Organogenesis and Somatic Embryogenesis in Passionfruit ...

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Organogenesis and Somatic Embryogenesis in Passionfruit (Passiflora sp.)

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Wagner Campos Otoni*,1, Daniela Lopes Paim Pinto1, Diego Ismael Rocha1, Lorena Melo Vieira1, Leonardo Lucas Carnevalli Dias1, Maurecilne Lemes da Silva2, Crislene Viana da Silva1, Elisonete Ribeiro Garcia Lani1, Luzimar Campos da Silva1and Francisco André Ossamu Tanaka3 1

Departamento de Biologia Vegetal, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brasil. Departamento de Ciências Biológicas, Universidade do Estado de Mato Grosso-UNEMAT, 78300000 Tangará da Serra, MT, Brasil. 3 Departamento de Fitopatologia e Nematologia, Núcleo de Apoio à Pesquisa em Microscopia Eletrônica na Pesquisa Agropecuária, ESALQ, 13418-900 – Piracicaba, SP, Brasil. *Correspondence author: [email protected])

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Abstract Biotechnology applied to passionfruit has attracted a lot of interest from various research teams worldwide. Tissue culture studies in Passiflora was initiated in mid60s, and since then, an ever increasing number of reports on tissue culture basedtechniques applied to the genus have been published. In this chapter some past and ongoing achievements in passionfruit tissue culture are highlighted, with emphasis in those aspects related to somatic embryogenesis. The protocol for somatic embryogenesis of passionfruit was recently developed in our laboratory. Embryoderived plants have been successfully recovered and acclimatized under greenhouse conditions. Further studies were reported to be directed at synchronizing hystodifferentiation of the embryogenic cultures, and to produce synthetic seeds. This morphogenic pathway has been exploited to generate fully acclimatized transgenic plants of P. cincinnata expressing gus and gfp genes, by means of sonicated-assisted Agrobacterium transformation (SAAT). Indeed, somatic embryogenesis plays an important role among tissue culture techniques as an efficient means for plant regeneration and large-scale propagation, as well as contributing to the understanding of the physiological and molecular aspects of cell differentiation.

Keywords: Agrobacterium transformation, Organogenesis, Passionfruit (Passiflora sp.), Somatic embryogenesis, Tissue culture

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INTRODUCTION Passionfruit has many names around the world. Some of the common names are: fleur de la passion, fiore della passion, maracuyá, maracujá, passiflore bleue, mburukuja, maracujá, and passionsfrucht. The economic importance of passion fruit has increased significantly over the past three decades, reflecting worldwide production of approximately 805,000 tons (ITI Tropicals, 2010). Passionfruit cultivation extends all across the Brazilian territory, but the low productivity of commercial orchards is seen as the result of a lack of breeding programs for genetic improvement (Zerbini et al., 2008). Besides, it has remarkable ornamental and medicinal importance (Tommonaro et al., 2007; Pipino et al., 2010). Propagation of passionfruit is performed predominantly by means of sexual reproduction. This is a consequence of floral characteristics and self-incompatibility, which lead to a practice that produces plants with high genetic variability and nonuniformity with regard to agronomic traits. However, most passionfruit species can also be propagated by cuttings, grafting, and layering (Alexandre et al., 2009). Plant tissue culture, via micropropagation technique is an important method to rapidly multiply superior or high-yielding genotypes and disease-resistant rootstocks, particularly with species where selfing occurs (Drew, 1997). Tissue culture studies in Passiflora were initiated as early as 1966 (Nakayama, 1966), with shoot production from stem segments of mature plants of P. caerulea. Micropropagation of yellow passionfruit and P. molissima was reported by Moran Robles (1979). Since then, an increasing number of reports on tissue culture based techniques applied to the genus have been published as reviewed elsewhere (Vieira and Carneiro, 2004; Vieira et al., 2005; Zerbini et al., 2008). The various tissue culture-based techniques that have been applied to passionfruit are: micropropagation (Dornelas and Vieira, 1994; Nhut et al., 2007), physiological studies (Desai and Mehta, 1995; Dias et al., 2010), protoplast and somatic hybridization (Otoni et al. 1995; Davey et al., 2005), genetic transformation (Manders et al., 1994; Reis et al., 2007; Silva, 2009; Paim Pinto, 2009), synthetic seeds (Paim Pinto, 2009), gynogenesis (Rêgo et al., 2011a), androgenesis (Silva, 2009), in vitro conservation (Passos and Bernacci, 2005; Faria et al., 2007), and in vitro selection for disease resistance (Flores et al., 2012). The tissue culture techniques have proven to be effective tools for breeding programs of several plant species (Zerbini et al., 2008). The desirable traits can be introduced from non-commercial to commercial varieties via tissue culture techniques and maintained via asexual propagation methods (Alexandre et al., 2009). For breeding purposes, somatic hybridization has enabled the production of hybrid combinations not possible by sexual means (Otoni et al., 1995; Anthony et al., 1999; Davey et al., 2005), and some of the generated somatic hybrids were grown to maturity and further characterized under field conditions (Barbosa et al., 2007). Genetic transformation is another important strategy for obtaining diseaseresistant plants of yellow passionfruit and P. edulis f. edulis, which is especially relevant since passionfruit woodiness (caused by Passionfruit woodiness virus or Cowpea aphid-borne


