EFFECTS OF CARBENDAZIM ON IN VITRO MICROPROPAGATION AND FLOWER- ING OF PEPPER ELDER HERBAL PLANT

INTRODUCTION Peperomia pellucida is a medicinal plant belongs to the family Piperaceae and commonly known as pepper elder, shining bush plant and man to man. It is a tropical annual fibrous shallow-rooted herb, which grows as succulent and erect plant (Wagner et al. 1999; Majumder 2012). The stem part is fleshy, round, delicate and glabrous (Majumder 2012), whereas bud like structures attached to several fruiting spikes. shiny heart-shaped fleshy leaves are arranged alternately. The flowers are numerous, scattered on the spike with only stamens EFFECTS OF CARBENDAZIM ON IN VITRO MICROPROPAGATION AND FLOWERING OF PEPPER ELDER HERBAL PLANT


INTRODUCTION
The aim of this study was to investigate the potential of in vitro culture of P. pellucida, i) Surface sterilization with suitable surfactant for mass propagation of clonal material, in vitro milieu to engage in other applied biotechnologies; ii) PGRs induced the multiple shoot initiation and proliferation with suitable organic elicitors followed by root induction and acclimatization from shoot tip and axillary node explants; iii) this study deals with the development of in vitro flower system from shoot tip explants and the subsequent transplantation of the plantlets. Furthermore, we examined the safety of the in vitro and ex vitro plant material in pharmacological studies, in comparison with field grown plants of P. pellucida.

MATERIALS AND METHODS Surface sterilization of explants
Plants of P. pellucida (Voucher Specimen No: KLU47794, University of Malaya Herbarium) were collected from the Botanical garden of Institute of Biological Science, Faculty of Science, Kuala Lumpur. Surface sterilization techniques were tested for the explants used inshoot multiplication and in vitro flowering studies. Initially, axillary node and shoot tip explants of P. pellucida were immersed in a2% Teepol solution (4 min) and washed with running tap water (20 min) to remove adherent particles followed by a rinsed off with sterile distilled water (SDW). Further sterilization was done under aseptic conditions of laminar air flow cabinet. Both explants were treated with 70% ethanol (1 min), soaked in 5% sodium hypochlorite v/v (8 min) washed with DW (5 min). Then the explants were treated with a freshly prepared mixture of 0.1% HgCl2 (w/v) for 2 min. In addition, 0.4% (v/v) Carbendazim (Carbendazim powder; dissolved in 70% ethanol), a broad-spectrum systemic fungicide, for 5 min. After each treatment, explants were thoroughly washed with SDW for removal of disinfectants traces. Finally, washed with the distilled water with continuous shaking of explants upto 5 min. 18 1999; Sheikh et al. 2013). The plant is used in traditional medicine to cure conditions like abdominal pain, boils, colic fatigue, abscesses, acne, convulsions, fever, gout, headache, renal disorders, , conjunctivitis, skin diseases, breast cancer rheumatic joint pain and to lower blood cholesterol level (Khan et al. 2010).
Plant tissue is widely used for conservation, commercial propagation and production bioactive compounds. The common challenges for plant tissue culture, especially stock plants derived from mature plants have been elimination of culture contaminants and failures of micropropagation protocols. Aseptic conditions has always been a key factor towards successful in vitro plant culturing and mass multiplication (Mug'omba et al. 2012). A wide range of elicitors have been employed to modify plant metabolism in order to enhance shoot regeneration and pharmaceutical compounds in plant tissue culture (Baskaran et al. 2013). In vitro flowering approach has proven to be a very useful strategy for the investigation of flowering physiology and the important factors influence the processes such as plant growth regulators (PGRs) , light, carbon source and pH of the medium (Heylen and Vendrig 1998) as well internal factors like explant position, genotype and gene expressed during flowering (Jumin and Nito 1996). Hence, no reports on in vitro propagation and in vitro flowering of P. pellucida.
The MS powder dissolved and fortified with 30 g L-1 sucrose (carbon source) and gelled with 0.8% (w/v) agar and the pH of the medium was adjusted to 5.7 ± 0.2 using 0.1N NaOH (Sodium hydroxide) or 0.1 N HCl (Hydrochloric acid) after addition of PGRs. The medium was autoclaved at 121ºC and pressure for 30 min and maintained. The explants were inoculated on MS (medium prepared in culture tube (25 X 150 mm) and plugged tightly with non-absorbent cotton. All the cultures were maintained at 25 ± 2ºC under 16/8 (light/dark) regime provided by cool white, fluorescent light (60 µmol-2 light intensity) with 55-60% relative humidity.

