JBA-06824; No of Pages 11 Biotechnology Advances xxx (2014) xxx–xxx

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Research review paper

Phaseolus vulgaris — Recalcitrant potential Katarzyna Hnatuszko-Konka a,⁎, Tomasz Kowalczyk a, Aneta Gerszberg a, Aneta Wiktorek-Smagur b, Andrzej K. Kononowicz a a b

Department of Genetics, Plant Molecular Biology and Biotechnology, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland Department for Good Laboratory Practice, Bureau for Chemical Substances, Dowborczykow 30/34, 90-019 Lodz, Poland

a r t i c l e

i n f o

Article history: Received 31 January 2014 Received in revised form 4 June 2014 Accepted 5 June 2014 Available online xxxx Keywords: Phaseolus vulgaris Common bean Regeneration Transformation Legumes

a b s t r a c t Since the ability to genetically engineer plants was established, researchers have modified a great number of plant species to satisfy agricultural, horticultural, industrial, medicinal or veterinary requirements. Almost thirty years after the first approaches to the genetic modification of pulse crops, it is possible to transform many grain legumes. However, one of the most important species for human nutrition, Phaseolus vulgaris, still lacks some practical tools for genomic research, such as routine genetic transformation. Its recalcitrance towards in vitro regeneration and rooting significantly hampers the possibilities of improvement of the common bean that suffers from many biotic and abiotic constraints. Thus, an efficient and reproducible system for regeneration of a whole plant is desired. Although noticeable progress has been made, the rate of recovery of transgenic lines is still low. Here, the current status of tissue culture and recent progress in transformation methodology are presented. Some major challenges and obstacles are discussed and some examples of their solutions are presented. © 2014 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . Obstacles and solutions in regeneration protocols . . . . . . . Indirect approaches as a rare promising phenomenon . . . . . The recapitulation of the tissue culture status in common bean . Transformation . . . . . . . . . . . . . . . . . . . . . . . . . Straight and combined Agrobacterium-mediated transformation Direct methods of gene transfer . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction Legumes, the third largest family of higher plants, are notoriously recalcitrant both to regeneration and transformation. Grain legumes (that rank third behind cereals and oilseeds in world production) have lower responsiveness to in vitro regeneration compared to the forage legumes (Veltcheva and Svetleva, 2005). This is also the case for Phaseolus vulgaris, an economically important crop. The common bean is the most ⁎ Corresponding author. Tel.: +48 42 635 42 19, +48 692 434 221 (mobile); fax: +48 42 635 44 23. E-mail addresses: [email protected] (K. Hnatuszko-Konka), [email protected] (T. Kowalczyk), [email protected] (A. Gerszberg), [email protected] (A. Wiktorek-Smagur), [email protected] (A.K. Kononowicz).

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important food legume for direct human consumption in several countries of Latin America and Africa, however its position cannot be overestimated in the USA, Canada or India. Even the Common Market of the European Union, focused rather on cereals, admits to cropping more than 1300 its varieties specified in the Common Catalogue of Varieties of Vegetable Species (2011) (including dwarf and climbing ones). It seems completely justified as beans combined with cereals assure a balanced diet of energy and protein. Bean seeds provide important minerals, vitamins, dietetic fibre but no unsaturated fatty acids (De LaFuente et al., 2011). As P. vulgaris represents a major protein source in the population's diet, it is obvious that it is still of high agronomic interest worldwide. Among over 30 species of the genus Phaseolus (according to different

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Please cite this article as: Hnatuszko-Konka K, et al, Phaseolus vulgaris — Recalcitrant potential, Biotechnol Adv (2014), http://dx.doi.org/10.1016/ j.biotechadv.2014.06.001

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authors it is difficult to estimate how many Phaseolus species exist, the number may reach even 50 or 60 species) the common bean is the most widely distributed crop, occupying more than 90% of the area intended for beans in general (Broughton et al., 2003; Morales, 2006). Having been adapted to diverse environmental conditions, the common bean is not free from biotic and abiotic constraints. It suffers from six widespread major diseases and some unfriendly abiotic conditions such as soil toxicities, drought stress or nutritional deficiencies (Beaver and Osorno, 2009; Popelka et al., 2004). It is a challenge on which both plant biotechnology and conventional breeding methods have been focused on legume improvement for several years. And there may be many targets for such improvement. Apart from the above, they may concern for example the enrichment of the seed proteins of pulse crops in sulphur-containing amino acids, changing the plant anatomy or reducing the time needed for flowering and seed setting in long duration crops (Eapen, 2008). Also the usage of legumes as ‘green factories’ seems completely justified. Consequently, several international initiatives (the Medicago Genome Consortium; International Conferences on Legume Genomics and Genetics ICLGG) that concentrate primarily on the field of legume genomics and genetics (Colpaert et al., 2008). For P. vulgaris studies, an international consortium — ‘Phaseomics’ was established in 2000 in Sevilla, Spain (Broughton and Aguilar, 2005). The main purpose of this initiative was to establish the necessary framework of knowledge for the advancement in studies of bean. Phaseomics gathers a number of scientists from all over the world that focus on different aspects of widely understood Phaseolus biology. Due to these efforts plant regeneration and transformation in the legume family have been achieved for several species, however one of the most important food legumes, P. vulgaris, remains recalcitrant to both routine in vitro breeding and genetic engineering. It is still difficult to determine whether beans are generally not amenable to regeneration or transformation only because of their indigenous lack of competence or how to crush their resistance simply remains undiscovered. However, in the 1970s and 1980s, a similar situation existed regarding cereals, that were considered to have low potential for regeneration and transformation processes and then the concentrated efforts of plant scientists enabled success in the field of cereal engineering (Shrawat and Lorz, 2006). The number of researchers interested in legume biology and the undoubtedly observed dynamic development of knowledge, justify the opinion that also in the case of Phaseolus it is a question of the time to devise repeatable and efficient procedures. Some recent and promising reports, both on regeneration and transformation protocols, are presented here. Whether any of them may become a base for the routinely used procedure is open to question. It should be pointed out that the reported outcomes are presented rather in the form of confrontation among the trends in P. vulgaris research than of direct comparisons. Regeneration Tissue culture of P. vulgaris is repeatedly considered to be difficult. It is particularly inconvenient as the lack of a rapid and efficient regeneration system hampers possibilities of its genetic improvement. Although the number of papers is available, the proposed methods of regeneration still seem to be not easily reproducible. The utility of the tissue culture achievements established for other representatives of the Phaseolus genus also seems rather exaggerated. For example, application of the procedures successfully used for regeneration of whole plants of different Phaseolus species (Phaseolus acutifolius, Phaseolus coccineus, Phaseolus polyanthus) results only in shoot production in the case of P. vulgaris (Delgado-Sánchez et al., 2006). That suggests at least a species-specific protocol. Apart from the physiological state of the explant, cell or tissue specialisation of the culture and cultivation conditions, a plant genotype is the basic factor responsible for regeneration processes (Svetleva et al., 2003). Thus, genotype limitations

