AOBPreview published online on June 27, 2008
Annals of Botany, doi:10.1093/aob/mcn108
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Vindoline Formation in Shoot Cultures of Catharanthus roseus is Synchronously Activated with Morphogenesis Through the Last Biosynthetic Step
Unidad de Bioquímica y Biología Molecular de Plantas and Graduate Program in Plant Sciences and Biotechnology, Centro de Investigación Científica de Yucatán, Calle 43 No. 130 Chuburná, Mérida Yucatán 97200, México
* For correspondence. E-mail felipe{at}cicy.mx
Received: 6 March 2008 Returned for revision: 8 May 2008 Accepted: 4 June 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Background and Aims: The Madagascar periwinkle (Catharanthus roseus) produces the monoterpenoid alkaloid vindoline, which requires a specialized cell organization present only in the aerial tissues. Vindoline content can be affected by photoperiod and this effect seems to be associated with the morphogenetic capacity of branches; this association formed the basis of the study reported here.
Methods: Vindoline-producing in vitro shoot cultures were exposed either to continuous light or a 16-h photoperiod regime. New plantlet formation and alkaloid biosynthesis were analysed throughout a culture cycle.
Key Results: In cultures under the photoperiod, the formation of new plantlets occurred in a more synchronized fashion as compared to those under continuous light. The accumulation of vindoline in cultures under the photoperiod occurred in co-ordination with plantlet formation, in constrast to cultures under continuous light, and coincided with a peak of activity of deacetylvindoline acetyl CoA acetyltransferase (DAT), the enzyme that catalyses the last step in vindoline biosynthesis. When new plantlet formation was blocked in cultures under the photoperiod by treatment with phytoregulators, vindoline synthesis was also reduced via an effect on DAT activity. No association between plantlet formation and other biosynthetic enzymes, such as tryptophan decarboxylase (TDC) and deacetoxyvindoline 4-hydroxylase (D4H), was found. Effects of light treatment on vindoline synthesis were not mediated by ORCA-3 proteins (which are involved in the induction of alkaloid synthesis in response to elicitation), suggesting that the presence of a different set of regulatory proteins.
Conclusions: The data suggest that vindoline biosynthesis is associated with morphogenesis in shoot cultures of C. roseus.
Key words: Catharanthus roseus, deacetylvindoline acetyl CoA acetyltransferase, DAT, in vitro shoot cultures, morphogenesis, vindoline
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The route leading to the biosynthesis of monoterpenoid indole alkaloids (MIAs) in the Madagascar periwinkle (Catharanthus roseus) is one of the best characterized (Fig. 1; De Luca and Laflamme, 2001). The availability of several biosynthetic genes (both structural and regulatory) has allowed the complexity of the mechanisms involved not only in their formation but also in the control of the process to be established (van der Fits and Memelink, 2000). In cell cultures, biosynthesis of MIAs is induced by fungal elicitors through the participation of the octadecanoic pathway and the involvement of protein phosphorylation (Pauw et al., 2004). The induction of alkaloid synthesis in response to jasmonate requires the participation of ORCA proteins, a family of transcriptional factors that co-ordinately activate genes of primary and secondary metabolism (van der Fits and Memelink, 2000). Jasmonate also activates MIA biosynthesis in developing seedlings (Vázquez-Flota and De Luca, 1998) and rootless shoot cultures (Hernández-Domínguez et al., 2004), although differing in the type of alkaloids produced. Corynanthea alkaloids, such as ajmalicine, are the predominant type in cell cultures, while vindoline, an aspidospermae-type alkaloid, is the main one in seedlings and shoot cultures (El-Sayed and Verpoorte, 2004; Hernández-Domínguez et al., 2004). Among the more than 100 MIAs produced in C. roseus, only the biosynthesis of vindoline has been shown to require a particular cell organization, involving the participation of specialized cell types that are detected exclusively in aerial tissues (Murata and De Luca, 2005; Mahroug et al., 2007). Furthermore, only vindoline synthesis is induced by light (Aerts and De Luca, 1992; Vázquez-Flota and De Luca, 1998), and these effects appear to be independent of cytomorphogenesis (Vázquez-Flota et al., 2000). The tissue and light regulation of vindoline synthesis is mainly exerted through the last two enzymes, deacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline acetyl CoA acetyltransferase (DAT; De Luca and Laflamme, 2001; Fig. 1). Both enzymes respond similarly to light treatments and jasmonate exposure in developing seedlings and rootless in vitro shoot cultures (Vázquez-Flota and De Luca, 1998; Hernández-Domínguez et al., 2004). In addition, their transcripts display identical cell distribution in leaves (St-Pierre et al., 1999), suggesting the operation of common regulatory elements. However, high levels of d4 h transcripts, but not of dat, were detected in transgenic cell cultures constitutively over-expressing ORCA3, a protein required for the jasmonate induction of MIA synthesis (van der Fits and Memelink, 2000), indicating the existence of certain differences.