Shoot proliferation and rooting
Shoot tips (3-4 cm) and axillary nodes (4-5 cm length) collected from 4-5 months old P. pellucida plants were inoculated into MS medium with different concentrations of plant growth regulators and organic elicitors after surface sterilization and maintained under culture room conditions. The MS medium was supplemented with different concentrations of cytokinins 0.5-2.0 mgL -1 6-benzyl adenine (BA) and kinetin (KN) with 0.1-2.0 19 mgL -1 NAA, IBA or NAA for multiple shoot initiation and development. For the maximum proliferation of multiple shoots, the explants were treated with optimum concentration (KIN and NAA) combine with organic elicitors of 50-200 mgL -1 casein hydrolyte and glutamine. The number of regenerated shoots per explant was observed and the shoots were visually evaluated after 12 weeks. For root induction, the full-strength MS medium supplemented with different Conc. of auxins, 0.5-2.5 mgL -1 IAA, IBA and NAA were used. Root induction (%), root number (mean ± SE), root length (cm), shoot length (cm) and morphological characters were observed at the end of eight weeks.

In vitro flowering and rooting
A rapid and economic in vitro flowering protocol was developed from axillary node explants of P. pelludica. Explants (axillary nodes) were cultured on MS medium supplemented with 0.5-2.0 mgL -1 KIN and 0.1-1.0 mgL -1 ABA (Abscisic acid) with sucrose treatment (1-6% sucrose). Flowered shoots were transferred to a MS medium supplemented with 0.5-2.5 mgL -1 IAA with 3% sucrose for rooting.
Flowered shoots with a welldeveloped root system were acclimatized and introduced to the field.

Acclimatization
For acclimatization, the rooted plantlets (From both experiments: micropropagation and in vitro flowering) were removed from the cultures vessels and rinsed with SDW to remove the adhering agar. Plantlets were established in pots containing sterile soil content mixture of manure, sterile soil and vermiculite [1:1:1 in ratio]. Survival rate (%), flower initiation and maturation were recorded after 6 weeks of hardening. Plantlets were initially covered with a polythene sheet to maintain relative humidity (90%). All survived plantlets were transferred to a greenhouse after 10 weeks and water sprayed in two days interval.

Statistical analysis
In vitro shoot induction, rooting, hardening, in vitro flowering and acclimatization were conducted in completely randomized design. The experiments were repeated thrice with a total of 30 replicates per treatment. Experimental data were recorded after two weeks from shoot regeneration of P. pellucida. All the data obtained were analyzed using analysis of variance. The means were compared using Duncan's Multiple Range Test at P= 0.05 using SPSS 14.0. The mean values with similar alphabets within columns are not significantly different. The values are the means ± standard error.

RESULTS AND DISCUSSION Explant Surface sterilization
Explants (axillary nodes and shoot tip) were surface sterilized with different surfactants and carbendazim (0.1, 0.2, 0.4, 0.6 and 0.8% prepared by 70% ethanol). Surface sterilization with a soap solution (Teepol), 70% etha-nol (1 min), 5% NaoCl2 (8 min), 0.1% HgCl2 (2 min) and followed by 0.4% Carbendazim (5 min) was suitable for explant survival (Fig.  1a). The surfactants can prevent initial contamination, but fungi often find their way into cultures at a later date (2 weeks) ( Table 1). The contaminated explants were discarded after autoclaving. We have observed that fungal contaminants are faster than the growth of in vitro shoot multiplication and are easy to eliminate from explants using fungicides (0.4% carbendazim) and induced the multiple shoots without contamination.
Despite of using sterile techniques and aseptic conditions, contamination of plant cell and tissue cultures remains a persistent problem (Orlikowska et al. 2016). Carbendazim is the active ingredient derived from thiophanate methyl and benomyl breakdown process, which are commercially available as Topsin M and Benlate. Earlier days, benomyl has been added to the media and autoclaved; the media contain benomyl degraded to form car-

