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WSN 47(2) (2016) 318-328 EISSN 2392-2192

Comparative foliar micromorphological studies of in vitro and field transferred of erecta L.

M. Manokari*, Mahipal S. Shekhawat Biotechnology Laboratory, Department of Science, M.G.G.A.C. Mahe, Pondicherry - 673 311, India *E-mail address: [email protected]

ABSTRACT The micromorphological studies of in vitro and field transferred plants of Duranta erecta were conducted in the present investigation. The nodal shoot segments were used as explants for the establishment of cultures. The shoots were multiplied on MS medium (Murashige and Skoog) supplemented with 6-benzylaminopurine (BAP), kinetin (Kin) and Naphthalene acetic acid (NAA). In vitro multiplied shoots were rooted on half strength MS medium augmented with indole-3 butyric acid (IBA). The rooted plantlets were successfully hardened and field transferred. A comparative foliar micromorphological studies were made of in vitro grown and field transferred plants to understand the developmental changes of micro-external structures of plants towards environment. The differences in constants such as stomata, vein-islets and termination numbers and trichomes across the environments imply the developmental changes of micropropagated plants of Duranta erecta.

Keywords: Duranta erecta; micropropagation; micromorphology; stomatal index; trichomes

1. INTRODUCTION

Duranta erecta L. is a medicinally important horticultural , belongs to the family . The plant is also known as golden dewdrop, pigeon and skyflower. This is World Scientific News 47(2) (2016) 318-328

an evergreen shrub grows up to the height of 3 m. It is native to the United States and cultivated in tropical and subtropical gardens throughout the globe as an (Anis et al., 2001). The plant gains ornamental attention because of its pale green with lavender . Its leaves are elliptic, opposite and the flowers are arranged on terminal as well as axillary branches. The are globose orange berry and produced in tight clusters, hence it is known as Golden dew drops. This species is medicinally used to treat various illnesses such as neuralgic disorder, pneumonia, malaria, intestinal worms, insects repellent, vermifuge, diuretic, antidote to treat itches, infertility and abscesses (Castro et al., 1996; Whistler, 2000; Rahmatullah et al., 2011). It exhibits antioxidant, antibacterial and antimicrobial activities against human pathogens (Fu et al., 2010; Bangou et al., 2012; Prabhakar et al., 2015). The presence of saponins in the fruits and leaves are reported to be toxic at raw form (Thompson, 2008), and causes vomiting, fever, nausea, gastro enteric irritation, drowsiness, convulsions and dermatitis (Hardin and Arena, 1969; Westbrooks and Preacher, 1986). But at prescribed quantities, the fruits are used to cure intestinal worms (Whistler, 2000). D. erecta contains several valuable secondary metabolites such as coumarinolignoids, repenins A-D, durantin, cleomiscosin, iridoid glycosides as durantosides and lamiide, flavonoids, , saponins, acetosides and tannins (Anis et al., 2000; Ahmed et al., 2009; Khanal et al., 2014). It inhibits the growth of Staphylococcus epidermidis bacterium due to presence of total flavonoids in higher concentrations (Bangou et al., 2012). Recently this plant is explored for the synthesis of silver and zinc oxide nanoparticles using biogenic processes (Patil and Hooli, 2015; Ravindran et al., 2016). The ultimate success of in vitro propagation for plant improvement lies in the successful establishment of plantlets in the soil (Pospisilova et al., 2009), this is mainly restricted by the difficulties during transfer of the plantlets from in vitro to the in vivo conditions (Chandra et al., 2010). The application of micropropagation process is being hampered by the number of in vitro induced anatomical, physiological and biochemical challenges (Hazarika, 2006; Bairu and Kane, 2011; Ruffoni and Savona, 2013). This is due to the sudden change in the very special culture environment such as high relative humidity, low light intensity, readymade source of carbohydrate to relatively harsh environments like low relative humidity and high light intensity. The present study aims to study the comparative foliar micromorphology of in vitro and field transferred plants of D. erecta.

2. MATERIALS AND METHODS 2. 1. In vitro propagation For the establishment of cultures, nodal shoots segments were cultured on MS (Murashige and Skoog, 1962) medium supplemented with different concentrations of BAP and Kin (0.5-4.0 mg L-1). The pH of media was adjusted to 5.8 ± 0.02 prior to autoclaving for 15 min at 121 ºC and 1.1 kg cm-2 pressure. After four weeks, the in vitro regenerated shoots were multiplied by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium in the culture flasks. Agar gelled medium supplemented with various concentration and combination of BAP and Kin (0.1 to 3.0 mg L-1) and IAA/NAA ranging from 0.1 to 2.0 mg L-1 were used for the multiplication of shoots.

