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Research review paper
Phaseolus vulgaris Recalcitrant potential
Katarzyna Hnatuszko-Konka
, Aneta Gerszberg
Aneta Wiktorek-Smagur
, Andrzej K. Kononowicz
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
abstractarticle info
Article history:
Received 31 January 2014
Received in revised form 4 June 2014
Accepted 5 June 2014
Available online 20 June 2014
Phaseolus vulgaris
Common bean
Since the ability to genetically engineer plants was established, researchers have modied a great number of
plant species to satisfy agricultural, horticultural, industrial, medicinal or veterinary requirements. Almost thirty
years after the rst approaches to the genetic modication 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 re-
generation and rooting signicantly hampers the possibilities of improvement of the common bean that suffers
from many biotic and abiotic constraints. Thus, an efcient 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 statusof tissue culture and recent progressin transformation methodologyare presented. Some
major challenges and obstacles are discussed and some examples of their solutions are presented.
© 2014 Elsevier Inc. All rights reserved.
Introduction................................................................ 1205
Regeneration................................................................ 1206
Obstaclesandsolutionsinregenerationprotocols............................................. 1206
Indirectapproachesasararepromisingphenomenon........................................... 1208
Therecapitulationofthetissueculturestatusincommonbean....................................... 1208
Transformation................................... ............................ 1209
Straight and combined Agrobacterium-mediatedtransformation ...................................... 1210
Directmethodsofgenetransfer..................................................... 1211
Conclusions ................................................................ 1212
References................................................................. 1213
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 responsivenessto in vitro regeneration compared to the forage le-
gumes (Veltcheva and Svetleva, 2005). This is also the case for Phaseolus
vulgaris, an economically important crop. The common bean is the most
important food legume for direct human consumption in several coun-
tries 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 specied in the Common Catalogue of
Varieties of Vegetable Species (2011) (including dwarf and climbing
ones). It seems completely justied as beans combined with cereals as-
sure a balanced diet of energy and protein. Bean seeds provide impor-
tant minerals, vitamins, dietetic bre 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
Biotechnology Advances 32 (2014) 12051215
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42 635 44 23.
E-mail addresses: (K. Hnatuszko-Konka),
(T. Kowalczyk), (A. Gerszberg),
(A. Wiktorek-Smagur), (A.K. Kononowicz).
0734-9750/© 2014 Elsevier Inc. All rights reserved.
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authors it is difcult 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 diverseenvironmental 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 deciencies
(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 pro-
teins of pulse crops in sulphur-containing amino acids, changing the
plant anatomy or reducing the time needed for owering and seed set-
ting in long duration crops (Eapen, 2008). Also the usage of legumes as
green factoriesseems completely justied.
Consequently, several international initiatives (the Medicago Ge-
nome Consortium; International Conferences on Legume Genomics
and Genetics ICLGG) that concentrate primarily on the eld of legume
genomics and genetics (Colpaert et al., 2008). For P. vulgaris studies,
an international consortium —‘Phaseomicswas 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 re-
generation 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 difcult to determine
whether beans are generally not amenable to regeneration or transfor-
mation 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 regenerationand transforma-
tion processes and then the concentrated efforts of plant scientists en-
abled success in the eld 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 efcient 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 ques-
tion. 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.
Tissue culture of P. vulgaris is repeatedly considered to be difcult. It
is particularly inconvenient as the lack of a rapid and efcient regener-
ation system hampers possibilities of its genetic improvement. Al-
though 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, appli-
cation 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-specic 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 regener-
ation processes (Svetleva et al., 2003). Thus, genotype limitations
indirectly underliethe difculty in development of routine regeneration
procedure for legumes or even beans. It is unquestionable that beans
demonstrate extremely high diversity regarding regeneration respon-
siveness. All three classic pathways of in vitro propagation (organogen-
esis, somatic embryogenesis and proliferation of shoot meristems from
the regions surrounding the shoot bud) were described for the common
bean with limited efciency and low repeatability. In consequence the
necessity of a genotype-dependent and cultivar-specic procedure is
Most of the published procedures were based on direct organogen-
esis 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 embryogene-
sis it occurs rather sporadically (Jacobsen, 1999; Kwapata et al., 2010;
Martins and Sondahl, 1984; Nae et al., 2013). Nevertheless, until
now severaltypes of cells, tissues and organs (cotyledonary nodes, em-
bryonic axes, auxiliary shoots, cotyledon with split embryo axis, inter-
nodes, 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; Thoetal.,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 path-
ways 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 mor-
phogenesis 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 transforma-
tion, the true term regenerationfunctions in a narrower context. Re-
generation 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: organo-
genesis 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. cal-
lus) processes. As these terms are used in many different ways in the lit-
erature their precise usage in the scientic 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 problemsdiscussing
the ways they have been solved.
