Rollinia mucosa (Jacq.) Baill.: establishment of callus culture and lignan production

PhD. Solange Faria Lua Figueiredo,1 PhD. Vera Regina Campos Viana,1 MSc. Cláudia Simões,2 PhD. Luiz Carlos Trugo1 y PhD. Maria Auxiliadora Coelho Kaplan1

Summary

Callus cultures were established from different organs of seedlings and in vitro propagated plants of Rollinia mucosa. The growth rate, type of callus and furofuranic lignan biosynthetic pattern were significantly influenced by the origin of the plant material, the explant type and the growth regulators used: 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid, 6-benzyladenine, gibberellic acid, and picloram.The efficiency of callus production and lignan synthesis was significantly higher in foliar blade explants in the majority of media. The best biomass was obtained on media with picloram. In seedlings explants, naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid induced epiyangambin synthesis in calli from foliar blade and magnolin in calli from epicotyl and petiole. In in vitro propagated plants explants, the synthesis of epiyangambin was induced by picloram only in calli from stem. Calli from foliar blade cultured in 10,4, 20,8 and 31,2 µM of picloram presented a two-three fold increase in epiyangambin rates with regard to levels detected in the original plant.

Subject headings: ROLLINIA; CULTURE MEDIA.

]]> Rollinia mucosa, a tropical tree known in Brazil as "biribá", has been used in the traditional medicine for the treatment of tumors in the West Indies and Indonesia.20 Acetogenins, substances which have excellent potential for antineoplasic and cytotoxic activities, were isolated from different organs of this species.4,5 Furthermore, furofuranic lignans with a significant antagonist effect to platelet-activating factor (PAF),3,11 such as magnolin (MAG), epiyangambin (EPIY) and yangambin (YAN) were found. Yangambin also presents pharmacological properties related to cardiovascular function22. Epieudesmin (EPIE) another furofuranic lignan, has an inibitory activity of cAMP phosphodiesterase.24

Magnolin, yangambin and epieudesmin have been isolated from immature fruits20 and leaves of adult plants of R. mucosa6,20 and magnolin was also isolated from seeds.5 Lignan production in R. mucosa tissues was also observed both in in vivo grown seedlings and in in vitro propagated plants derived from hypocotyl explants.6 The pattern of accumulated lignans was dependent on the origin of the plant material as well as of the plant organ. Hence, in seedlings magnolin and yangambin were predominant in the leaves, while epiyangambin was only found in very small quantities in hypocotyls. In contrast, epiyangambin was the unique lignan produced by leaves excised from in vitro propagated plants.6

The potential of tissue culture techniques for the production of several secondary metabolites has been known for many years. The development of callus culture could provide an alternative supply of compounds for use in medicine, stimulating the production or inducing the biosynthesis of novel compounds not found in the intact plant.8,25 Although the lignan production on callus culture have been investigated successfully on species as Podophyllum peltatum14, P. hexandrum10, Forsythia intermedia21 and Ipomoea cairica19, similar studies have not been reported in R. mucosa so far. This work had as first objective the establishment of a protocol for callus culture of R. mucosa by manipulating different types and combinations of plant growth regulators and as second objective the increasing and/or inducing of furofuranic lignans production in calli derived from organs of seedlings and in in vitro propagated plants. The establishment of friable calli cultures also will allow its application in further studies with cell suspension cultures.

Methods

Plant material. Callus cultures were initiated from organs of two-month-old nursery-grown seedlings and from in vitro propagated plants derived from hypocotyl explants of R. mucosa, cultivated on medium with 2,3 mM KIN + 2,2 mM BA7. Callus cultures were initiated from hypocotyl, epicotyl, young leaves (2º and 3º nodes) and mature leaves (4º and 5º nodes) from seedlings. Leaves were separated into foliar blade and petiole. From in vitro propagated plants were only used foliar blade, independent of its development stage, and stem explants.