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We have induced rootless shoot cultures from seedlings of C. roseus. These cultures, which have been maintained for over 24 months, display an alkaloid pattern similar to that of leaves, with vindoline as the predominant one. Variation of vindoline content among different batches, commonly observed in in vitro shoots (Hirata et al., 1990), can be reduced by keeping the cultures under a 16-h photoperiod regime, and this effect seems to be associated with their morphogenetic capacity. Furthermore, increases in DAT enzyme activity coincide with morphogenetic periods, whereas D4H activity remains unaffected, suggesting that the association between vindoline biosynthesis and morphogenesis is mainly exerted through this step.
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MATERIALS AND METHODS |
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Plant material
Rootless shoot cultures of Catharanthus roseus (L.) G.Don were induced from 7 d-old seedlings as previously described by Hernández-Domínguez et al. (2004). Light-adapted cultures were maintained by transfers every 2 weeks on semi-solid PC medium supplemented with 4·5 µM BAP (equivalent to 1 mg L–1) and sucrose at 25 g L–1 (PC–P medium) at 27 °C, under continuous illumination with a photon flux of 60 µmol m–2 s–1 provided by a combination of 39-W fluorescent and 60-W incandescent lamps (Philips, Alto Collection and Studio series, respectively; Philips de México, Mexico DF). Prior to adapting shoots to the photoperiod regime, they were maintained for over 1 year under the conditions described above, without any callus or root formation. Shoots were adapted to a 16-h photoperiod for a 3-month period, being maintained under similar culture conditions with a temperature decrease of 4 °C after the transition from light to darkness.
Light treatment and effects on shoot formation
For the analysis of effects of light regime, plantlets from multiple shoot clusters, either from light- or photoperiod-adapted cultures, were individually excised and sown on semi-solid PC–P medium in glass culture jars, each jar containing four individual plantlets. Recently sown individual plantlets are termed ‘explants’ hereafter. The number of newly formed plantlets on the explant was recorded throughout a 36-d culture cycle. Formation of new shoots on the explant resulted in cluster development. In order to evaluate the extension of cluster development, these were assigned to different categories according to the number of newly formed plantlets on the initial explant: Category 1 corresponded to clusters presenting 1–3 plantlets (in addition to the original explant); Category 2, corresponded to those with 4–6 plantlets; Category 3, to those with 7–9; and Category 4 to those with 10–12 (see Table 1). Fresh and dry weights of the complete cluster were also recorded.
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Disruption of cluster morphogenetic development
In the photoperiod treatment, shoot clusters at the transition between Categories 1 and 2 (14 d) were transferred to fresh PC media, supplemented with 20 µM each of IAA and BAP (PC–DD), in order to arrest new plantlet formation. A separated set of shoot clusters was left undisturbed (undisturbed-control), whereas another set was simultaneously transferred to fresh PC–P medium (transfer-control).
Analytical procedures
Entire clusters of shoots were subjected to analysis. Ajmalicine and vindoline were quantified according to Hernández-Domínguez and Vázquez-Flota (2006). Enzyme activities of TDC, D4H and DAT were determined by radioassays, using desalted extracts (PD-10 columns, Amersham-Pharmacia), as described elsewhere (Hernández-Domínguez et al., 2004). Transcript accumulation corresponding to D4H and DAT was analysed by RT-PCR. Briefly, 1·0 µg of total RNA was retrotranscribed with 50 U of MLV-reverse transcriptase. Oligonucleotide primers employed for D4H were 5'GGATTTCAGTGTGTAGAG3' and 5'GATAAGGGAAGAGCTATCG3', as sense and antisense, respectively, which resulted in a 946-bp product. For DAT, primer sequences were 5'ACCAAACGTGCGTATCCC3' and 5'CCCATATCGGCTTTCCC3', (sense and antisense), producing a 1043-bp fragment. Transcript accumulation for ORCA 3 was also analysed using as specific primers 5'CTTCTCAGCGATAATTCTG3' and 5'GTTAATAATTTTACAATG3' (sense and antisense; van der Fits and Memelink, 2000). This combination of primers resulted in the amplification of a 651-bp product. Retrotranscribed RNA was also amplified for β-tubulin as an internal control, using 5'ACTACTGCTGAACGGGAAA3' and 5'ACATCTGCTGGAAGGTG3' as sense and antisense primers. Nucleic acid analysis and agarose gel electrophoresis were performed according to standard procedures.
