REGENERATION OF PLANTLETS FROM IN VITRO LEAF CULTURE OF RENADES ARUNODAY HYBRID ( AERIDES ROSEA LODDIGES EX PAXT. X RENANTHERA IMSCHOOTIANA ROLFE)

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1 J. Orchid Soc. ndia, 13(1-2): 19-24, 1999 REGENERATON OF PLANTLETS FROM N VTRO LEAF CULTURE OF RENADES ARUNODAY HYBRD ( AERDES ROSEA LODDGES EX PAXT. X RENANTHERA MSCHOOTANA ROLFE) ( S.K. Sinha* and Sadanand N. Hegde Division of Orchidology, State Forest Research nstitute, P.B. No. 159, tanagar-791 t t t, Arunachal. Pradesh, ndia. *Orchid Research Centre, SFR, Tipi. Bhalukpong , Arunachal Pradesh, ndia.,. ~,.... Abstract Leaves of in vitro raised 6 months old seedlings of Renades Arunoday hybrid were inoculated in 1/2 strength Murashige and Skoog (1962) medium supplemented with 2% sucrose and different growth regulators viz. AA, BAP, NAA, KN etc., alone and various combinations of BAP and NAA. Explants showed better response (90%) to protocorm-like bodies (PLBs) and callus initiation in 2 mql SAP whereas in the presence of 0.5 rnql NAA, only 20% of the explants responded within 7 wks of culture. Cent per cent explants responded in a combination containing 1 rnql BAP and 0.5 mql NAA. The morphogenetic potential was best shown in entire leaf culture followed by leaf base segment. The subsequent subculture in BM supplemented with 1 rnql each of BAP and NAA showed best differentiation of PLBs into plantlets with well developed roots and shoots. ntroduction Almost every morphological structure in. higher plants, owing to their cellular totipotency characteristics, can be induced to dedifferentiate into an unorganised callus and can be maintained indefinitely on a sterile nutrient medium of appropriate constitution. After Morel's (1960) successful attempts to obtain virus-free mericlones through shoot tip culture, researchers have successfully used shoot tip and axillary bud explants for micropropagation of large number of orchids in order to maintain true-to-type quality. However, as in case of monopodial orchids, excision of these explants result in sacrificing of the mother plant, many attempts have been successfully made to use alternative explants such as immature inflorescence bud (Zimmer and Pieper, 1978; Goh and Wong, 1990), flower stalk bud (Voneda and Sakamoto, 1983; Valmayor et al., 1986; Shirnasaki and Uemoto, 1991), leaf ( Hass-Von Schmude, 1984; Mathew and Received August 10, 1997; Accepted January 7, 1999 Rao, 1985; Vij et al., 1994) and root tip (Sanchez, 1988; Yoneda and Momose, 1988; Holter and Zimmer, 1990) in various orchid species and hybrids. Renades 'Arunoday' is an intergeneric vandaceous hybrid (Aerides rosea Loddiges ex Paxt. X Renanthera imschoatieae Rolfe) developed at the Orchid Research Centre, Tipi and registered by the Registrar of Orchid Hybrid at RHS London (Hegde, 1991; Hegde et al., 1988). Since, only seed culture techniques have been employed for multiplication of Renades Arunoday, the present investigation was undertaken so as to develop high frequency plantlet formation from in vitro culture of Renades Arunoday leaf explants. Materials and Methods Aseptic seedlings of Renades Arunoday at the Orchid Research Centre, Tipi were the source of explants. Leaves ( cm in length) were cultured on 1/2 strength of Murashige and Skoog

