Organogenesis from leaf and internode explants of Ophiorrhiza prostrata, an anticancer drug (camptothecin) producing plant
Keywords: camptothecin, medicinal plant, Ophiorrhiza prostrata, plant growth regulators.
Camptothecin (CPT) analogues and derivatives serve as a novel class of effective anticancer agents that exert their action against DNA topoisomerase I. This paper presents procedures for the rapid, high frequency regeneration of a camptothecin producing plant, Ophiorrhiza prostrata D. Don from leaf and internode explants via shoot organogenesis. The concentrations of plant growth regulators and explant types exhibited discrete roles in the efficacy of shoot induction. N6-benzyladenine (BA) was the most effective cytokinin for the induction of shoots. Murashige and Skoog (MS) medium with 8.87 µM BA and 2.46 µM indole-3-butyric acid (IBA) yielded the highest number of shoots from leaf and internode explants (76.0 and 90.8 shoots respectively). In the case of leaf explants, explants from the proximal end produced a higher number of shoots than those from the mid and distal end. Leaf and internode explants cultured on MS medium supplemented with α-naphthaleneacetic acid (NAA) and BA developed shoots, calli and roots. Calli subcultured onto medium supplemented with 8.87 µM BA and 2.46 µM IBA developed a mean of 20.1 shoots within 40 days. Excision and culture of internode and proximal leaf explants from the established cultures on MS basal medium significantly enhanced the number of shoots and yielded a mean of 18.3 and 13.7 shoots respectively within 40 days. Histological examination of leaf explants showed that the shoots were of sub-epidermal origin, confined to the sub-epidermal cells above the vascular traces. Shoots cultured on half-strength MS basal medium with 10.74 µM NAA and 2.32 µM Kn produced a mean of 48.2 roots per shoot. Direct transfer of rootless healthy shoots showed a 50% survival rate, whilst it was 100 percent in the case of in vitro rooted shoots.
isolated and characterized for the first time by Wall
et al. (1966), is a monoterpene indole alkaloid originally derived
from Camptotheca acuminata (Nyssaceae), a native of
The genus Ophiorrhiza belongs to the family Rubiaceae, which comprises 150 species. The roots of Ophiorrhiza species, O. mungos and O. pumila have been reported as the sources of CPT and 10-methoxycamptothecin (Tafur et al. 1976; Saito et al. 2001; Sudo et al. 2002; Watase et al. 2004). The Ophiorrhiza spp. is also used to provide remedies for ulcers, helminthiasis, snake poison, poisonous wounds, gastropathy, leprosy, and hydrophobia (Kirtikar and Basu, 1975). O. prostrata D. Don is an herbaceous perennial medicinal plant, exploited for the production of camptothecin, which is accumulated mainly in the roots. A comparative study of camptothecin content in Nothapodytes foetida, O. mungos and O. rugosa indicated highest yields of camptothecin and 9-methoxy camptothecin in N. foetida (Roja, 2006).
The rate of plant propagation is critical to meet the pharmaceutical demand for camptothecin. A slow propagation rate in O. prostrata, because of low seed viability and germination rate as well as a small number of propagules (stem cuttings), has restricted the natural dissemination of the plant. In addition, the destruction caused by harvesting the roots as a source of the drug has threatened the survival of the plant. Thus, the large-scale demand necessitates rapid multiplication of the plant within a short timeframe without a negative impact on the natural resources. In vitro morphogenesis without a callus phase is regarded as the most faithful strategy to obtain plants with high speed as well as genetic fidelity. Direct shoot induction as an easy way for the rapid plant propagation has been reported in several medicinal plants viz. Murraya koeningii (Rout, 2005), and Euphorbia nivulia (Martin et al. 2005). Micropropagation of O. mungo by using seedling shoot culture has been reported (Jose and Satheeshkumar, 2004). Callus mediated plant regeneration is an easy way to obtain somaclonal variants as has been emphasized in several plants (Dennis and Boban, 2005; Dhar and Joshi, 2005; Faisal and Anis, 2005; Agrawal and Sardar, 2006). So far, no protocol for in vitro propagation of O. prostrata has been published. The propagation of C. acuminata conventionally as well as through in vitro culture is not as easy as that of herbs such as O. prostrata. This study establishes rapid propagation protocols using leaf and internode explants in order to meet the demand, which could curtail the impact on the natural population and prevent the plant from becoming endangered.