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RESULTS |
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Effects of light regime on culture growth and shoot development
The term ‘explants’ is used for individual plantlets transferred to fresh media in order to distinguish them from newly formed shoots or clusters of shoots. Explants developed in clusters formed by multiple shoots (or new plantlets formed on the explant) throughout the culture cycle (Fig. 2). No evident morphological abnormalities were detected, either under continuous light (CL) or photoperiod (PP). Although CL clusters were bigger than the PP ones, at the end of the experiment both of them presented a similar number of plantlets (Fig. 2, inset), indicating that the light regime affected biomass gain rather than their morphogenetic potential. The transition from light to dark plays a critical role in plant differentiation processes since it allows the setting of internal circadian rhythms; indeed, photoperiod is frequently used in in vitro cultures to synchronize different physiological events, including shoot formation. The development of shoot clusters was followed in cultures exposed to both light regimes over a 36-d period (Table 1). Analysis of the early phase of the culture period (up to 8 d) revealed that morphogenetic events started earlier in CL than in PP cultures. After 4 d, 28 of 100 explants had developed shoots in CL cultures, and nine of them had more than three new shoots (Category >1; Table 1). By the day 8, 43 clusters had developed, with 25 of them belonging to Categories >1; on the day 12 this had increased to 74 clusters, with 33 being in Categories >1. In contrast, more than 80 % of the PP cultures did not form new shoots (i.e. in Categories <1) during the first 8 d (Table 1) and most of the explants (86 %) turned into clusters at day 12; interestingly, nearly all of them (76 %), remained in Category 1 at that point in time. These data showed that even when continuous exposure to light accelerated morphogenetic events, dark-to-light transition resulted in a more synchronous response. This response was still present during the mid-culture period (days 16–24), when most clusters moved from to Category 1 to 2 (Table 1). Even though both CL and PP cultures showed only slight changes between days 12 and 16, by the day 20 most clusters belonged to Category 2, both under CL and the PP regime. Nevertheless, clusters under the PP treatment were predominantly Category 2 (79 %) when compared to CL cultures, which had a significant proportion of both lower and upper Categories (<2 and >2; Table 1). No significant changes in these proportions were observed until to the beginning of the last third of the culture period, when clusters moved into Categories 3 and 4. At day 28, cultures under PP were mainly in Category 3 (80 %), whereas only 45 % of those under CL belonged to this Category. Four days later, 70 % of the PP clusters had developed to Category 4, while most of the CL ones (53 %) remained in Categories lower than 4.
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Thus, morphogenetic events under PP occurred synchronously, since nearly all of the clusters analysed (approx. 80 %) formed shoots simultaneously throughout the culture period on days 12, 20, 28 and 32 (Table 1). In contrast, even when a high proportion of the CL clusters presented simultaneous shoot formation, in a significant portion of them morphogenesis had either occurred already or it was delayed, as shown by the distribution of cluster Categories during the culture period.
Alkaloid accumulation
It has been shown previously that alkaloid accumulation in shoot cultures displays a pattern similar to that of leaves, with vindoline as the predominant alkaloid and lower amounts of ajmalicine and catharanthine (Hernández-Domínguez et al., 2004). Light regimes did not alter this distribution and in both cases vindoline represented over 60 % of the total amount of alkaloids. However, vindoline showed peaks of accumulation only in PP clusters at days 12, 20 and 28 (Fig. 3), coinciding with the phases of shoot formation (Table 1). It has been noticed previously that shoot cultures exposed continuously to light showed higher variation in vindoline content among different batches (Hirata et al., 1990). Interestingly, during a 3-month period with 2 week subcultures the vindoline content in PP shoots was between 1·76 and 2·51 µg g–1 d. wt, whereas in CL cultures it ranged from 0·42 to 2·78, indicating that photoperiod reduced the variation.