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mosaic virus), Fusarium wilt (caused by Fusarium oxysporum f. sp. passiflorae) and bacterial blight (caused by Xanthomonas axonopodis pv. passiflorae) are the major limiting factors for the crop (Zerbini et al., 2008). In vitro conservation based on shoot cultures (Faria et al., 2007) or zygotic embryo culture (Guzzo et al., 2004) is another important application to breeding, since it provides not only a continuous source of material, but also represents an interesting approach for germplasm collection and preservation. Flores et al. (2012) established an in vitro selection approach in passion fruit plants were examined in vitro for the phytotoxic effect of fusaric acid (FA) and toxin culture filtrate (TCF) of F. oxysporum in some concentrations. To confirm the stability of insensitivity of the putative regenerants, the resistant plants obtained by means of in vitro selection were cloned and further used for in vivo resistance evaluation assays against F. oxysporum. Interestingly, the sensitivity to the selective agents in vitro and the in vivo resistance response of plants were correlated (Flores et al., 2012). The development of the root system was the parameter most affected by the use of FA and TCF in the in vitro experiments and when inoculated with the conidial suspensions in vivo. Successful in vitro selection using FA and TCF to select resistant genotypes of passion fruit was proven to be effective. More recently, gynogenesis in passionfruit by means of ovule culture was reported for the first time (Rêgo et al., 2011a), and in vitro induction of autotetraploids (Rêgo et al., 2011b), representing additional potential applications to the breeding of the species. 1. MORPHOLOGICAL PATHWAYS FOR REGENERATION IN PASSIONFRUIT 1.1. Regeneration via Organogenesis Currently prevailing regeneration system in passionfruit is organogenesis-based (Nhut et al., 2007; Zerbini et al., 2008), although recent studies have demonstrated regeneration via somatic embryogenesis in few Passiflora species. Regeneration has been achieved by organogenesis from a wide range of passionfruit species, using several plant growth regulators combinations and type of explants such as leaf, hypocotyl, root, cotyledon, and tendril (Table 1). So far, it has been the main morphogenetic pathway applied to most tissue-culture based techniques applied to Passiflora. Table 1. Optimal parameters for Passiflora in vitro regeneration as determined by different authors (Adapted from Vieira and Carneiro, 2004, and Zerbini et al., 2008). Species P. cincinnata P. cincinnata

Source of explant Mature zygotic embryo Nodal explants

Hormone type and concentration

18.1 µM 2,4-D and 4.4 µM BA 4.44 µM BA

Reference Rocha et al., 2012 Flores et al., 2012

Otoni et al. P. foetida

P. edulis cincinnata P. edulis

Mature embryo and P.

