Indian Journal of Experimental Biology Vol. 39, July 2001 , pp. 697-704

Rapid in vitro multiplication and restoration of Celastrus paniculatus Willd. sub sp. paniculatus (Celastraceae), a medicinal woody climber

Lakshmi G Nair & S Seeni* Plant Biotechnology Division, Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram 695 562, India

Received 1 December 1999; revised 1 December 2000

Nodes, shoot tips, internodes and leaf bases (-1.0 cm) excised from young vines of the flowering woody climber, Celastrus palliculatus Wi lid. sub. sp. palliculatus (Celastraceae) were cultured in Murashige and Skoog (MS) medium containing agar (0.6%), sucrose (3%) and varied concentrations of 6-benzyl aminopurine (BAP) and kinetin. All the explant types were regenerative and maximum number (3.6) and frequency (94%) of axillary shoot formation of (5.08 cm long) was recorded in the nodes cultured in BAP (1 mg L-1

) after 6 weeks. Combinations of BAP (1 mg L· 1) and indole-3-acetic acid/l­

naphthalene acetic acid (0.Ql-1 mg L·1; IAAlNAA) tested with nodes induced formation of less number (3 and 2.2) of shoots

at same frequency (94%). All the explant types viz. node, shoot tip, internode and leaf base of ill vitro derived shoots responded earlier and better in lower concentrations of BAP (0.5-2 mg L-1

) with formation of 8,3.1, 6.4 and 1.8 shoots respectively during the same period. In spite of the advanced and increased caulogenic responses, differences in cytokinin requirements between different explants observed during culture initiation still persisted with the nodes, shoot tips, internodes and petiole segments responding best at 0.5, I and 2 mg L-1 BAP, respectively. The repeated reculture up to 10 cycles of the nodes from the shoot cultures each at 6-week intervals enabled multiplication and stocking of shoots without decline. Rooting of 3-7 cm shoot cuttings was induced in half-strength MS liquid medium containing IAA (1 mg L· 1

) with formation of 7.25 roots of 2.41 cm length within 6 weeks. Rooted plants were established at 84-96% rate in community pots without hardening, the least value (84%) being obtained with NAA- induced thick and calloid rooted plants. Four month old community potted plants were reintroduced into native forest habitats at 95% efficiency and 8 months after restoration, the plants were uniform in morphological, growth, cytological and peroxidase and esterase isozyme characteristics.

Celastrus paniculatus Willd. sub sp. paniculatus (Celastraceae) is a woody medicinal climber distributed at 1200m altitude in hilly tracts of India and other countries of south-east Asia. Seeds of this plant are the source of Ayurvedic drug jyothishmati used in treating rheumatism, gout and neurological disorders in India. A black empyreumatic oil, oleum nigrum, distilled from the seeds is a tranquiliser widely used to treat beri-beri and as rubifacient1

• The seed extract exhibits antibacterial, antifertility and antispermatogenic effects in male rats2

• Leaf sap is an emmenagogue and an antidote for opium poisoning3

.

Flower possesses analgesic and anti-inflammatory activities4

• Bark is reported to be abortifacient, depurative and a brain tonic and taken internally for snake biteS. Phytochemical investigations have revealed the presence of sesquiterpenoids of ~­dihydrogenic series in the seed oil6

.

Wild populations of C. paniculatus are severely depleted owing to injudicious exploitation in the places where they occur. Destructive harvesting of plants long before flowering has hampered natural regeneration through seeds. While, seeds exhibit poor

*Correspondent author

viability and germination, conventional propagation through vegetative cuttings is slow and cumbersome. The present investigation was undertaken to establish a reliable micropropagation method so that multiplication and stocking of plants for future supply will be possible.