indirectly underlie the difficulty in development of routine regeneration procedure for legumes or even beans. It is unquestionable that beans demonstrate extremely high diversity regarding regeneration responsiveness. All three classic pathways of in vitro propagation (organogenesis, somatic embryogenesis and proliferation of shoot meristems from the regions surrounding the shoot bud) were described for the common bean with limited efficiency and low repeatability. In consequence the necessity of a genotype-dependent and cultivar-specific procedure is suggested. Most of the published procedures were based on direct organogenesis or shoot development from meristematic cells (Arellano et al., 2009). Many examples of a direct organogenesis pathway may be found in the literature e.g. reported by Ahmed et al. (1998), Ahmed et al. (2002), Albino et al. (2005), Ebida (1996), McClean and Grafton (1989), Mohamed et al. (1992) or Quintero-Jiménez et al. (2010). Yet, to the best of our knowledge and belief, there are only a few protocols based on indirect organogenic regeneration of the common bean (Arellano et al., 2009; Collado et al., 2013; Mohamed et al., 1993; Zambre et al., 1998) as in the case of induction of somatic embryogenesis it occurs rather sporadically (Jacobsen, 1999; Kwapata et al., 2010; Martins and Sondahl, 1984; Nafie et al., 2013). Nevertheless, until now several types of cells, tissues and organs (cotyledonary nodes, embryonic axes, auxiliary shoots, cotyledon with split embryo axis, internodes, hypocotyls, leaves, leaf petioles or intact seedlings) have been used to induce all the regeneration pathways (Albino et al., 2005; Delgado-Sánchez et al., 2006; Franklin et al., 1991; Mahamune et al., 2011; Thảo et al., 2013). However, it should be noted that the protocols named did not always yield regeneration of the whole P. vulgaris plants. The number of published regeneration procedures of common bean is quite enormous and the reported approaches cover all pathways of in vitro regeneration: organogenesis, somatic embryogenesis and proliferation of shoot meristems from the regions surrounding the shoot bud (Eapen, 2008). Such a general description of regeneration can be found in the literature. However, the above depiction of it is very wide and describes each way of plant development except natural morphogenesis of a generative origin. From this point of view, it should be rather characterized as the ways in which plants can be propagated through tissue culture. Thus, it is very important to keep it in mind that in tissue culture practice especially focused on plant transformation, the true term “regeneration” functions in a narrower context. Regeneration comprises plant development from somatic tissue section lacking preformed meristems (i.e. leaf, calluses) while proliferation/ micropropagation occurs using meristematic tissues like axillary bud regions. Plant regeneration itself can occur by two pathways: organogenesis or somatic embryogenesis (Phillips and Hubstenberger, 1995). At this level it is also very important to make a clear distinction between wider terms – organogenesis and somatic embryogenesis – and their subtypes: direct (adventitious) and indirect (de novo origin via i.e. callus) processes. As these terms are used in many different ways in the literature their precise usage in the scientific reports would greatly simplify the comparison of the results and determination of the current status of the research on the regeneration of P. vulgaris plants. Here we present the examples of different approaches to common bean regeneration and attempt to refer to the main problems discussing the ways they have been solved. Obstacles and solutions in regeneration protocols It may be concluded from scientific reports that some obstacles in the regeneration process have been identified and currently efforts are made to eliminate them. According to many authors it is possible to influence the plant competency (Cruze de Carvalho et al., 2000; Mohamed et al., 1992; Veltcheva and Svetleva, 2005; Zhang et al., 1997). Precultivation of parent plants on a medium enriched with BAP (benzylaminopurine), TDZ (thidiazuron) or CPPU (forchlorfenuron) may stimulate the division of competent cells and indirectly influence

Please cite this article as: Hnatuszko-Konka K, et al, Phaseolus vulgaris — Recalcitrant potential, Biotechnol Adv (2014), http://dx.doi.org/10.1016/ j.biotechadv.2014.06.001

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the regeneration potential (Cruze de Carvalho et al., 2000; Svetleva et al., 2003). For example, in comparison to the control medium, Veltcheva and Svetleva (2005) achieved a 5–7-fold increase in the number of regenerants per explant by precultivation of seedlings on 1 μM (0,2 mg/L) BAP containing MS (Murashige&Skoog) medium. This alternation of in vitro response yielded the enhancement of indirect organogenesis from leaf petioles in all three tested Bulgarian varieties. The same scientists showed the determinative role of the age factor. Explants originating from 7-day old seedlings retained the potential for regeneration while those from 14-day old ones — lost it (Veltcheva and Svetleva, 2005). That may prove that young cells have greater potential to respond while being elicited by stimulation with exogenous hormones. The question of the precultivation role was earlier mentioned in 1991 when Malik and Saxena obtained seven-fold increase in shoot regeneration frequency after donor seedlings had been placed on 5 μM (1,1 mg/L) BAP containing medium. The same scientists showed that induction of shoot regeneration occurred more efficiently if a leaf explant included both petiole and a portion of lamina (Malik and Saxena, 1991) or the intact seedling was used as primary material (Malik and Saxena, 1992). Their observation is reflected in the report by Ahmed et al. (2002) in which the question of influence of morphological integrity of the donor plant was raised. It was shown that the number of buds and shoots produced by intact seedling (without roots) was noticeably greater than that from cotyledonary node explants (MS + 1 mg/L BA and 0.1 mg/L NAA). Furthermore, the same protocol addressed rooting problems indicating that only large (at least 2-cm long) shoots with two trifoliate leaves possessed the capability of root development (Ahmed et al., 2002). Concentrating on the analysis of the medium composition in 2006 a promising direct organogenic bud induction protocol was established by Delgado-Sánchez and colleagues. The regeneration of whole common bean plants from embryonic axes of mature seeds was reported. Its efficiency varied noticeably between the two Mexican cultivars (90% of bud cluster formation and 83% of full plant regeneration versus 18% of bud cluster formation and 50% of full plant regeneration). Interestingly, the winning response was achieved on MS medium supplemented with 5 or 10 mg/L BAP (and adenine, but its influence was not revealed). This observation varied considerably from the previous papers as lower BAP concentrations (0.1–2.5 mg/L) were already used for the response induction. The efficiency obtained was then much lower, which suggests positive correlation between BAP concentration and the number of organogenic buds (Delgado-Sánchez et al., 2006; Thảo et al., 2013). Although the reported in vitro system was species and genotype-dependent the authors were compelled to overcome an inherent obstacle — the fact that most seed legumes are more prone to root formation than to shoot formation. For that reason higher concentrations of cytokinins are needed for shoot regeneration compared to other plant families (Veltcheva et al., 2005). As the procedure developed by Delgado-Sánchez brought the promising efficiency of whole plant production (from 25% to 83% depending on the cultivar), the same research group reported an improved direct organogenesis protocol in 2010. The common bean shoot induction from embryonic axes was achieved in four cultivars through modification of medium conditions (here, it should be noted that only one of the cultivars – Flor de Mayo Anita – investigated by Delgado-Sánchez et al. was retested by Quintero-Jiménez et al.; however, the organogenic response in those approaches was consistent in providing evidence of repeatability in different common bean cultivars). The Gamborg medium supplemented with 10 mg/L BA yielded the highest regeneration efficiency (from 13% to 100%) (Quintero-Jiménez et al., 2010). Interestingly, contrary to the above-mentioned protocol, the Quintero-Jiménez group showed that the Murashige and Skoog (1962) and Gamborg et al. (1968) basal media might differ considerably regarding regeneration efficiency. The difference in response from embryonic axes is evident: while Gamborg induced high organogenic shoot formation (98–100%) and whole plant regeneration (93%), the Murashige and Skoog medium initiated lower,