Obstacles and solutions in regeneration protocols
It may be concluded from scientic reports that some obstacles in
the regeneration process have been identied and currently efforts
are made to eliminate them. According to many authors it is possible
to inuence 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 inuence
1206 K. Hnatuszko-Konka et al. / Biotechnology Advances 32 (2014) 12051215
the regeneration potential (Cruze de Carvalho et al., 2000; Svetleva
et al., 2003). For example, in comparison to the control medium,
Veltchevaand Svetleva (2005) achieved a 57-fold increase in the num-
ber of regenerants per explant by precultivation of seedlings on 1 μM
(0,2 mg/L) BAP containing MS (Murashige&Skoog) medium. This al-
ternation of in vitro response yie lded the enhancement of i ndirect or-
ganogenesis 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 oc-
curred more efciently if a leaf explant included both petiole and a por-
tion of lamina (Malik and Saxena, 1991) or the intact seedling was used
as primary material (Malik and Saxena, 1992). Their observation is
reected in the report by Ahmed et al. (2002) in which the question
of inuence 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 coty-
ledonary node explants (MS + 1 mg/L BA and 0.1 mg/L NAA). Fur-
thermore, 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 theanalysis of the medium composition in 2006 a
promising direct organogenic bud induction protocol was established
by Delgado-Sánchez and colleagues. The regeneration of whole com-
mon bean plants from embryonic axes of mature seeds was reported.
Its efciency 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). Inter-
estingly, the winning response was achieved on MS medium supple-
mented with 5 or 10 mg/L BAP (and adenine, but its inuence was not
revealed). This observation varied considerably from the previous pa-
pers as lower BAP concentrations (0.12.5 mg/L) were already used
for the response induction. The efciency obtained was then much
lower, which suggests positive correlation between BAP concentration
and the number of organogenic buds (Delgado-Sánchez et al., 2006;
Tho 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 con-
centrations of cytokinins are needed for shoot regeneration compared
to other plant families (Veltcheva et al., 2005). As the procedure devel-
oped by Delgado-Sánchez brought the promising efciency of whole
plant production (from 25% to 83% depending on the cultivar), the
same research group reported an improved direct organogenesis proto-
col in 2010. The common bean shoot induction from embryonic axes
was achieved in four cultivars through modication of medium condi-
tions (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 dif-
ferent common bean cultivars). The Gamborg medium supplemented
with 10 mg/L BA yielded the highest regeneration efciency (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 efciency. The
difference in response from embryonic axes is evident: while Gamborg
induced high organogenic shoot formation (98100%) and whole plant
regeneration (93%), the Murashige and Skoog medium initiated lower,
inconsistent organogenic shoot formation (1573%) and whole plant
regeneration (29%) (Quintero-Jiménez et al., 2010). While most of sci-
entists use MS medium exclusively Mukeshimana et al. (2013) have re-
cently 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 ade-
nine (A) or adenine sulphate (AS) (respectively) for organogenic
shoot formation in P. vulgaris. The obtained results are consistent as, re-
gardless of basal medium, no signicant 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 obtain-
ed 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 justied, 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 ad-
enine 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 efciency 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 ve Costa Rican
varieties and the number of shoots and leaves noticeably differed
among them again suggesting a cultivar-specic protocol (Gatica Arias
et al., 2010). The morphology of shoot regeneration induced from em-
bryonic 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 conrmed the stimulating inuence 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 inef-
cient in vitro shoot production remains one of the most signicant obsta-
cles 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; Tho et al., 2013).