P. suberosa P. edulis f. flavicarpa P. alata

4.5µM BA combined with 13.6, 18.1, 22.6 or 27.1 µM 2,4-D

Rosa and Dornelas, 2012

4.44 µM BA

Silva et al., 2011 Paim Pinto et al. 2011 Garcia et al., 2011 Rêgo et al., 2011 Pinto et al., 2010

Root segments Mature zygotic embryo Internodal segments Unfertilized ovules

18.1 µM 2,4-D and 4.4 µM BA 44.4 µM BA 0.21 µM 2,4-D + 0.088 µM BA

Leaf discs and hypocotyl segments Mature zygotic embryo

4.43 µM BA, 2.27 µM TDZ or 4.43 µM BA + 2.27 µM TDZ + AgNO3 18.1 µM 2,4-D and 4.4 µM BA

Paim Pinto et al., 2010

P. caerulea

Leaf

4.4 µM BA

Busilacchi al., 2008

P. cincinnata

Mature zygotic embryo Young tendrils

18.1 µM 2,4-D and 4.4 µM BA 4.43 µM BA + 11.41 µM IAA, or 49.20 µM 2iP + 2.68 µM NAA

Silva et al., 2009 Pipino et al., 2010

P. edulis f. edulis, P. giberti and P. laurifolia

Stem apices and nodal segments (from greenhouse)

No growth regulators

Faria 2007

P. edulis

Leaf and root

2.22 µM BA

P. incarnata

Hypocotyl leaf

P. edulis f. flavicarpa

Stem (thin layer)

4.4 µM BA


Hypocotyl segments vertically cultured

3.08 µM BA

Alexandre, 2009

4.4. µM BA

Reis et al., 2007

4.4 µM BA + 5% CW

Fernando, 2007

P. cincinnata

Hybrid “Guglielmo Betto” M. Vecchia (P. incarnata L. × P. tucumanensis L.)

P. edulis f. flavicarpa P. edulis f. flavicarpa

zygotic

and

Hypocotyl segments Hypocotyl and leaf

4.4 µM BAP + 5% coconut water (CW)

et

et

al.,

Lombardi al., 2007 Fernando al., 2007

et

Nhut 2007

et

et al.,


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Passiflora spp.

Endosperm and zygotic embryos (matures seeds)

2,4-D, kinetin, NAA, GA3, BA, IAA and IBA in several combinations

Guzzo et al., 2004

P. edulis f. flavicarpa P.giberti P. mollissima P. edulis f. flavicarpa

Leaf segments

13.2 BA + 9.3 µM kinetin + Pluronic F-68 (PF) 4.4 µM BA + 2.32 µM kinetin + PF No growth regulators + ACC, AVG or STS

Davey et al., 2005


Shoot cultures

5.02 µM IAA and inhibitors of ethylene

Barbosa et al., 2007

P. caerulea

Leaves

10 µM BA + 0.1 µM IAA

Jasrai and Mudgil, 1999


Leaf

4.4 or 8.8 µM BA

Barbosa, 2007

P. mollissima P. edulis f. flavicarpa P. giberti

Nodal segments

BA and kinetin at various concentrations and combinations

Cancino et al., 1998


Leaf

4.4 µM BA and transference to 9.9 µM kinetin + 5 µM IAA

Silva 2009

P. giberti

Embryogenic suspension

Picloram

Otoni, 1995

P. edulis x P. edulis flavicarpa

Adult and juvenile buds

10 µM kinetin + 5 µM IAA

Drew, 1997

Shoot tips

cell

precursors or

Reis et al., 2007

et

al.,

Despite this no protocol has proven to be efficient in generating higher number of plants. In addition, plantlets belonging to this genus have difficulty growing under in vitro conditions with reduced gas exchange. As a climacteric species, it is expected that ethylene sensitivity is maintained under in vitro culture systems. Therefore, several regeneration protocols have considered the use of ethylene inhibitors as beneficial for enhancing morphogenic responses (Dias et al., 2010; Pinto et al., 2010), suggesting that an improvement in the number of gaseous changes between the internal atmosphere of the culture vessels and the surrounding atmosphere may be beneficial to better manipulate the growth and differentiation of micropropagated passionfruit, as well as lessening the negative effects of ethylene.