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inconsistent organogenic shoot formation (15–73%) and whole plant regeneration (29%) (Quintero-Jiménez et al., 2010). While most of scientists use MS medium exclusively Mukeshimana et al. (2013) have recently reported the potential of two basal media, Lloyd and McCown's (1980) woody plant medium (WPM) and Quoirin and Lepoivre medium (QL) (Quoirin and Lepoivre, 1977), for further improvement of shoot production from embryo axes. However this precise data were not shown (Mukeshimana et al., 2013). Nevertheless, both, the approach of Delgado-Sánchez and of Quintero-Jiménez tested the utility of adenine (A) or adenine sulphate (AS) (respectively) for organogenic shoot formation in P. vulgaris. The obtained results are consistent as, regardless of basal medium, no significant effect of A or AS on shoot organogenic formation or shoot development was reported in any experimented variant. This remains in contrast with the results obtained by Gatica Arias et al. (2010) that suggested that BAP combined with AS, improve the process of organogenesis. According to van Staden et al. (2008) such a divergence among results may be justified, as the effect of adenine on shoot induction is rather unpredictable. It depends not only on species and cultivar but also on the nature and rate of cytokinin used. For example, different concentrations of BA may induce stimulation or decrease in propagation. That may happen because the mechanism of action of adenine has not yet been fully explained. It is possible that adenine enhances natural cytokinin biosynthesis or acts as its precursor (van Staden et al., 2008). In the case of the procedure established by Arias Gatica the highest efficiency for shoot formation was achieved when the MS induction medium was supplemented with 5 mg/L BAP and 20 or 40 mg/L AS. The research was conducted on five Costa Rican varieties and the number of shoots and leaves noticeably differed among them again suggesting a cultivar-specific protocol (Gatica Arias et al., 2010). The morphology of shoot regeneration induced from embryonic axes of the above-mentioned Costa Rican cultivars was also studied using scanning electron microscopy. The observations did not reveal the morphological differences between plants treated (or not) with BAP but confirmed the stimulating influence of cytokinin. This ultra structural analysis showed that BAP caused the formation of a great number of shoots that started on the second day of the culture (Jiménez et al., 2012). In the light of numerous publications it seems that a relatively inefficient in vitro shoot production remains one of the most significant obstacles at the stage of actual regeneration. According to many authors this phenomenon seems to result from the presence of phenolic compounds secreted from injured sites. Their oxidation by polyphenoloxidases, peroxidases or air causes characteristic browning of the explant and the surrounding culture medium (Gatica Arias et al., 2010). Formation of brown callus at the base of a plant explant hinders shoot regeneration (Barikissou and Baudoin, 2011; Kwapata et al., 2010; Thảo et al., 2013). Such limitations in the establishment of tissue culture were reported for many species like Pinus sylvestris (Laukkanen et al., 1999) or Musa spp. (Titov et al., 2006). According to previously described protocols the supplementation with cytokinin should overcome this obstacle, however it was also reported that the presence of some plant growth regulators such as BAP, kinetin, TDZ or IAA intensified the extent of browning (Gatica Arias et al., 2010). Thus, as the synthesis of phenolic compounds may be also stimulated by cytokinins, the use of high concentrations (5–10 mg/L−1) of these hormones to enhance the number of shoots induced should be reconsidered. For example, such an enhanced accumulation of phenolic compounds during in vitro growth was reported by Schnablová et al. (2006) in transgenic tobacco overproducing endogenous cytokinins. A promising solution to the ‘phenolic problem’ was reported by Kwapata et al. (2010). When compared to the control medium with no antioxidants, the supplementation of charcoal, silver nitrate, glutathione or ascorbic acid significantly increased the regeneration frequency and development of multiple shoots in vitro. The best results were achieved when silver nitrate and activated charcoal were used (an increase in regeneration frequency of 18% and 16% respectively). This modification of the induction medium enabled

Please cite this article as: Hnatuszko-Konka K, et al, Phaseolus vulgaris — Recalcitrant potential, Biotechnol Adv (2014), http://dx.doi.org/10.1016/ j.biotechadv.2014.06.001

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development of an efficient system for in vitro apical meristem shoot proliferation of different varieties of common bean. Also the constraint of poor rooting of in vitro grown shoots was overcome in these studies. The shoots were dipped in IBA and moved to the rooting media containing IBA, IAA or NAA. It is significant that cytokinins should be removed from the rooting medium since they may delay root establishment (Kwapata et al., 2010). In this context it was presented by DelgadoSánchez et al. (2006) also that low BAP concentration (0 and 0.1 mg/L) induced only formation of roots and elongation of stems. The question of root development was probably indirectly resolved also by elimination of phenolic compound activity and use of activated charcoal. The beneficial effect of activated charcoal for in vitro rooting was also reported by Barikissou and Baudoin (2011) where the role of activated charcoal in the micro cutting medium was evaluated. The use of activated charcoal was shown to effectively increase the regeneration rate of rooted in vitro plantlets to ca 90% (compared to 43% achieved on the control medium) (Barikissou and Baudoin, 2011). In 2009 high frequency direct plant regeneration from mature seeds of common bean was reported by Dang and Wei. The established in vitro system obtained over ten regenerated plantlets from one explant. The seeds germinated for 6 days on Murashige and Skoog (MS) medium supplemented with thidiazuron or N6-benzylaminopurine (BA). Using cotyledonary nodes, multiple buds were induced by Dang and Wei on the MS medium enriched with 5.0 mg/L BA with the induction frequency 71.9% after 4-week culture. This bud frequency increased from 12.1% (pure control MS medium) to 71.9% when 1 mg/L of TDZ was added (it is possible that thidiazuron exert its impact by modifying the metabolism of endogenous cytokinins). The subsequent shoot formation frequency varied from 61.3 to 87.6% depending on AgNO3 presence and the average root frequency was 84.3% (Dang and Wei, 2009). This study seems compatible with the research conducted by e.g. Cruze de Carvalho et al. (2000) or Veltcheva and Svetleva (2005) who used the plant explant precultivation stage to enhance regeneration efficiency. Also the beneficial influence of silver nitrate was emphasised by Kwapata who presented the increase in regeneration frequency and development of multiple shoots in vitro initiated by its addition (Kwapata et al., 2010). Indirect approaches as a rare promising phenomenon Although, due to the presented attempts some critical steps in the regeneration process were perceived, many scientists still believe that to obtain a maximum in vitro regeneration response, the medium formulation must be made specifically for a particular cultivar (Gatica Arias et al., 2010; Kwapata et al., 2010). Since a routine genotype-independent pathway of regeneration is the most important requirement for successful genetic transformation of Phaseolus spp., such an approach would mean a significant deceleration in common bean improvement. As indirect pathways (organogenesis mostly) occur rather sporadically in regenerating of common bean plants the occurrence of genotype-independent procedures among them is on the rise. A genotype-independent pathway of indirect organogenesis in P. vulgaris was recently reported by Arellano et al. (2009). Apical meristems and cotyledonary nodes dissected from embryonic axes of Negro Jamapa cultivar were used as the parent explants. The most efficient callus production was achieved on 1.5 μM (0.3 mg/L) 2,4-dichlorophenoxyacetic acid (2,4-D) containing MS medium. Two-week old calli were transferred to a shooting medium containing 22.2 μM (5 mg/L) BAP. After four weeks of incubation the number of well developed shoots (stem with leaves 1.0–2.0 cm long) regenerated per callus was evaluated and shoots were placed on the rooting medium consisting of MS macro and micronutrient salts, B5 vitamins and 7 g/L agar and low concentrations of growth factors: 0.444 μM (0.1 mg/L) BAP and 0.054 μM (0.01 mg/L) NAA, pH 5.7 ± 0.1. Except for a Negro Jamapa cultivar, the other nine P. vulgaris varieties were successfully

