Ex vitro multiple shoot regeneration potential of hypocotyls of four Rhizophoraceae mangroves

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1 Scientia Agriculturae E-ISSN: X / P-ISSN: DOI: /PSCP.SA Sci. Agri. 14 (1), 2016: PSCI Publications Ex vitro multiple shoot regeneration potential of hypocotyls of four Rhizophoraceae mangroves Pramodini Rout 1, Pradipta Kumar Mohapatra 2, Uday Chand Basak 1* 1. Seed Bank and Seed Biology Division Regional Plant Resource Centre (R & D Institute of Forest and Environment Department) Bhubaneswar-15, Odisha, India 2. Department of Botany Ravenshaw University Cuttack , Odisha, India *Corresponding author uc_basak07@yahoo.co.in Paper Information Received: 15 January, 2016 Accepted: 18 March, 2016 Published: 20 April, 2016 Citation Rout P, Kumar Mohapatra P, Chand Basak U Ex vitro multiple shoot regeneration potential of hypocotyls of four Rhizophoraceae mangroves. Scientia Agriculturae, 14 (1), Retrieved from (DOI: /PSCP.SA ) Key words: Bud primordia; Histology; Multiple shoot; Rhizophoraceae A B S T R A C T Day by day true mangrove species belonging to the family Rhizophoraceae occurring mangrove forests of Odisha coast, India are struggling hard for survival due to habitat loss and fragmentation. To arrest such problems, application of various scientific methods and techniques are needed to augment vegetative propagation systems with all Rhizophoraceae mangroves species. In this context, mass multiplication through shoot regeneration by decapitation from a single hypocotyl is a targeted tool. The present study revealed that the hypocotyls of four Rhizophoraceae mangroves viz. Kandelia candel, Bruguiera gymnorrhiza, Bruguiera parviflora and Rhizophora apiculata showed good potential for ex vitro shoot regeneration through decapitation and bud primordia formation from a histological point of view. Among four studied species, the best multiple shooting ability was recorded in the hypocotyls of K. candel at 40-Day of decapitation. The hypocotyl of K. candel gave rise to multiple shoots with 5.5±2.0 nos. per hypocotyl regenerated at 40-day followed by B. gymnorrhiza (4.0±1.5), B. parviflora (3.4±1.17) and R. apiculata (3.0±1.2). From histological analysis, at day-20 shoot primordia were found initiated from meristem cells in the region of the severed vascular bundles near the exposed surface of the hypocotyls whereas induced meristematic cells gave rise to vascular connected multiple shoots at 40-day of decapitation in case of B. parviflora. Similarly, shoot or bud primordia were developed with tunica layer at 20-day and fully developed shoot initials possessing vascular connection was recorded at 40-day in both B. gymnorrhiza and R. apiculata. Shoot or bud primordial with dense cytoplasmic cells was observed at day-20 and emerging shoot initials having vascular connection were noticed at day-40 in K. candel. The fact that additional bud primordia were present within the hypocotyls of Rhizophoraceae family suggests that further implementation of the hypocotyls is helpful to maximize the potentiality of shoot regeneration and conservation of the mangrove forest PSCI Publisher All rights reserved. Introduction Mangroves are unique coastal ecosystems situated at the interface between land and sea of tropical and subtropical latitudes having great ecological and economic values (Kathiresan and Qasim, 2005). Unfortunately, despite the enormous benefits they provide to us, mangroves in general and true mangrove species of the family Rhizophoraceae occurring in Odisha coast are largely continue to shrink due to restricted natural regeneration, habitat destruction, fragmentation, depletion of growing stock, post-dispersal damages of hypocotyls by crabs and insects, sporadic flowering and poor seed set (Basak et al. 1995). In order to mitigate such problem, application of various scientific methods and techniques are needed to facilitate vegetative propagation systems for artificial regeneration of Rhizophoraceae mangroves. Through natural regeneration, a single hypocotyl gives rise to only one individual plant. However, numerous shoots may be produced artificially from a single hypocotyl through breaking of apical dominance by decapitating the hypocotyls at collar region. Then the multiple shoots are subjected to vegetative propagation through stem cutting and/or air-layering to produce many plantlets ex vitro. Few encouraging reports are available on multiple shoot regeneration in hypocotyls of Rhizophoraceae mangroves under in vitro condition. The in vitro method described by Satuwong et al., (1995) for Bruguiera gymnorrhiza indicates that upper region of