Materials and Methods Top cuttings with 5-7 leaves emanating from the

woody trunk of a - 20 year-old flowering plant in a natural forest segment of the botanic garden of our institute were collected in November-December 1995. The cuttings were washed in 1 % (v/v) Labolene (Glaxo India Pvt. Ltd., Mumbai) detergent for 8-10 min and then in running tap water for 15 min. Surface sterilisation was done by immersion in 0.1 % (w/v) HgCh for 5-7 min and followed by 4-5 washes in sterile distilled water. Different explants viz. shoot tip (- 0.5 cm), single node explants (1-2 cm), internodes (0.5-1.0 cm) and youngest 1-3 folded leaves were dissected out, washed once in sterile distilled water and blotted over sterile filter paper discs before transfer to nutrient medium. The leaves were used intact or dissected into - 1.0 cm leaf segments. The explants were implanted either vertically or horizontally on the medium.

698 INDIAN J EXP BIOL, JULY 200 I

The nutrient medium contained salts and vitamins of MS medium7 supplemented with 3% (w/v) sucrose and varied concentrations (0.1-S.0 mg L-') of the cytokinins, 6- benzyl amino purine (BAP) and kinetin and combinations of BAP (1.0 mg L-') and indole-3-acetic acid (lAA) or I-naphthalene acetic acid (NAA), (0.01-1.0 mg L-'). The medium was adjusted to pH S_8 before adding 0.6% (w/v) agar (Hi Media Laboratories Pvt. Ltd. Mumbai), dissolved and dispensed in culture tubes and then autoclaved at 121 °C and 1.1 kg cm-2 pressure for 18 min. All the cultures were incubated in a culture room maintained at 2So ± 2°C, RH SO-60% and 12hr photoperiod. A photon flux density of SO-60 )l Em-2 sol was provided by cool white fluorescent tubes (Philips India Ltd., Mumbai).

After 6 weeks, shoot tips, nodes, internodes and young leaves (- 1.0 cm) were dissected out of - S cm shoots formed on the primary nodal explants and recultured in MS medium containing different concentrations of BAP (O.I-S.O mg L-'). To test the continued caulogenic response of the explants, upto 10 reculture cycles each at 6-week intervals were tried. For rooting, the shoots (- 4 cm) were harvested and transferred individually to MS half and full strength agar or liquid medium with filter paper bridges containing 3% (w/v) sucrose, and varied concentrations (0.01-2.0 mg L-') of IAA, indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) for rooting under 12hr photoperiod.

The rooted plantlets obtained after 6 weeks were weaned and transplanted in pots (to cm diam.) containing a mixture of sand, soil and farmyard manure (1: I : I). The potted plants were well irrigated under greenhouse conditions without hardening for 3-4 months before transferring to the field . For reintroducing the pot-established plants, undergrowth in an approximate S m2 area within the forest segment of the institute were thinned and pits of 30 x 30 x30 cm size were made in the floor at I m spacing from the support tree trunk, filled with top soil and leaf mold (I: 1). Since the canopy consisted of such trees as Termillalia pallicuiata, Caraya arborea and Pterocarpus marsupium, the planting site received only diffused light. The de potted plants were planted in May 1997 and watered twice a day until they were established in southwest monsoon rains of June 1997.

Statistical analysis was performed on the results of each experiment and data were compared using Analysis of variance (ANOY A) and Least Significant Difference (LSD) multiple range test.

Actively growing root tips were collected randomly at 11.30 A.M. from S-6 month old pot established micropropagated plants and the mother plant and were prefixed in 8-hydroxy quinoline at ISoC in a refrigerator for 2hr, washed in distilled water and then fixed in Carnoy's fluid (1:3 glacial acetic acid: 9S% ethanol). Root tip squashes prepared in acetocarmine (2%) were examined using Leitz Orthoplan Microscope.

Polyacrylamide gel electrophoresis of crude enzyme extracts of young leaves from randomly selected 4-month old mericlones was dones followed by activity staining of gel for isoperoxidase'l and isoesterase '0.