Establishment of callus cultures. The seedling explants were disinfested with 1% NaOCl containing 0,05 % (v/v) Tween 80 for 10 min with agitation and rinsed three times (5,5 and 10 min under agitation) with sterile distilled water. Explants were excised and immersed in 12,5 mM PVP-40 for 5 min and cultured on MS medium17 supplemented with 30 g/L-1 sucrose and different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D); naphtaleneacetic acid (NAA); 6-benzyladenine (BA) and gibberellic acid (GA3) (Table 1), supported by literature used for other annonaceous, species1,12,13,18. Was also tested the effect of auxin picloram aiming to establish the callus culture (Table 1). Explants from in vitro propagated plants were cultured on the same media mentioned above. The pH value was adjusted to 5,8 prior to autoclaving (121 ºC, 15 min) and the medium was solidified with 7g/L-1 agar (Sigma). Five explants were placed in each culture glass flask (4 x 4 x 4.5 cm) containing 10 ml of medium. The foliar blade was used independent of its developmental stage for the experiments with picloram (PIC) cultures were kept in a growth chamber at 27 ± 1 ºC in the dark. Thirty explants were used in each experimental assay. Subcultures of calli to fresh medium with the same composition (Table 1) were made at monthly intervals during a period of 3 months. After this period the characteristics of color, type and growth rate [dry weight (DW)-50 ºC/24h] were evaluated.

Table 1. Plant growth regulators used in the establishment of callus culture of R. mucosa

Lignan analysis. Calli were powdered and extracted with 95% EtOH in a Soxhlet apparatus (16h). Thirty calli were used for each treatment. The extracts were filtered and evaporated to dryness under vacuum, suspended in EtOH:H2O (7:3) and partitionated with CHCl3. The fractions were evaporated to dryness and ressuspended in MeOH. The extracts were chromatographed on HPLC using a RP-C18 column (Spherisorb ODS2; 5 µm; 250 x 4,6mm) with a pre-column of the same packing material. Isocratic operation with a mobile phase of CH3 CN: H2O (48:52) was used, flow rate of 1 ml.min-1 for analytical work and UV detection at 254 nm. Lignans were identified by comparison with authentic material isolated from intact plant tissues, and characterized by 1H-NMR (Paulo et al., 1991). Ethyl ferulate was used as a external standard. Absorption integrated signals were identified according to the retention times and by samples co-eluting with standards. The absorption signals areas were determined by integration. Triplicate injections were made from every sample (extract) and the average of the peak areas was used to quantify lignans concentration, expressed in mg/g dry weight.6 ]]>

Statistical analysis. Data were arranged in a completely randomized design and evaluated by analysis of variance (ANOVA). Differences between means were tested using the Tukey test with a significance level of 5 %. Data presented in tables correspond to the mean values ± standard deviation.

Results

Seedling -callus culture and lignan production

Callus induction and maintenance. Besides the media shown in Table 1 were also used other combinations between 2,4-D (0; 0,4; 2,3; 4,5; 135,7 mM), NAA (0; 0,5; 1,3; 2,7; 5,4 mM), BA (0; 0,9; 2,2; 4,4; 8,8 mM) and GA3 (0; 2,9; 28,9 mM).

However as was observed, on these media, calli with reduced fresh weight and hard oxidation (data not shown) they were not considered in this study. All media presented in Table 1 induced callus growth without organogenic response and a similar pattern of development, regardless of the medium used. Calli were visible within 10 days on media supplemented with PIC and between 15-21 days in media supplemented with 2,4-D or NAA.

In media with 2,4-D, NAA, BA and GA3, beige compact calli were produced mainly from leaf explants and the highest yield mass was obtained from mature foliar blade explants on medium M3 (Table 2). The concentration of 2,4-D did not significantly influence callus yield from young foliar blade and petiole explants. Hypocotyl and epicotyl-derived calli cultured on media M2, M3, M4 and M7 showed different morphological types: friable, compact and an intermediate between these two types. Addition of GA3 in medium containing 2,4-D (M4) did not have any significant effect in callus growth compared with medium M2, except for epicotyl explants (Table 2).

