Final SPH -JHS Coverpage 17-1 Jan 2022 single 255 J. Hortl. Sci. Vol. 17(1) : 255-259, 2022 This is an open access article d istributed under the terms of Creative Commons Attribution-NonCommer cial-ShareAl ike 4.0 International License, which permits unrestricted non-commercial use, d istribution, and reproduction in any med ium, provide d the original author and source are credited. Short Communication The coconut palm (Cocos nucifera L.) is one of the most beautiful and useful trees in the world and all parts of this ‘wonder palm’ are useful in one way or other. Coconut, an out-breeding perennial tree, is seed propagated, exhibits great variation in morphological and agronomic characters. Vegetative multiplication of elite coconut palms is a promising possibility for producing uniform planting material with high yield a nd disea se-r esista nce. Pr otocols for coconut micropropagation have been developed in various laboratories using different explant sources (Nguyen et al., 2015). Among various explants, the most extensively studied are the rachillae from inflorescence and plumule from zygotic embryos. Flowering is a complex phenomena regulated by both internal and external factors and induction of in vitro flowering is very rare in most of the crops. Under natura l conditions, flower forma tion norma lly commences when a plant attains maturity. Juvenile phase of a plant is genetically controlled and is species specific which means that a plant flowers only when genetic factors including photoperiodic response are congenial. However, these conditions can often be altered so that the plant can be induced to undergo an early reproductive phase. Such an attempt to induce flowering in vitro has been attempted in many plant systems. In vitro cultur e pr ovides a n idea l experimental system for studying the molecular mechanism of flowering. In vitro flowering studies has been conducted in many perennial crops e.g., bamboo (Joshi and Nadgauda, 1997), red hot pokers (Taylor et al., 2005), date palm (Allouche et al., 2010), oil palm (Nizam and Te-chato, 2012) etc. However, in vitro flowering in coconut has not yet been reported. Reducing duration of juvenile phase is an advantage especially in coconut with long pre-bearing period of 6-10 years. Here, in the process of establishing in vitro regeneration of coconut using immature inflorescence explants, strikingly, a few cases of in vitro flowering in coconut plantlets was observed. This paper aims Occurrence of in vitro flowering in coconut (Cocos nucifera L.) Shareefa M.*, Thomas R.J., Sreelekshmi J.S. and Anitha K* ICAR-Central Plantation Crops Research Institute, Regional Station, Kayamkulam, Alappuzha-690533, Kerala, INDIA ICAR-CPCRI, Kudlu P.O., Kasaragod-671124, Kerala *Corresponding author E-mail : hishareefa@gmail.com ABSTRACT Immature inflorescence with outer spathe length of 5.5 cm size collected from West Coast Tall cultivar of coconut was used as the explant and rachillae bits were inoculated in Y3 media supplemented with 2, 4-D (1 mg L-1). The cultures were incubated in dark for eight months and sub-cultured into the same media at monthly interval. The white shoot like outgrowths formed were sub cultured to ½ MS media fortified with 1 mg L-1 each of NAA and BAP and subsequently transferred to light condition. After three months, the emerging shoot like structure was transferred to Y3 media fortified with NAA and BAP. Upon developing 3 - 4 leaves, the cultures were transferred to rooting media and root initiation was observed after two months. The transition of vegetative shoot to reproductive state was accompanied by some morphological changes including rapid emergence of long and thin leaves followed by emergence of