2 medium (1962; MS) supplemented with 2 % sucrose which served as basal medium. Culture media were supplemented with auxins (NAA and AA) and/or cytokinins (BAP and KN) at 0.5 m9"1 to 2 mql' concentration and coded accordingly as M 1 to M21. ph of the medium was adjusted to 5.6 and the medium was solidified with 0.8% agar. n order to as~ess the morphogenetic potential of segmented leaf explants, leaf bases and leaf tips ( cm) were inoculated in BM supplemented with 0.5 rnql:' each of NAA and BAP. Five flasks with 4 explants in each were maintained as replicates. PLBs and plantlets were subcultured in 1/2 strength MS medium containing 1rnql' each of NAA and BAP. Culture flasks were incubated under 2,000 lux of 14 hr illumination at 25 ± 2 C temperature. Observations were recorded SNHA AND HEGDE from time to time and comparison of response was made at the end of 7th wk of culture. The maximum proliferation of PLBs, callus or leaf primordia was graded as excellent ( moderate as good (+ +), least as poor (+) and if none of the explant showed any particular response none (-) was coded. Results Leaf explants started swelling at their bases within 4 wks of culture, and these produced PLBs, callus and leaf primordia in 28 wks depending on the type of growth regulator used (Table 1, Figs. 1-3) from leaf base, lamina, leaf tip and even on all over the surface of leaf. The media supplemented with varying concentration of NAA (M1 to M3) and AA (M4 to M6) have shown the least response (below t, Table 1. Regeneration response of Renades Arunoday leaf explants on 1/2 strength Murashige and Skoog medium (MS) and its various conbinations with growth regulators Media Growth regulators (mgl 1) used Per cent Code NAA AA BAP KN response (M) Type of response PLB Callus Leaf primordia Control, ' Excellent, + +. Good, + Poor, ' None 20

3 N VTRO LEAF CULTURE ;, , C 0 Co.... e 0., c., o 0; Q Cl ~ 20 a ,0 Concentration rnql') ) mmaa c::::.:j BAP 11 KN Fig 1. Response of leaf explants to various concentrations of NAA, AA, BAP and KN. 20%) for the formation of PLBs, callus and leaf primodia. On the other hand, the media supplemented with BAP (M7 to M9) and KN (M 10 to M 12) showed comparatively better response ranging from 10% to 90% (Table 1, Fig. 1). However, the response was better when NAA and BAP were used in combination; the medium (M 14) containing 0.5 mql' NAA and 1.0 rnql:' BAP supported PLB induction in cent per cent explants. t is significant, however, to note that 2 rnql' BAPsupplemented medium (M9) showed 90% response with respect to leaf primordia. Similar response was also seen on medium M 18 supplemented with both NAA (1 rnql'] and BAP (2 rnql') with respect to the formation of maximum callus (Fig. 3). The experiment carried out to assess the response of different portions of leaf segments such as leaf base, leaf tip and entire leaf on the medium M 13 showed that entire leaf produced maximum PLBs in comparison to leaf base and leaf tip. Fifty per cent leaf explants ( basal) regenerated PLBs and callus whereas leaf tip explants produced only leaf primordia (Table 2). t was further observed that Table 2. Comparative response of entire/or segmented leaf explants on M13 medium Explant Per cent Type of response response PLBs Callus Leaf primordia Entire leaf Leaf base Leaf tip + + good, + Poor, - None

4 SNHA AND HEGDE t=igs Leaf culture of Renades Arunoday: 2 (A-D). Callusing and plantlet regeneration {1/2 MS + NAA (0.5 rnql') + BAP (1mql'} } after 6 wks of culture: A, Plantlets on entire leaf surface; B, Callus on leaf lamina and base; C, Callus on leaf tip and base; D, Callus on leaf base. 3, Callusing on leaf tip and base {1/2 MS + NAA (1 mql' ) + BAP (2 rnql') } after 6 wks of culture; 4, PLBs with differentiating plantlets (1/2 MS + NAA(0.5 rnql' ) + BAP (1 mgl ')} after 6 wks of subculture; 5, Seedlings with well developed roots and shoots {1/2 MS + BAP (1 mql') + NAA (1 mgl ')} after12 wks of culture. prolonged culture of PLBs (exceeding 4 months) in the same medium, resulted in their differentiation into plantlets (Fig. 4). However, when these PLBs were subcultured on M17 medium, they got differentiated into well developed plantlets within 12 wks of subculture (Fig. 5). Discussion n the present study, the. explants in 22 media containing auxins alone responded least (below 20%) whereas those in medium supplemented with cytokinins responded better (10% to 90%) with respect to formation of PLBs, callus and leaf primordia. Tanaka and Sakanishi (1980) also reported similar findings in the. case of Phalaenopsis. However, Mathews and Rao (1985) reported that treatment of auxin or cytokinin alone failed to induce proliferation in the leaf