1.2. Regeneration via Somatic Embryogenesis

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Somatic embryogenesis plays an important role efficient means for plant regeneration and large-scale propagation, as well as contributing to the understanding of the physiological and molecular aspects of cell differentiation (Yang and Zhang, 2010). The first report on the induction of somatic embryogenesis in the genus was for P. giberti by Otoni (1995). The author reported the establishment of embryogenic cell suspensions from embryogenic leaf-derived cultures initiated in Picloram-supplemented medium. Cell suspensions, maintained in AA2 medium were used to establish a regeneration protocol based on isolated and cultured protoplasts (Otoni, 1995; Anthony et al., 1999).The induction of embryogenic calli lines from hypocotyls, anthers and zygotic embryo explants of P. cincinnata has been recently achieved in our laboratory. Silva (2007) reported anther culture-derived somatic embryogenesis of P. cincinnata. Embryogenic lines were maintained in semi-solid or liquid media, established from primary or secondary embryos, remaining highly proliferative. In addition, a reproducible protocol for somatic embryogenesis using mature zygotic embryo was proposed for P. cincinnata (Silva et al., 2009), and plants have been successfully recovered and acclimatized under greenhouse conditions (Silva et al., 2009; Paim Pinto et al., 2010). To induce somatic embryogenesis, zygotic embryos of P. cincinnata were cultured on medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6benzyladenine (BA). After 30 days of culture, primary calli were formed, some of which contained sectors with pro-embryogenic masses (Silva et al., 2009; Paim Pinto et al., 2010). Embryogenic calli were yellowish, with friable texture, and the pro-embryogenic masses were easily distinguishable by their clear and bright color. Some torpedo-stage somatic embryos were observed during the induction phase. The addition of the growth regulators BA and 2,4-D was essential for the formation of pro-embryogenic masses from P. cincinnata mature zygotic embryos. The importance of auxins, particularly 2,4-D, for the induction of embryogenic competence is well known. The use of 2,4-D is necessary for the initiation of embryogenesis, specifically for the dedifferentiation of somatic cells (Fehér, 2005). Therefore, the combination of 2,4-D with other growth regulators is a common practice in the initial induction phase of somatic embryogenesis in several species, including grape (Pinto-Sintra, 2007), Brazilian grape tree (Motoike et al., 2007), rose (Kim et al., 2003), annatto (Paiva Neto et al., 2009), among others. Despite the advantages of in vitro propagation, genetic instability is commonly observed in plants derived from in vitro cultures and may limit the use of somatic embryogenesis, particularly in massive plant propagation and genetic transformation. The occurrence of somaclonal variation is influenced by culture conditions and growth regulators (Karp, 1991). To evaluate the genetic stability of the P. cincinnata plantlets derived from primary and secondary somatic embryogenesis, flow cytometric analysis was used (Paim Pinto et al., 2010). Embryogenic calli obtained from culturing zygotic embryos on Murashige and Skoog (MS) medium containing 18.1 µM 2,4-D and 4.4 µM BA were transferred to a differentiation medium, and torpedo and cotyledonary embryos were obtained. These primary embryos were maintained on the differentiation medium to generate secondary embryos. Conversion of primary and secondary embryos yielded 305 and 138 normal plants, respectively. Almost 90% of plantlets survived following acclimatization. Flow cytometric analysis revealed that seed-derived plants