Such limitations in the establishment of tissue culture were reported
for many species like Pinus sylvestris (Laukkanen et al., 1999)orMusa
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 intensied 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 con-
centrations (510 mg/L
) of these hormones to enhance the number
of shoots induced should be reconsidered. For example, such an en-
hanced 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 phe-
nolic problemwas reported by Kwapata et al. (2010).Whencompared
to the control medium with no antioxidants, the supplementation of
charcoal, silver nitrate, glutathione or ascorbic acid signicantly in-
creased 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 modication of the induction medium enabled
1207K. Hnatuszko-Konka et al. / Biotechnology Advances 32 (2014) 12051215
development of an efcient 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 contain-
ing IBA, IAA or NAA. It is signicant 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 Delgado-
Sánchez 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 elimina-
tion of phenolic compound activity and use of activated charcoal. The
benecial effect of activated charcoal for in vitro rooting was also report-
ed by Barikissou and Baudoin (2011) where the role of activated charcoal
in the micro cutting medium was evaluated. The use of activated char-
coal was shown to effectively increase the regeneration rate of rooted
in vitro plantlets to ca 90% (compared to 43% achieved on the control me-
dium) (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 N
-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 frequen-
cy 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 metabo-
lism of endogenous cytokinins). The subsequent shoot formation fre-
quency varied from 61.3 to 87.6% depending on AgNO
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 efciency.
Also the benecial inuence of silver nitrate was emphasised by
Kwapata who presented the increase in regeneration frequency and de-
velopment 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 medi-
um formulation must be made specically for a particular cultivar
(Gatica Arias et al., 2010; Kwapata et al., 2010). Since a routine
genotype-independent pathway of regeneration is the most impor-
tant requirement for successful genetic transformation of Phaseolus
spp., such an approach would mean a signicant 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 efcient callus production was achieved on 1.5 μM(0.3mg/L)
2,4-dichlorophenoxyacetic acid (2,4-D) containing MS medium.
Two-week old calli were transferred to a shooting medium contain-
ing 22.2 μM (5 mg/L) BAP. After four weeks of incubation the number
of well developed shoots (stem with leaves 1.02.0 cm long) regenerat-
ed per callus was evaluated and shoots were placed on the rooting me-
dium 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 approxi-
mately 0.5 shoots per callus, the procedure may be a subject of further
improvement and should be regarded as a very promising basis for es-
tablishing a protocol for P. vulgaris.
It is well known that plant regeneration from different varieties of
P. vulgaris callus cultures in vitro is difcult to achieve. Two proce-
dures of indirect organogenesis reported by Mohamed et al. (1993)
and by Zambre et al. (1998) appeared to be highly genotype-specic.
Thus far, the Arellano's (2009) protocol for indirect regeneration was
the rst genotype-independent approach to plant regeneration of com-
mon 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) coty-
ledons dissected from the embryonic axis of three-day-old germinated
seeds of ve 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 path-
way. 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 per-
centage of callus formation in explant from 12-month-old seeds was sig-
nicantly lower (97%, 91%, 89%, and 56%, respectively) (Collado et al.,
In the studies conducted in the Department of Genetics, Plant Mo-
lecular 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). Thestudies were conducted on randomly chosen cultivars, Casa-
blanca, Laponia and Plus (data not published)(Fig. 1). The already men-
tioned 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 phytohor-
mone 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 briey presented below (Table 1).
Numerous attempts were made to develop in vitro efcient and re-
peatable regeneration by direct and indirect organogenesis or
direct and indirect somatic embryogenesis (Veltcheva et al., 2005). Con-
sequently, reports on common bean in vitro regeneration are available,
however mostly with results differentiated among genotypes. Accord-
ing to Hammatt et al. (1986) the common recalcitrance observed
among large seeded legumes results from reduction of genetic variabil-
ity. 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 inter-
mediate. The rst 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
1208 K. Hnatuszko-Konka et al. / Biotechnology Advances 32 (2014) 12051215
manipulation, direct pathways are more suitable as they do not enter
genetic variation.
Nevertheless, at the moment it is still difcult to designate a routine
in vitro system for common bean, which would be fully genotype-
It has been almost thirty years since the rst attempts to produce
transgenic pulse crops were undertaken. Since the 1980s brought a
breakthrough in genetic engineering some papers on legume transfor-
mation were also reported (Eapen et al., 1987; Kohler et al., 1987a,
1987b). The rst publication on the successful development of a trans-
genic member of Leguminosae family (Vigna unguiculata) was presented
by Garcia et al. (1986). Although attempts at whole plant regeneration
failed, it was conrmed that a transgenic callus had originated from
transgenic mesophyll cells. The protocol that adopted leaf disc transfor-
mation 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 trans-
genic 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
rst 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 repre-
sents one of the few species within the grain legumes, which show rea-
sonable potential for genetic engineering. Hence, P. acutifolius was
suggested for use as a bridge speciesto introgress transgenes into the
P. vulgaris plants (Dillen et al., 2000; Popelka et al., 2004). Earlier, at-
tempts to genetically modify the common bean via Agrobacterium-
mediated 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, inde-
pendent of transformation techniques. And almost three decades later
this observation still appears to be true.