young shoots of O. prostrata were collected from potted plants
and used as the starting material. Young leaves and internode segments
were washed separately under running tap water followed by 5% (v/v)
solution of Extran (a neutral liquid detergent, Merck India Ltd.,
Mumbai) for 5 min followed by surface sterilization using 0.1% (w/v)
mercuric chloride. Leaf segments were sterilized for 7-9 min, while
internode segments were treated for 10-12 min. Decanting of the mercuric
chloride was followed by repeated washes (3 times 5 min each) in sterile
water. The sterilized tissues were cut into appropriate sizes (leaf
explants of 10 mm2 and internode of 7-
of shoot induction was studied by taking random sections at different
growth stages. Sections were stained with a dilute solution of safranin
and observed under a Leitz Dialux 20 (
(derived through in vitro rooting) as well as well-grown shoots
without roots (ex vitro rooting) from the shoot multiplication
medium were transferred to small pots containing soil and sand (1:1),
covered with moistened polyethylene bags and kept at room temperature
experiments were performed in a completely randomized design. Twenty
replicates were used for each treatment, and all the treatments were
repeated twice for confirmation. The mean values of different treatments
were analyzed using
and internode explants cultured on MS basal medium produced directly
a mean of 2.9 and 4.0 shoots respectively. Addition of PGRs induced
shoot formation and the number of shoots depended on the types and
concentrations of PGRs, BA in particular (Table
1) as well as the explant types. MS medium containing BA alone
was superior to that containing Kn alone for the induction of shoots
(Table 1). On medium with 8.87 µM BA, internode
explants developed a mean of 51.5 shoots, while the proximal leaf
explants produced a mean of 38.0 shoots within 40 days. Upon medium
with 8.87 µM BA, leaf explants initiated shoots within 14 days, while
the internode explants induced shoots within 12 days. Lower concentrations
of BA induced callus with a reduction in number of shoots. Higher
levels of BA increased the amount of callus. The calli later developed
shoot buds. Increasing BA concentrations above 8.87 µM exhibited a
negative effect on the height of shoots i.e., the shoot height
decreased as BA concentrations increased. Kn containing media favoured
better shoot elongation than that on BA alone supplemented media,
and the shoots on medium having Kn attained >
Synergy between BA and auxins exhibited a positive effect in the induction of shoots. MS medium with 8.87 µM BA and 2.46 µM IBA developed the highest number of shoots; 76.0 and 90.8 shoots from leaf and internode explants respectively (Table 1; Figure 1a and Figure 1b). As on medium with other PGRs, the shoots on leaf explants were developed adaxially, however, with a few shoots on the abaxial side. Of the different regions in the leaf, explants from proximal region were superior in the induction of shoots. Increasing IBA concentration favoured callus induction especially from the cut ends of explants, which later regenerated into shoots. The shoots were longer on media containing BA and auxins than those in media containing BA alone. The regenerated shoots developed roots following further culture. The combination of BA and NAA was inferior for shoot formation (Table 1) but facilitated the formation of a higher amount of callus.
Direct organogenesis is regarded as the most reliable method for clonal propagation because it upholds genetic uniformity among the progenies. As in the present study, direct formation of shoots without an exogenous trigger has been reported in different explants/species, e.g. from internodes of Bacopa monniera (Tiwari et al. 1998; Shrivastava and Rajani, 1999), and leaf explants of Drosera binata (Kawiak et al. 2003). Watase et al. (2004) has reported spontaneous formation of shoots from hairy roots of Ophiorrhiza pumila. The development of shoots on basal media may be due to stimulation by endogenous hormones or some signals related to wounding, which play a vital role during the induction of regeneration, or the ratio of ions present in the medium. The difference in the number of shoots formed in leaf and internode explants may be a result of differences in the regeneration potential of different explants, which is attributed by the physiological state, age and cellular differentiation among the constituent cells (Murashige, 1974). Enhancement in the induction of shoots by the synergy of BA and auxins observed in the present study has been documented in Stevia rebaudiana (Sivaram and Mukundan, 2003), Aloe vera (Liao et al. 2004), Murraya koeningii (Rout, 2005) and Euphorbia nivulia (Martin et al. 2005). Nevertheless, adventitious shoot regeneration has been accomplished with a mean 10.4 of shoots per shoot explants of Ophiorrhiza mungo cultured on MS medium with 2.22 µM BA (Jose and Satheeshkumar, 2004). Though Kn was inferior for shoot formation in O. prostrata, efficacy of Kn or other cytokinins either alone or in combination with BA/auxins in direct shoot formation has been demonstrated in Asparagus maritimus (Stajner et al. 2002), Robinia pseudoacacia (Shu et al. 2003), and Bixa orellana (De Paiva et al. 2003).
