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WSN 49(2) (2016) 283-294 EISSN 2392-2192

Plant growth regulators and sucrose requirements for in vitro induction of shoots from different explants of (L.) Corr. Serr.

M. Manokari*, Mahipal S. Shekhawat Biotechnology Laboratory, Department of Science, M.G.G.A.C. Mahe, Pondicherry, India *E-mail address: [email protected]

ABSTRACT An efficient in vitro regeneration and conservation system depend on the healthy culture induction from the suitable explants. Culture induction is a significant stage when very small plant material exists from the rare species. It is necessary to develop culture induction protocol from various explants to conserve the valuable plant species. Atalantia monophylla is a rare species with life giving properties. Shoots were induced from the shoot tip, node and internode explants. Among these the nodal shoot segments were reported most appropriate explant for the induction of shoots from the nodal meristems. MS medium (Murashige and Skoog) proved better than the Woody (WP) medium in bud breaking. Sucrose at 3% level was optimum concentration for the establishment of cultures than the other concentrations evaluated. Shoot tips responded on MS medium supplemented with 0.5 mg L-1 each of BAP and Kin, nodal shoot segments responded better on MS medium augmented with 1.0 mg L-1 BAP . Half strength MS medium supplemented with 2,4-D (1.0 mg L-1) induced maximum responsive callus (87%) from the internode explants.

Keywords: Atalantia monophylla; in vitro; rare; explants; PGRs

1. INTRODUCTION

Atalantia monophylla (L.) Corr. Serr. (syn: Limonia monophylla L.) is one of the important species of the family . It is commonly known as Wild lemon, Jungli World Scientific News 49(2) (2016) 283-294

Nimbu, Aranyanimbuka, Banjamir nimbu, Bannimbu, Kattunarakam, Kattuelumichai etc. (Kandappa et al., 2015). This species is reported as rare and endemic to southern peninsular India (Sukumaran and Raj, 2007). It is a large thorny shrub grows up to 2.5 meters in height. Leaves are simple, alternate, oblong and entire with crenulate margin. Flowers are white, small and arranged in axillary racemes. Fruits are small berries with minute seeds (Sankaranarayanan et al., 2010). The leaves and bark of this plant are traditionally used in the treatment of vitiated kapha, vata, flatulance, hemiplegia, arthritis, skin diseases, bacterial infections and malignancy (Panda, 2004; Kumar and Narayana, 2010). The essential oil obtained from the berries is reported to cure chronic rheumatism, paralysis and inflammations (Sukumaran and Raj, 2010). The herbal extract made from the leaves is used in hemiplegia due to the presence of an active ingredient compound liniment. The boiled leaves are used to cure glandular swelling (Sankaranarayanan et al., 2010). The essential oil contains higher terpene esters (azulene group). The plant roots are antispasmodic and exploited due to the presence of alkaloids, atalaphylline and atalaphyllidine. The root bark yields limonoid and atalantin (Kirtikar and Basu, 1999). A. monophylla is used to control Spodoptera litura, Helicoverpa armigera, Earias vittella (Baskar et al., 2009; Muthu et al. 2010), Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti (Sivagnaname and Kalyanasundaram, 2004). Besides, the plant is reported to posses larvicidal and pupicidal (WHO, 1975), insecticidal (Grainge and Ahamed 1988, Sukumar et al. 1991), mosquitocidal (Sivagnaname and Kalyanasundaram, 2004), ovicidal (Baskar and Ignacimuthu, 2012), antifungal, antioxidant and cytotoxic (Kandappa et al., 2015) activities. The conventional propagation through seeds and stem cuttings of this species is not efficient as the rate of seeds germination and rooting ability is very poor. Vegetative propagation requires some special climatic conditions than the existing harsh environmental conditions. Therefore, the distribution of this species is limited and restricted in certain geographic zones. The biotechnological interventions have been used to conserve endangered, rare, ornamental and medicinal species. In vitro conservation methods gained significance for vegetatively propagated and non-orthodox seed yielding plant species for conservation and production of pathogen-free plantlets (Engelmann, 2011). Bud breaking or induction of shoots is an initial but very significant stage in the case of certain plant species when very small plant material exists in nature. The present communication describes the chemical factors affecting shoots induction in rare and endemic plant A. monophylla using different types of explants.

2. MATERIALS AND METHODS 2. 1. Plant material selection The field surveys were conducted and the plants were procured from the southern districts of the Coromandel Coast of India, lies on the geographical coordinates of 11° 55' 48" N, 79° 49' 48" E. The plants were identified using standard floras (Gamble, 1921; Matthew, 1982). Healthy and actively growing plants were collected and maintained in the greenhouse to get disease free planting materials.