Table 2. Effect of 2,4-D, NAA, BA, GA3 on callus culture derived from seedling explants of Rollinia mucosa and lignan production, after 90 days of culture

Hyp-hypocotyl; Epi-epicotyl; Yfb-young foliar blade; Mfb-mature foliar blade; Pet-petiole; C-Compact; I-Intermediate; F-Friable; - not detected.

According to the medium and the plant organ, the means followed by the same letter, lowercase in columns and capital in rows (for each evaluated parameter), are not significantly different by the Tukey test at 5 % probability level. Column-different organs in each medium; Row- each organ in all media.

* M5,M6,M8 - Calli with poor growth and brown color so they were not considered.

The use of NAA (M5-M8) did not stimulate callus yield except on medium M7 (Table 2). Calli produced on media M5, M6 and M8 exhibited poor growth and brown color after 2 months of culture. Thus, the calli obtained in these media were not considered.

In contrast to the observed with calli produced in media with 2,4-D and NAA, the presence of PIC, in general, induced friable and light-beige calli with a high growth rate. A direct correlation between DW and PIC concentration was observed until 20,8 µM PIC (medium M12), except for calli derived from hypocotyl (Table 3). In this concentration, the highest callus biomass was reached in cultures derived from foliar blade. This growth pattern has been maintained during 3 years.

Table 3. Effect of PIC on callus culture derived from seedling explants of Rollinia mucosa and lignan production, after 90 days of culture

Hyp-hypocotyl; Epi-epicotyl; Fb-foliar blade; Pet-petiole; C-Compact; F-Friable; - not detected.

According to the medium and the plant organ, the means followed by the same letter, lowercase in columns and capital in rows (for each evaluated parameter), are not significantly different by the Tukey test at 5 % probability level. Column- different organs in each medium; Row- each organ in all media.

Before the data observed in Tables 2 and 3 it was possible to verify that in all media used the best yield rates of callus were observed in foliar blade explants.

Lignan production

Higher level of 2,4-D (medium M3) and the interaction of GA3 with 2,4-D (medium M4) or NAA (medium M7) induced EPIY synthesis in leaf-derived calli (Table 2). The highest EPIY yields were obtained in calli derived from young (1,87 ± 0,18 mg/g DW) and mature foliar blade (1,70 ± 0,16 mg/g DW) cultured in presence of 80,7 µM NAA + 0,4 µM BA + 14,4 µM GA3 (M7) and 45,2 µM 2,4-D + 0,4 µM BA + 14,4 µM GA3 (M4), respectively, without significant differences between them.

MAG was the predominant lignan in almost all callus tissues, regardless of the consistence, original vegetative part and media used (Tables 2 and 3). All media, except M4, induced the synthesis of this lignan in epicotyl-derived calli. In petiole-derived calli, MAG induction was observed on media M3, M4 and M10 (Tables 2 and 3). The combination of NAA, BA and GA3 (medium M7) stimulated the highest MAG yield in mature foliar blade calli (0,.87 ± 0,08 mg/g DW) and in epicotyl calli (0,40 ± 0,05 mg/g DW). The use of PIC in spite of to act positively in the production of calli biomass, did not significantly influence the lignan content (Table 3). Lower levels were verified when compared with media supplemented with 2,4-D or NAA (Table 2).

Only the medium M3, with high 2,4-D concentration, favoured the biosynthesis of the three lignans (EPIE, EPIY, MAG) during the development of the leaf-derived calli (Table 2).

Biosynthesis of YAN was completely inhibited in all media supplemented with 2,4-D or NAA (Table 2). Media M10 and M11 were unique to preserve YAN synthesis capacity in foliar blade-derived calli (Table 3).

In vitro propagated plants organs-callus culture and lignan production

Callus induction and maintenance. Callus growth rates from explants derived from in vitro propagated plants were lower than those observed on calli obtained from seedling explants. In the presence of 2,4-D or NAA the majority of calli showed a intermediate consistence (Table 4) whereas those induced by PIC were friable and presented higher growth rates, mainly when derived from the foliar blade (Table 5).