pearly white inflorescence. Unlike normal inflorescence, the inflorescence emerged was terminal and was devoid of spathe. Prolonged subculture in the same media might have resulted in pH variation and subsequent reduction in organic and inorganic constituents of the media. The chemical stress experienced by the plantlet might have induced in vitro flowering. Key words: Cocos nucifera, immature inflorescence, hapaxanthic, prolonged subculture 256 Shareefa et al J. Hortl. Sci. Vol. 17(1) : 255-259, 2022 to present some observations connected with in vitro flowering of coconut palm and also tries to explain the possible factors involved. The procedure followed by Shareefa et al. (2019) was used for immature inflorescence culture of coconut. Immature inflor escence expla nts with outer spathe length of 5.5 cm size were collected fr om 25 yea r old West Coast Tall var iety a nd rachillae bits of 1 mm which were inoculated in Y3 media s u p p lement ed wit h 1 mg L -1 2 , 4 - dichlor ophenoxya cetic acid (2,4-D). The basal media also contained sucrose 40 g L-1, charcoal 1 g L-1 and agar 6 g L-1. The cultures were incubated in dark condition at 27° ± 2°C and sub cultured in same media. After eight months, cultures were tr a nsf er r ed to ½ Mur a shige a nd Skoog (MS) medium with 1mg L-1 each of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). The cultures were initially kept in diffused light for one month followed by incubation in light condition for about 16 hours light (45-60 µmol m-2 s-1 PPFD) provided by white Light Emitting Diode (LED) tubes. After 4-6 months in light, the multiple shoots wer e sepa r a ted f r om t he pa r enta l clump a nd transferred for shoot regeneration to Y3 media with 1 mg L-1 each NAA and BAP. After developing 3- 4 leaves, the cultures were transferred to rooting media containing Y3 with NAA (2 mg L-1) and BAP (2 mg L-1) and Indole 3-acetic acid (2 mg L-1) along with sucrose 30 g L-1 for root initiation. Within one month of dark incubation, the rachillae ex pla nts s welled a nd whit e out gr owths wer e observed in culture initiation media. The cultures when tr ansferr ed to light conditions gr adua lly turned green and developed multiple shoots which could be easily detached from parental clump. The s ep a r a t ed s hoot s wer e t r a ns f er r ed t o s hoot regeneration media for formation of well developed leaves. Root initia tion was obser ved a fter two months in the rooting media. In vitro flowering was observed in few plantlets cultured in the rooting media and such plantlets developed had four leaves and few root initials. In order to develop secondary roots, the plantlets were kept in the same media for a period of six months. The onset of in vitro flowering was accompanied by some morphological changes in the plantlets which include rapid emergence of long and thin lea ves befor e the a ppea r a nce of pea r ly white inflorescence. Unlike normal inflorescence, the emergence of inflorescence was terminal in the in vitro raised plantlets and the inflorescence was devoid of spathe (Figure.1). The ability of explants to form flowers in vitro depends on nu mer ou s inter na l a nd ext er na l, physical and chemical factors and virtually all these factors interact in various complex ways (Compton and Vielleux, 1992). In the present study, induction of flowering was observed in plantlet cultured on Y3 media fortified with NAA (2 mg L-1) and BAP (2 mg L-1) and IAA (2 mg L-1). The combined effect of auxin and cytokinin on in vitro flower induction has also described in a number of previous studies (Handro, 1983; Wang et al., 2002; Ammar et al., 1987; Jeyachandran and Bastin, 2013; Lin et al. 2005; Saritha and Naidu, 2007a; Sudhakaran and S iva s a n ka r i, 2 0 0 2 ; Ta ylor e t a l . 