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All the cultures were incubated at 28 ±2 °C temperature, 60-70% Relative Humidity (RH) and 30-35 µmol m-2s-1 photon flux density (PFD) light for 12 h/d using cool white fluorescent tubes (Phillips, India). The in vitro produced shoots were isolated of appropriate size and cultured on half and one-fourth strengths of MS medium containing auxins (IAA, IBA and NAA) ranging from 1.0 to 4.0 mg L-1 and incubated under diffused light conditions for the induction of roots from the cut ends. The in vitro rooted plantlets were washed with autoclaved distilled water to remove adhered nutrient agar and then transferred to soilrite® and moistened with one-fourth strength of MS basal salts. The in vitro produced plantlets were hardened in the greenhouse and finally transferred to the field.

2. 2. Foliar micromorphological studies Experiments were conducted to study the foliar micromorphological changes (leaf constants) during plant developmental processes from in vitro to field environments, such as vein density, vein-islets and veinlet terminations, venation pattern, types of stomata, stomatal density, stomatal frequency and stomatal index, and the trichomes of the leaves of plants developed in vitro after 4th subculture in multiplication phase, and hardened plants transferred to the field (after 6th week). Plants were randomly selected for the micromorphological experiments from both the environments. The entire leaves at third to seventh leaves from the base were excised manually for all the experiments. To observe the developmental changes in structure and functioning of the stomata, stomatal morphology and trichome distribution the epidermal peelings were obtained manually by standard method (Johansen, 1940). The stomatal density, frequency and index were calculated by the method suggested by Salisbury (1927, 1932). Types of stomata have been described by following the classification and terminology as suggested latest by Croxdale (2000) and Prabhakar (2004). For venation study, leaves required from each stage were excised and fixed primarily in formalin acetic acid alcohol (FAA in the ratio of 1:1:3) solution. In addition, fixed as well as fresh leaves were cleared in 70% ethanol (v/v) until chlorophyll was completely removed (12- 24 h), bleached with 5% (w/v) NaOH for 24-48 h, rinsed three times in distilled water and allowed to remain in saturated Chloral hydrate solution for 24-48 h (Sass, 1940). The cleared leaves were used for the study of venation pattern, vein-islets and veinlet terminations and trichomes. The terminology adopted for venation pattern was of Hickey and Wolfe (1975). The materials were stained with 1 % (v/v) safranine (Loba chemie, India) aqueous solution for 4–8 min and rinsed carefully in distilled water to remove excess stain and then mounted in water and examined under the microscope (Labomed iVu 3100, USA) and analyzed using software Pixelpro.

2. 3. Observations and Data analysis The experimental design consisted of two treatments (in vitro and field transferred plants) with ten replicates (consisting of plants from both environments per replication) and repeated thrice. The data were subjected to analysis of variance and the significance of differences among mean values was carried out using Duncan’s Multiple Range Test (DMRT) at P < 0.05 using SPSS software, version 16.0 (SPSS Inc., Chicago, USA).

