In vitro propagation of Dendrobium hybrids using flower stalk node explants

Indian Journal of Experimental Biology Vol. 43, March 2005, pp. 280-285 In vitro propagation of Dendrobium hybrids using flower stalk node explants K P Martin, Julie Geevarghese, Dominic Joseph & Joseph Madassery* Department of Biotechnology, University of Calicut, 673 635, India Received 4 June 2004; revised 7 December 2004 Large-scale in vitro propagation protocol for Dendrobium hybrids Sonia 17 and 28, two hi ghly prized commercial cut flower cultivars through shoot multiplication using flower stalk node explants and protocorm-like bodies (PLBs) formation was accomplished. Both hybrids did not exhibit significant differences in initiation, multiplication, rooting, and field establishment. Flower stalk nodes cultured on half strength Murashige and Skoog (MS) medium supplemented with 6.97 ij.m kinetin (Kn), or 15% coconut water (CW) or 13.3 ij.m of N 6 -benzyladenine (BA) evoked bud break. Kn showed better growth of the initiated bud. Excision and culture of the initiated shoots on medium having same amount of Kn developed more than 5 shoots per shoot directl y from the base. Subsequent culture enhanced the rate of shoot induction. Transfer of isolated shoots onto 44.4 ij.m of BA enriched medium displayed induction of more than 6 PLBs from the base within 60 days. PLBs underwent rapid multiplication upon transferral to medium having the same concentration of BA (44.4 ~tm ). Subsequent culture increased the proliferation of PLBs. No decline was observed in the proliferation of shoots as well as PLBs up to I 5'h subculture. PLBs transferred onto half strength MS medium with 6.97 ij.m of Kn underwent conversion of more than 90% PLBs to shoots. The shoots were rooted at the best on half strength MS medium with 2 g 1" 1 activated charcoal. Survival rate of the plantlets of the two hybrid cultivars after acclimatization was more than 80%. Keywords: Clonal propagation, Orchids, Protocorm-like bodies, Shoot regeneration IPC Code: lnt Cl 7 A 01 H/00 Orchids consist of a number of ornamental species having global importance as cut flowers and pot plants. The demand for their cut flowers increased rapidly over the years. Because of the higher number of flowers per inflorescence and recurrent flowering, Dendrobium hybrids remain as a favourite orchid of cut flower industry. Cut flower industry depends on elite cultivars and the hybrids, which in turn depends on the availability of true-to-type planting propagules. Absence or restricted seed setting in hybrids curbs the propagation via seeds. Conventional propagation of the hybrids by division of offshoots and plants are very slow (due to the development of low number of new plants from the base), and consume years to develop commercial quantities of elite clones. Since Morel's 1 mericloning of Cymbidium, in vitro propagation of orchids progressed as an attractive alternative for en masse multiplication of highly prized culti vars at faster rates than conventional procedures. Clonal propagation of orchids using leaf (either from seedling or mature plants), flower stalk and stem node and root explants has been documented *Correspondent author: E-mail: hdbiotech@sify.com; marttin@ sify.com on several ornamental orchids, Paphiopedilum 2 3 PI l. 4-6 D d b. 7-10 c l II w aenops1s, en ro zum, att eya, 0 ncl. c. I zum. tz, C ym b zc. l. tum tl t4. Th e present stu d y emphasizes large-scale in vitro propagation of two commercial cut flower hybrids of Dendrobium, Sonia 17 and 28 through shoot multiplication a~d PLBs induction and subsequent plant regeneration. Materials and Methods Flower stalk segments of Dendrobiwn hybrids Sonia 17 and 28 collected from plants grc'.vn in the net-house of the Department were washed under running tap water and followed by a neutral liquid detergent, Extran (5% v/v; Merck India Ltd., Mumbai), for 5 min. After thorough wash in double distilled water, the flower stalk segments were sterilized using 0.1 % (w/v) me: curic chloride solution for 9-12 min. They were then washed thoroughly with sterile double distilled water. Sterilized flower stalk segments cut into a size of 10-15 mm with one bud were cultured on half strength Murashige and Skoog 15 medium. The medium was fortified with various concentration::; of N 6 -benzyladenine (BA), kinetin (Kn), or coconut water (CW) singly or in combination, and also in combination with a-naphtha-