2 0 0 5 ; Thiruvengadam and Jayabalan, 2001). The role of cytokinins on in vitro flowering has been well documented (Wang et al., 2001; Saritha and Naidu, 2007b). Cytokinins alone do not a ppea r to be responsible for floral initiation. It is reported that cytokinins are known to interact with sucrose to cause the shift in the apical mer istem fr om a vegetative phase to a reproductive one (Bernier et al. 2002; Bernier and Pe´rilleux, 2005). Sugars are primary sources known for reliable induction and development of flowers in many plant species such as r ose (Vu et al. 2006), Passiflora suberosa ( S c or z a a nd J a nic k, 1 9 8 0 ) , Vi g n a m u n g o (Ignacimuthu et al., 1997) indicating that presence of ca r bon s ou r ces on the c ult ur e medium is necessary for floral stimulation. There are many other physico-chemical factors which affected the in vitro flowering mechanism. Kolar and Senkova (2008) reported that reduced mineral nutrient availability accelerated in vitro flowering in Arabidopsis thaliana. The effect of Paclobutrazole, LEDs and sucrose on flowering of Euphorbia milli plantlets in vitro was studied by Dewir et al. (2007). In tobacco, important factors influencing in vitro flowering were light, growth regulators, carbohydrates and pH of the culture medium (Heylen and Vendrig, 1988). The most essential part of plant tissue culture is the media which supplies hormones a nd necessa ry nutrients for growth and development. In the present investigation, maintaining cultures for six months in 257 same media resulted in good root growth in plantlets, which also resulted in floral initiation. Prolonged culture of rooted shoots in media containing NAA and PBZ together with higher concentration of sucrose at 7% was reported to induce floral development in oil pa lm (Niza m a nd Te-cha to, 2012). Dela ying subculture may lead to hormone alternation and depletion of nutrients in the culture media. Therefore the altered chemical composition might have created a stress due to the increa sed passage time for subculturing. It was interesting to note that in vitro flowering did not r esemble flower ing ex vitro, in tha t the inflorescences in vitro never matur ed a nd they subsequently senesced indicating that other factors, excluding cytokinins and a carbohydrate source, are required for continued normal development of the inflorescences. Cytokinins and sucrose therefore seem to act in the initial stages of floral initiation and development, however, full differentiation and maturation of the resulting flower bud requires involvement of other physiological factors. The results of the current study revealed that contrary to natural flower formation, in vitro neoformed inflorescences were completely uncovered, ie., lacking spa the. T her e a re two types of developmenta l processes namely hapaxanthic and pleonanthic, in palms (Tomlinson, 1990). In hapaxanthic type, the growth of the axis of palm is determinate due to conversion of the vegetative shoot apical meristem (SAM) to the reproductive state, resulting in a short flowering phase and this phenomenon is observed only in less than 5% of palm species. The rest of the palm species are pleonanthic, with an indeterminate SAM, in which the vegetative growth continues while producing a reproductive meristem at each leaf axil. According to the Tomlinson model, under in vivo conditions, flower ing in coconut is nor ma lly pleonanthic. However, in the present study, in vitro flowering was hapaxanthic as the inflorescence emergence wa s ter mina l r esulting fr om the development of the apical bud which was devoid of any bract, which consequently gave rise to uncovered inflorescences. The