regenerated, suggesting a genotype independent procedure (Arellano et al., 2009). Since the shoot regeneration frequency was of approximately 0.5 shoots per callus, the procedure may be a subject of further improvement and should be regarded as a very promising basis for establishing a protocol for P. vulgaris. It is well known that plant regeneration from different varieties of P. vulgaris callus cultures in vitro is difficult to achieve. Two procedures of indirect organogenesis reported by Mohamed et al. (1993) and by Zambre et al. (1998) appeared to be highly genotype-specific. Thus far, the Arellano's (2009) protocol for indirect regeneration was the first genotype-independent approach to plant regeneration of common bean. However, recently the in vitro plant regeneration via indirect organogenesis was reported by Collado et al. (2013) that could be widely applicable for different bean cultivars. As primary explants cotyledonary nodes (CN) and cotyledonary nodes with one (CN1) or two (CN2) cotyledons dissected from the embryonic axis of three-day-old germinated seeds of five commercial cultivars were used. The optimum proliferation of calli was achieved on MS medium containing 0.04 mg/L of TDZ, while a shoot regeneration frequency of approximately 3.0 shoots/callus was achieved on medium supplemented with 2.25 or 4.50 mg/L of BAP. The obtained plants were fertile and showed normal developmental pathway. Interestingly, it was found that formation of morphogenetic callus was strongly affected not only by primary explant type but also by seed age. In comparison with fresh, 4- and 8-month-old seeds, the percentage of callus formation in explant from 12-month-old seeds was significantly lower (97%, 91%, 89%, and 56%, respectively) (Collado et al., 2013). In the studies conducted in the Department of Genetics, Plant Molecular Biology and Biotechnology (University of Łódź, Poland) similar constraints have been met in establishment of regeneration procedure for P. vulgaris. We followed Malmberg's (1979) suggestion assuming that the screening of a large number of genetic lines might become a source of useful information for plant regeneration (Svetleva et al., 2003). The studies were conducted on randomly chosen cultivars, Casablanca, Laponia and Plus (data not published) (Fig. 1). The already mentioned predisposition for root formation that is characteristic of seed legumes was also revealed in our research. Moreover, similar to Rubluo and Kartha (1985) a diverse response of genotype to different regeneration media was observed. It was elicited both by phytohormone combination or type of basal medium used (Murashige&Skoog or Gamborg) (data not published). In the case of our bean explants the results were consistent with the observations by Quintero-Jiménez et al. (2010) suggesting that Gamborg medium induces stronger in vitro response. The recapitulation of the tissue culture status in common bean Based on the research reports some repeatable problems that occur during in vitro regeneration of P. vulgaris plantlets can be indicated. The troublesome stages and factors are briefly presented below (Table 1). Numerous attempts were made to develop in vitro efficient and repeatable regeneration by direct and indirect organogenesis or direct and indirect somatic embryogenesis (Veltcheva et al., 2005). Consequently, reports on common bean in vitro regeneration are available, however mostly with results differentiated among genotypes. According to Hammatt et al. (1986) the common recalcitrance observed among large seeded legumes results from reduction of genetic variability. Modern varieties might have lost their diversity because of the long history of legume inbreeding (Hammatt et al., 1986). Hence, there may be a certain divergence in the estimation of systems using callus intermediate. The first protocol for organogenic shoot induction from dedifferentiated callus cells was reported by Mohamed et al. (1993). Since callus shows low genetic stability, the indirect approaches via somaclonal changes can de novo broaden the genetic diversity simultaneously developing a system for regeneration of P. vulgaris. From the point of view of the research on heredity or genetic

Please cite this article as: Hnatuszko-Konka K, et al, Phaseolus vulgaris — Recalcitrant potential, Biotechnol Adv (2014), http://dx.doi.org/10.1016/ j.biotechadv.2014.06.001

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Fig. 1. Stages of regeneration of Polish cultivar of common bean (Laponia) on the medium supplemented with growth regulators (medium compositions not published). A — seed germination, B — hypocotyl explants, C — non-morphogenic callus induction, D, E — shoot multiplication, F, G — plantlet rooting, H — regenerated Phaseolus vulgaris plant.