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2. 2. Explants and sterilization Different types of explanting materials namely leaves, apical shoot tips, nodal shoot segments and internodes were used to establish the cultures. The explants were washed thoroughly with 2% solution (v/v) of Tween-20® followed by running tap water for 10 min and treated with 0.1% solution (w/v) of Bavistin (systemic fungicide; BASF India Ltd., India) for 6-8 min, then washed thrice with distilled water. The explants were then dipped in 70% ethanol for 1 min, followed by surface sterilized with 0.1% solution (w/v) of HgCl2 for 4-5 min under laminar air flow cabinet. The sterilized explants were washed with autoclaved double distilled water for 5-6 times to remove the adhered traces of HgCl2. The explants were inoculated and cultured on different basal media supplemented with different concentrations and combinations of plant growth regulators (PGRs).

2. 3. Nutrient media and culture conditions Two types of nutrient media were used for the present study. These include MS basal medium (Murashige and Skoog, 1962) and Woody Plants (WP) medium (Llyod and McCown, 1980). Sucrose (Hi-Media, Mumbai) was added as a source of carbohydrate. Different concentrations of sucrose (1%, 2% and 3%) were tested to find out optimum concentration of carbon requirement for the establishment of cultures. Additives (50 mg L-1 of ascorbic acid and 25 mg L-1 each of arginine, adenine sulphate and citric acid) were incorporated in the culture medium. Culture medium was solidified by 0.8% agar to support the proper position of the plant material in the medium. The pH of the medium was adjusted to 5.8 ±0.02 using 0.1 N NaOH or 0.1 N HCl prior to autoclaving for 15 min at 121 ºC and 1.1 kg cm-2. The cultures were kept in growth room under controlled conditions at 23 ±2 °C to 28 ±2 °C temperature with illumination of 20-50 µmol m-2s-1 Spectral Photon Flux Density (SPFD) and 60-70% relative humidity (RH). The light was provided by fluorescent tubes and incandescent bulbs (Philips, India). Positive air pressure and temperature was maintained by air conditioning system.

2. 4. Effect of plant growth regulators on culture induction To investigate the effect of plant growth regulators on bud break and the establishment of cultures in vitro, the explants were inoculated vertically and horizontally on MS medium containing different concentrations of plant growth regulators (BAP; 6-benzylaminopurine, Kin; Kinetin, IAA; indle-3 acetic acid and NAA; α-naphthalene acetic acid), ranging from 1.0 to 3.0 mg L-1. Leaf explants (1 cm long) were inoculated directly into the medium (maximum one per culture tube) after trimming both the ends to induce callus. The medium employed for callus induction was augmented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). The explants inoculated on medium devoid of growth regulators were served as control. The culture vessels were properly capped and sealed after inoculation. Explants were harvested throughout the year to study the seasonal response on establishment of the cultures.

2. 5. Observations and data analysis All the experiments were set up in randomized block design with a minimum of 20 replicates per treatment, and experiments were performed three times. The observations were taken after four weeks of inoculation. The culture induction response represents the efficiency of explants on a specific medium after number of days of inoculation as mentioned in the

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results. The data were statistically analyzed using analysis of variance (ANOVA), and differences among the mean values were compared with Duncan’s multiple range test (P< 0.05) using SPSS ver. 16. The results were expressed as mean ±Standard Error (SE).

3. RESULTS AND DISCUSSION 3. 1. Seasonal collection of explants for culture induction The in vitro response of explants was greatly affected by the season/month of collection of the explants in the establishment of cultures of A. monophylla. Maximum percentage of response (95%) was observed during the months of October-December from all the explants evaluated (Fig. 1). Less percentage of culture responsiveness was observed during summer months (April-June) of the year. The physiological state of explants under special in vitro conditions was determined by the season of explants collection and the influence of the plant growth regulators. The seasonal response of explants in cultures were reported in Azadirachta indica (Arora et al., 2010), Celastrus paniculatus (Phulwaria et al., 2013), Schleichera oleosa (Saha, 2013), Morinda citrifolia (Shekhawat et al., 2015b), Hemidesmus indicus (Shekhawat and Manokari, 2016b) and Blyttia spiralis (Patel et al., 2016).

Fig. 1. Seasonal effect of explants collection on induction of shoots.