MARTINet al. : PROPAGATION OF DENDROBIUM HYBRIDS 281 leneacetic acid (NAA) as described in the following paragraphs. For in vitro rooting, half strength MS medium was supplemented with 1.0-3.0 g r' of activated charcoal (AC). All media were supplemented with sucrose (2%), and gelled with 0.7% (w/v) agar. The ph of the medium was adjusted to 5.5 before the addition of agar. The media were sterilized in an autoclave at a pressure of 1.06 kg cm 2 (121 C temp.) for 20 min. Test tubes (25x200 mm) and jam bottles (53x125 mm; 2 mm thick) were used as culture vessels. Twenty and 50 ml medium were dispensed to test tubes and jam bottles, respectively. Culture tubes were closed by cotton plugs, while polypropylene autoclavable lids (14 mm htx48 mm inner diam with 2 mm thick) were used for jam bottles. All cultures were incubated at 25 ±2 C under 16.8 hr light/dark (at an irradiance of 25 ).!mol m 2 s '). The harvested plantlets were washed thoroughly under tap water and the root portions dipped in 0.2% solution of fungicide (Bavistin) for 2 min. The plantlets, then planted in a mixture of sand, brick or tile and charcoal pieces and coir fiber (1:4:4:2), were acclimatized in Biotron LPH 200 (Japan) with a temperature at 25 ±2 C, humidity 85±5% and 12 hr light period (25 ).!mol m 2 s '). After 30 days in Biotron, the plantlets were transferred to net-house and subsequently to small pots. The experiments were set up in a completely randomised design. During the initiation and first subculture, 10 replicates were raised for each treatment. In all other cases (i.e. multiplication of shoots, induction of PLBs and its conversion to shoots and rooting of shoots) 20 replicates were raised for each treatment and were repeated twice. Mean values of different treatments were compared using Duncan's multiple range test. Results and Discussion Both the hybrids of Dendrobium did not exhibit significant differences at all stages [i.e. establishment, initiation and multiplication of shoots, induction of protocorm-like-bodies (PLBs) and its conversion to shoots, rooting of shoots and field establishment] of in vitro propagation. Half strength MS basal medium did not evoke bud break from the flower stalk nodes. But, half strength MS medium fortified with Kn (2.32-23.2 )lm), CW (5-20%), and BA (2.22-22.2 )..lm) either singly or in combination and also in combination with NAA (0.54 or 2.69 )lm) induced bud break. Initiation of axillary buds from node explants as in the present study by BA, Kn, and CW was reported in D. macrostachyum 16 Medium with 6.97 ~ Kn, 10 or 15% CW and 13.3 ~ BA when used singly was found superior to other concentrations, and these concentrations respectively induced a mean of 1.4 (50%), 1.3 (60% ), and 1.1 (50%) shoots per flower stalk node explant of the hybrid Sonia 17 within 60 days. On all other concentrations, the mean number of shoots per explant was < 1. The hybrid Sonia 28 did not exhibit significant difference in number of shoots as well as in the percentage of shoot inducing explants. The shoots initiated on medium having Kn showed better growth (> 2 em), and followed by medium with CW (> 1.5 em). The shoots on BA containing medium were short ( < 1.5 em) with small leaves. Addition of NAA at 0.54 or 2.69 )lm along with BA or Kn was not stimulative to shoot induction in both hybrid cultivars. No influence of NAA when added in combination with Kn or BA in the induction of shoots was demonstrated in other Dendrobium species 16 17. Culture of isolated shoots from primary culture increased the induction of shoots upon transfer to medium with different levels of Kn (2.32-9.29 )lm) or BA (2.22-44.4 )..lm). As in primary culture, Kn at 6.97 )lm was superior, and developed a mean of 5.4 and 5.2 shoots per shoot in Sonia 17 and Sonia 28, ' respectively within 60 days (Table 1; Fig. la). In primary culture, while the developed shoots were from the axillary bud, during subculture, shoots were induced directly from the base (Fig. la). Increase or decrease of Kn reduced the number of