Lignan production

NAA and 2,4-D favoured only MAG synthesis (Table 4), whereas in cultures with PIC the production of EPIY was the most significant (Table 5). The EPIY content was directly correlated with PIC concentrations, until 20,8 µM (medium M12). The highest PIC levels (10,4, 20,8 and 31,2 µM) were more effective for stimulating EPIY synthesis in foliar blade-derived calli, without statistical difference among them (Table 5). PIC, in all concentration used, induced EPIY synthesis, in calli produced from stem explants

Table 4. Effect of 2,4-D, NAA, BA, GA3 on callus culture derived from in vitro propagated plants explants of Rollinia mucosa and lignan production, after 90 days in culture

S- stem; F- foliar blade; C-Compact; I- Intermediate; F-Friable; - not detected. According to the medium and the plant organ, the means followed by the same letter, lowercase in columns and capital in rows (for each evaluated parameter), are not significantly different by the Tukey test at 5 % probability level. Column- different organs in each medium; Row- each organ in all media.
* M5, M6, M8 - Calli with poor growth and dark beige to brown color, so they were not considered.

Table 5. Effect of PIC on callus culture derived from in vitro propagated plants explants of Rollinia mucosa and lignans production, after 90 days in culture

S- stem; F- foliar blade; C-Compact; I- Intermediate; F-Friable; - not detected.
According to the medium and the plant organ, the means followed by the same letter, lowercase in columns and capital in rows (for each evaluated parameter), are not significantly different by the Tukey test at 5 % probability level. Column- different organs in each medium; Row- each organ in all media.

Discussion

The stablishment of callus culture an furofuranic lignan production were clearly demonstrated by this study. Both parameters were influenced by the growth regulator type and concentration under different organs.

Explants of R. mucosa seedling needed NAA and GA3 in levels significantly higher than those used in other annonaceous species, such as Annona squamosa,18 A. cherimola12,13 and A. muricata1, to induce biomass callus. Similarly to the observations described on those reports, the presence of BA was also an important factor for callus growth in R. mucosa, although lower concentration was required. These results support the idea of distinct metabolic mechanisms of plant growth regulators in different species in the same family. ]]>

Due to the greatest callus growth rates with friable consistence obtained in media supplemented only with PIC, we opted not to use it in combination with other growth regulators. These characteristics presented by the calli in media with PIC demonstrate their potentiality to be used for the establishment of a rapid system of friable callus and suspension cultures. The higher efficiency of PIC as compared to other auxins was also observed in callus culture of Zea mays,16 in six cultivars of Solanum tuberosum using 20,8 µM PIC9, similar level to the one used on medium M12 with R. mucosa. In Taxus x media var. Hatfieldii an increase on callus growth factor up to 5,8 fold of DW8 occurred when was used a concentration of PIC similar to medium M11 (10,4 µM).

With regard to lignan production in seedling callus culture although 22,6 µM 2,4-D plus 0,4 µM BA (M1) have favored EPIE production in calli from mature foliar blade, the production rate did not reach the same level yielded from seedling organ (2,54±0,38 mg/g DW)6. This medium was also capable to induce EPIE synthesis, in spite of reduced level, in calli from hypocotyl since this lignan was not detected in the original explant6.

The efficacy of 90,4 µM 2,4-D + 0,4 µM BA (M3); 45,2 µM 2,4-D + BA + 14,4 µM GA3 (M4) and 80,7 µM NAA + BA + GA3 (M7) to induce the EPIY synthesis was observed in calli from leaf, once this lignan was only produced in low concentration by the hypocotyl of seedlings6.

Previous work reported that MAG was not yield by epicotyl and petiole of seedling.6 However, in the present work, was verified the induction of MAG synthesis in some calli from these organs, being the total production of this lignan, greatest on media with 2,4-D and NAA than in presence of PIC. However, the type and concentration of plant growth regulators used were not efficient to stimulated the YAN biosynthesis in R. mucosa calli, whereas this lignan was produced in all seedling organs6. In spite of only 5,2 and 10,4 mM PIC had preserved the synthesis capacity of YAN in foliar blade-derived calli, the lignan content were inferior than those produced from seedling leaves.6 From in vitro propagated plants, although NAA and 2,4-D have favoured only MAG synthesis, the content in calli originated from the stem and the foliar blade remained below the amounts found in the original explants, 0,37 ± 0,04 and 0,60 ± 0,06, respectively.6 On the other hand, the highest PIC concentrations (10,4, 20,8 and 31,2 µM) reached rates of EPIY approximately 2-3 fold of those found in foliar blade of plants.6