manipulation, direct pathways are more suitable as they do not enter genetic variation. Nevertheless, at the moment it is still difficult to designate a routine in vitro system for common bean, which would be fully genotypeindependent. Transformation It has been almost thirty years since the first attempts to produce transgenic pulse crops were undertaken. Since the 1980s brought a breakthrough in genetic engineering some papers on legume transformation were also reported (Eapen et al., 1987; Kohler et al., 1987a, 1987b). The first publication on the successful development of a transgenic member of Leguminosae family (Vigna unguiculata) was presented by Garcia et al. (1986). Although attempts at whole plant regeneration failed, it was confirmed that a transgenic callus had originated from transgenic mesophyll cells. The protocol that adopted leaf disc transformation technique using Agrobacterium tumefaciens was followed by many other scientists (Garcia et al., 1986). The usage of Agrobacterium for transformation of stem segments of forage legume resulted in transgenic Medicago sativa calli and plants (Shahin et al., 1986). Eapen and colleagues developed a regeneration and transformation system of Vigna aconitifolia protoplasts also through co-cultivation with A. tumefaciens (Eapen et al., 1987). Among the Phaseolus genus the first approach testing Agrobacterium-mediated transformation device was used to transform tepary beans (P. acutifolius A. Gray) (Dillen et al., 1997). That appeared to be a good choice as P. acutifolius represents one of the few species within the grain legumes, which show reasonable potential for genetic engineering. Hence, P. acutifolius was suggested for use as a ‘bridge species’ to introgress transgenes into the P. vulgaris plants (Dillen et al., 2000; Popelka et al., 2004). Earlier, attempts to genetically modify the common bean via Agrobacteriummediated gene transfer were described by Mariotti et al. (1989) or Franklin et al. (1993), but the production of stably transformed plants was not reported (Svetleva et al., 2003). It resulted in chimeric tissues or non-regenerable transgenic callus, respectively (Franklin et al., 1993; Mariotti et al., 1989). It has to be emphasised that even in the pioneering investigations in many cases the cultivar used was recognised as an important factor for transformation frequencies, independent of transformation techniques. And almost three decades later this observation still appears to be true. Among the target species no efforts are spared to achieve an efficient protocol for stable transformation of legumes, which contribute almost

30% of the world's major crop production (Arellano et al., 2009). Although it is difficult to select a model plant for such a large family, P. vulgaris was suggested as a diploid model species within the legume family. The common bean is a diploid species with 2n = 22 chromosomes and a medium-sized genome (the haploid genome size ranges from 588 to 637 Mbp (Gepts et al., 2008; McClean et al., 2008)). Meanwhile however, P. vulgaris proved to be one of the most recalcitrant species both in regeneration and engineering. Consequently, two other models were developed, Medicago truncatula and Lotus japonicus exhibiting two developmental systems for nodulation (Dita et al., 2006). Although at present numerous reports are available on successful transformation of a number of P. vulgaris cultivars, common bean has been transformed genetically with limited success. Scientists have no doubt that basic difficulty concerning achieving efficient transformation of that ‘key species’ (and legumes in general), which is related to their low responsiveness to in vitro regeneration. Efficient differentiation, shoot development and whole plant regeneration processes based on a reliable in vitro culture system are an essential requirement for studies on gene expression, comparative genomics and common bean improvement (Gatica Arias et al., 2010). Among the factors contributing to the lack of progress in developing transgenic pulse crops the lack of competent totipotent cells for transformation is listed (Beaver and Osorno, 2009). In order to determine them various explants were reported as a target for transformation: seed meristems (Russell et al., 1993), callus cultures (Franklin et al., 1993), intact shoot tips of germinating bean seeds (Lewis and Bliss, 1994), mature embryos (Aragão et al., 1992), leaf discs (Genga and Ceriotti, 1990); apical meristems (Brasileiro et al., 1996), shoot apexes (Aragão and Rech, 1997), seedlings, cotyledonary nodes and hypocotyls (Eissa et al., 2000; Kumar et al., 2004; McClean et al., 1991), intact leaf tissues (Kapila et al., 1997), embryo axis (Dillen et al., 1995), protoplasts (Leon et al., 1991), leaves and immature seeds, cotyledons and embryo axes of immature bean seeds (Genga et al., 1991, 1992), and shoot apexes of embryogenic axes (Kim and Minamikawa, 1995). As the young embryonic tissues display greatest potential for regeneration, embryonic axes and cotyledonary nodes are among the most often used for transformation (Eapen, 2008). Of course, also the chosen method itself necessitates the control of many specific parameters. For example Agrobacterium-mediated transformation depends on the type and physiological state of an explant tissue, its maturity, vector and the Agrobacterium strain used, preculture and co-cultivation conditions and the interactions between Agrobacterium and its host (Zhang et al., 1997). In the case of P. vulgaris the choice of cultivar is a crucial factor affecting

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Table 1 Troubleshooting: problems occurring during Phaseolus vulgaris in vitro regeneration process. Problem

Factor to be affected

Reasons and potential solutions

Comments

Low seed germination index

Seed age

Fresh seeds show higher germination index (probable related to seed quality and cultivar used)

Seed quality (e.g. vigour) Presence of endophytic or pathogenic bacteria

Reliable seed origin Certified seeds free from pathogens, bactericide treatment

Common bean cultivar The regeneration potential

Screening Seed age — the significance of seed age for in vitro regeneration has been reported suggesting fresh seed usage Preculture of parental plants on a medium stimulating proliferation of competent cells The age factor — young cells/tissues have higher potential of in vitro respond (also while being elicited by stimulation with exogenous hormones) Preservation of the morphological integrity of the donor plant — the number of buds and shoots produced by intact seedling is greater than that from cotyledonary node explants Medium — concerns both basal medium type and hormonal composition Explant type

Some of the plant pathogens do not survive long storage conditions thus the older seeds may be favourable (Trapiello and González, 2012) – Obstacle usually not mentioned in scientific papers, however visual detection of contamination is unreliable, seeds may appear healthy in spite of contamination (Gent and Ocamb, 2014) – E.g. formation of morphogenic callus occurred more frequent when explants originate from fresh seeds E.g. medium supplemented with BAP, TDZ or CPPU Experimental evaluation required — according to many reports too young explants are more susceptible to necrosis

Low regeneration competency

Inefficient in vitro shoot production

Prone to root formation rather than to shoot formation

Presence of phenolic compounds and their connection with brown callus formation

Poor rooting

Common bean cultivar Shoot size/stage of shoot development

Rooting medium composition

Presence of phenolic compounds Common bean cultivar

Common bean cultivar — screening Manipulation of medium composition — match of nature and rate of cytokinin used (however effect on shoot induction may be unpredictable) Charcoal, silver nitrate, glutathione or ascorbic acid supplementation was proven to stimulate development of multiple shoots in vitro (indirectly via limitation in brown callus development) Screening The choice of shootlets competent to rooting — according to some reports only large (N2-cm long) shoots are capable of root development The choice of basal medium type and hormonal composition pretreatment of shoots with IBA and transfer to the rooting media Use of activated charcoal was shown to increase efficiency of root development Screening

transformation rate and it cannot be underestimated. Until now, its influence seems to be inevitable regardless of an engineering technique. Also the age and origin of a common bean explant are similarly significant in agroinfection as they are in biolistic devices. Namely, according to Aragão and Rech (1997) the morphology of explants may affect the generation of modified plants due to the fact that some cultivars have the central area of the meristematic region covered by the primordial leaves (Gepts et al., 2008). That can reduce efficiency of the common bean transformation via the bombardment of apical meristematic regions. Indirectly one returns to the genotype/variety dependent procedures. As has been mentioned, a number of researchers work to find solutions to bean recalcitrance to genetic engineering. Some of them investigate the regeneration capacity of different common bean explants; others examine the influence of various phytohormones and/or different sorts of basal media on in vitro potential. For instance, Cruze de Carvalho et al. (2000) exploited the transverse thin cell layer (tTCL) technique to increase the frequency of shoot regeneration without an intermediate callus stage (Albino et al., 2005). Consequently, mainly in