5 N VTRO LEAF CULTURE i culture of Vanda hybrid. Further, a combined treatment with NAA and BAP showed synergistic response over the individual treatment of NAA or BAP. Media containing NAA and BAP responded 10% (M20) to 100% (M14) and this is in conformity with the earlier findings of Mathews and Rao (1985) in Vanda hybrid. Presently, the entire leaves regenerated PLBs,'callus and leaf primordia along their base, lamina', tip and even on all over the leaf surface frequently (Fig. 2). However, the frequency of proliferation from leaf base is higher than other leaf parts. This may be due to the reason that the monocot leaf base is highly meristematic (Zimmer and Pieper, 1975). On the other hand, comparative study of leaf segment cultures in isolation, culture leaf base responded upto 50% whereas the leaf tip responded only upto 5 %. Similar findings have also been reported earlier by Mathews and Rao (1985) in Vanda hybrid. The extent of response of in vitro explants cultured on media containing a combination of NAA and BAP (M 14) was maximum (100%) as compared to their effect when used alone. PLBs, upon prolonged culturing, in the same medium differentiated into plantlets (Fig. 4). However, the rate of differentiation was better in the medium supplemented with 1 rnql' each of NAA and BAP (Fig. 5). These data indicate that 1/2 strength MS medium supplemented with 0.5 rnql" NAA and lrnqr ' BAP appears to be suitable for PLB induction from leaf culture. Thereafter, these are to be sub-cultured on 1/2 strength MS medium containing 1 mql' each of NAA and BAP for differentiation into plantlets. Acknowledgments Thanks are due to Government of ndia, Department of Biotechnology, Ministry of Science and Technology, New Delhi for the financial assistance to carry out this work. Thanks are also due to Shri G.P. Shukla, PCCF, Shri K. Namchoom, CCF and Shri M.L. Deori, Director, State Forest Research nstitute, tanagar, Arunachal Pradesh for the encouragement and facilities. References Goh, C. J. and P.F. Wong Micropropagation of the monopodial orchid hybrid Aranda 'Deborah' using inflorescence explants. Scientia Horticulturae, 44 : Hass-Von Schmude, N.F Tissue culturing Phalaenopsis using leaves and leaf segments. n: Proc. 11th World Orchid Conference (ed. W. T. Kiat) pp Miami, Florida. Hegde, S. N Orchids from lab to field in Arunachal Pradesh. Arunachal Forest News, 9(1) : Hegde, S. N., A. N. Rao, R. S. nghalhali, and N. Ahmed Culture Notes from OROC, Tipi-3 : Breeding and in vitro culture of vandaceous orchids. Aerides rosea X Renanthera imschootiana (RenadesJ. Arunachal Forest News, 7 (1,?) : Holter J. and K. Zimmer Shoot regeneration from root tips of orchids in vitro-. Effect of shoot parts on root system and length and width of Mormodes his trio roots on regeneration ability. Gartenbauwissenschaft, 55(4) : Mathews, V. Helena and P. S. Rao n vitro culture of Vanda hybrid (Vanda TMA X Vanda Miss JoaquimJ. Studies on seedling explants. Pioc. ndian Natn. Sci. Acad., 51 (4) : Morel, G Producing virus-free Cymbidiums, Am. Orchid Soc. Bull., 29 : 495,97. M}uashige, T. and F. Skoog A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15:

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