Results and Discussion Initial swelling and emergence of only a single

axillary shoot from the node and elongation of shoot tip to attain 1-2 cm length in 3 weeks occurred on MS basal medium. The shoots ceased to grow thereafter. Exogenous addition of cytokinins induced the formation of callus-free buds at varied frequencies. Maximum number (3.6) of axillary shoots was formed in 94% of the nodal explants within 6 weeks in presence of 1.0 mg L-' BAP (Fig. 2). Internode, shoot tip and petiole explants required more time (8 weeks) to produce maximum of 2.4, 2.0 and 1.1 shoots at 94, 94 and 83% respectively at relatively higher concentration (2.0 mg L-') of BAP (Fig. la and 2). Intact leaves became enlarged and curled in but stopped short of shoot initiation. Higher concentration of BAP (2.0 mg L-') and longer induction period required for maximum caulogenic response in explants other than node may be attributed to di fferential physiological status and endogenous concentration of growth regulators of different explant types as revealed by the studies in Quercus". It is also possible that the qualitatively different meristems present in different explant types make all the difference. The resident meristem present in the node was induced more easily and effectively than the adventitious meristems. which were to be organised from the epidermal and sub-epidermal cells in internodal and petiolar segments. Despite the presence of resident meristem, the shoot tips were a poor substitute for nodes owing to increased apical dominance and consequent elongation of the shoot tip with infrequent formation of an additional axillary shoot. The preference of the nodal explants over the apically dominant shoot tips is well documented in Fagus'2 and chestnut 13

.

NAIR & SEENI: RAPID IN VITRO MULTIPLICATION AND RESTORATION OF CELASTRUS PANICULATUS 699

"lI' ··· ... · .. ··-li Fig. I-Micropropagation of Celastrus paniculatus. (a). Caulogenic responses of flowering plant derived (top - bottom) shoot tip, node, internode and petiole explants after 10 (L) and 20 (R) days of culture in MS medium containing 1.0 mg L'I (node) and 2.0 mg L'I (internode, shoot tip and petiole) BAP; (b) Caulogenic responses of shoot culture derived (top - bottom) node, internode, shoot tip and petiole explants after 10 (L) and 20 (R) days of culture in MS medium containing 0.5 mg L' I (node) and 1.0 mg L' I (internode, shoot tip and petiole) BAP; (c). Multiple shoots emanated from recultured nodal explants after 6 weeks of culture in MS medium containing 0.5 mg L,IBAP; (d). Rhizogenic response of shoots 6 weeks after transfer to half-MS liquid medium supplemented with 1.0 mg L'I IAA, IBA and NAA; (e). Micropropagated plants established in community pots 8 weeks after deflasking; (t). Micropropagated plants 3 months after reintroduction into natural forest segment; (g). Root tip cell from a regenerated plant showing diploid chromosome number (2n = 46); and (h-i) Isozyme profile of peroxidase (h) and esterase (i) obtained after electrophoresis and activity staining of crude leaf extracts of randomly selected mericlones.

Table 1-Relative levels of shoot formation at optimal concentrations of BAP during culture initiation in different explant types of field-grown plants and shoot cultures. Observations were made after 6 weeks

Source of explant Explant MS+BAP Mean no, of shoot after Shoot length (mg L'I) culture(%) (cm)

Field grown plant Node 1.0 3.6(94) 5.08 Shoot tip 2.0 2.0(94) 1.53 Internode 2.0 2.4(94) 2.37 Petiole segment 2.0 1.1(83) 2,02

Shoot culture Node 0.5 8.0(100) 3,14 Shoot tip 1.0 3.1(95) 2,17 Internode 1.0 6.4(100) 2.32 Petiole segment 2.0 1.8(95) 2,01

700 INDIAN J EXP BIOL, JULY 2001

High frequency formation of shoots was obtained when the nodes and shoot tips were planted vertically and others in horizontal position. The influence of explant position on caulogenesis, also reported in certain other cultures, is often related to polarity as well as relative level of supply of nutrients and other factors to prospective meristematic centres of the explant that may unfold the morphogenetic potential 14

• In all the explant types and positions, higher concentrations (2.0-5.0 mg L") of BAP induced the formation of shoots with short internodes thereby confirming the well known inhibitory influence of cytokinins on shoot elongation and consequent rosette type crowding of shoots 15.16. In such cases, transfer and subculture of the shoots in basal medium to nullify this effect and to promote shoot elongation became inevitable I7

•18

. Most of the combinations of BAP 0 .0 mg L") and IAN NAA (0.01-1.0 mg L" ) tested in nodal explant cultures,

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induced the formation of less number (3.0 and 2.2) of shoots though the percentage response (94%) was not significantly altered. Higher concentrations (> 0.05 mg L 'I) of IAA and NAA induced callusing and inhibited shoot formation. These results presumably indicated a threshold level of endogenous auxin 19 in the explants.