2 the hypocotyls can give rise to shoots. Similarly, formation of multiple shoots and their histological basis also reported in hypocotyls of Kandelia candel by Ogita et al., (2004) following in vitro methods. They hypothesized that a wound induced meristem may be present and responsible for the formation of the adventitious shoots. Ex vitro multiple shoot regeneration through Decapitation method using hypocotyls of Rhizophoraceae mangroves i.e Bruguiera gymnorrhiza was reported earlier by Basak and Das (2002). However, there is no scientific report on histological basis of ex vitro multiple shoot regeneration in hypocotyls of Rhizophoraceae mangroves. Hence, the aim of the present study was to report histological basis of ex vitro multiple shoot regeneration in hypocotyls of four Rhizophoraceae mangroves. Materials and Methods Collection of sample (Propagules) Propagules of Bruguiera gymnorrhiza (L.) Savigny (Local Name-Bandari), Bruguiera parviflora (Roxb.) Wight &Arn.exGriff. (Local Name-Kaliachua), Rhizophora apiculata Bl. (Local Name-Rai) and Kandelia candel L. (Druce) (Local Name-Sinduka) were collected from the mangrove forest of Mahanadi Delta of Odisha, India ( N latitude and E longitude. The species were authenticated using institutional herbaria. Multiple shoot regeneration through decapitation Propagules of all studied species of nearly equal length and diameter were decapitated in the collar region (Basak and Das, 2002) and grown in polybags containing a mixture of garden soil and sand (1:1) and then kept under mist system of green house. The propagules were fed everyday with fresh water. Observations were made on the development of multiple shoots from the decapitated surface of the hypocotyls. To analyze histological changes during multiple shoot regeneration, samples were collected at different time intervals i.e at Day-0, Day-20 and Day-40 of decapitation in order to obtain time-bound and species-specific histological changes during multiple shoot regeneration. Histological analysis The junction portion (2-3cm, called collar zone) of the decapitated hypocotyls were sampled at 0-day, 20-day and 40- day to record differential stages of shoot regeneration i.e from initiation to emergence of adventitious shoot formation. Samples were preserved in 70% ethanol. Uniform and thin transverse sections (40-50µm) of the shooting zone were cut in sledge microtome (Spenser make, model 1010-SMT-009). Selected sections were stained in safranin followed by fast green (double staining) following a series of alcohol dehydration and mounted in DPX Dwivedi and Singh (1885). The sections were examined under compound microscope to record anatomical changes during initiation and formation of multiple shoots and documented through photomicrography (Nikon Microscope, Eclipse 50I, Japan). Results and Discussion Induction of multiple shoot in hypocotyls The hypocotyls of Rhizophoraceae mangroves especially Bruguiera gymnorrhiza, Bruguiera parviflora, Kandelia candel and Rhizophora apiculata had profound multiple shoot inducing ability under ex vitro condition. The ex vitro shoot regeneration exhibited by the hypocotyls of studied species summarized in Table-1, Figure.1. A, B, C and D. Highest shoot induction ability was observed in K. candel hypocotyls at 40 day of decapitation followed by B. gymnorrhiza, R. apiculata and B. parviflora. Maximum shooting percentage (95±1.05) and shoot number per hypocotyl (5.5±2.0) were recorded in K. candel at 40 day of decapitation and R. apiculata scored lowest. Shooting percentage (90±2.66) and shoot number (4.0±1.5) were achieved in B. gymnorrhiza followed by B. parviflora with shooting percentage (80±2.05) and shoot no (3.4±1.17) and R. apiculata with shooting percentage (3.0±1.2) and no of shoot (70±2) at 40-day. Our observation is nearly similar to that described by Basak and Das (2002) for B. gymnorrhiza (3.5±0.1). Ogita et al, (2004) reported that the hypocotyls of K. candel gave rise to 3 to 5 visible buds in in vitro conditions. Table 1. Performance of multiple shoot regeneration from hypocotyls of Bruguiera gymnorrhiza, Bruguiera parviflora, Rhizophora apiculata and Kandelia candel. Name of species Average no of visible shoots appeared per hypocotyls % of multiple shoot induction 20-Day 40-Day Bruguiera gymnorrhiza 2.1± ±1.5 90±2.66 Kandelia candel 2.4± ±2.0 95±1.05 Rhizophora apiculata 1.8± ±1.2 70±2 Bruguiera parviflora 2.0± ± ±2.05 Values were expressed Mean±SD 211