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had on average 3.01 pg of nuclear DNA (2C) and all plants, except for a single plant regenerated via primary embryogenesis, maintained their ploidy. This single plant contained more than twice the average DNA content - 6.21 pg (4C). Epidermal stomata of leaves of the tetraploid plant were larger but lower in density than those of diploid plants; this indicated that stomatal characteristics were useful in distinguishing between diploid and tetraploid plants of passion fruit (Paim Pinto et al., 2010). Based on the results of this study, it was found that the ploidy of P. cincinnata plants regenerated via somatic embryogenesis was found to remain stable, as assessed by flow cytometry, indicating the usefulness of this regeneration protocol for passionfruit clonal propagation in diverse biotechnological approaches. In summary, embryo-derived plants have been successfully recovered and acclimatized under greenhouse conditions. Further studies were reported to be directed at synchronizing hystodifferentiation of the embryogenic cultures, and to produce synthetic seeds, and to extend the protocol to P. edulis (Paim Pinto et al., 2011). Furthermore, this morphogenic pathway has been exploited to generate fully acclimatized transgenic plants of P. cincinnata expressing gus and gfp genes, by means of sonicated-assisted Agrobacterium transformation (SAAT) (Paim Pinto, 2009). 1.3. Production of Synthetic Seeds Synthetic seeds are defined as encapsulated somatic embryos or other types of propagules with or without artificial endosperm supplemented with nutrients, substances for control of contamination and growth regulators (Nieves et al., 1998) that can be converted into plantlets via in vitro or ex vitro cultures (Standardi and Piccioni, 1998). The use of artificial seeds has been described for a wide range of species of agronomic interest (Nieves et al., 1998; West et al., 2006). Several types of propagules are used for the production of synthetic or artificial seeds, and the successful use of these different propagules has already been reported for cereals, fruits, ornamental and medicinal plants and conifers (Nieves et al., 1998; West et al., 2006). Nevertheless, the production and viability of artificial seeds on a commercial scale are still restricted. To generate synthetic seeds, somatic embryos were obtained from the in vitro culture of mature zygotic embryos of P. cincinnata. Somatic embryos at precotyledonary and cotyledonary stages and mature zygotic embryos were used as source for encapsulations studies. Somatic embryos were mixed with a 2.5% sodium alginate solution (w/v). The beads were prepared by picking up the embryos suspended in the gel matrix using cut 0.5-mL pipette tips and then dropped individually into a sterile 0.1M CaCl2.2H2O solution for complexation, for 20 minutes, resulting in the formation of hydrogel beads with the encapsulated embryos. The beads were then washed in deionized and autoclaved water and placed on filter paper to remove excess water. There were high rates of germination and plantlet survival for encapsulated mature zygotic embryos cultured in flasks, in both medium types, lacking nutrients and

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supplemented with nutrients and activated charcoal, without significant differences among the treatments. No germination of encapsulated somatic embryos at the precotyledonary stage was recorded after 60 days of in vitro culture in flasks. The encapsulated embryos at the cotyledonary stage managed to break the sodium alginate matrix. There were no significant differences among the treatments for synthetic seeds produced from somatic embryos of P. cincinnata. No significant differences were found for the synthetic seed germination rate of cotyledonary-stage somatic embryos in cellulose plugs and other treatments. In the treatments where the cellulose plugs were wetted with half-strength MS medium, approximately 40% of encapsulated somatic embryos maintained competence. The higher germination rate (42%) of synthetic seeds ex vitro cultured in Gerbox was obtained by using Florialite™ substrate with embryos encapsulated in sodium alginate (Paim Pinto, 2009).

