Methods for large scale multiplication of Piper longum are reported. Shoot tips cultured on Murashige &amp; Skoog's medium supplemented with 0.5 mgl-1 benzyl adenine or kinetin in combination with 0.5 mgl-1 I-naphthalene acetic acid developed multiple shoots at very low frequency. Multiple shoots originated in large numbers, only from nodes of vertically planted explants, while shoots dnveloped&nbsp;de novo&nbsp;from all over the surface, when planted horizontally in the same medium containing 2·5 mgl-1 benzyl adenine. Leaf segments also developed adventitious buds in large numbers in Murashige &amp; Skoog's medium containing 3 mgl-1 benzyl adenine, I mgl-1 kinetin and 10% (v/v) coconut water. Callus induced from leaf and stem explants in Murashige &amp; Skoog's medium with 1-2 mgl-1 2, 4 dichlorophenoxy acetic acid in combination with Imgl-1 benzyl adenine developed shoot buds when transferred to medium containing 3 mgl-1 benzyl adenine. All the shoots were rooted in Murashige &amp; Skoog's basal medium. The isolated plantlets, hardened in vermiculite were transferred to pots containing garden soil and sand.
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LAWRENCE, G M NAIR, V SARASAN, ELIZABETH THOMAS, BEENA

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Journal of Spices & Aromatic Crops 2 (1&2): 34·40, 1993 34 Plant regeneration in Piper longum L. (Piperaceae) through direct and indirect adventitious shoot development v SARASAN, ELIZABETH THOMAS, BEENA LAWRENCE & G MNAIR! Plant Tissue Culture Laboratory, Department of Botany University of Kerala, Kariavattom Campus Trivandrum . 695 581, India. ABSTRACT Methods for large scale multiplication of Piper longum are reported. Shoot tips cultured on Murashige & Skoog's medium supplemented with 0.5 mgl·! benzyl adenine or kinetin in com· bination with 0.5 mgl·! I·naphthalene acetic acid developed multiple shoots at very low frequency. Multiple shoots origi· nated in large numbers, only from nodes of vertically planted explants, while shoots dnveloped de novo from all over the sur· face, when planted horizontally in the same medium containing 2·5 mgl·! benzyl adenine. Leaf segments also developed adven· titious buds in large numbers in Murashige & Skoog's medium containing 3 mgl·! benzyl adenine, Imgl·! kinetin and 10% (v/v) coconut water. Callus induced from leaf and stem explants in Murashige & Skoog's medium with 1·2 mgl·!2, 4· dichlorophenoxy acetic acid in combination with Imgl·! benzyl adenine developed shoot buds when transferred to medium containing 3 mgl·! benzyl adenine. All the shoots were rooted in Murashige & Skoog's basal medium. The isolated plantlets, hardened in ver· miculite were transferred to pots containing garden soil and sand. Key words: direct organogenesis, long pepper,multiple shoots, Piper longum, regeneration. Abbreviations BA CW 2,4·D 2·ip Kin MS NAA benzyl adenine coconut water (liquid endosperm) 2,4·dich lorophenoxy acetic acid 2·isopentenyl adenine kinetin Murashige & Skoog (1962) l·naphthalene acetic acid ICorrespondent author Plant regeneration in long pepper Introduction Tissue culture techniques in recent years have made easy propagation of many economically and medicinally important plants. Such techniques are a lso suc-cessfully used for experimenting and standardizing methodologies for im-provement of crops. Ma ny of the tropi-cal spices earning a large revenue have not received adequate attention for im-provementoftheir yield and performance utilizing tissue culture techniques, ex-cept for a few main spices (Hosoki & Sagawa 1977; Kuruvinashetti, Haridasan & Iyer 1982; Rao et al. 1982; Nadgauda, Mascarenhas & Madhu-soodanan 1983; Mathews & Rao 1984; Kumar et al. 1985; Babu, Samsudeen & Ravindran 1992; Sarasan 1992). Piper longlll/! L. (Long pepper, Piperaceae) , an uni sexual perennial climber commonly found in Asian coun-tries, is a valuable source of active sub-stances of medicinal value besides being used as a spice (Anonymous 1969). The fru its and roots contain major alkaloids such as piperin e and piplartine in addi-tion to sterol s and glycosides (Anony-mous 