3 Changes in histological interpretation during shoot regeneration Results showed that hypocotyls were capable of giving rise to multiple shoot buds in all the studied species. These findings clearly indicate that the hypocotyls of Rhizophoraceae mangrove have high shoot regeneration potentials. The hypocotyls of Kandelia candel is composed of a single layer of epidermis with a thick cuticle at the time of decapitation (Day 0) (Fig. 4. A). Shoot primordia were found initiated from meristem cells in the region of the severed vascular bundles near the exposed surface of the hypocotyls by day 20 (Fig.4. B). Though, the exact origin of the shoot buds was difficult to trace, it was appeared that shoot buds were of endogenous in origin i.e. developed from underneath of the cut-surface of the cortex of the propagules because of activation of decapitation-induced meristematic cells which tends to formation of vascular-connected multiple shoots by day 40 (Fig. 4. C). The wounding induces the formation of a meristem in K. candel when placed under in vitro culture conditions and that this meristem has organogenic potential reported by Ogita et al., (2004). Hypocotyls of B. gymnorrhiza were developed new meristem which was close to the exposed surface of the hypocotyls (Fig. 3. B) and this meristem develops vascular bundles leading to multiple shoot formation (Fig. 3. C). Shoot formation from the cut surface of the hypocotyls is common; however new shoots often initiate from parenchyma cells associated with severed vascular tissues Miranda et al., (1999). The formation of a wound periderm is a normal developmental event which has been reported by Romberger et al., (1993). In one of the mangrove species, Avicennia germinans, the cork cambium appears rapidly at the base of the hypocotyl about the fifth day after seedling liberation and by the seventh day it extends along half its length as reported by Chapman The development of shoot bud primordials were found at day-20 and visible shoot buds were developed at day 40 in case of B. parviflora (Fig. 2.B and C). The rapid formation of wound periderm may be an inherent character of mangrove plants Ogita et al., (2004). Besides few large shoot buds appearing at cut surface of the hypocotyl, many more bud primordia are present underneath the wound-induced cork cell layers. It is possible that the tightly packed corks cells 212

4 might have physically ostracted the expansion of the small, developing bud primordia. By day 20, shoot bud primordia with tunica layer was formed in cortical region and fully developed shoot initials were developed at 40-day in R. apiculata (Fig. 5.B and C). In our study, shoot meristems always initiate just underneath the cork cells. Then structural evidence proves beyond a doubt that the site of shoot meristem formation is the wound-induced meristem. Since shoot meristems are always formed in association with the vascular tissue, it is likely that material such as additional nutrients or a higher concentration of certain metabolites may be present in or near the vascular bundles which could induce shoot organogenesis from the wound induced meristem cells. 213

5 Conclusion In conclusion, it could be suggested that the hypocotyls of Rhizophoraceae family are capable of producing adventitious multiple shoot through decapitation for augmenting vegetative propagation. Thus, through this method, 2 to 3 times more planting materials could be produced. Better shooting potential in terms of percentage of shooting and quantity of shoot bud per hypocotyls were obtained in K. candel. The advantages of organogenic potential of the wound-induced meristem near the exposed surface of the hypocotyl of Rhizophoraceae mangrove may thus be availed to increase the number of shoots produced by the explants for mass propagation purposes. 214

6 Acknowledgements The authors are thankful to the Ministry of Environment and Forests & Climate Change (formerly MoEF), Government of India for financial assistance vide project No.22-11/2006-CS (M). The authors are also thankful to the Department of Forest and Environment (Mangrove Wildlife Division), Government of Orissa for providing samples in the buffer zone of mangrove forest of Mahanadi Delta of Odisha coast, India. References Basak UC, Das P In vivo induction of multiple shoots for scaling up of propagation of tree mangrove Bruguiera gymnorrhiza (Linn.) Sav. (Rhizophoraceae). Indian Journal of Marine Science. 31(3): Ogita S, Yeung EC, Sasamoto H Histological analysis in shoot organogenesis from hypocotyls explants of Kandelia candel (Rhizophoraceae). Journal of Plant Research. 117: Basak UC, Das AB, Das P Metabolic changes during rooting in some cuttings of 5 mangrove species of Orissa. Plant Growth Regulation. 17(2): Dwivedi JN, Singh RB Essential of plant techniques. Scientific Publishers, Jodhpur. India pp Satuwong I, Ninomiya I, Ogino K Callus and multiple shoot formation of Bruguiera gymnorrhiza. Bulletin Ehime University For. 32:25 33 Miranda J, Konschuh MN, Yeung EC, Chinnappa CC In vitro plantlet regeneration from hypocotyls explants of Stellaria longipes (Caryophyllaceae). Cannadian Journal of Botany. 77: Romberger JA, Hejnowicz Z, Hill JF Plant structure: function and development. Springer-Verlag, Berlin Heidelberg New York Chapman VJ Mangrove vegetation. Cramer, Leuterhausen, Germany Kathiresan K, Qasim SZ Biodiversity of Mangroves. Hindustan Publishing Corporation (India), New Delhi. pp