Standardized production of Phyllanthus tenellus Roxb . by plant tissue culture 1 Produção padronizada de Phyllanthus tenellus Roxb . por cultura de tecidos vegetais

Exigencies as ethic plant raw material are part of the needs of modern phytotherapy. Micropropagation offers opportunities to obtain mass propagation of superior genotypes in short time. This study aimed to develop a protocol of direct and indirect organogenesis of Phyllanthus tenellus Roxb. Nodal segments from plantlets obtained by in vitro germination were subcultured in modified Murashige and Skoog medium added with different plant growth regulators: IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), GA 3 (3-giberelic acid) and KIN (kinetin). The highest proliferation rate was obtained using the combinations: IBA, KIN + GA 3 (3.5 mg L) and IBA + KIN (2.4 mg L). Rooting was intensified after 40 days, reaching 100% for all media with indole-3-butyric acid. Addition of 2,4 dichlorophenoxyacetic acid (2,4D) provided the best results for production of friable calli. Acclimatization was 100% effective for plantlets cultured in control medium, with decrease in survival rate in grown plantlets from media added with growth regulators.


Introduction
Nowadays, there is an increasing interest in the use of medicinal plant, but insufficient information, quality and control of some phytotherapeutical products offered is evident, and may not bring the results expected by professionals and users (VICTÓRIO;LAGE, 2008).Tissue culture techniques enable high efficiency and quick production of ethic and pathogens-free plants; besides provoke a considerable interest as a potential alternative to produce bioactive compounds.Plant growth regulators are fundamental to integrate and regulate plant development and their addition in culture media have been widely employed to improve morphogenesis and plant development (NAMDEO, 2007).
Phyllanthus tenellus R o xb. is a member of Phyllanthaceae family, widely found in several places in Brazil, especially in Rio de Janeiro city (HOFFMANN et al., 2006).This species has been exploited as a source of herb medicines.Popularly known as "quebra-pedra" and "erva-pombinha", in folk medicine, its infusion is used as antibacterial and to treat kidney and urinary bladder disturbances, diabetes and intestinal infections (CALIXTO et al., 1997;LORENZI;MATOS, 2008).Morphological similarity among some Phyllanthus s p ecies hinders their identification and may explain in part the use of different species in popular medicine for the same purpose (GARCIA et al., 2004).In view of this, the current study aimed to establish a system of standardized production of raw material of P. tenellus using different tissue culture techniques to guarantee the supply of ethic and qualified plant material properly identified.

Plant material
Seeds were collected from plants of Phyllanthus tenellus Roxb.maintained in a greenhouse at Universidade Federal do Rio de Janeiro (Brazil) and were used as source of plant material to initiate tissue cultures.Voucher specimen was identified and is deposited at the Herbarium of National Museum of Rio de Janeiro (R 200872).
Root segments were excised from 60-day-old plantlets, and then inoculated in 250 mL erlenmeyer containing 50 mL of liquid MS½N.Treatments consisted of MS½N (control), MS½N + 0.4 mg L -1 IAA and MS½N + 1.0 mg L -1 2,4D.Root cultures were maintained on a shaker at 100 rpm for 60 days under the same physical conditions above.

Leaf anatomy analysis
P. tenellus l e aves from plantlets cultured in MS½N medium were used in histological analysis.Leaf samples were diaphanized and stained with safranin to verify the leaf blade venation and the stomata types at the Standardized production of Phyllanthus tenellus Roxb.by plant tissue culture epidermal surfaces.The leaf clearing technique initially involved placing leaves in 50% sodium hypochloride.Leaves were then washed in distillated water for three times and after, they were run through 5% acetic acid for 1 min and 30% ethanol.Lastly, leaves were dyed with safranin (5 min), placed rapidly in 1% chloridric acid and washed in distillated water.The material was prepared on slides with 50% glycerin.Venation description was done according to Hickey terminology (1973).
For observation of leaf structure, plants were fixed in ethanol (70%) during 48 h.Three replicates of leaves and petioles samples from the third node were dehydrated in an ethanol series (80; 90 and 95% each hour), infiltrated and embedded in basic Historesin (Leica Microsystems, Germany) in eppendorf tubes (1 mL) and sectioned with rotary microtome (820 Spencer Microtome, American Optical Corporation, USA).Leaf sections of 8 μm thickness were stained in toluidine blue (0.05%) and prepared on permanent slides.The stained sections were examined and photographed by a Carl Zeiss optical microscope (model 4746.20-990).

Acclimatization
Sixty-day-old plantlets were transferred to commercial soil in seed plots of isopor with 9 cm 2 area, and kept in a greenhouse.Plants were protected from direct water with a transparent plastic film for 1 month.Survival percentage and plant height were evaluated after 30 days.After growth regulator treatment, some plantlets were subcultured in MS½N for eliminating any growth regulator residues and then be acclimatized.

Statistical analysis
Each experiment was done in a completely randomized design.Data were subjected to analysis of variance (ANOVA) and statistical average comparisons were made by Tukey's test at the 5% significance level.To infer if exist difference between two percentages; the Student's test (P < 0.05) was applied.