E.g. divergence in the requirements: intact seedling display stronger regeneration respond while cotyledon nodes appear to be better transformation target Experimental evaluation required Divergence in the requirements: explant suitable for regeneration may not be an efficient as a transformation target – Exemplary question of BAP (already discussed in the text) — different concentrations may induce stimulation or decrease of propagation Synthesis of phenolic compounds may be stimulated by cytokinins, therefore application of cytokinin high concentrations for shoots induction is open to question – –

Experimental evaluation required

– –

the last eight–ten years, a promising advance has been made also in Phaseolus engineering. Although many papers on successful engineering of Phaseolus spp. were published, until 2005 no efficient routine transformation method for any consumable large-seeded beans was established. To date, there is only a reproducible genetic transformation system for the tepary bean (P. acutifolius A. Gray) (Zambre et al., 2005). At present, reports are available on successful transformation of a number of P. vulgaris cultivars, using both Agrobacterium and biolisticmediated methods or even combination of different methods (Aragão et al., 2011; Brasileiro et al., 1996; Espinosa-Huerta et al., 2013; Liu et al., 2005). Straight and combined Agrobacterium-mediated transformation The use of Agrobacterium rhizogenes-mediated transformation brought the construction of semi-transgenic plants consisting of transgenic roots on wild type de-rooted seedlings (Colpaert et al., 2008; Estrada-Navarrete et al., 2006; Estrada-Navarrete et al., 2007).

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Estrada-Navarrete et al. (2006) established such a reproducible and efficient procedure for P. vulgaris. The seedlings of several cultivars, landraces and accession of common bean were infected and transformation efficiency varied between 75 and 90%. Among the four tested strains of A. rhizogenes hairy roots were most effectively induced by the strain K599 (cucumopine type). The host plant genotype also had an influence on hairy root formation (Estrada-Navarrete et al., 2006). In 2007 the same group published a more detailed protocol concerning P. vulgaris transformation with A. rhizogenes K599 (Estrada-Navarrete et al., 2007). The complementary system for production of semi-transgenic common bean plants was reported by Colpaert et al. (2008). Hairy roots were induced in two cultivars, Xan and Carioca, and obtained in 50% and 75% of the plants, respectively (Colpaert et al., 2008). Due to this alternative solution new possibilities for the exploration of Phaseolus genus (research on functional genomics, on root biology, root–microbe interactions) are opened for scientists. However, such a pathway of transgenic common bean production is not without complications. Since the progenies of composite plants do not inherit the transgenic traits, the utility of A. rhizogenes may be diminished (Mukeshimana et al., 2013). Nevertheless, the susceptibility of Phaseolus spp. to A. rhizogenes was already tested by Brasileiro's group (Brasileiro et al., 1996). Two strains, 8196 and A4, were used to transform six Brazilian Phaseolus cultivars: three of the common bean and three representing the tepary bean species. The results proved the crucial role of a genotype in determining susceptibility and demonstrated the potential of the 8196 strain for genetic engineering as it caused formation of hairy roots on all investigated plant genotypes. In the same paper Brasileiro et al. tested eight A. tumefaciens strains using all six above-mentioned Phaseolus varieties. The general observations relating to plant genotype were identical and all strains (AT 2553, AT 8196, T37, 82.139, Bo524, Ach5, R10, 15955) were shown to be virulent in the chosen cultivars. Additionally, common bean appeared to be more susceptible to A. tumefaciens strains than tepary bean. Interestingly, as the Agrobacterium cocultivation strategy had not envisaged its reproducible use, the new approach was established. The classic Agrobacterium-mediated transformation of bean was preceded by wounding embryo meristems with microprojectiles (Brasileiro et al., 1996). The fact that the bombardment pre-treatment enhanced the percentage of tumour formation when compared with the inoculation without bombardment (from 30% to 50%–70%) clearly weighs in favour of the combined strategy. Hence, another paper describing successful transformation of common bean by A. tumefaciens has been published presenting a protocol based on the combination of sonication and vacuum infiltration methods. To the best of our knowledge this approach (Sonication assisted Agrobacterium-mediated Transformation, SAAT) developed by Liu et al. (2005) brought one of the highest efficiencies of transformation (12%) of P. vulgaris plants. The optimal conditions included Agrobacterium-mediated transformation of seedlings followed by 5 min sonication and 5 min vacuum infiltration and it enabled generation of transgenic kidney bean with late embryogenesis abundant gene from Brassica napus (Liu et al., 2005). It prevails even over more recent reports on biolistics-based systems, however until now no subsequent studies using this protocol have been reported. Nevertheless, it is a very promising procedure, especially that Agrobacterium-mediated transformation of Phaseolus species was performed with limited success. In 2013 Mukeshimana et al. (2013) presented the study evaluating factors influencing transient and stable transformation of common bean using A. tumefaciens. The transformation was preceded by optimisation of regeneration of four cultivars representing various commercial classes of P. vulgaris. The capacity of leaf explants, stem sections, and embryo axes for in vitro response was tested using 30 MS based media supplemented with different combinations of plant growth regulators. Among the chosen explants only embryo