flowers were malformed and never matured indicating that optimum interaction of light, temperature, plant growth regulators and nutrients are essential for flowering and normal maturation of flowers. Similarly, undersized and malformed flowers have been observed previously in other species (Ramanayake et al. , 2001). T he malformation Occurrence of in vitro flowering in coconut J. Hortl. Sci. Vol. 17(1) : 255-259, 2022 Fig.1a. Initial stage of in vitro flowering in coconut (arrow) Fig. 1b. Fully emerged in vitro inflorescence 258 occasionally observed in the flowers produced in vitro may have been partially due to competition and or nutritional deficiencies as reported in Pentanema indicum (Sivanesan and Jeong, 2007). Summary In the present study, prolonged subculture in the same media might have resulted in changes in the pH and reduction in concentration of organic and inorganic constituents of the media. The resulting chemical stress might have induced in vitro flowering in coconut. The interesting observation was that in vitro neoformed inflorescences were completely uncovered, lacking spathe and were terminal. The flowers were malformed and never matured indicating that optimum interaction of light, temperature, plant growth regulators and nutrients are essential for flowering and normal maturation of flowers. However, in vitro flowering can be efficiently used to understand the snapshots of physiological, hormonal and molecular regulation of flowering and such information gathered can be used to save time in future genetic improvement programs. ACKNOWLEDGEMENTS We thank Indian Council of Agricultural Research, New Delhi for supporting the work which was carried out as a part of the Institute Funded research project. We also thank Dr. George V. Thomas (former Director, ICAR-CPCRI), Dr P. Chowdappa (former Director, ICAR-CPCRI), Dr. V. Krishnakumar (former Head, ICAR-CPCRI, Regional Station, Kayamkulam) and Dr. S. Kalavathy (Acting Head, ICAR-CPCRI, Regional Station, Kayamkulam) for providing the necessary facilities for carrying out this study. REFERENCES Allouche, F. M, Meziou. B., Kriaa, W., Gargouri, R., Drira, N. 2010. In vitro flowering induction in date palm (Phoenix dactylifera L.). J. Plant Growth Regul.,29: 35-43. Ammar, S., Benbadis, A., Tripathi, B. K. 1987. Floral induction in date palm seedling (Phoenix dactylifera L. var. Deglet Nour) cultured in vitro. Can. J. Bot., 65: 137-142. Bernier, G. and Pe ŕilleux, C. 2005. A physiological overview of the genetics of flowering time control. Plant Biotechnol. J., 3: 3-16. Bernier, G., Corbesier, L., Pe ŕilleux, C. 2002. The flowering process: on the track of controlling factor s in Sinapsis alba. Russ. J. Plant Physiol., 49: 445-450. Compton, M. E. and Vielleux, R. E. 1992. Thin cell layer morphogenesis. Hortic Rev., 14: 239-264. Dewir, Y. H., Chakrabarty, D., Hahn, E. J., Paek, K. Y. 2007. Flower ing of Euphorbia millii plantlets in vitro as affected by paclobutrazol, light emitting diodes (LEDs) and sucrose, Acta Hortic, 764: 169-173. Handro, W. 1983. Effects of some growth regulators on in vitro flowering of Streptocarpus nobilis. Plant Cell Rep., 2: 133-136. Heylen, C. and Vendrig, J. C. 1988. The influence of different cytokinins and auxins on flower neoformation in thin cell layers of Nicotiana tabacum L. Plant Cell Physiol., 29: 665-671. Ignacimuthu, S., Franklin, G., Melchias, G. 1997. Multiple shoot formation and in vitro fruiting of Vigna mungo L. Hepper. Curr. Sci., 73: 733- 735. Jeyachandran, R. and Bastin, M. 2013. An efficient protocol for in vitro flowering and fruiting in Micrococca mercurialis (L.) Benth. Int. J. Appl. Nat. Sci., 2: 18-22. Joshi, M. S. and Nadgauda, R. S. 1997. Cytokinin and in vitro