a
8.1 ± 1.0 3.7 ± 0.2 b 2.7 ± 0.1 b 2.6 ± 0.8 b 2.4 ± 0.2 bc 1.6 ± 0.5 bc 0.4 ± 0.1 cd 0.1 ± 0.1 d
16.5 ± 0.4 0.6 ± 0.0 b 0.5 ± 0.0 b 0.4 ± 0.0 b e e e e
B. campestris a
a
4.7 ± 0.3 1.2 ± 0.1 b 0.3 ± 0.1 c e e e e e
I. aquatica a
3.5 ± 0.1 1.8 ± 0.1 b 1.3 ± 0.1 c e e e e e
Z. mays
S. bicolor a
8.1 ± 1.0 4.0 ± 0.3 b 2.9 ± 0.3 b 2.6 ± 0.2 b 1.0 ± 0.2 c 0.4 ± 0.0 c 0.3 ± 0.0 c 0.2 ± 0.0 c
16.2 ± 0.5 a 1.3 ± 0.1 b 0.7 ± 0.1b c 0.2 ± 0.1 c e e e e
Mean values (±SE) with different lowercase, superscript letters within each column denote significant (p 0.05) differences between groups.
that L. camara L could not produce shoots when cultured on a medium with a low concentration of TDZ (Affonso et al., 2007; Samani et al., 2014). In addition, it was found that a high concentration of TDZ (12.0e40.0 mM) inhibited shoot elongation, though a low concentration of 2.0 m M TDZ, which had a low multiplication rate, did promote shoot height. This was contradictory to the reports of Affonso et al. (2007) and Samani et al. (2014) where low concentrations of TDZ (0.44 mM) inhibited shoot elongation of L. camara L. Many auxins has been reported for rooting of lantana, such as indole acetic acid (Affonso et al., 2007; Samani et al., 2014), indole butyric acid (IBA, Charan and Kamlesh, 2015; Veraplakorn, 2016) and NAA (Veraplakorn, 2016). In the current research, high root formation was found on the MS medium containing 5.0 m M or 10.0 m M NAA which gave 9.2 and 6.7 roots/shoot, respectively. However, there was no significant difference in root length on rooting media containing 0.0e20.0 mM NAA, though the medium with 0.5 mM NAA produced long roots with a mean of 2.5 cm. In comparison, lantana rooted on the MS medium with 5.0 mM IBA induced 9.9 roots/shoot with a mean length of 8.4 cm (Veraplakorn, 2016), which suggested that IBA is better than NAA for lantana root induction. The experiment was conducted to determine an optimized medium for callus induction. In previous research, lantana leaf explants were suitable for callus induction and media containing 40.0 mM BA in combination with 0.0e40.0 mM NAA or only 4.0 m M or 20.0 mM BA has efficiently induced callus formation (Veraplakorn, 2016). The current results using callus cultured on NB medium which contained 21.5 m M NAA and 22.5 m M BA showed the highest RGR. While B1 and B2 produced small callus, their RGR did not differ from the control medium. This was similar to many reports that the media supplemented with BA in combination with NAA promote callus in a range of species within the Verbenaceae for example Tectona grandis L.f. (Widiyanto et al., 2005),
Clerodendrum indicum (L.) Kuntze (Mukherjee et al., 2012) and Lippia citrodora L. (Boustani et al., 2016). Lantana callus contained high allelopathic potential by producing allelochemicals under in vitro conditions, similar to lantana growing in the field (Saxena et al., 2013). In the current study, the extract of lantana leaf and callus had allelopathic effects of inhibiting seed germination and seedling growth of B. campestris var. chinensis, I. aquatica Forsk., Z. mays L. and S. bicolor L. Seed of the four test species expressed significantly decreased germination and seedling growth compared to the control treatment. The efficacy of lantana extract has been reported as 1% of callus extract being toxic to the growth of Salvinia molesta Mitchell and seedlings that were treated with callus extract showed blackening of the fronds and died within 14 d (Saxena et al., 2013). In addition, lantana extract has a different allelopathic efficiency on the germination and seedling growth of each species (Ahmed et al., 2007; Hussain et al., 2011; Tadele, 2014). Many reports indicated that lantana leaf extract can inhibit germination in plants, such as Triticum aestivum (Hossain and Alam, 2010), Lathyrus sativus L. (Talukdar, 2013), Phaseolus