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3. RESULTS AND DISCUSSION

In the present report, very quick response was observed from nodal shoot segments cultured on MS medium supplemented with BAP and Kin (within a week of inoculation). Highest numbers of shoot buds per explants were observed at BAP 2.5 mg L-1. Culture induction on higher concentrations of cytokinins was reported in number of Verbenaceae members. In Tectona grandis, shoot organogenesis has been achieved by the use of BAP and -1 GA3 (Widiyanto et al., 2005). Rathore and Shekhawat (2011) reported that BAP at 2.0 mg L was appropriate for culture initiation in Vitex negundo. Braga et al. (2012) regenerated shoots using nodal explants on MS medium with BAP and NAA in litoralis. Comparatively less number of shoots was noted on MS medium supplemented with Kinetin. The success in establishment of in vitro culture of Verbenaceae members are hindered mainly due to phenolic exudation in the medium. Continuous subculture on fresh medium can reduce the level of exudation from the explants (Rathore and Shekhawat, 2011). Braga et al. (2012) reported addition of poly N-vinylpyrrolidone in the culture medium to exempt phenolic exudation. The shoots were better multiplied on full strength MS medium +1.0 mg L-1 BAP + 0.5 mg L-1 Kin + 0.1 mg L-1 NAA. The combined effects of cytokinins with lower concentrations of auxins for better multiplication of shoots was also reported in Vitex negundo (Rathore and Shekhawat, 2011). In vitro multiplied shoots (4-5 cm) were rooted on half strength MS medium supplemented with IBA 1.5 mg L-1. The reduced mineral salts concentration and IBA for root induction has also been reported in V. negundo (Rathore and Shekhawat, 2011). The in vitro rooted shoots were transferred to soilrite® and maintained in the greenhouse for hardening and the plants were transferred to the field after two month with 86% of survival rate. The application of microscopy with plant tissue culture offers a perfect platform for internal as well as morphological assessments in plant growth and developmental studies (Moyo et al., 2015). The physiological disorders are often manifested in the anatomy of the tissue cultured regenerants could be studied by micromorphological investigations. Rocha et al. (2012) characterized the anatomical events and ultrastructural aspects of direct and indirect organogenesis in Passiflora edulis. Negi et al. (2011) compared the anatomical structures of shoot and root developed through micropropagation with the in vivo plants of Cassia auriculata. Comparative micromorphological study of in vitro and wild plants of Couroupita guiagensis was reported by Shekhawat and Manokari (2016a). The external morphology of in vitro and the field transferred plants were similar in resemblance. Both the epidermises were single layered with wavy walls. The leaves were hypostomatic, the abaxial epidermis only stomatiferous and the cells are highly undulated but the adaxial cells are less undulated (Fig. C and D). The stomata were regularly distributed except intercostal regions and mainly facing toward upward direction (Fig. A and B). The stomata were always opened irrespective of time under in vitro conditions and the contiguous stomata were frequent. Anisocytic stomata were prominent but paracyic, anomocytic and diacytic stomata were observed occasionally under in vitro environment (Fig. A). the subsidiary cells become clear, distinct and large in size in the field transferred plants. Anisocytic and anomocytic stomata were prominent but contiguous stomata were rarely observed. Peltate hairs were observed on adaxial and abaxial epidermises, which were totally absent in the in vitro grown plants (Fig. B and D).

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A B

C D

Fig. A. Stomatal frequency, contiguous stomata (arrows) in abaxial epidermis of in vitro grown plants. Fig. B. Stomatal frequency, contiguous stomata (black arrow) and peltate hair (red arrow) in abaxial epidermis of field transferred plants. Fig. C. Adaxial epidermis of in vitro grown plants. Fig. D. Adaxial epidermis with peltate hair (red arrow) in adaxial epidermis of field transferred plants.

The stomatal density of in vitro and field transferred plants was 76.2 and 50.6 respectively. The stomatal frequency was 32.9 in the in vitro which reduced to 24.1 after field transfer. The stomatal index was also considerably decreased from in vitro (36.8) to the field environment (30.2) (Fig. 1). The reticulate type of venation was observed in D. erecta. The lateral veins were fairly thick from the midrib and the veinlets were thin and profusely branched in the field transferred plants. The vein-islets and veinlet terminations were distinct. The venation pattern in the in vitro leaves was opened, and the veinlets were closed and often terminate in areoles in the field transferred plants. Veins and vein-islets were fewer in vitro leaves as compared to the field transferred plantlets. Number of vein-islets and veinlet terminations were increased, and becomes distinct in shape during the hardening period. The vein-islets of in vitro plants were 9.3 which increased to 22.5 after six weeks of transfer to the field. The veinlet terminations were simple as well as branched into two with 1-6 per vein-islet. The number of veinlet terminations were 12.0 in the in vitro leaves, which were increased to 15.3 in the field transferred plants (Fig. E and F, Fig. 2).

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Fig. 1. Comparative stomatal parameters of in vitro and field transferred plants.

Fig. 2. Comparative leaf architectural parameters (vein islet and veinlet terminations) of in vitro and field transferred plants.

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V V V

V

E F

G H Fig. E. Venation pattern in leaves of in vitro raised plants. Fig. F. Venation pattern in leaves of the field transferred plants (VI-Vein-Islet, VT- Veinlet Termination). Fig. G. Contiguous peltate hairs and conical hair (surface view) under in vitro grown plants. Fig. H. Peltate hairs (surface view) in field transferred plants.