Among the target species no efforts are spared to achievean efcient
protocol for stable transformation of legumes, which contribute almost
30% of the world's major crop production (Arellano et al., 2009). Al-
though it is difcult 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 chromo-
somes and a medium-sized genome (the haploid genome size ranges
from 588 to 637 Mbp (Gepts et al., 2008; McClean et al., 2008)). Mean-
while however, P. vulgaris proved to be one of the most recalcitrant spe-
cies 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). Althoughat present numerous reportsare 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 difculty concerning achieving efcient transformation
of that key species(and legumes in general), which is related to their
low responsiveness to in vitro regeneration. Efcient 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, comparativegenomics and common beanimprove-
ment (Gatica Arias et al., 2010). Among the factors contributing to the
lack of progress in developing transgenic pulse crops the lack of compe-
tent 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, cotyle-
donary 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 im-
mature 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 specicparameters. For example Agrobacterium-mediated
transformation depends on the type and physiological state of an ex-
plant 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
Fig. 1. Stages ofregeneration of Polish cultivar of common bean (Laponia) on the medium supplemented with growth regulators (medium compositions not published). A seed germi-
nation, B hypocotyl explants, C non-morphogenic callus induction, D, E shoot multiplication, F, G plantlet rooting, H regenerated Phaseolus vulgaris plant.
1209K. Hnatuszko-Konka et al. / Biotechnology Advances 32 (2014) 12051215
transformation rate and it cannot be underestimated. Until now, its in-
uence seems to be inevitable regardless of an engineering technique.
Also the age and origin of a common bean explant are similarly signi-
cant in agroinfection as they are in biolistic devices. Namely, according
to Aragão and Rech (1997) the morphology of explants may affect the
generation of modied 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 efciency of the common
bean transformation via the bombardment of apical meristematic re-
gions. Indirectly one returns to the genotype/variety dependent proce-
dures. As has been mentioned, a number of researchers work to nd
solutionsto bean recalcitrance to genetic engineering. Some of them in-
vestigate the regeneration capacity of different common bean explants;
others examine the inuence of various phytohormones and/or differ-
ent 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
the last eightten 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 efcient routine trans-
formation 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 num-
ber of P. vulgaris cultivars, using both Agrobacterium and biolistic-
mediated 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 trans-
genic roots on wild type de-rooted seedlings (Colpaert et al., 2008;
Estrada-Navarrete et al., 2006; Estrada-Navarrete et al., 2007).
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
Some of the plant pathogens do not survive
long storage conditions thus the older seeds
may be favourable (Trapiello and González,
Seed quality (e.g. vigour) Reliable seed origin
Presence of endophytic or pathogenic
Certied seeds free from pathogens,
bactericide treatment
Obstacle usually not mentioned in scientic
papers, however visual detection of
contamination is unreliable, seeds may appear
healthy in spite of contamination (Gent and
Ocamb, 2014)
Common bean cultivar Screening
Low regeneration
The regeneration potential Seed age the signicance of seed age for
in vitro regeneration has been reported
suggesting fresh seed usage
E.g. formation of morphogenic callus occurred
more frequent when explants originate from
fresh seeds
Preculture of parental plants on a medium
stimulating proliferation of competent cells
E.g. medium supplemented with BAP, TDZ or
The age factor young cells/tissues have
higher potential of in vitro respond (also
while being elicited by stimulation with
exogenous hormones)
Experimental evaluation required according
to many reports too young explants are more
susceptible to necrosis
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
E.g. divergence in the requirements: intact
seedling display stronger regeneration
respond while cotyledon nodes appear to be
better transformation target
Medium concerns both basal medium type
and hormonal composition
Experimental evaluation required
Explant type Divergence in the requirements: explant
suitable for regeneration may not be an
efcient as a transformation target
Common bean cultivar screening
Inefcient in vitro shoot
Prone to root formation rather than to
shoot formation
Manipulation of medium composition
match of nature and rate of cytokinin used
(however effect on shoot induction may be
Exemplary question of BAP (already discussed
in the text) different concentrations may
induce stimulation or decrease of propagation
Presence of phenolic compounds and
their connection with brown callus
Charcoal, silver nitrate, glutathione or ascorbic
acid supplementation was proven to stimulate
development of multiple shoots in vitro
(indirectly via limitation in brown callus
Synthesis of phenolic compounds may be
stimulated by cytokinins, therefore
application of cytokinin high concentrations
for shoots induction is open to question
Common bean cultivar Screening
Poor rooting Shoot size/stage of shoot development The choice of shootlets competent to rooting
according to some reports only large (N2-cm
long) shoots are capable of root development
Rooting medium composition The choice of basal medium type and
hormonal composition pretreatment of shoots
with IBA and transfer to the rooting media
Experimental evaluation required