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axis explants displayed limited recalcitrance to regeneration. Interestingly, of several media enabled multiple shoot production, the optimal one was again genotype-dependent. Nevertheless, the ability of three A. tumefaciens strains to transfer gusA gene to common bean was tested (GV3101, LBA4404 and EHA105). P. vulgaris plants were again proved to be susceptible to Agrobacterium spp. and factors influencing gene delivery were shown to be dependent on various parameters (bacterial strain, co-cultivation time, explant type or plant genotype) (Mukeshimana et al., 2013). Such observations appear to correspond to some earlier reports (e.g. Zhang et al., 1997). Among the strains used, GV3101 appeared to be the most effective, which suggests the highest susceptibility of common bean to the nopaline Agrobacterium strain. Although the embryo axes of common bean were shown to be optimal transformation targets, none of the calli or plantlets obtained developed into normal plants (Mukeshimana et al., 2013). However, just recently a glimmer of hope for A. tumefaciensmediated transformation of common bean has appeared. To the best of our knowledge, the research by Espinosa-Huerta et al. (2013) yielded one of the highest transformation efficiencies ever — 10–28% with certain variations due to the cultivar tested, the Agrobacterium strain and selection agent used. Like Mukeshimana's group, Espinosa-Huerta et al. (2013) analyzed variables involved in stable and efficient agroinfection. The whole procedure of in vitro breeding, starting from seed germination, hypocotyl dissection to media composition was based on reports by Delgado-Sánchez et al. (2006) and QuinteroJiménez et al. (2010). Having regenerated the common bean cultivars (Flor de Mayo Anita and Pinto Saltillo), Espinosa-Huerta et al. successfully introduced two genes of agronomic importance: defensine and proton pump pyrophosphatase genes that confer drought tolerance and resistance to fungal pathogens respectively. Two A. tumefaciens strains, GV3101 and GV2260, were shown to be virulent in the chosen cultivars, although the GV3101 strain appeared to cause some degree of necrosis and to inhibit full regeneration. Nevertheless, a transformation frequency of at least 10% regardless of strain used suggests that the reported protocol is a viable alternative to other transformation systems. Moreover, molecular analysis of several generations (T0 to T3) confirmed the presence and activity of transgenes, simultaneously proving the genetic stability of transformed lines of P. vulgaris (Espinosa-Huerta et al., 2013). Direct methods of gene transfer Since the usage of the straight Agrobacterium strategies in the case of P. vulgaris did not at first yield high frequencies of transformation, the applicability of direct methods of gene transfer was also tested. In the early 1990s reports appeared describing transformation of beans using particle bombardment or electroporation (Aragão et al., 1992, 1996; Dillen et al., 1995; Genga et al., 1992; Russell et al., 1993). The first DNA introduction into navy bean seed meristems via electric discharge-mediated particle acceleration was followed by shoot induction. However that approach to genetic modification of P. vulgaris (cv. Seafarer) brought a low rate (0.03%) of recovery of transgenic plants (Russell et al., 1993). Earlier, Aragão et al. (1992) presented transient expression of Brazil nut 2S-albumin gene in cells of the embryonic axis. The particle bombardment however, did not enable the establishment of reproducible system for stable transformation. Another paper by the same research group (Aragão et al., 1996) reported expression of 2S-albumin gene in transgenic common bean plants. Similar average frequency of stable transgenic plant production (0.9%) was achieved via particle bombardment using a high-pressure helium method and embryogenic axes as a target explant of common bean (Aragão et al., 1996). Continuation of the research on the introduction of the be2s1 gene (one of the Brazil nut's 2S-albumin genes) to improve the methionine content of the common bean seeds brought a promising biolistic procedure. Two of five transgenic lines displayed the increased methionine content (by 14 and 23%) compared to that in non-

Please cite this article as: Hnatuszko-Konka K, et al, Phaseolus vulgaris — Recalcitrant potential, Biotechnol Adv (2014), http://dx.doi.org/10.1016/ j.biotechadv.2014.06.001

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transformed plants (Aragão et al., 1999). Three years later the same research group again used bombardment techniques to transform P. vulgaris cultivars Carioca and Olathe with the bar gene. The bar gene from Streptomyces hygroscopicus encodes phosphinothricin acetyl transferase (PAT) that confers tolerance to the herbicide glufosinate ammonium (GA). Then only 0.5% of the regenerated plants (To) were resistant to the herbicide. However, tests under greenhouse and field conditions evaluation showed that the plants were tolerant to GA (Aragão et al., 2002). This was the first field release of a transgenic line of common bean. The previously reported rates of efficiency of such transformations varied between 0.2 and 0.9%, depending upon common bean variety. Thus, the biolistic technique is still considered as a labour-intensive approach that yields low transformation frequencies. Moreover, the biolistic methods of gene delivery may display drawbacks in the form of complex and uncontrolled pattern of DNA integration and of lack of efficient selection of transformed cells. However, the increase in the recovery of fertile transgenic plants became greater due to the use of the selective herbicidal agent, imazapyr. Firstly, it was reported in soybean (Aragão et al., 2000) and later in dry bean plants (Bonfim et al., 2007). Nevertheless, due to particle bombardment technique some desirable traits were introduced into P. vulgaris plants. The transgenic P. vulgaris lines were incorporated into the breeding programme to test gene expression under greenhouse and field conditions (Gepts et al., 2008). Recently, partial resistance to bean golden mosaic virus (BGMV) in a transgenic common bean (P. vulgaris L.) line expressing mutated rep and bar genes was reported. The BGMV rep gene is essential for virus replication. One of 17 T0 lines displayed tolerance to herbicide and partial resistance to the virus (Faria et al., 2006). A year later, Bonfim et al. (2007) published research aimed at generation of transgenic common bean plants with high resistance to BGMV. The particle bombardment technique was used to enter an RNA interference construct to silence the sequence region of the AC1 viral gene, however the rate of transformation efficiency was reported to be low (0.66%). Eighteen transgenic lines were produced through bombardment of embryonic axes and of them only one line presented high resistance (93% of the plants free of symptoms). Significant progress in the transformation via particle bombardment was reported by Rech et al. (2008). The established protocol combined resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature common bean seeds (cultivars: Olathe Pinto, Pontal) and biolistic techniques. The average frequency of transformation (measured as the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) was 2.7% (Rech et al., 2008). Since the early 1990s the Brazilian researchers have been working on engineering of BGMV-resistant lines of common bean (Aragão and Faria, 2009). After the exploration of different concepts, they developed two lines that showed high BGMV-resistance. In 2009 Aragão and Faria (2009) described their attempts to obtain the first transgenic plant in Latin America. Field trials supported the first evaluation under greenhouse conditions. Moreover, homozygous lines were crossed with non-transgenic common bean plants to gain a hemizygous population. In order to test gene flow both types and the control plants were inoculated using viruliferous whiteflies. It resulted in observations of 100% symptomless homozygous plants and only 28.7% of hemizygous plants showing mild symptoms while wild plants showed severe symptoms. Additionally, transgenic plants and seeds displayed no significant differences in morphological parameters in comparison to wild ones (Aragão and Faria, 2009). Recently, another paper on biolistic bombardment of common bean plants has been published by Kwapata et al. (2012). Shoot apical meristem primordia of five P. vulgaris L. varieties (Condor, Matterhorn, Sedona, Olathe, Montcalm) were genetically modified. Kwapata and colleagues presented the introduction of Barley HVA1 gene and reported development of drought tolerance of transgenic plants at under greenhouse conditions. The use of gus colour marker gene enabled standardisation of the biolistic bombardment to strike