induction of flowering in bamboo: Bambusa arundinacea (Retz.) Willd. Curr. Sci., 73: 523-526. Kolar, J. and Senkova, J. 2008. Reduction of mineral nutrient availability accelerates flowering of Arabidopsis thaliana, J. Plant Physiol., 165: 1601-1609. Lin, C. S., Chen, C. T., Hisao, H. W., Chang, W. C. 2005. Effects of growth regulators on direct flowering of isolated ginseng buds in vitro. Plant Cell Tissue Organ Cult., 83: 241-244. Nguyen, Q. T., Bandupriya, H. D., Lopez, V. A., Sisunandar, S., Foale, M., Adkins, S. W. 2015. Tissue culture and associated biotechnological interventions for the improvement of coconut (Cocos nucifera L.): a review. Planta, 242: 1059-1076. Shareefa et al J. Hortl. Sci. Vol. 17(1) : 255-259, 2022 259 Occurrence of in vitro flowering in coconut J. Hortl. Sci. Vol. 17(1) : 255-259, 2022 Nizam, K. and Te-chato, S. 2012. In vitro flowering and fruit setting of oil palm Elaeis guineensis Jacq). J. Agric. Sci. Technol., 8(3): 1079-1088. Ramanayake, S., Wanniarachchi, W., Tennakoon, T. 2001. Axillary shoot proliferation and in vitro flower ing in a n a dult gia nt ba mboo, Dendrocalamus giganteus Wall. Ex Munro. In Vitro Cell. Dev. Biol. Plant ., 37: 667-671. Saritha, K. V. and Naidu, C. V. 2007a. High frequency plant regeneration and in vitro flowering of regenerated plantlets of Spilanthes acmella Murr-an important threatened bio-insecticide medicinal plant. Acta Hortic., 756: 183-198. Saritha, K. V. and Naidu, C. V. 2007b. In vitro flowering of Withania somnifera Dunal.-an important antitumor medicinal plant. Plant Sci., 172: 847-851. Scorza, R. and Janick, J. 1980. In vitro flowering of Passiflora suberosa L. J. Am. Soc. Hortic. Sci., 105: 892-897. Shareefa, M., Thomas, R. J., Sreelekshmi, J. S., Rajesh, M. K., Anitha Karun. 2019. In vitro regeneration of coconut plantlets from immature inflorescence. Curr. Sci., 117 (5): 813-820. Siva nesa n, I. a nd Jeong, B. R. 2007. Micropropagation and in vitro flowering in Pentanema indicum Ling. Plant Biotechnol., 24: 527-532. Sudhakaran S, Sivasankari V. 2002. In vitro flowering r esponse of Ocimum basilicum L. Plant Biotechnol., 4: 181-183. Taylor, N.J., Light, M. E., Van-Staden, J. 2005. In vitro flowering of Kniphofia leucocephala: influence of cytokinins. Plant Cell Tissue Organ Cult., 83: 327-333. Thiruvengadam, M. and Jayabalan, N. 2001. In vitro flowering of Vitex negundo L.- A medicinal plant. Plant Cell Biotechnol. Mol. Biol., 2: 67- 70. Tomlinson, P. B. 1990. The Structural Biology of Palms. Clarendon Press, Oxford, London. Vu, N. H., Anh, P. H., Nhut, D. T. 2006. The role of sucrose and different cytokinins in the in vitro floral morphogenesis of rose (Hybrid tea) cv. ‘First Prize’. Plant Cell Tissue Organ Cult., 87: 315-320. Wang, G., Yuan, M., Hong, Y. 2002. In vitro flower induction in roses. In Vitro Cell. Dev. Biol. Plant., 38(5): 513-518. Wang, S., Tang, L., Chen, F. 2001. In vitro flowering of bitter melon. Plant Cell Rep., 20: 393-397. (Received: 01.10.202; Revised:06.05.2022; Accepted: 02.08.2022) 00 A Final SPH -JHS Coverpage First 2 pages.pdf 00 Content and in this issue.pdf 01 Mohan Kumar G N.pdf 02 Meera Pandey.pdf 03 Biradar C.pdf 04 Varalakshmi B.pdf 05 Vijayakumari N.pdf 06 Barik S.pdf 07 Sajid M B.pdf 08 Ranga D.pdf 09 Usha S.pdf 10 Manisha.pdf 11 Amulya R N.pdf 12 Akshatha H J.pdf 13 Adak T.pdf 14 Sujatha S.pdf 15 Gowda P P.pdf 16 Subba S.pdf 17 Dhayalan V.pdf 19 Ahmed S.pdf 20 Vishwakarma P K.pdf 21 Deep Lata.pdf 22 Udaykumar K P.pdf 23 Nayaka V S K.pdf 24 Sahel N A.pdf 25 Bayogan E R V.pdf 26 Rathinakumari A C.pdf 27 Yella Swami C.pdf 28 Saidulu Y.pdf 29 Sindhu S.pdf 30 Neeraj.pdf 31 Sivaranjani R.pdf 32 Rashied Tetteh.pdf 34 Sangeetha G.pdf 35 Shareefa M.pdf 36 Last Pages.pdf