In vitro shoots induction
Axillary buds and shoot tip explants were induced 2 or 3 shoots with roots and shoots are tall (3-4 cm) in free MS basal medium (Control) (Figure 1b). Explants of P. pellucida were cultured on MS medium with different cytokinins [benzyl adenine (BA) and kinetin (KIN); 0.5-3.0 mgL -1 ] with auxins (NAA, IAA and IBA; 0.5-2.0 mgL -1 ) to evaluate their potency on shoot multiplication (Table 2; Figure 1c-e). The shoot regenerated rate varied significantly between treatment after 12 weeks of culture initiation (Table 2). However, axillary node explants induced shoots which were taller (3.4 cm/ explant) (Figure 2a) than shoot tip explants (Table 2). On the other hand, BA alone, BA combine with NAA, KIN combine with NAA concentration induced the multiple shoots after 12 weeks of culture ( Axillary node explants cultured on MS medium with 2.0 mgL -1 KIN and 0.5 mgL -1 NAA exhibit the multiple shoots (11.6 /shoot) and produced tall shoots with less number (Figure  2 b-c) than shoot tip explants (Table 2). After 12 weeks, cultured shoot tip explants 2.0 mg L-1KIN with 0.5 mgL -1 NAA produced the multiple shoots 14.4/ explant (Figure d and e) and the results were better than axillary node explants ( Table 2). As well, we have tested without cytokinins of shoot multiplication in shoot tip and axillary node explants; explants were produced single shoot with branched roots (Figure 1f and g) in MS medium supplemented with IAA, IBA and NAA treatments (data not shown).
So far, no in vitro propagation report in the plants, but species protocols are different from the previous reports. A low auxin concentration combination with high cytokinins concentration promoted shoot proliferation in Crambetataria (Piovan et al. 2011)andP. Obtusifolia (Hany and Amira 2014)belongs to the family Piperaceae. In vitro propagation is an excellent technique to obtain maximum number of plants within a short period of time . Our studies showed that axillary node developed shoots were tall as well less shoot indicating that cytokinins-auxin have shown the synergistic effect in shoot proliferation (Table  2).Results were agreed with the previous reports of KIN and NAA was the most appropriate concentration for in vitro shoot multiplication of other species medicinal herbs of Bauhinia racemosa (Sharma et al. 2017).

Multiple shoots proliferation with organic elicitors
Present study established the multiple shoot proliferation protocol with organic elicitors (casein hydrolysate and glutamine), cytokinins of KIN and BA combination with auxins of NAA was examined in shoot tip explants of P. pellucida (Table 3). The prolonged culturing in the same medium did not increase the shoot regeneration frequency (Table 2) and in order to achieve a higher shoot proliferation, effects of casein hydrolysate and glutamine were tested (Table 3). Optimum response was (21.5 shoots per explant after 12 weeks of culture; Figure 1i) obtained in MS medium supple-mented with 2.0 mgL -1 KIN and 0.5 mgL -1 NAA with 100 mgL -1 glutamine and 100 mgL -1 casein hydrolysate. The results were significantly higher than the results of the treatment with 1.5 mgL-1 BA and 0.5 mgL -1 NAA (Fig.  1h). In many studies, the organic elicitors are regulating the biochemical entities which have prolific influence on plant growth and multiple shoot development as they control vital cellular processes including DNA replication, fruit development, senescence and secondary metabolite production under abiotic and biotic stress conditions (Baskaran and Van Staden, 2011;Cvrckova et al. 2014).

In vitro rooting
In this study, the effect of different auxins (IAA, IBA, NAA and 2,4-D) were evaluated for in vitro rooting of P. pellucida. In vitro generated shoot tips were transferred to rooting MS medium supplemented with IAA, IBA, NAA and 2,4-D treatment. Our results showed that IAA induced the highest rooting percentages (94.2%), greater root numbers (17.6), longer root length (2.9 cm) and shoot length (10.5 cm) than NAA and IBA treatment after 8 weeks (Table 3; Fig. 1j-m). As well, root induction, proliferation and shoot length was observed in axillary node explants ( Figure 2 d-h); whereas NAA, IBA and 2,4-D treatment induced roots were lesser ( Fig. 2e-f) than IAA treatment in axillary buds (data not shown). As well, multiple shoots of axillary node explants were tried for root induction and proliferation (Fig. 2g-h) and the results were significantly lower than shoot tip explant rooting treatment (data not shown). Without PGRs, free MS media induced the less roots at the end of 8 weeks (Table 3). The highest rooting percentage, up to 94.2%, occurred in 1.5 mg L-1 IAA (Figure 1l and m); while IAA concentrations either lower or higher 1.5 mg L-1 had significantly lower percentages (Table 4) 2,4-D induced basal callus and the shoot was shorten with less root number (data not shown). We have observed the root numbers (17.5 roots per shoot induced by 1.5 mg L-1 IAA and the mean root length 2.9 cm (Fig.  1m) and the shoot length (10.54 cm) / culture and the similar type of results were observed in Salvadorapersica (Kumari et al. 2017).