Retention of the explants with proliferated shoots in the initiation medium for more than 8 weeks resulted in decline as evidenced from the formation of smaller new leaves and premature leaf fall. Shoot tip necrosis was also observed in 10-15% of the shoot cultures. These responses are often related to nutritional, mainly calcium deficiencies in the immediate vicinity of the growing shoots2o

. Although further reinforcement of the medium with adenine sulphate, aminoacids, calcium or iron is

d d21 22 . C . l I ' recommen e . In . pantcu atus as a so In

Hemidesmus indicus 17, subculture of the tissues at 6

week intervals helped to circumvent this problem.

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Fig. 2-Shoot formation in different explant types derived from flowering plants of Celastrus paniculatus cultured in MS agar medium containing 3% sucrose and varied concentrations of BAP. Observations were made after 8 weeks. (a) node, (b) interneode . (c) shoot tip and (d) petiole. Bar followed by the same letter(s) is not significantly different (P$O.05) . • Shoot number 0 Length (cm) -D.- % Response]

NAIR & SEENI: RAPID IN VITRO MULTIPLICATION AND RESTORATION OF CELASTRUS PANICULATUS 701

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Fig. 3-Shoot formation in recultured explants of Celastrus paniculatus cultures in MS agar medium containing different concentrations of BAP. Observations were made after 6 weeks of reculture. (a) node, (b) internode, (c) shoot tip and (d) petiole. Bar followed by the same letter(s) is not significantly different (P:SO.05) .• Shoot number 0 Length (cm) -il- % Response]

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702 INDIAN J EXP BIOL, JULY 2001

All the explants of in vitro shoots responded rapidly with initiation of buds within 2 weeks (Fig. 1 b) and formation of maximum number of shoots at higher frequencies (95-lO0%) than those of the flowering plants within 6 weeks (Fig. 3). The concentration of the cytokinin (BAP) required for optimal shoot initiation was reduced to half (0.5-1.0 mg L-1

) except in petiolar segments (2.0 mg L· 1).

Maximum number (8.0) of shoots owing to increased axillary bud proliferation was formed in nodal segments (Fig. Ic). The earlier and greater capacity of morphogenesis in such recultured explants is not entirely unexpected due to the absence of a lag period between explanting and adaptation of explants to in vitro conditions and significantly higher activity of meristematic centres of micropropagated plants23

•

Inspite of the advanced and increased caulogenic responses, differences in cytokinin requirement between different explants observed during culture initiation still persisted with the nodes, shoot tips, internodes and petiolar segments responding best at 0.5, 1.0 and 2.0 mg L-1 BAP, respectively (Table 1). It appeared that even after adaptation and considerable period of culture in the best nutritive and hormonal milieu, some physiological compartmentalisation occurred within the shoots causing the observed differences in explant response. It was also of interest to note that many shoot buds of less than 2 mm size were formed along with other shoots along the entire length of the internodes but did not develop into shoots during reculture. After the harvest of well­grown shoots (3-5 cm), these buds were induced to develop into normal shoots during subculture of the explants. Published reports I 8,24 suggest that the auxin synthesised in the larger shoots is translocated basipetally thereby inhibiting further growth of the buds into shoots.