Presence of phenolic compounds Use of activated charcoal was shown to
increase efciency of root development
Common bean cultivar Screening
1210 K. Hnatuszko-Konka et al. / Biotechnology Advances 32 (2014) 12051215
Estrada-Navarrete et al. (2006) established such a reproducible and ef-
cient procedure for P. vulgaris. The seedlings of several cultivars, land-
races and accession of common bean were infected and transformation
efciency 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 inuence
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,
rootmicrobe 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 engi-
neering 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 varie-
ties. The general observations relating to plant genotype were iden-
tical and all strains (AT 2553, AT 8196, T37, 82.139, Bo524, Ach5, R10,
15955) were shown to be virulent in the chosen cultivars. Addition-
ally, common bean appeared to be more susceptible to A. tumefaciens
strains than tepary bean. Interestingly, as the Agrobacterium co-
cultivation strategy had not envisaged its reproducible use, the
new approach was established. The classic Agrobacterium-mediated
transformation of bean was preceded by wounding embryo meri-
stems with microprojectiles (Brasileiro et al., 1996). The fact that
the bombardment pre-treatment enhanced the percentage of tu-
mour formation when compared with the inoculation without bom-
bardment (from 30% to 50%70%) clearly weighs in favour of the
combined strategy.
Hence, another paper describing successful transformation of com-
mon bean by A. tumefaciens has been published presenting a protocol
based on the combination of sonication and vacuum inltration
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 efciencies of transforma-
tion (12%) of P. vulgaris plants. The optimal conditions included
Agrobacterium-mediated transformation of seedlings followed by
5 min sonication and 5 min vacuum inltration and it enabled genera-
tion of transgenic kidney bean with late embryogenesis abundant
gene from Brassica napus(Liu et al., 2005). It prevails even over more re-
cent reports on biolistics-based systems, however until now no subse-
quent 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
In 2013 Mukeshimana et al. (2013) presented the study evaluat-
ing factors inuencing transient and stable transformation of com-
mon bean using A. tumefaciens. The transformation was preceded
by optimisation of regeneration of four cultivars representing vari-
ous 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
axis explants displayed limited recalcitrance to regeneration. Interesting-
ly, 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 inuencing gene deliv-
ery 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 re-
ports (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 em-
bryo 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. tumefaciens-
mediated 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 efciencies ever 1028% with cer-
tain 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 efcient
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 Quintero-
Jiménez et al. (2010). Having regenerated the common bean cultivars
(Flor de Mayo Anita and Pinto Saltillo), Espinosa-Huerta et al. success-
fully 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 transforma-
tion frequency of at least 10% regardless of strain used suggests that the
reported protocol is a viable alternative to other transformation sys-
tems. Moreover, molecular analysis of several generations (T0 to T3)
conrmed 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 rst yield high frequencies of transfor-
mation, the applicability of direct methods of gene transfer was
also tested. In the early 1990s reports appeared describing transforma-
tion 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 rst DNA introduction into navy bean seed meristems via
electric discharge-mediated particle acceleration was followed by
shoot induction. However that approach to genetic modication of
P. vulgaris (cv. Seafarer) brought a low rate (0.03%) of recovery of trans-
genic plants (Russell et al., 1993). Earlier, Aragão et al. (1992) presented
transientexpression of Brazil nut 2S-albumin gene incells of the embry-
onic axis. The particle bombardment however, did not enable the estab-
lishment of reproducible system for stable transformation. Another
paper by the same research group (Aragão et al., 1996) reported expres-
sion of 2S-albumin genein transgenic common bean plants. Similar av-
erage 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 ve transgenic lines displayed the increased
methionine content (by 14 and 23%) compared to that in non-
1211K. Hnatuszko-Konka et al. / Biotechnology Advances 32 (2014) 12051215
transformed plants (Aragão et al., 1999). Three years later the same re-
search 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 eld
conditions evaluation showed that the plants were tolerant to GA
(Aragão et al., 2002). This was the rst eld release of a transgenic
line of common bean. The previously reported rates of efciency 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 frequen-
cies. Moreover, the biolistic methods of gene delivery may display
drawbacks in the form of complex and uncontrolled pattern of DNA in-
tegrationand of lack of efcient 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 (Bonmetal.,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 pro-
gramme to test gene expression under greenhouse and eld conditions
(Gepts et al., 2008). Recently, partial resistance to bean golden mosaic
virus (BGMV) in a transgenic common bean (P. vulgaris L.) line express-
ing mutated rep and bar genes was reported. The BGMV rep gene is es-
sential 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, Bonm et al. (2007) published research aimed at generation of
transgenic common beanplants withhigh resistance to BGMV. The par-
ticle 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 efciency was reported to be low (0.66%).