3. 2. Effect of different types of explants in culture establishment Selection of appropriate explants is a significant step to avoid exploitation of somatic tissues from the rare and conservation prioritized species. For the establishment of cultures, different explants like leaves, shoot tips, nodes and internodes were cultured on different media (MS and WP medium) supplemented with different combinations and concentrations of growth regulators. The in vitro growth and morphogenesis is largely governed by plant tissue

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culture medium, and it generally comprises of inorganic salts, organic compounds, vitamins, additives such as ascorbic acid, adenine sulphate, arginine and citric acid, and gelling agent (agar) etc. The problem of phenolic exudation was tried to control by incorporation of additives, activated charcoal and by periodic subculture. Among the different methods tried, subsequent subculture with 8 days interval on fresh medium amended with additives (50 mg L-1 of ascorbic acid and 25 mg L-1 each of arginine, adenine sulphate and citric acid) resulted with better response. Addition of activated charcoal in the medium delayed the culture induction repose from the explants.

3.3. Culture establishment from shoot tip explants Two different basal media (MS, WP) were tested to optimize the appropriate culture medium for A. monophylla. Shoot tips showed elongation up to 3.0 cm within 10 days of incubation on MS medium (Fig. 2A and B). However, shoot tips cultured on WP medium responded after 20 days for elongation up to 2.4 cm and only 72% of explants responded on WP medium. Shoot tip elongation was observed on MS medium supplemented with 0.5 mg L- 1 each of BAP and Kin. Higher percentage of response (92%) with three shoots was observed from the shoot tip explants when cultured on MS medium augmented with 0.5 mg L-1 each BAP and Kin (Table 1). The combined effect of cytokinins for shoots induction was reported in Artemisia absinthium (Shekhawat and Manokari, 2015) and Glinus lotoides (Teshome and Feyissa, 2015). At higher concentration of cytokinins, the induction response was suppressed. Shoot tips were reported better explants in micropropagation of hystrix (Eng et al., 2014) and Vitex trifolia (Ahmed and Anis, 2014).

Fig. 2A. Culture induction using shoot tip explants. Fig. 2B. Shoot bud elongation -Microscopical view (scale bar 50µm).

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Table 1. Effect of different concentrations of cytokinins (BAP and Kin) on shoot induction response of shoot tip explants.

Conc. of cytokinins Response Number of shoots Shoot length (cm) (mg L-1) (%) (Mean ±SE) (Mean ±SE)

Control 0.00 0a 0.0±0.00a 0.00±0.00a BAP 0.1 56d 2.3±0.00d 1.12±0.12b 0.5 83g 2.8±0.12e 2.60±0.07c 1.0 80g 2.4±0.20d 2.03±0.20c 1.5 69f 2.0±0.21c 1.19±0.24b 2.0 51c 1.7±0.19c 1.04±0.11b Kin 0.1 50c 1.6±0.12c 1.00±0.14b 0.5 59e 2.1±0.13b 1.93±0.20c 1.0 55d 2.0±0.20c 1.22±0.19b 1.5 52c 1.8±0.00c 0.93±0.11b 2.0 46b 1.2±0.13b 0.85±0.00b BAP + Kin 0.1 60e 2.0±0.10c 2.13±0.15c 0.5 92i 3.0±0.17e 3.04±0.11d 1.0 89h 2.5±0.21b 2.59±0.33c 1.5 73f 2.2±0.11b 2.10±0.10c

Note: Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance of variation between the concentrations was studied using DMRT at 5% level (P < 0.05). Mean values represented in corresponding column followed by same alphabets are not significantly different.

3.4. Culture induction from nodal shoot segments Freshly sprouted nodal shoot segments were found most suitable explants for the induction of multiple shoots in A. monopylla. Similar findings regarding suitability of nodal shoot segments as explants were reported in Citrus limon (Rathore et al., 2007), Terminalia

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catappa (Phulwaria et al., 2012), Salvadora oleoides (Shekhawat et al., 2012) and Morinda coreia (Shekhawat et al., 2015a). Among different concentrations and combinations of cytokinins tested, 1.0 mg L-1 BAP was observed more effective with respect to bud break from the nodal explants. Cent percentage bud break was achieved within ten days of inoculation on full strength MS medium supplemented with 1.0 mg L-1 BAP. The highest (12.4±0.20) number of shoot buds was observed with 3% sucrose and additives when incorporated with 1.0 mg L-1 BAP (Fig. 3A to C). The influence of additives in the culture medium in establishment of cultures and better multiple shoots induction was reported in number of plant species such as Stevia rebaudiana (Sridhar and Aswath, 2014), Blyttia spiralis (Patel et al., 2016) and Hemidesmus indicus (Shekhawat and Manokari, 2016b). Comparatively less percentage of response was observed on Kin alone and BAP+Kin in shoots induction from the nodal explants. The superiority of BAP over Kin for bud breaking response from nodal shoot segments was reported in Citrus limon (Rathore et al., 2007). The percentage of response and number of shoots differentiated were less on WP medium at higher concentrations of cytokinins with additives (Table 2 and Fig. 4).