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Vindoline biosynthetic enzymes
The activities of TDC, D4H and DAT were analysed throughout the culture period (Fig. 4). TDC showed a peak of activity early during the culture cycle, both in CL and PP (Fig. 4A). Maximum TDC activity preceded that of D4H and DAT (Fig. 4A–C), as has been detected previously in shoot cultures and unfolding seedlings (Hernández-Domínguez et al., 2004), and it was lower than in root cultures (Islas et al., 1994). D4H presented a significant level of activity during the middle phase of the culture cycle, with light regime having no influence on its profile (Fig. 4B). In contrast, DAT activity was affected by the light treatment (Fig. 4C). In shoots maintained under CL, DAT activity followed a similar trend to that of D4H and exhibited its maximal activity in the mid phase of the culture period (Fig. 4C). However, in PP cultures DAT displayed three peaks of activity at days 12, 20 and 28 (Fig. 4C), coinciding with both vindoline accumulation (Fig. 3) and periods of shoot formation (Table 1).
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Low levels of TDC transcript were detected in the early phases of the culture period, both in CL and PP shoots (data not shown). D4H transcript levels showed similar trends in both CL and PP shoots (Fig. 5A). In contrast, DAT transcripts showed high levels towards the end of the culture period only in PP shoot, while decreasing markedly after 20 days in CL cultures (Fig. 5B). The amounts of DAT transcripts were not modified in PP cultures, regardless of variations in enzyme activity (Fig. 5B).
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Vindoline biosynthesis in shoots is not related to ORCA proteins
In cell cultures, MIA biosynthesis is induced by jasmonate and derivatives, and it requires protein phosphorylation (Pauw et al., 2004). It also involves the participation of transcriptional activators termed ORCA, a small group of proteins belonging to the AP2 family (van der Fits and Memelink, 2000). Orca3 functions as a master regulator gene, activating transcription of several genes involved not only in MIA biosynthesis, but also in primary metabolic branches, supplying carbon skeletons required for the process (van der Fits and Memelink, 2000). Interestingly, even when in vitro shoots were biosynthetically active in terms of vindoline, ORCA 3 transcripts remained undetectable (Fig. 6A, lane C). Exposure to methyl jasmonate (MeJA), which increases vindoline biosynthesis in shoots of C. roseus (Hernández-Domínguez et al., 2004), also induced ORCA 3 transcript accumulation (Fig. 6A, lane M) in a similar manner to undifferentiated cell cultures (Fig. 6B). These data suggest that although in vitro shoots were MeJa responsive, basal vindoline synthesis in these cultures does not require the participation of ORCA 3.
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DAT activity is reduced in undifferentiating plantlets
In order to confirm that the association between vindoline synthesis and morphogenesis occurs specifically at the DAT enzymatic step, the regular morphogenetic development of shoot clusters was disrupted by exposure to equimolar concentrations of auxins and cytokinins. Fourteen-day-old PP clusters (at the transition from Category 1 to 2) were cultured on PC–DD medium. Such treatment induced stem shortening in shoots within 6 d and the formation of callus-like structures in tissues directly in contact with the medium after 10 d. Furthermore, culture of shoot clusters on DD medium avoided the formation of new plantlets, and after 2 weeks organized structures were practically absent (Table 2). Morphology of shoots in both undisturbed- and transfer-controls (see Materials and Methods) did not present abnormalities, except for lower plantlet formation in the transfer-control. Vindoline content in undisturbed controls increased 6 d after the experiment was initiated, as previously observed (Fig. 3, day 20 of PP shoot). These high vindoline contents coincided with the formation of new shoots as well as with an increase of DAT activity (see day 20 in Table 1 and Fig. 4C), and were maintained at the same level until the end of the experiment. In contrast, vindoline content in clusters on PC–DD medium was reduced within the first 6 d, coinciding with a severe decrease in DAT activity (Table 2). Although D4H activity was also reduced, values of over 60 % of the activity of those observed in controls were still detected (Table 2). Other effects observed during tissue dedifferentiation included increases in ajmalicine content and TDC activity (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The requirement of cell organization for alkaloid synthesis is well documented. In Catharanthus roseus, different cell types are involved in alkaloid biosynthesis, particularly in that of vindoline (Mahroug et al., 2006). Indeed, the lack of vindoline in undifferentiated in vitro cultures has been attributed precisely to the absence of such cell organization (St-Pierre et al., 1999) and this is supported by the reduction in its content during callus formation (Constabel et al., 1982; Aerts et al., 1992). Light also plays a critical role for vindoline biosynthesis and these effects are exerted through the last two enzymes in the process, D4H and DAT, which require the involvement of phytochrome (Aerts and De Luca, 1992; Vázquez-Flota and De Luca, 1998).