Eighteen transgenic lines were produced through bombardment of em-
bryonic axes and of them only one line presented high resistance (93%
of the plants free of symptoms). Signicant progress in the transforma-
tion 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 (mea-
sured as the total number of fertile transgenic plants divided by the
total number of bombarded embryonic axes) was 2.7% (Rech et al.,
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 rst transgenic plant in
Latin America. Field trials supported the rst evaluation under green-
house conditions. Moreover, homozygous lines were crossed with
non-transgenic common bean plants to gain a hemizygous population.
In order to test gene ow both types and the control plants were inoc-
ulated using viruliferous whiteies. 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 signicantdiffer-
ences 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 ve P. vulgaris L. varieties (Condor,
Matterhorn, Sedona, Olathe, Montcalm) were genetically modied.
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 signicant
difference in the response of both cultivars tothe 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 sufciently effective to routinely utilise them for an analysis
of the gene function, and thus impeded the advancement in plant
Taking into consideration gene functional analysis in the species
not amenable to stable genetic transformation, a promising turning
point was reached by the rst report on efcient 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 efcient protocol to transform
common bean has not yet been established. However, there is a
great need and pressure for establishment of both repeatable regen-
eration and transformation procedures that would yield a reasonable
number of transgenic P. vulgaris plants. Thus, considering the scien-
tic community involvement, it seems that overcoming of the
existing obstacles is only a question of time.
Improvement of P. vulgaris with the use of genetic engineering is
inevitable. Sooner or later its regeneration and transformation recal-
citrance shall be overcome. In a way, it already is, apart from its eco-
nomic feasibility. The scientic attempts and numerous publications
have offered some general directions that in recent years resulted in
the development or optimisation of both regeneration and transfor-
mation processes. However, it is obvious that genetic engineering is
an option, which necessitates the availability of a repeatable and ef-
cient 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 inuence 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 signicance of basal
medium and/or hormone combinations used is under permanent eval-
uation (Barikissou and Baudoin, 2011; Gatica Ariaset al., 2010; Kwapata
et al., 2010). And here one also perceives divergence in reports describ-
ing varied responses depending on variety (Delgado-Sánchez et al.,
2006; Mukeshimana et al., 2013; Quintero-Jiménez et al., 2010). The
issue of mediumformula 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 cul-
ture 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
1212 K. Hnatuszko-Konka et al. / Biotechnology Advances 32 (2014) 12051215
seems that the screening and development of cultivar-specic regener-
ation protocol would be more probable and attainable than achieve-
ment of one model bean within Phaseolus genus. Since regeneration
determines the efcient transformation process it would be optimal to
use a plant explanthighly 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 ge-
notypes 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 conrmed
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 sup-
port in the form of sonication or particle bombardment that yields
one of the best results (Brasileiro et al., 1996; Liu et al, 2005). Interest-
ingly, 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
rst eld trials were modied by biolistic bombardment methods
(Aragão and Faria, 2009; Bonmetal.,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 sufcient
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 reected by the example of report by Espinosa-Huerta et al.
(2013) that caused that the efcient 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 engi-
neering is especially important as genetic transformation is a powerful
tool to gain valuable prole on gene expression and functions. It
would also enable research on common bean diversity that seems nar-
row, 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 modication
should reect the needs of the farmers, who will use the cultivars
(Beaver and Osorno, 2009).