2. INVOLVEMENT OF ETHYLENE IN PASSIONFRUIT MORPHOGENESIS Ethylene is a gaseous hormone that is produced in almost all plant tissues, although the production rate depends on the type of tissue and on the developmental stage (Bleecker and Kende, 2000). Although frequently associated with maturation, ethylene is involved in several other stages of the plant life cycle, including germination, seedling growth, leaf and petal abscission, organ senescence, and responses to biotic and abiotic stresses (Schaller and Kieber, 2002). The hormone is not usually added to in vitro cultures, but does invariably accumulate in the culture recipients. The quantity accumulated depends on the production rate of ethylene in the tissues and the gas exchange rates in the cultures; although the culture flasks do allow exchange of ethylene, its production frequently exceeds its loss, resulting in accumulation in the culture flasks (Pua, 1999). Ethylene greatly influences the regeneration capacity of plant material, either promoting or inhibiting morphogenetic processes in several species, such as Nicotiana tabacum (Biondi et al., 1998), Malus domestica (Ma et al., 1998), Passiflora edulis f. flavicarpa (Reis et al., 2007), Carica papaya (Magdalita et al., 1997), Bixa orellana (Paiva Neto et al., 2009) Actinidia deliciosa (Arigita et al., 2003), Arachis hypogaea (Ozudogru et al, 2005), Capsicum chinense and Cydonia oblonga (Marino et al., 2008). Several studies have investigated different compounds that affect the biosynthesis of ethylene or its action on the morphogenic responses of in vitro cultures (Ozudogru et al., 2005; Joshi and Kothari, 2007). As a climacteric fruit, Passiflora spp. shows high rates of ethylene production (Shiomi et al., 1996), which may also limit the in vitro morphogenic potential of the explanted developmental processes (Biondi et al., 1998). The yellow passion fruit (P. edulis f. flavicarpa) is very sensitive to ethylene, which affects shoot apex development, rhizogenesis, axillary shoot development from nodal segments, and adventitious shoot regeneration from hypocotyl explants (Barbosa et al., 2001). A better knowledge of biosynthesis and ethylene accumulation dynamics, as well as control of ethylene levels within the culture flasks, is essential for an adequate establishment of in vitro culture protocols for this species.


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The effects of ethylene on in vitro development of passionfruit axillary buds have been investigated, and the sensitivity of P. edulis f. flavicarpa shoot apices to this growth regulator has been demonstated (Barbosa et al., 2001). Intense chlorosis, as detected by the decrease in pigment content, was observed with 3, 10, and 30 mM 1aminocyclopropane-1-carboxylic acid (ACC), which led to plant senescence. Similarly, in the ACC treatments, a reduced number and length of differentiated roots was observed. Likewise, it was stated that passionfruit cotyledonary explant regeneration was improved when ethylene inhibitors were added to the culture medium. The inclusion of silver thiosulfate (STS) in the culture medium significantly increased the differentiation and development of adventitious shoots from hypocotyl and leaf explants of P. edulis f. flavicarpa. The effects of the ethylene precursor ACC and two inhibitors, silver thiosulfate (STS) and aminoethoxyvinylglycine (AVG), were tested in yellow passionfruit (P. edulis f. flavicarpa) axillary buds cultured in vitro (Reis et al., 2001). The organogenic responses were assessed by the number of buds per explant, mean leaf area per explant, and shoot length. ACC-supplemented medium significantly inhibited all evaluated responses at both concentrations tested. When ethylene action and biosynthesis were inhibited, a significant increase in the number of developed buds and average leaf area was observed. Accumulated ethylene and its accumulation rate were significantly greater at 10 mM ACC, with a maximum production rate detected: at the 14th day and a decline at the 21st day. The results suggest beneficial effects of ethylene inhibitors on in vitro development of axillary buds and their reliability for use as an alternative approach to evaluate sensitivity of Passiflora species to ethylene. Even though shoot elongation did not differ from that of the control, the inhibition of the ethylene action and its biosynthesis by AVG and STS, respectively, significantly enhanced the number of buds per explant and leaf area. According to Dias et al. (2010), the highest ethylene levels were observed in the treatments with either ACC or STS added separately, and in the ACC/AVG and ACC/STS combinations, and the ethylene levels for the ACC/STS treatment were greater than with the other treatments. Mercury perchloride (MP) was efficient in capturing the ethylene produced, and the accumulation levels were considerably lower than the control treatment. The addition of AVG permitted ethylene accumulation levels similar to the control treatment, and a reduction was observed in the levels after the sixth day, as observed in the treatment with MP (Dias et al., 2010). These results, taken together, reinforce the high sensitivity of passionfruit tissues to ethylene and induction of culture senescence by its accumulation, although it may limit in vitro morphogenesis of this species. As a climacteric species, the inhibition of ethylene biosynthesis or action will most likely benefit morphogenesis of the species by enabling an increase in the number of axillary shoots associated to a higher leaf area per shoot. The flask atmosphere must be taken into account during the establishment of a reliable micropropagation system for passionfruit, which is not available at the present time. In spite of excellent morphogenic responses in vitro, the species has a very limited

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multiplication rate, which limits the development of an efficient protocol for micropropagation. Ethylene could be one of the factors associated with this limitation.