High shoot regeneration potential of proximal end explants as to leaf tip explants as in the present study has been emphasized in Beta vulgaris (Zhang et al. 2001), Anthurium andraeanum cultivars Tinora Red and Senator (Martin et al. 2003), and Euphorbia nivulia (Martin et al. 2005). In the view of Welander (1988), the high potential of the proximal end to the distal may be due to the difference in the maturity between proximal and distal end of the leaf, and which is supported by the fact that leaves reach maturity first at distal (tip) and subsequently in a basipetal progression. High frequency shoot induction at the proximal region may also be due to the higher level of IAA and abscisic acid (Rajasekharan et al. 1987).
Leaf and internode explants cultured on MS media with different levels of NAA either alone or in combination with BA or Kn produced calli, roots, and shoots (< 4) (data not shown). MS medium with 5.37 µM NAA alone induced a mean fresh weight of 219 and 188 mg calli per explants respectively from internode and leaf explants. Combinations of NAA with BA or Kn favoured increased numbers of shoots along with callus formation. MS medium with 5.37 µM NAA and 2.22 µM BA induced the highest amount of callus (310 and 295 mg from internode and leaf respectively) with < 6 shoots. The calli developed were pale green to dark green and semi-hard.
initiated on MS medium with NAA alone or in combination with BA or
Kn upon subculture developed adventitious shoots. MS medium with
The internode and leaf explants derived from established in vitro cultures on MS basal medium produced high numbers of shoots with means of 18.3 and 13.7 respectively. A high yield of shoots was achieved by culturing stem and leaf (proximal) segments as well as shoot clumps excised from the primary cultures on medium optimal for direct shoot multiplication (MS medium with 8.87 µM BA and 2.46 µM IBA). The number of shoots developed was difficult to count and was further increased in subsequent cultures (Figure 1c). The shoots cultured on MS medium without PGRs developed long shoots with roots. The roots turned to reddish brown from white through golden yellow. The colour of the roots may be due to the accumulation of secondary metabolites (CPT and its derivatives).
The shoots were initiated from sub-epidermal cells especially from the region above the vascular bundles of the explants (Figure 1d and Figure 1e). Both palisade and spongy cells developed shoots. The shoots were shown to contain an extension of the vascular bundles from the source tissue. In the case of leaf explants, the shoots originated from subepidermal cells above the vascular traces. Shoots originating from subepidermal cells have been reported in tobacco stem explants (Creemers-Molenaar et al. 1994). The ability of different tissues to form shoots directly in other species has been demonstrated including leaf epidermis (Lo et al. 1997), vascular cambium (Wenzel and Brown, 1991), cortex (Arai et al. 1997) and cortical, subcortical (Twyford and Mantell, 1996) or epidermis or cortex of root (Knoll et al. 1997).
vitro as well as ex vitro root induction was preceded by
transfer of healthy shoots with more than
Transfer of rootless shoots directly into pots containing sand and soil (ex vitro rooting) resulted in a survival rate of 50% shoots. The shoots resumed growth after 15 days of transplantation. Ex vitro rooting may reduce the micropropagation cost and also the time from laboratory to field. Rooting extra vitrum has been reported in Rotula aquatica (Martin, 2003a), Eupatorium triplinerve (Martin, 2003b) and Prunus spp. (Pruski et al. 2005). In the present study, ex vitro shoots exhibited only 50% survival. Liu and Li (2001) noticed a high survival rate of plantlets through ex vitro propagation. This study showed ex vitro rooting gave a lower percentage of survival than in vitro rooting though ex vitro rooting looks promising considering the reduction in cost by avoiding the in vitro rooting and use of auxins and the reduction in labour and time of plantlet establishment from laboratory to land.
Plantlets derived after in vitro rooting showed 100% survival in field conditions. The plantlets transferred revived growth after 9 days and grew well (Figure 1g). The field established plants were identical in morphology and growth pattern to that of source plant.
The protocol described in this study enables production of more than 75 plants within two months using single internode explant. This may realize the demand of pharmaceutical industries and minimize the impact of over exploitation of the plants. Besides the propagation of elite cultivars and conservation of this rare medicinal plant, a highly efficient regeneration protocol opens a way for improvement of the plant through genetic transformation strategies.
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