Results and discussion
The effects of plant growth regulators in plantlets development, in callogenesis, in root cultures; and, leaf anatomy description of P. tenellus leaves were the main results achieved in this research.
A simple reduction of salt solutions NH 4 NO 3 and KNO 3 of MS improved rooting and plant development, besides to avoid glassy aspect of P. tenellus cultures (Table 1).Hyperhydricity is related to nitrate concentration (HAZARIKA, 2006).Comparing MS and Different letters denote statistical differences (p<0.05,Tukey`s test).The results are the average ± SD.IAA, indole-3-acetic acid; IBA, indole-3butyric acid; KIN, kinetin; BAP, 6-benzylaminopurine; GA MS½N, the same combination of IAA 0.2 mg L -1 and KIN 0.5 mg L -1 induced significant differences in shoot height and rooting.Previous researches with other medicinal species so as Calendula officinalis also showed that reduced concentration of NH 4 NO 3 and KNO 3 of MS salts resulted in better lateral shoot production and rooting (VICTÓRIO et al., 2008).
Additions of KIN and IBA or KIN, IBA and GA 3 in medium improved the best proliferation rate and shoot elongation in P. tenellus (Table 1), that are important parameters to consider in large-scale production.Media without growth regulators produced plantlets with satisfactory height and rooting to acclimatization, however the regenerative incidence of shoots per explant was low.The use of growth regulators may induce about 2.5 times more number of shoots than control medium (MS½N).KIN plus IBA and GA 3 improved shoot number production of P. tenellus in about 50%.The maximum production of shoots per explant was 3.5 and 3.2 in the media containing the following growth regulators 0.2 mg L -1 IBA, 0.8 mg L -1 KIN and 0.3 mg L -1 GA 3 or 0.2 mg L -1 IBA and 0.5 mg L -1 KIN, respectively.Better results were observed when growth regulators were used in combinations.A higher level of KIN enhanced adventitious root formation.Still, for KIN and IBA combination, the increasing in KIN concentration up to 0.5 mg L -1 improved shoot number from 1.32 to 3.21 (Table 1 and Figure 1 and 2).Bhattacharyya e Bhattacharya (2001) verified that KIN and IAA in combination were more efficient than BAP to induce new shoots per explant in P. amarus cultures.In tissue cultures of P. caroliniensis, KIN induced higher number of shoots than BAP at same concentrations (CATAPAN et al., 2001).
Combination of KIN, IAA and GA 3 resulted in reduced rooting and shooting, probably due to auxin type and its concentration since replace IAA to IBA (0.2 and 0.4 mg L -1 ) improved rooting, number of shoots and the growing in height of P. tenellus plantlets.The smallest concentration of IBA was enough to improve rooting of P. tenellus in 100% (TAB. 1 and FIG. 3 and 4), despite of 98% incidence of basal callus.Rooting was intensified from 40 days of culture.P. tenellus plantlets showed fragile aspect when compared with ex vitro plants probably due to anatomical and physiological epigenetic alterations resulted of the in vitro conditions.The fragile aspect was characterized by thick stems and small leaves that easily fall.Fragility has been reported as an inherent feature of plantlets obtained by tissue culture techniques as result of low light intensity and changes of abiotic factors under in vitro condition.
Acclimatized plantlets from IBA, KIN and GA 3 or KIN and IBA media showed 81.9% and 13.6% survival respectively, after 30 days of transplantation to field.The low survival may be due to basal callus, short shoots and ineffective rooting.Plantlets cultured in medium added of auxin, cytokinin and gibberellin achieved 8.1 ± 3.7 cm height, while plantlets from medium contained cytokinin and auxin reached 4.0 ± 1.0 cm.Sixty-day-old plantlets cultured in control medium (MS½N) showed 100% of adaptation to the ex vitro environment (Figure 5).These plantlets achieved the height of 11.0 ± 5.6 cm.Flowers and fruits were verified in all plants after one month of acclimatization.Within 50 days of acclimatization, plants should be transferred to soil.
Callus production was achieved from 20 day of culture and it was intensified during 60 days from nodal segment and leaf explants.IAA and GA 3 induced the formation of small and compact callus of P. tenellus in white color (Table 2).Callogenesis reached 100% in medium with addition of 1.0 mg L -1 2,4D in both types of explants (Figure 6 and 7).2,4D is generally related to the callogenesis induction in culture of different species by promoting high cell division rate with consequent differentiation of the plant tissues.The addition of 2,4D at 0.1 or 1.0 mg L -1 concentration stimulated shoot development and rooting in darkness.The use of 2,4D resulted in friable callus in green, brown, gray or yellow colors (Table 2).At 1.0 mg L -1 callogenesis was not effective, while 0.8 mg L -1 KIN concentration induced white and compact calli.Color changes may be related to secondary metabolites production.The culture condition established in the current work induced the formation of friable fast-growing calli from nodal segment explants resulting in a suitable system for in vitro biomass and metabolite production and a resource to initiate suspension cell cultures.
With respect to root cultures, it was observed the production of friable callus as cited by Victório and Lage (2007).In addition, several small nodules were observed in roots (Figure 8 and 9).Morphological and anatomical descriptions are the first parameters of botanic authentication for guarantee quality control of herbal medicines.Such characteristics are considerable aspects to confirm Phyllanthus s p ecies and to contribute to raw material supplementation for phytotherapics.In order to certificate P. tenellus species, the leaves were analyzed in their morphology and anatomy.Leaf venation of P. tenellus is pinnate type, camptodromous pattern, characterized by gradual decreasing in secondary venations in the leaf apical section (Figure 10).The leaf is amphistomatic and the greatest concentration of stomata occurred on the abaxial surface.The following stomata types were observed, paracytic, anisocytic and anomocytic (Figure 11).According to Metcalfe (1963) the occurrence and distribution of the stomata types present high value in taxonomic identification.Transverse sections from leaf blade showed uniestratified epidermis (Figure 12 and 13).The pattern of leaf epidermis and parenchymas were similar of those revealed to ex vitro plants (Garcia et al., 2004).Palisade parenchyma is unistratified and the spongy parenchyma

Table 2 -
Callogenesis obtained from nodal segments of Phyllanthus tenellus Roxb.after 60 days in culture on MS medium supplemented with 2,4D