3. INVOLVEMENT MORPHOGENESIS

OF

POLYAMINES

(PAS)

IN

PASSIONFRUIT

Polyamines operate in regulation of many biological processes, such as macromolecules biosynthesis, cell division and differentiation, embryogenesis and organogenesis (Handa and Mattoo, 2010). The importance of polyamines has become clear from the changes in polyamine levels that accompany certain developmental transitions or exposure to stress conditions (Vera-Sirera et al., 2010). The main PAs founded in superior plants are putrescine (Put), spermidine (Spd) and spermine (Spm), occurring in free forms and conjugated with phenolic acids and low molecular weight molecules (Kuznetsov et al., 2006; Takahashi and Kakehi, 2010). Researches linking the PAs with somatic embryogenesis (Paul et al., 2009; Wu et al., 2009) have increased in last decade, although the mechanism of action are not yet clarified (Niemi et al., 2002). According Shoeb et al. (2001), the precise regulation of polyamine levels is required during critical stages of plant development. Several studies have shown a different morphogenic responses associated with Put/(Spd + Spm) rates. Studies involving Nicotiana tabacum protoplasts showed that high levels of Put in relation to Spd + Spm could be associated with totipotency (Papadakis et al., 2005) and as biomarkers for germination in gymnosperms and angiosperms (Pieruzzi et al., 2010). Interactions of PAs/auxin (Nag et al., 2001), and PAs/ethylene (Dias et al., 2010) have been described as a determinant factor for cell division and differentiation. Cui et al. (2010) suggests that polyamines may play their roles in regulating the plant architecture through affecting the homeostasis of cytokinins and sensitivities to auxin and cytokinin. In passionfruit we examined the behavior of ethylene and polyamines throughout morphogenic response of hypocotyl explants of two species of passion fruit (P.cincinnata and P. edulis f. flavicarpa ‘FB-100’), and studied biosynthesis competition between the two hormones. Ethylene and polyamine levels were measured over a period of organogenesis induction (Dias et al., 2010). Concomitantly, histology was carried out and the ontogenesis of the shoots was observed as they formed, to characterize the different events involved in cell redifferentiation and regulation of polyamine and ethylene levels. Jointly a study was accomplished with polyamines application and its inhibitor (MGBG) in the culture medium, in order to observe possible alterations in the morphogenic patterns. A delay was observed in morphogenic responses in P. edulis as compared to P. cincinnata, which coincided with observations of the polyamine and ethylene levels produced in these species. Cells at differentiated stages showed high expansion and elongation rates, as well as high ethylene levels associated with high polyamine levels, suggesting that the two biosynthesis pathways are highly regulated and plastic, and their interaction could be an important factor to define differentiation. The addition of polyamines did not promote a great organogenic response, however the incorporation of MGBG in the culture medium lessened shoot bud differentiation,


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indicating the need of minimum concentrations of polyamines for the morphogenic events. The studies of effects of PAs in somatic embryogenesis in Passiflora will allow an establishment of efficient protocols for other passionfruit species. 4. ANATOMICAL ASPECTS OF ORGANOGENESIS EMBRYOGENESIS IN PASSIONFRUIT

AND

SOMATIC

To better characterize the structural aspects involved in organogenic cultures, anatomical studies have been carried out. Studies involving highly comprehensive and descriptive anatomical analyses of in vitro organogenesis in Passiflora species have been published (Fernando et al., 2007; Lombardi et al., 2007). Structural analyses of in vitro organogenesis systems show that the formation of meristematic centers involved in regeneration have different origin, varying according to the explants type and its development stage (Lombardi et al., 2007). In some explants, clusters of meristematic cells may become isolated from the other cells, forming meristemoids. These cell clusters give rise to leaf primordia or continue to develop forming buds or protuberances (Fernando et al., 2007). Regeneration of leaf structures is a common feature in Passiflora, however, it has been misinterpreted as non-elongating buds (Fernando et al., 2007). This organogenic route compromises the success of the in vitro culture, because the presence of the shoot apical meristem is essential for bud formation. Protuberance formation was observed in hypocotyls and leaves (Fernando et al., 2007) and, recently, in root explants of P. edulis f. flavicarpa Degener (unpublished data). Protuberances can be formed directly or through callus phase. They are vascularized structures formed by a continuous uniseriate epidermis which together with the subepidermal layers form the peripheral region of the protuberance. In some parts of these structures, the peripheral cells have meristematic appearance, showing competence to form buds and/or leaf primordia on its surface (Fernando et al., 2007).