The repeated subculture of the nodes of shoot cultures upto 10 cycles each at 6 week intervals enabled continuous production of shoots without loss of vigor, callusing and morphological and growth abnormalities (data not shown). Thus as practised in Eucalyptui5 and Xanthosoma caracu26

, the clonal multiplication of this woody climber could also be accelerated by axillary meristem proliferation using successive nodal explant cultures over 15 months. These results together with the demonstrated regenerative ability and use of all the explant types for multiplication were sugg~tive of the amenability of this woody climber for in vitro handling on par with the established herbaceous model systems like tobacco, Brassica and tomato.

Roots were not induced in chorus with shoot formation during culture initiation and multiplication in the cytokinin regime. When individual shoots were implanted in half and full strength MS liquid media free of growth regulators, root formation occurred only in the former within 5 weeks but the number (2-3) and frequency (46%) were poor. Addition of auxins enhanced the rate of rhizogenesis. Of the three auxins (lAA, IBA and NAA) tested, (1.0 mg L-1 IAA) was preferred as it stimulated the development of 7.25 (92%) roots of maximum length (2.41 cm) after 4 weeks compared to less vigorously growing short, calloid roots with considerable callusing at cut ends of shoots in media fortified with IBA (85%) and NAA (80%; Fig. 4,Id). Among the cultures incubated under light and dark regimes, rapid rhizogenesis (2 weeks) occurred in the latter. However, prolonged incubation in the dark inhibited the growth of both shoots and roots. An initial dark period of one week and transfer to I2hr photoperiod accelerated both root initiation and elongation. The stimulatory effect of reduced salt concentrations (Y2 MS), dark condition and IAA on in vitro rooting observed in the study are well documented27

,28.

It was striking to note that rooting of shoots transferred to agar medium was delayed to 4 weeks with profuse callusing at cut ends of the shoots. Feathery roots were formed in the medium supplemented with IBA and NAA, which remained on the surface with only a few penetrating the gel matrix. In liquid medium, the roots were easily and profusely formed. Roots formed in presence of IAA and IBA were long and dark in colour and those induced by NAA were thick and calloid in nature29

•30

.

The enhanced rooting in the liquid medium also reported in such woody species as sweet gum31 and jujube32 is attributed to differences of imbibing substances viz. CO2 and increased partial pressure of 0 2

33• This attribute is of particular significance as the

expensive agar may be dispensed with to reduce the cost of production and the rooted plants were readily transferred from liquid medium to soil.

The establishment of the rooted plants even without hardening was achieved with ease under the greenhouse conditions and most of the plants showed signs of growth as evidenced from the emergence of new leaf within 3-4 weeks after transplantation in pots (Fig. Ie). Despite the induction of roots in an aqueous environment, the plants were healthy, free of vitrification and the roots were hardy to facilitate high percentage (96%) establishment. The auxin type-induced changes in rooting were not without significance as the

t.,

.. , NAIR & SEENI: RAPID IN VITRO MULTIPLICATION AND RESTORATION OF CELASTRUS PANICULATUS 703

thick and calloid rooted plants obtained in presence of NAA showed relatively poor establishment (84%) and leaf fall even after establishment. Similar findings were reported in Hemidesmus indicus17

• Four months after establishment, the vigorously growing community potted plants were free from morphological and growth defects and was suitable for reintroduction into natural forest segments. More than 95% establishment rate was recorded after 12 months in the field trials conducted in May 1997 (Fig. If). Microscopic examination of the root tip squashes from these plants revealed the presence of 2n = 46 chromosomes in all the randomly collected samples thereby indicating the cytological stability of the plants micropropagated from the nodal explants (Fig. 19). Activity staining of gels for isoperoxidases and isoesterases revealed identical banding pattern with 2 and 6 anodic isozymes respectively (Fig. 1h, i) possibly suggesting the genetic uniformity of the micropropagated plants.

Overall, the data presented reinvigorate the feasibility of using axillary meristem for clonal multiplication of C. paniculatus, an overexploited medicinal climber at a desirable pace. Judicious use of nodal explant may facilitate conservation, cultivation and sustainable utilization through bulk supply of raw material for local drug industry.

Acknowledgement This work was supported in part by the award of a

Senior Research Fellowship to Ms Lakshmi G Nair by the University Grants Commission, New Delhi.

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