However, the effect of light on this process is not exerted via promotion of the required cell organization in tissues, since it is already present in etiolated cotyledons (Vázquez-Flota et al., 2000). In the C. roseus rootless-shoot line, the repetitive light/dark transition to which the PP cultures were exposed allowed a more synchronized development in comparison to CL cultures (Table 1). Furthermore, stages of plantlet formation in PP cultures coincided with vindoline synthesis, and they were specifically associated with DAT activity but not with D4H activation (Figs 3 and 4B, C). The specific association of DAT activation was further demonstrated by the effects on the enzyme of the hormonal treatments used to induce de-differentiation (Table 2). Genes controlling morphogenesis have been described and isolated from different species. Recently, RAP2·6L, a member of the B4 subfamily of the ERF/APETALA2 transcription factor gene family controlling shoot formation was isolated from Arabidopsis (Che et al., 2006). RAP2·6L knockdown mutants displayed a reduced expression of several genes normally up-regulated during shoot development, including some involved in key metabolic pathways (Che et al., 2006). Catharathus is not an amenable species for genetic transformation; although a few methods have been described, low regeneration efficiencies have hampered the development of knockdown or over-expressing mutants (Zárate and Verpoorte, 2007). Hence the effects of genes, such as RAP2·6L, on vindoline biosynthesis and DAT could not be assessed. Nevertheless, RAP2·6L is activated when tissues are under hormonal conditions leading to shoot formation, such as a high cytokinin to auxin ratio, and repressed in conditions used for callus induction, such as equimolar hormone concentrations (Che et al., 2006). In this way, cultures on BAP and exposed to photoperiod allowed co-ordination of the morphogenetic program with vindoline synthesis, suggesting the operation of controlling genes. Interestingly, when cultures were exposed to conditions that directed tissues to callus formation and were shown to repress genes controlling shoot formation, vindoline synthesis was also repressed, and this effect was specific for DAT (Table 2). It is interesting to note that in a low-vindoline-accumulating mutant recently isolated, DAT activity was similar to those with normal contents (Magnotta et al., 2006).
Changes in the alkaloid profile during callus induction from Catharanthus tissues depend on the explant used. For example, leaf tissues, which accumulate vindoline and catharanthine, changed their profile to a more root-related pattern, with ajmalicine and serpentine as the predominant alkaloids (Morris, 1986). In contrast, root explants tend to maintain the original alkaloid profile (Endo et al., 1987). Data shown in Table 2 are consistent with these observations since DAT activity decreased in cultures exposed to conditions for callus formation, whereas TDC increased.
The co-ordination of events such as plantlet formation and vindoline synthesis requires the participation of regulators, such as the Arabidopsis RAP2·6L (Che et al., 2006). Unfortunately, no potential orthologues to RAP2·6L have been reported in Catharanthus, although some AP2 proteins without a defined function have been isolated (Murata et al., 2006; Shukla et al., 2006). On the other hand, genes controlling alkaloid biosynthesis in Catharanthus include the AP2 type ORCA proteins (van der Fits and Memelink, 2000). In Catharanthus cell cultures, ORCA3 mediates the jasmonate induction of MIA synthesis, co-ordinating the expression of genes involved in primary and secondary metabolisms (van der Fits and Memelink, 2000). However, even when D4H transcripts can be detected in cell lines constitutively expressing ORCA3, this is not the case for DAT (van der Fits and Memelink, 2000; Rischer et al., 2006). Interestingly, ORCA3 was not related to vindoline synthesis in shoot cultures (Fig. 6), suggesting the occurrence of different regulatory genes specific for DAT transcriptional activation in this system. Nevertheless, it should be pointed out that DAT expression is not sufficient to produce vindoline, since this alkaloid was not accumulated in roots cultures transformed with dat (Magnotta et al., 2007).
In summary, the exposure of Catharanthus shoots to a photoperiod regime allowed the synchronization of the morphogenetic event of plantlet formation with the biochemical process of vindoline biosynthesis. Furthermore, this connection is related specifically to DAT among all the biosynthetic enzymes involved in this pathway.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The authors thank Dr M.L. Miranda-Ham for a critical review of this manuscript, Ms M. Monforte-González for her technical assistance with the alkaloid measurements and Ms Mildred Carrillo-Pech for maintaining the stocks of shoot cultures. This work was supported by the National Council for Science and Technology (CONACYT, México), grants 31608 B and 060746.
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FOOTNOTES |
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Present address: Hospital General Agustín O'Horán, Calle 59 A S/N Mérida Yucatán 97000, México. 
Present address: Unidad de Investigación en Biotecnología Vegetal, Instituto Tecnológico Superior de Acayucán, Carretera Costera del Golfo Km. 216·4 Col. Agrícola Michapa, Acayucán Veracruz 96100, México
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LITERATURE CITED |
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