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Figure 1: Electron micrographs of the nucleus of cells involved in process of in vitro plant regeneration of the Passiflora species. A) Nuclear envelope invaginations (arrows) of cells formed on root-callus Passiflora edulis (indirect organogenesis). B) regular shaped nucleus and no membrane invaginations in embryogenic cells derived from embryogenic calli of P. cincinnata (somatic embryogenesis). Abbreviations: n, nucleus; nu, nucleolus. Bars = 2µm.

Comparative studies reveal differences in the cytological organization of cells involved in in vitro organogenesis and somatic embryogenesis (Verdeil et al., 2007). In P. edulis, hypocotyl cells involved in regeneration via indirect organogenesis showed the nuclear membrane with numerous pores and progressive formation of invaginations producing irregularly shaped nuclei. These characteristics can be correlated with the occurrence of amitoses and may compromise the genetic stability of primary regenerants (Fernando et al., 2007). In contrast, embryogenic cells derived from embryogenic calli of P. cincinnata zygotic embryos have large, centered, regularly shaped nuclei and a single nucleolus (Figure 1) (Rocha et al., 2012). These results corroborate the findings of PaimPinto et al. (2010), in which flow cytometry analysis found no differences in ploidy level in plants of P. cincinnata originated from primary embryogenesis. These anatomical characterizations and the better knowledge of the cell and tissue layers involved in the morphogenic events leading to regeneration may contribute positively to increase the efficiency of transformation, as well as laying the basis for the genetic transformation of different Passiflora species by means of biolistics- and Agrobacterium-mediated gene transfer techniques.

Conclusions A lot of progress has been done in passion fruit tissue, cell and organ culture since the first report. However, the protocols for micropropagation based on organogenesis still need to be improved, as well as the one based on somatic embryogenesis does need to be tested and extended to other members of Passifloraceae. As far as the current protocols for genetic transformation are concerned there is also a need to be revisited in order to improve and optimize the protocols for increasing the transformation efficiencies and reproducibility. We believe that regeneration protocols based on somatic embryogenesis may be beneficial to reach those needs. Histocytological analyses have proven very valuable for studying the process of in vitro plant regeneration. In Passiflora, the characterization of cell development and the identification of cell clusters involved in morphogenic processes have contributed to the optimization of protocols for in vitro propagation and their subsequent application to genetic transformation. Additionally, studies on the molecular aspects involving the characterization of gene expression during organogenesis and somatic embryogenesis of Passiflora will lead to a better understanding of such morphogenic processes, and may also contribute positively to increasing the multiplication rates and efficiencies of transformation protocols.


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Acknowledgments The authors would like to thank to FAPEMIG, CAPES and CNPq, for financial support; to Viveiros Flora Brasil Ltda. (Araguari, MG) for generously providing Maguary seed populations, and to our various collaborators (Dr. Marcelo Carnier Dornelas, Unicamp, São Paulo; Dr. Francisco Murilo Zerbini, UFV, Viçosa, MG; Dr. Maria Catarina Megumi Kasuya, UFV, Viçosa, MG; Dr. Lyderson Facio Viccini and Dr. José Marcello Salabert Campos - UFJF, Juiz de Fora, MG; Dr. Eny Iochevet Segal Floh, USP, São Paulo; and Dr. Miguel Luis Pedro Guerra, UFSC, Florianópolis, SC) who kindly made available their facilities to be shared with our group.

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