shoots. The shoots transferred onto half strength MS medium fortified with 13.3 )lm of BA developed a mean of 3.3 and 3.0 shoots per shoot in Sonia 17 and Sonia 28, respectively. The shoots developed on BA supplemented medium were very small. Medium having BA at above 15.5 )lm (up to 44.4 )..lm) during the first subculture showed no shoot induction. As in primary culture, combination of Kn or BA with NAA was not stimulatory to shoot induction when compared to medium supplemented with Kn or BA alone. Isolation and culture of shoots in succession on medium with 6.97 )lm of Kn increased the number of shoots per shoot (Fig. lb), and showed no reduction in number up to 15th subculture. Shoot induction and multiplication by Kn supplemented medium was documented In Phalaenopsis 4, Dendrobium

282 INDIAN J EXP BTOL, MARCH 2005 macrostachyum 16, and lpsea malabarica 18. Similar to previous cultures, during the 3'd culture, medium having BA up to 15.5 )...tm favoured induction of shoots. The shoots developed showed a tendency to remain stunted corresponding to the increase of BA. Shoots transferred onto medium with 44.4 )...tm of BA facilitated the best induction of PLBs from the basal region of the shoots and developed more than 6 PLBs within 60 days (Table 2). Addition of NAA along with BA did not enhance PLBs formation. The PLBs were cream or pale green in colour. Subculture of PLBs onto medium containing the same concentration of BA (44.4 )...tm) resulted in rapid proliferation, and induced more than 30 PLBs within 60 days. During 5th subculture, more than 200 PLBs were developed per culture (Fig. lc). About 5% of the PLBs differentiated into shoots on this medium. No decline in PLB proliferation was observed up to 15th subculture. Induction of PLBs from different explants by the addition of BA was reported among orchids 4 6. Reduction of BA in subsequent cultures did not significantly reduce the proliferation of PLBs, however it depended on the number of subcultures. After loth subculture, the PLBs underwent rapid Table!-Efficacy of different growth regulators in induction of shoots during first subculture [Values are mean of 10 replicates] Growth regulators (JlM) Cultures induced shoots(%) Number of shoots per shoot BA Kn NAA Sonia 17 Sonia 28 Sonia 17 Sonia 28 0 0 0 0 0 og or 2.22 0 0 50 50 0.8r 0.5" 4.44 0 0 70 60 l. 3 er l.ld 6.66 0 0 70 70 1.9d 1.8c 8.87 0 0 70 60 2.3d 2.!c 13.3 0 0 80 80 3.3b 3.0b 15.5 0 0 60 50 2. ld 2.0c 0 2.32 0 70 60 2. l d 2.0c 0 4.65 0 70 70 3.5b 3.2b 0 6.97 0 90 80 5.4" 5.2" 0 9.29 0 70 60 2.9c 2.9b 2.22 6.97 0 70 70 3.2bc 3.0b 4.44 6.97 0 70 60 2.ld 2.0c 8.87 6.97 0 70 60!.2cf l.ld 0 6.97 2.69 80 80 3.6b 3.4b 0 6.97 5.37 80 70!.4c!.3d 13.3 0 2.69 70 70 l. 8 dc 1. 7c 13.3 0 5.37 60 60 1.2cf!.Oct Mean values followed by di fferent letters within column are significantly different at 5% level. Growth period is 60 days. Table 2-Effi cacy of different growth regulators in induction of PLBs during third subculture [Values are mean of 20 rep li cates) Growth regulators (JlM) Cultures induced PLBs (%) Number of PLBs per shoot BA Kn NAA Sonia 17 Sonia 28 Sonia 17 Sonia 28 17.7 0 0 0 0 oe oe 22.2 0 0 0 0 oe oe 3 1.1 0 0 0 0 oe oe 39.9 0 0 35 30 2.4c 2.2c 44.4 0 0 55 50 6.4" 6. 1" 48.8. 0 0 50 40!.3d 1. 1 d 44.4 0 0.54 50 50 3.5b 3.3b 44.4 0 2.69 45 45 1.4d 1. 1 d 44.4 0 5.37 0 0 oe oc Mean values fo llowed by different letters within column are significantly different at 5% level. Growth 12eriod is 60 dal's.

MARTINet al.: PROPAGATION OF DENDROBIUM HYBRIDS 283 Fig. 1 (A-G)---/11 vitro propagation of Dendrobiwn hybrids through induction of shoots and PLBs. (A)--Shoots induced from base of the in vitro derived shoots on half-strength MS medium with 6.97 11M of Kn (during first subculture); (B)--Increased number of shoots on the above medium during second subculture; (C)---PLBs proliferation on half-strength MS containing 44.4 11M of BA (5 1 h subculture); (D)---PLB derived shoots developed on half-strength MS containing 6.97 11M of Kn (within 20 days); (E)--Well grown shoots on halfstrength MS containing 6.97 11M of Kn; (F)--Rooted shoots on 2 g r' of activated charcoal supplemented half strength MS medium; and (G)--Established PLB derived plantlets (60 days).