The hairy covering is varied in the leaf surface of D. erecta. Two types of trichomes were observed, (i) short conical hairs and (ii) the short silicified peltate trichomes. The peltate trichomes were embedded on the thickened walls of the epidermal cells. The conical hairs were composed of short thin-walled stalk cell seated on the epidermis and long terminal cells with thick walls (Fig. G, H, I, J, K). The density of the trichomes was found maximum in field transferred plants (12) than the in vitro plants (17) (Fig. E-H). The trichomes were less and underdeveloped under in vitro environments which become fully developed after transferring to the field condition (Fig. E-H). Contiguous peltate hairs were observed in the in vitro grown plants but absent in field transferred plants (Fig. G and H). The morphology, physiology and internal anatomy of plants are intrinsic to the environmental conditions where they survive (Hazarika et al., 2000). The in vitro saturated atmosphere, vessel headspace, sucrose as readymade source of carbon and energy, contentious source of water, growth hormones, decrease air turbulence, high air humidity and lower temperature limits the inflow of CO2 and outflow of gaseous plant products from the vessels, which causes the abnormal internal structures compared with in vivo plants (Lamhanedi et al., 2003; Lebedev and Schestibratov, 2013). The altered morphology, anatomy and physiology of plants under in vitro culture environment are reviewed by many researchers in number of plants (Yokota et al., 2007; Chandra et al., 2010; Soares et al., 2012; Darwesh and Rasmia, 2015, Shekhwat and Manokari, 2016b).

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I J

K

Fig. I. Fully developed silicified conical hair on abaxial surface of field transferred plants. Fig. J. Developed peltate hair on adaxial epidermis of field transferred plants. Fig. K. Underdeveloped peltate and conical hairs (arrows) of in vitro plants.

The abnormalities such as opened and contiguous stomata, contiguous and underdeveloped trichomes were repaired, reduced and developed, when the plantlets fully adapt the field environment. It has been reported that the gradual acclimatization process help the micropropagated plantlets to impair the abnormalities and undergo adaptive changes to their environment, which are necessary for survival and the maintenance of normal growth in ex vitro environment (Yang and Miao, 2010). The substantial changes in morphology and anatomy of in vitro regenerated plants under ex vitro conditions were studied by various researchers (Osorio et al., 2013; Rathore et al., 2013a; Lodha et al., 2015; Shekhawat and Manokari, 2016a and b). Chirinea et al. (2012) reported that the field grown plants of Ficus carica possessed higher number of stomata, greater epicuticular wax thickness and leaf tissue than the in vitro grown plants. Adaptability of in vitro regenerated plants in the field was studied through foliar anatomical features in Elaeis guineensis (Luis et al., 2010), Bletilla striata (Lesar et al., 2012), Solenostemon scutellarioides (Sahu and Dewanjee, 2012), Couroupita guianensis (Shekhawat and Manokari, 2016a) etc. Plants systematically develop certain changes internally as well as externally while shifting from in vitro to in vivo conditions, due to the decrease in relative humidity and high temperature of the field environment. The development of veinlets, vein-islets and increase in

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vein density of the in vitro regenerated plants towards in vivo environment explains the gradual developments of vascular tissues as reported in Cleome gynandra (Rathore et al., 2013a), Cadaba fruitcosa (Lodha et al., 2015) and Hemidesmus indicus (Shekhawat and Manokari, 2016b). The vein clearing studies could explain developmental changes in vein density during the hardening period based on gradual acclimatization process. Foliar micromorphological studies of in vitro and field transferred plants could correlate physiological aspects such as photosynthesis, mineral transport, respiration with anatomical and developmental patterns of differentiation and photomorphogenesis. This knowledge could help in understanding the responses of plants towards different environmental conditions.

4. CONCLUSION

The present study describes micromorphological studies of in vitro and field transferred plants of Duranta erecta. The nodal shoot segments responded better on MS medium supplemented with BAP. The shoots were multiplied on MS + BAP + Kin + NAA. In vitro multiplied shoots were rooted on half strength MS medium supplemented with IBA. The rooted plantlets were successfully hardened and field transferred. The comparative micromorphological study reveals the structural development of micropropagted plants from in vitro to field environment. The difference in leaf stomatal density, vein-islets, veinlet termination numbers and trichomes in different environment helps to understand the adaptation and developmental changes occurring in plants from in vitro to in vivo conditions.

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( Received 23 April 2016; accepted 08 May 2016 )

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