radiatus (Gantayet et al., 2014) and Eragrostis tef (Tadele, 2014). However, lantana extract has also been reported to induce germination and growth of seedling in some species, such as Zea mays and Eleusine coracana (Tadele, 2014). This was similar to the current results where leaf and callus extract caused different allelopathic effects on each test species. However, lantana extract did not promote germination and seedling growth of Z. mays in the current study; but leaf and callus extracts showed the lowest effect when compared to the other species. In addition, the inhibitory effect of lantana on garden pea was proportional to the extract concentrations. It has been reported that the higher concentration of the extract caused a stronger effect on seedling growth while lower concentrations showed a variation in effect between inhibition and stimulation (Kar et al., 2014). In the current study, the concentration of the extract from 1.25% to 5.0%
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showed no different effect on seed germination of Z. mays and the highest germination inhibition was found with 7.5% or 10.0% extract. The highest concentration of extract at which the four test species (B. campestris; I. aquatica; Z. mays; S. bicolor) could germinate was 0.6%, 1.25%, 10.0%, and 1.25%, respectively. In addition, seedling growth of each test species was different when they were exposed to the same concentration of the extract. This suggested that lantana extract has specific allelopathic effects on each plant species to suppress seed germination and seedling growth (Ahmed et al., 2007; Hossain and Alam, 2010; Talukdar, 2013; Tadele, 2014). The MS medium supplemented with TDZ from 12.0 to 20.0 mM promoted high shoot numbers of lantana. However, the longest shoot length was found on medium containing 2 m M TDZ. Shoots rooted well on media containing 5.0 mM or 10.0 mM NAA. In addition, the best medium for callus induction was NB which had the highest RGR. Allelopathic effects, both from leaf and callus extracts showed effective inhibition on seed germination and seedling growth of the test species. Each of the test species expressed varying responses to the extracts. Leaf and callus extracts had the lowest potential to inhibit germination and growth of Z. mays when compared to the other species. The results indicated that lantana extract will be of interest to examine for its potential as a natural herbicide. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research was supported by the Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand. Sorghum bicolor seed was derived from the National Research Center of Millet and Corn (Suwan Farm). The author would also like to acknowledge Dr. Ian Bennett for comments that greatly improved the manuscript. References Affonso, V.R., Bizzo, H.R., Lima, S.S., Esquibela, A., Sato, A., 2007. Solid phase microextraction (SPME) analysis of volatile compounds produced by in vitro shoots of Lantana camara L. under the influence of auxins and cytokinins. J. Braz. Chem. Soc. 18, 1504e1508. Ahmed, R., Uddin, M.B., Khan, M.A.S.A., Mukul, S.A., Hossain, M.K., 2007. Allelopathic effects of Lantana camara on germination and growth behavior of some agricultural crops in Bangladesh. J. For. Res. 18, 301e304. Bhojwani, S.S., Razdan, M.K., 1996. Plant Tissue Culture: Theory and Practice, a Revised Edition. Elsevier Science B.V, Amsterdam, the Netherlands. Boustani, A., Omidi, M., Torabi, S., Zarekarizi, A.R., 2016. Callus induction and plant regeneration in lemon verbena (Lippia citrodora L.), an important medicinal plant. Trakia J. Sci. 1, 30e38. Charan, S., Kamlesh, C., 2015. Micropropagation and analysis of phytochemical profile of lantana camara whole plant extraction. World J. Pharm. Pharm. Sci. 4, 1907e1919.
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