These results confirm that the production of secondary metabolites in callus culture is not dependent only of the callus growth rate, but that their biosynthesis is also related with the origin of plant material, type and concentration of plant growth regulators used. The biosynthetic capacity was different between media supplemented with 2,4-D, NAA or PIC. As previously observed, NAA and 2,4-D induced the highest rates of lignan production in callus cultures of Podophyllum hexandrum,10 Forsythia intermedia21 and Ipomoea cairica.19 PIC also promoted the induction of EPIY synthesis, in stem-derived calli yielded from explants of in vitro propagated plants of R. mucosa, once this lignan was not produced by the intact organ.6 PIC has been reported to enhance callus growth, but did not stimulate metabolite production on cultures of Taxus x media var. Hatfieldii8. Nevertheless, in studies reported for Taxus sp2,15 PIC was used to produce high yields of taxanes.

The differences between in vivo and in vitro lignan production reflect variations on the secondary metabolism in these conditions and may be due to either a lack of induction or repression/inhibition of specific enzymes.23

The biochemical mechanisms of induction and stimulation of secondary metabolites synthesis by growth regulators are not clear. They may act by repressing, stimulating or inducting a common precursor or transforming an intermediate compound involved in the biosynthetic pathway of these lignans. This genetic expression variation could control lignan biosynthesis, resulting in differentiated metabolite production patterns with regard to the original explants.

The protocol for establishing of R. mucosa callus culture from organs of seedlings and in vitro propagated plants described here can be used for the production of furofuranic lignans. It is evident that callus tissues of R. mucosa are an alternative source of EPIE, EPIY and MAG being a attractive technique for production, mainly of EPIY, on a commercial basis. Moreover the procedure reported here is also an useful tool to study dynamic aspects of secondary metabolism related to the activation of furofuranic lignan biosynthetic pathways or the establishment of cell suspension cultures.

Acknowledgements: The authors acknowledge Dra . Elisabeth Mansur for suggestions and english correction.

Resumen

Se establecieron cultivos de callo de diferentes órganos de semilleros y de plantas de Rollinia mucosa propagada in vitro. La tasa de crecimiento, el tipo de callo y el patrón biosintético de lignan furoforánico estuvieron significativamente influenciados por el origen del material vegetal, el tipo de explante y los reguladores de crecimiento usados: 2,4 ácido diclorofenoxiacético, ácido naftaleneacético, 6-benziladenina, ácido giberélico, y picloram. La eficiencia de la producción de callo y de la síntesis de lignan fue marcadamente mayor en los explantes de hoja foliar en la mayoría de los medios de cultivo. La mejor biomasa se obtuvo en los medios de cultivo con picloram. En los explantes en semilleros, el ácido naftaleneacético y el ácido 2,4-diclorofenoxiacético indujeron la síntesis de epiyangambina en callos de hoja foliar y de magnolina en callos de epicotilo y petiolo. En los explantes de plantas propagadas in vitro, la síntesis de epiyagambina se indujo mediante picloram sólo en callos del tallo. Los callos de hoja foliar cultivados en 10, 4, 20,8 y 32 µM de picloram presentaron un incremento de 2 a 3 veces en las tasas de epiyagambina con respecto a los niveles detectados en la planta original.

DeCS: ROLLINIA; MEDIOS DE CULTIVO.

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Recibido: 25 de agosto de 2003. Aprobado: 16 de septiembre de 2003 ]]> PhD. Solange Faría Lua Figueiredo. Avenida Epitácio Pessoa, 2900/204-Lagoa. Rio de Janeiro. Brazil. E-mail: solangelua@openlink.com.br

1 PhD. Adjunct Professor.
2 Master. Biologist.