In vitro flowering and rooting
A rapid andeconomici vitro flowering protocol was developed from shoot tip explants of P. pelludica (Fig. 3a-j). Sterile explants (shoot tip) were cultured on MS medium supplemented with KIN, BA, and ABA with sucrose treatmentsand flowerswere induced after 6 weeks of cultureinitiation (Fig. 3a). Maximum number of flowerswere induced on MS mediumcontaining 2.0 mgL-1 KIN and 0.5 mgL-1 ABA (Abscisic acid) with 5% sucrose after 12 weeks (Table 5; Fig. 3d-e). As well, we have noticed the flower bud induction on MS medium supplemented with 2.0 mg L -1 KIN and 0.5 mg L -1 ABA with 5% sucrose (Fig. 3b-c). This combination yielded the highest number of flowers (4.7) / shoot and response (75.4%) (Fig. 3d-g) after 12 weeks. Whereas KIN and ABA with different percentage of sucrose were (4% and 6%) induced flower buds which did not mature until 12 weeks. Flowers were emerged from the branch region of the developing shoots of P. pellucida. On the other hand, the flower production was absent in MS medium supplemented with KIN and ABA with 2% and 3% sucrose treatments (data not shown). Maximum number of roots were obtained from the medium supplemented with 1.5 mgL-1 IAA and 3% sucroseafter 16 weeks (Fig. 3h-i).
In the present study, the flower inducing agent ABA combination with KIN with 5% sucrose was found to be responsible for in     (Cheruvathur et al. 2015). We have observed 5% sucrose was suitable for the flower initiation and maturation in P. pelluci-tained in in vitro flower development in Brachystelma glabrum (Revathi et al. 2017     da than other sucrose treatments. Similar result was observed in Morusindica (Gogoi et al. 2017).

Acclimatization
Acclimatization was divided in to two experiments: i) Acclimatization of plantlets obtained from axillary node and shoot tip explants. (Fig. 1/p); ii) Acclimatization of plantlets obtained from the in vitro flowering experiment. The hardened plantlets were covered with transparent plastic polythene bags in order to retain moisture and avoid transient shock. To allow proper aeration, small pores were made on plastic bag and water sprayed at regular intervals.
Each set consisted of 25 pots (100 x 125 mm) (Experiment 1: without flower/pot; Experiment 2: with flower/pot. Shoot tip and axillary node derived plantlets were hardened and acclimatization in the green house ( Fig. 1n-p and Fig. 2i) after 2 month , the acclimatized plants showed flowers (Fig. 1n-p and Fig.  2i).
In vitro flowered plantlets were produced new leaves and new flowers with shoot development (Fig. 3j). All plantlets were transferred to pots containing a mixture of manure, sterile soil and vermiculite [1:1:1 in ratio] and grown under culture room conditions with humidity level regulated at 50%, temperature at 25±2°C and photoperiod of 16 hours light and 8 hours dark. Experiment 1, the hardened plantlets survival rate was significantly higher than experiment 2 of in vitro flowering studies after 2 months. Plant field growth (%) and survival rate were recorded until 6 months.

CONCLUSION
The present work studied in vitro shoot induction and multiplication from axillary and shoot tip explants of P. pellucida. A combination of 2.0 mgL -1 KIN, 0.5 mgL -1 NAA, 100 mgL -1 glutamine and 100 mgL -1 casein hydrolysate promoted the maximum shoot produc-tion. Successful rooting was achieved on full strength MS medium fortified with 1.5 mgL -1 IAA and the rooted plantlets were successfully hardened in tissue culture conditions. In vitro flowering protocol succeed in 2.0 mgL -1 KIN, 0.5 mgL -1 ABA with 5% sucrose and showed 4.7 flowers/shoot followed by rooting and hardened and the characters compared with mother plant. Acclimatization protocols were uniform, healthy and high survival rate after transplantation to field of environment.