284 INDIAN J EXP BIOL, MARCH 2005 Table 3----Effect of different growth regulators in conversion of PLBs to shoots [Values are mean of200 PLBs] Growth regulators (~tm ) Conversion of PLBs (%) BA Kn NAA Sonia 17 Sonia 28 0 0 0 47.0( 45.0f 0.22 42.0g 40.5g 0.44 37.5" 35.5" 2.32 59.5d 57.0d 4.65 83.0" 8 l.ob 6.97 93.5" 92.0" 9.29 85.0" 83.5b 0.54 53.0" 54.0c 2.69 46.0f 44.0( 6.97 0.54 83.5b 8 1.5" 6.97 2.69 68.5c 67.5" Mean values fo llowed by different letters within column are significantly different at 5% level. Growth peri od is 60 days. Table 4-lnduction of roots on half strength MS medium with activated charcoal [Values are mean of 200 shoots] Activated Charcoal (g r') 0.0 1.0 1.5 2.0 2.5 3.0 Number of roots per shoot Sonia 17 Sonia 28 Mean va lues followed by different letters wi thin column are significantly differe nt at 5% level. Growth period is 60 days. proliferation even on half strength MS basal medium (data not shown). Conversion of PLBs to shoots relied on growth regulators supplemented to the medium (Table 3). Half strength MS basal medium facilitated conversion of 35% PLBs to shoots. The conversion of PLBs to shoots on half strength MS medium containing 2.69 )lm of NAA was slow, and the shoots were very small. NAA supplemented medium thus also exhibited proliferation of PLBs, but the roots developed on this medium were < 1.5 em long. Kn at 6.97 )lm was th e best for conversion of PLBs to shoots, on which 93.5 and 92% PLBs in Sonia 17 and 28, respectively progressed to shoots (Table 3; Fig. I D, E). The shoots developed also underwent proliferation. About 30% of shoots developed 2-5 roots per shoot. Auxin supplemented medium was inferior to Kn (Table 3). In vitro propagation of orchids using flower stalk node explants through axillary bud proliferation and also by PLB formation was reported in Phalaenopsis 5, Oncidium 12 and Cymbidium 19 The shoots were rooted well upon transfer to halfstrength MS medium supplemented with 2 g r 1 activated charcoal (Table 4; Fig. 1 F). On this medi um, more than 9 roots per shoot were developed within 60 days, and the roots were >3 em in length. The shoots grew well with 3-7 dark green leaves. Efficacy of rooting and growth of the plantlets on activated charcoal containing medium was also demonstrated earlier in Phalaenopsis 4. The plantlets after treatment with fungicide were planted in a mixture of sand, brick or tile, charcoal pieces and coir fi ber ( 1 :4:4:2), which exhibited 81 and 84% survival respectively in Sonia 17 and 28 after acclimation in Biotron LPH 200 (.lapan). The plantlets transferred after 30 days to nethouse grew well (Fig. lg) and were subsequently transplanted to pots. The present protocol enables to fulfil the worldwide orchid growers' demand for clonal saplings of these hybrids. Acknowledgement The authors gratefully acknowledge the financial support provided by the Department of Biotechnology (Govt. of India), New Delhi in the form of a research project (No. BT/PR 1689/SPD/16/120/99. Dt. 14 March 2000). References I Morel G, Producing virus-free Cymbidiums, Am Orchid Soc Bull, 29 ( 1960) 495. 2 Hu ang L-C, Lin J-C, Kuo C-I, Huang B-L & Murashige T, Paphiopedilum cloning in vitro, Sci Hart, 9 1 (2003) Ill. 3 Chen T-Y, Chen J-T & Chang W C, Plant regeneration through direct shoot bud formation from leaf cultures of Paphiopedilum orchids, Plant Cell Tissue Organ Cult, 76 (2004) I I. 4 Tanaka M, Micropropagati on of Plwlaenopsis spp., in Hightech and micropropagation IV. Biotechnology in agriculture andfores/ly, vol. 20, edited by Y P S Bajaj (Springer-Verlag, Berlin, Heidelberg, New York) 1992,246. 5 Chen Y C & Piluek C, Effects of thidiazuorn and N 6 - benzylaminopurine on shoot regeneration of Phalaenopsis, Plant Growth Regul, 16 ( 1995) 99. 6 Park S-Y, Murthy H N & Paek K- Y, Rapid propagation of Phalaenopsis froni floral stalk-deri ved leaves, In Vitro Cell Dev Bioi Plant, 38 (2002) 168. 7 Arditti J, Mosich S K & Ball E A, Dendrobiwn node cultures: A new means of clonal propagation, Aus Orchid Re v, 38 ( 1973) 175. 8 Soediono N, Use of coconut water NAA, 2,4-D and vitamins in shoot tip cultures of Dendrobiwn cv. Jaqueline Thomas White, Orchid Rev, 9 1 ( 1983) 86.

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