1969). The conventional method of propagation of this plant is through layeri ng of mature branches which is time consuming. In the present paper we describe rapid multiplication of this species through direct and indirect or-ganogenesis. Materials and methods Shoot tip s, nodal, internodal and leaf segments from greenhouse grown ma-ture-ph ase P. longlll/! plants were used as explants. The explants were washed throughly in running water for 2 hand soaked in Teepol (a commercial neutral 35 detergent) solution before surface steril-ization with 0.1% (w/v) mercuric chlo-ride solution. Shoot tips and young leaves were sterili zed for 7 min and stem seg-ments for 10 min. The explants, after sterili zation were washed several times in sterile water. The culture medium consisted ofMS nutri ents (Murashige & Skoog 1962) containing 2% sucrose and soldified with 0.8% agar. The pH of the medium was adjusted to 5.8 before au-toclaving at 121°C for 20 min at 15 Ibs per sq inch pressure. The cultures were incubated at 25±2°C under 16 h photo-period at light intensity of3000 lux from cool white nuorescent tubes. Shoot tips (0.5-1.0 em) were inoculated on MS basal medium supplemented with 0.1-1 mgl-l NAA and 0.1-1 mgl-l Kin or BA. Nodal segments and internodes of 1-1.5 em were cultured on MS medium supplemented with 2-5 mgl-l BA alone. The nodal segments were planted either vertically or horizontally in the medium to find out differences in response, if any. One em long stem segments and 0.5 cm2 leaf di scs were cultured on MS medium supplemented with 1-4 mgl-l2, 4-D and 1 mgl -l BA, 2-3 mgl- l BA+l mgl- l Kin/2-ip with 10% CW for callus regeneration and adventitious bud in-duction. After 30 days of primary cul -ture, the explant/callus was subcultured in medium containing 0-2 mgl-l 2, 4-D andl-2 mgl-l BA. Morphogenic callus, selected from these cultures, was sub-cultured in a medium containing 2-3 mgl-l BA for shoot induction whil e leaf di scs with developed adventitious buds were transferred to MS medium con-taining 3mgl-l BA, Imgl -l Kin and 10% CWo The regenerated shoots from dif-ferent experiments were subcultured in MS basal medium (full and half strength) Sarasan et a 1. for rooting. The plantlets rooted and hardened for 15 days each in basal liq-uid medium and vermiculite were sub-sequently transplanted to garden soil and sand mi xture (1:3). The plants were initi a lly covered with pl astic bags (minimum 5 days) to retain humidity and nouri shed with 1/10 strength modi-fi ed Hoagland's solution (Epstein 1972). Results and discussion Shoot tips cultured on MS medium supplemented with 0.4-1 mgl'! NAA in combination with 0.1-0.4 mgl'! BA or Kin developed multiple shoots after 20 days. Further proliferation of shoots and de-velopment of roots occurred simulta-neously upon transferring to MS con-taining 0.5 mgl-1 BA or Kin in combina-tion with 0.5 mgl'! NAA. The number of multiple shoots developed was less (2 or 3) in each subculture. This may be be-cause of the strong apical dominance present in this plant due to high level s of endogenous auxins. Apical dominance as reasons for redu ction in shoot pro-ducing cultures is reported in few sys-tems (Newbury 1986; Selva et al. 1989). Multiple shoots developed within 25 days from nodal segments explanted on MS medium fortifi ed with 2-5 mgl·! BA and elongated furth er in about 40-50 days(Fig. 1). Shoots originated from the nodal region only when the ex plant was oriented vertically in the medium. However, buds originated adventitiously from all over the surface of the ex plant when planted horizontally in the medium after an initi al phase of slight callussing (Fig. 2). The initiation of buds from the horizontally placed nodal segment oc-curred after 45 days and there was a quicker establishment into shoots after this. Among the concentrations tried, 3 36 mgl'! BA was the most effective in pro-ducing maximum shoots (Table 1). The isolated shoots readily developed roots when tranferred to MS basal medium. The multipl e shoots also developed roots when left in the same medium for a longer period (45-90 days after shoot