Fig. 3A. Bud breaking in nodal shoot explant. Fig. 3B. Microscopical view of buds (scale bar 50µm). Fig. 3C. Multiple shoots induction from nodal explants.

Table 2. Effect of different concentrations of cytokinins (BAP and Kin) and IAA (0.1 mg L-1) on shoot induction response of nodal explants.

Conc. of cytokinins Response Number of shoot Shoot length (cm) (mg L-1) (%) buds (Mean±SE) (Mean±SE) Control 0.00 0a 0.0±0.00a 0.00±0.00a BAP 0.1 89d 10.5±0.00c 2.19±0.13c 0.5 93e 11.2±0.12f 2.66±0.27d

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1.0 100f 12.4±0.20g 4.36±0.31f 1.5 91e 12.0±0.21g 3.00±0.20e 2.0 86d 10.0±0.19e 2.12±0.14c Kin 0.1 72c 7.16±0.07c 1.47±0.10b 0.5 79e 8.31±0.11d 1.98±0.29c 1.0 87d 8.50±0.10d 2.10±0.11c 1.5 74c 7.94±0.22c 1.90±0.15c 2.0 66b 6.29±0.15b 1.74±0.21b BAP + Kin 0.1 63b 7.00±0.27c 2.13±0.15c 0.5 79d 7.93±0.10d 3.04±0.11e 1.0 85d 6.18±0.10b 2.59±0.33d 1.5 75c 5.22±0.00a 2.10±0.10c Note: Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance of variation between the concentrations was studied using DMRT at 5% level (P < 0.05). Mean values represented in corresponding column followed by same alphabets are not significantly different.

Fig. 4. Effect of different media on response of different explants.

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3. 5. Callus induction from leaf and internode explants Callus formation was observed in leaf and internodal explants on full strength MS medium supplemented with different concentrations of 2,4-D within week. The initial dark incubation of explants on 2,4-D for 2 days was found as limiting factor in callus induction. The callus was induced from the leaf explants within 3 weeks and after 10 weeks from the internode explants. Among the different strengths of MS medium and concentrations of growth regulators tested, half strength MS medium augmented with 1.0 mg L−1 2,4-D with additives was reported superior. All the concentrations of 2,4-D induced callus, from leaf and internode explants. Maximum percentage (87%) of callus was regenerated from internode explants than leaf explants (73%). Callus produced on this medium was fast growing, green, friable and had the potential to regenerate shoots on shoot differentiation medium (Table 3 and Fig. 5A and B).

Fig. 5A. Callus induction from internode explants. Fig. 5B. Callus tissues under photomicroscope (scale bar 50µm).

Table 3. Effect of 2,4-D on induction of callus from the leaf and internode explants.

Callus induction response Conc. of 2,4-D (mg L-1) (%) Callus characteristics Leaf Internode 0.0 0a 0a No callus induction 0.25 47c 74b Creamy white, slow growing 0.5 52d 79c Pale white, slow growing 0.75 69e 84d Pale green, slow growing 1.0 73f 87e Green, friable, fast growing 1.5 63e 80d Brown, poor growth

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2.0 58d 78c Slow growing 3.0 43b 71b Brown, no growth Note: Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance of variation between the concentrations was studied using DMRT at 5% level (P < 0.05). Mean values represented in corresponding column followed by same alphabets are not significantly different.

Callus initiated with leaf explants on 2,4-D medium was creamy white, unorganized and less proliferative which turned brown and died within 4 weeks. Further increase in concentration of 2,4-D in the medium did not show any progressive callus proliferation. Successful regeneration of plantlets from callus using cotyledons explants on MS medium supplemented with 2,4-D was reported in Citrus jambhiri (Savita et al., 2011), leaf explants in Citrus limon (Kasprzyk-Pawelec et al., 2015) and seeds, internode and apical shoot tip explants in Citrus sinensis (Azim et al., 2011). Reduced strength of MS salts for the induction of callus was reported in Ulex europaeus (Ramirez et al., 2012).

4. CONCLUSION

Culture establishment being the first step of any micropropagation protocol requires clear understanding of plant responses on various physiochemicals, in vitro culture environments including nutrient media, growth regulators, types of culture vessel etc. The culture establishment from the appropriate explants is challenging in development of a regeneration system for the rare species. The present study explains the suitable explant type, seasonal response of explants and optimum medium for culture initiation in A. monophylla. The developed method could be used as a tool for the conservation of this species which is endemic in nature with limited distribution.

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( Received 15 May 2016; accepted 05 June 2016 )

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