the primordial cell layer. Due to this optimisation the highest transient transformation frequency of gus expression achieved 8.4% (Kwapata et al., 2012). Summarising, before 2013 the biolistic system appeared to be the main effective option for generating fertile transgenic plants of the common bean. Only the report by Espinosa-Huerta et al. (2013) gave primacy to the A. tumefaciens-mediated transformation strategy. However, since to the above paper suggests that there was a significant difference in the response of both cultivars to the overall transformation process (nearly 50%) and area as yet no reports based on this protocol, the question of whether the system by Espinosa-Huerta et al. may be recognised as repeatable and genotype-independent remains open. Therefore, because of the noticeable resistance of the common bean to genetic engineering, all the systems presented have not been sufficiently effective to routinely utilise them for an analysis of the gene function, and thus impeded the advancement in plant improvement. Taking into consideration gene functional analysis in the species not amenable to stable genetic transformation, a promising turning point was reached by the first report on efficient and stable silencing of endogenous genes in common bean by VIGS. Moreover, it should be pointed out that its success also includes the ability of viral vectors to infect the plant species of interest, including P. vulgaris (Díaz-Camino et al., 2011). In the light of these efforts, current knowledge and protocols available it is clear that the attempts to engineer common bean is no minor task. A routine, fast and efficient protocol to transform common bean has not yet been established. However, there is a great need and pressure for establishment of both repeatable regeneration and transformation procedures that would yield a reasonable number of transgenic P. vulgaris plants. Thus, considering the scientific community involvement, it seems that overcoming of the existing obstacles is only a question of time. Conclusions Improvement of P. vulgaris with the use of genetic engineering is inevitable. Sooner or later its regeneration and transformation recalcitrance shall be overcome. In a way, it already is, apart from its economic feasibility. The scientific attempts and numerous publications have offered some general directions that in recent years resulted in the development or optimisation of both regeneration and transformation processes. However, it is obvious that genetic engineering is an option, which necessitates the availability of a repeatable and efficient in vitro system. Some critical tissue culture steps appeared to have been initially perceived. To increase the regeneration potential various approaches were used. Scientists endeavour to influence plant competency by precultivation of parent plants to stimulate the division of competent cells (Cruze de Carvalho et al., 2000; Veltcheva and Svetleva, 2005). The question of the impact of morphological integrity of the donor plant was also raised (Ahmed et al., 2002). As seed legumes are more prone to root than to shoot formation, the significance of basal medium and/or hormone combinations used is under permanent evaluation (Barikissou and Baudoin, 2011; Gatica Arias et al., 2010; Kwapata et al., 2010). And here one also perceives divergence in reports describing varied responses depending on variety (Delgado-Sánchez et al., 2006; Mukeshimana et al., 2013; Quintero-Jiménez et al., 2010). The issue of medium formula was raised also due to “the phenolic problem” that is thought to hamper the in vitro response and in connection with the rooting handicap that hinders the whole plant regeneration and the soil breeding stage (Kwapata et al., 2010). Consequently it often causes plant decay. Fortunately, the establishment or improvement of several tissue culture approaches have been presented recently for various common bean genotypes (Arellano et al., 2009; Gatica Arias et al., 2010; Kwapata et al., 2010). However, in the light of numerous reports it

Please cite this article as: Hnatuszko-Konka K, et al, Phaseolus vulgaris — Recalcitrant potential, Biotechnol Adv (2014), http://dx.doi.org/10.1016/ j.biotechadv.2014.06.001

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seems that the screening and development of cultivar-specific regeneration protocol would be more probable and attainable than achievement of one model bean within Phaseolus genus. Since regeneration determines the efficient transformation process it would be optimal to use a plant explant highly competent for both tissue culture and genetic engineering. Such convergence would be helpful as in the case of Mukeshimana et al. (2013) who reported that leaf explants of common bean showed the highest susceptibility to A. tumefaciens among all genotypes tested but they were not yet regenerable. Hence, the embryo axes of common bean remain the optimal explants that enable Agrobacterium-mediated gene transformation and subsequent in vitro response (Mukeshimana et al., 2013). However, despite the clearly displayed susceptibility to A. tumefaciens strains, there is one confirmed report of stably transgenic P. vulgaris plants after the use of an A. tumefaciens based system (Amugune et al., 2011; Espinosa-Huerta et al., 2013). It is the combination of indirect transformation and support in the form of sonication or particle bombardment that yields one of the best results (Brasileiro et al., 1996; Liu et al, 2005). Interestingly, in works by Brasileiro et al. (1996) the tepary bean showed lower susceptibility to classic agro-infection but currently it is actually the Phaseolus acutifolius that can be routinely transformed by A. tumefaciens (De Clercq et al, 2002; Zambre et al., 2005). Nevertheless, it may appear that in a short term consideration the biolistic devices might outrun the straight Agrobacterium strategies in the case of P. vulgaris. The transgenic plants that have undergone the first field trials were modified by biolistic bombardment methods (Aragão and Faria, 2009; Bonfim et al., 2007) while there are only few reports about Agrobacterium-mediated transformation that ended with the whole plant regeneration. Thus, for now it would seem more probable that the increase in transformation frequency will occur through further optimisation of biolistic devices. However, the number of laboratories that work on agroinfection of common bean is sufficient to believe that it is only a question of time and the optimal parameters for Agrobacterium approach will be established. And that has already been reflected by the example of report by Espinosa-Huerta et al. (2013) that caused that the efficient and robust protocol by Estrada-Navarrete et al. (2006, 2007) that uses A. rhizogenes to induce hairy root formation is no longer the only exception in the production of transgenic plants via indirect strategy. The overcoming of P. vulgaris recalcitrance towards genetic engineering is especially important as genetic transformation is a powerful tool to gain valuable profile on gene expression and functions. It would also enable research on common bean diversity that seems narrow, which is a serious problem for breeders who must overcome these limitations. Hence, both tissue culture and genetic transformation might become sources of new diversity giving breeders more useful genetic variants. However, as common bean cultivated in a particular region has a unique set of biotic and abiotic constraints genetic modification should reflect the needs of the farmers, who will use the cultivars (Beaver and Osorno, 2009). The question of future prospects in the field of common bean research remains open. On one hand the number of laboratories working on the know-how of Phaseolus regeneration and transformation together with combined funds is quite large. That enables to believe that it is a question of time as both the regeneration potential of P. vulgaris and its recalcitrance towards genetic engineering will be controlled. On the other hand after a dozen or so years of scientific efforts the level of achievements is not spectacular. Now, what should be our expectations in this case? What trends should be executed? The establishment of the economically feasible protocols for regeneration that would be fully common bean genotype-independent seems rather a long-distance perspective. As we have already mentioned we believe rather in concentrating efforts to wide screening and cooperation in optimisation of procedures for selected narrow pool of cultivars. Such optimisations should include determination of a plant explant highly competent for both tissue culture and transformation, conducted rather according to

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the protocols of supported agroinfection. Among other possible approaches there is a subject of our present investigations — searching for the alternative bacterial vectors of transformation (data not shown). Nevertheless, such concentrated activities require certain precision and convergence in results reporting. That would extremely simplify their comparison and monitoring of the actual levels of regeneration and transformation frequencies. For now, the determination of the current status of the research on those processes, while we use various cultivars, various basal media, various phytohormones, various explants, various frequency definitions and several other various parameters, is not a trivial undertaking. References Ahmed EE, Bisztray G, Velich I. Promoting shoot organogenesis on different explant seedlings of common bean. Bull Veg Crop Inst 1998;28:33–8. Ahmed EE, Bisztray G, Velich I. Plant regeneration from seedling explants of common bean (Phaseolus vulgaris L.). 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