initiation ). The number of regenerated plantlets was maximum in horizonta lly planted explants. Generally, multipl e shoots a re estab-lished only around the nodal portion. However, P.longltm appears to have a natural tendency of developing buds de novo from the explant irrespective of a node, if placed horizontally in the me-dium. This may be due to a reversal of polarity because of altered position and also to a certain extent tri ggered by phytohormones in the medium. Leaf di scs cultured in MS medium supplemented with 3 mgl'! BA, 1mgl-! Kin and 10% CW developed small buds on the petiolar region initially and sub-sequently along the main vein within 40 days (Fig. 3). These cultures when di-vided and subcultured into the same medium developed a la rge number of shoots (Figs. 4 & 5). Direct organogenesis is reported from explants of juvenile phase (Wong & Loh 1987; Nair et al. 1984) and from mature phase in a few plants (Choo 1988; Mark & Lineberger 1988). Multiple shoots were also induced directly from leaf segmen ts in P. longltm using a combination of BA and CW in MS medium (Sarasan & Nair 1991). The regeneration of plantlets from mature phaSe explants is reliable only when it is required to select plants of superior quality for regeneration (Rao & Lee 1986). Such explants can give rise to regenerants of the same phenotype if the parent plant was not subjected to Plant regeneration in long pepper 37 .Figs. 1~9. In vitro response of V81ious explants of Piper IongltJn 1. Nodal segments planted vertically in MS medium containing 3 mgl-! BA showing multiple shoots developed from the node. 2. Stem segments planted horizontally on MS medium containing 3rngl-1 BA showing profuse shoot development from the entire surface of the explant. 3. Leafsegment cultured in MS medium with 3 mgl-! BA, 1mgl- j Kin and 10% CW showing initial development of adventitious buds from the petiolar and midrib regions. 4. Later stages of the same shovving large number of developed shoots. 5. Isolated de novo developed shoots at various stages of development. 6_ Callus developed in 2mgl- j BA and 1 mgl- j 2, 4-D cultured in 3mgl-! BA containing medium showing development of shoots_ 7_ Isolated shoot rooted in MS basal medium. 8. Plantlets in vermiculite. 9. Plantlets in pots after 3 months. Table 1. In vitro response of stem and leaf explants of Piper longum Medium Explant Hormone (mgJ-I ) 2,4-D BA MS S 0 2 S 0 3 S 0 4 S 0 5 Sand L 1 1 Sand L 2 1 Sand L 3 1 Sand L 4 1 * From single cultured explant ** After 6 months in the field S - stem; L - leaf Response Direct regeneration Direct regeneration Direct regeneration . Direct regeneration Nodular callus No. of plantlets regenerated* Mean ± SD 32.9± 3.8 47.3 ± 5.5 37.6 ± 4.7 27.6 ± 3.7 20.7 ± 3.7 Profuse nodular callus 22 .9 ± 3.6 Friable callus 9.4 ± 2.9 Friable callus 9.4± 2.9 Rate of survival (%)** 85.6 88.1 98.9 99 .1 74.2 73.9 69.0 69.0 u, e .; '" ;> ;:: '" ~~ w 00 Plant regeneration in long pepper altered environmenta l condition or stress. Organogenesis from ln at';'re phase explants in this species is impor-tant because of its dioecious nature. Stem segments and leaf discs produced white callus from the cut ends after 20 days in MS medium containing 1-2 mgl·1 2, 4-D and 1mgl-l BA Callus induced from stem segments and leaf discs in MS basal medium containing 3-4 mgl-1 2, 4-D with 1 mgl-1 BA was friable. Nodular callus subcultured in 2 mgl-1 BA with 0-1 mgl·1 2, 4-D turned green, and subsequently produced shoot buds when transferred to MS medium containing 3-4 mg 1-1 BA (Fig. 6). It is interesting to note that 2, 4-D at lower concentra-tions induced compact nodular callus which on subculturing produced shoot buds, while callus derived at higher con-centrations of 2, 4-D was always friable and did not show morphoge'nesis even at altered combinations of hormones. This confirms the earlier observations t hat 2, 4-D at higher concentrati on blocks mor-phogenesis (Halperin & Jenson 1967; Negrutiu 1978). Shoot buds produced from the callus when isolated and sub-cultured in MS basal medium (both full and half strength) rooted within 7 days (Fig. 7). Transplantation of in vitro grown plantlets to field conditions is an impor-tant step in any t issue cu lture work. There are severa l techniques for aclimatization of in vitro grown plant-lets from the tube to the pots and subse-quently to the field (George & Sherrington 1984; Selvapandiyan et al. 1988). Plantlets of P. longum trans-planted directly to the soil became stunted, probably due to lack of suffi-cient root development. However, the percentage of survival was much higher when transplanted initially to vermicu-lite 'and subsequently to soil (Figs. 8, 9 39 & Table 1). Nourishing the plants with 1110 strength Hoagland's modified solu-tion while in vermiculi te enhanced their initial establishment. References Anonymous 1969 Wealth ofindia. Vol. 8. CSIR, New Delhi. Babu K N, Samsudeen K & Ravindran P N 1992 Direct regeneration of plantlets from immature inflores-cence of ginger (Zingiber officinale Rosc.) by tissue culture. J. Spices & Aromatic Crops 1 : 43-48. Choo l' M 1988 Plant regeneration in zig zag clover (Trifolium medium L.). Plant Cell Reports 7 : 246-248. Epstein E 1972 Mineral Nutrition of Plants. John Wiley, New Delhi. George E F & Sherrington P D 1984 Plant Propagation by Tissue Cul-ture. Exegetics, England. Halperin W & Jenson W A 1967 Ultra-structural changes during growth and embryogenesis in carrot cell cultures. J. Ultrastruct. Res. 18 : 428-443. Hosoki l' & Sagawa Y 1977 Clonal propagation of ginger (Zingiber officinale Rosc.) through tissue culture. Hort. Sci.l2 : 451-452. Kumar K B, Praka sh Kumar P, Balachandran S M & Iyer R D 1985 Development of clonal plantlets from immature panicles of cardamom. J. Plant. Crops 13 : 31-34. Kuruvinashetti M S, Haridasan P & Iyer R D 1982 'fissue culture studies in turmeric. Proc. Nat. Plant regeneration in long peppel' Sem., April 8-9 1980, Calicut (pp.39-41). Central Plantation Crops Research Institute, Kasargod. Mark H B, Linberger D R 1988 In vitro adventitious shoot formation on mature phase leaves and petioles of Liquidamber styracifluu L. Plant Science 57 : 173-179. Mathews V H & Rao P S 1984 In vitro response of black pepper (Piper nigrulll). Curl'. Sci. 183-186. Murashige T & Skoog F 1962 A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15 : 473-497. Nadgauda R S, Mascarenhas A F & Madhusoodanan K J 1983 Clonal multiplication of cardamom (Eletteria curdulllomum Maton) by tissue culture. J. Plant. Crops 11 : 60-64. Nair S, Gupta P K, Shirgurkar M V & Mascal'enhas A F 1984 In vitro organogenesis from leaf explants of Annona squamosa Linn. Plant Cell Reports 4 : 29-40. Negrutiu I 1978 Plant Tissue Culture. Proceedings of the Beijing (Peking) Symposium, Pitman, London. Newbury H J 1986 Multiplication of Antirrhinullllllajus L. by shoot tip 40 culture. Plant Cell, Tissue & Or-gan Culture 7 : 39-42. Rao S N & Lee S K 1986 Plant tissue culture and its agricultural appli-cations . In: Withers L A & Alderson P G (Eds.) (p.123) . Butterworths, London. Rao S N K, Swamy S N, Chacko E K & Swamy R D 1982 Regeneration of plantlets from callus of Elettal'ia cUl'dulllolllulll Maton. Proc. In-dian Acad. Sci. (Plant Sci.) 91 : 37-41. Sarasan V & Nair G M .1991 Tissue culture studies of medicinal plants: Morphogenesis, direct regenera-tion and somatic embryogenesis. Curl'. Plant Sci. Biotechnol. Agric. 12 : 237-240. Selva E, Scouffs B & Briquet M 1989 In vitro propagation of Vicia {aba L. by micro-cutting and multiple shoot induction. Plant Cell, Tis-sue & Organ Culture 18 : 167-179. Selvapandiyan A, Subramani J, Bhatt P N & Mehta A R 1988 A simple method for direct transplantation of cultured plants to the field. Plant Science 56 : 81-83. Wong K W & Loh C S 1987 In vitro regeneration of plantlets in Brassica alboglabra. Plant Cell, Tissue & Organ Culture 10 : 143-148. 

Plant regeneration in Piper longum L. (Piperaceae) through direct and indirect adventitious shoot development

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Plant regeneration in Piper longum L. (Piperaceae) through direct and indirect adventitious shoot development

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