The question of future prospects in the eld of common bean re-
search 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 scientic 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 beangenotype-independent seems rather a long-distance per-
spective. As we have already mentioned we believe rather in concen-
trating 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
the protocols of supported agroinfection. Among other possible ap-
proaches there is a subject of our present investigations searching
for the alternative bacterial vectors of transformation (data not
shown). Nevertheless, such concentratedactivities require certain preci-
sion and convergence in results reporting. That would extremely simpli-
fy their comparison and monitoring of the actual levels of regeneration
and transformation frequencies. For now, the determination of the cur-
rent status of the research on those processes, while we use various cul-
tivars, various basal media, various phytohormones, various explants,
various frequency denitions and several other various parameters, is
not a trivial undertaking.
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... Some recent examples of direct organogenesis may be found in the literature [10]. 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 the regeneration pathways [1,12,19]. Cotyledons with part of the embryonic axis as explained have been used to regenerate whole plants [20]. These protocols are very efficient. ...
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The study was conducted to study the growth and development of root-knot nematode, Meloidogyne incognita of Phaseolus vulgaris in tissue culture media. The data revealed the developmental common bean cv. Paulista of M.S media as the following: the seed swelling stage after three days, the radical appearance stage after five days. The cotyledons leaves appear after six days, the cotyledon leaves appear after eight days, and the seedling stage after twelve days. The life cycle of M. incognita completed about thirty-seven days in root tissues of Paulista cultivar. Microscopic examinations of the infected bean roots revealed the following: Second-stage juveniles of M. incognita penetrated the roots within 24 hrs after nematode inoculation. Third-stage juveniles were observed in roots (six-eight) days after nematode inoculation. While, fourth stage juveniles were observed in roots 12-14 days after nematode inoculation. The pre-adult females were seen inside roots within twenty days after nematode inoculation. The adult females were seen inside roots within twenty-seven days after nematode inoculation. Egg laying females with deposit egg masses of M. incognita was observed thirty-seven days after nematode inoculation.
... The validation of candidate genes in dry bean is challenging because the lack of an efficient transformation pipeline and its recalcitrance to in vitro regeneration (Baloglu et al., 2022;Hnatuszko-Konka et al., 2014). However, promising candidate genes were identified by integrating MQTL-YC and reported QTN. ...
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Increasing seed yield in common bean could help to improve food security and reduce malnutrition globally due to the high nutritional quality of this crop. However, the complex genetic architecture and prevalent genotype by environment interactions for seed yield makes increasing genetic gains challenging. The aim of this study was to identify the most consistent genomic regions related with seed yield components and phenology reported in the last 20 years in common bean. A meta-analysis of quantitative trait locus (QTL) for seed yield components and phenology (MQTL-YC) was performed for 394 QTL reported in 21 independent studies under sufficient water and drought conditions. In total, 58 MQTL-YC over different genetic backgrounds and environments were identified, reducing threefold on average the confidence interval (CI) compared with the CI for the initial QTL. Furthermore, 40 MQTL-YC identified were co-located with 210 SNP peak positions reported via genome-wide association (GWAS), guiding the identification of candidate genes. Comparative genomics among these MQTL-YC with MQTL-YC reported in soybean and pea allowed the identification of 14 orthologous MQTL-YC shared across species. The integration of MQTL-YC, GWAS, and comparative genomics used in this study is useful to uncover and refine the most consistent genomic regions related with seed yield components for their use in plant breeding.
... Common bean (Phaseolus vulgaris L.) is one of the most important legume for human consumption in various countries worldwide (Hnatuszko-Konka et al., 2014). Common bean is the most consumed species of the Phaseolus genus due to its good nutritional values and its socioeconomic importance (Broughton et al., 2003;Companhia Nacional de Abastecimento [CONAB], 2019;Chimenez-Franzon et al., 2022). ...
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The present work aimed to select populations for the breeding of common bean targeting fusarium wilt resistance and grain yield. Twelve carioca bean lines, which mainly differ in fusarium wilt resistance and grain yield, were crossed in a 6x6 partial diallel scheme. The parents and their 36 F1’s hybrids were evaluated for fusarium wilt severity caused by FOP isolate UFV 01 (FWS), and grain yield (YIELD). 34 F4’s populations, 12 parents and three other lines were also evaluated for grain yield per plant. The data of F1's parents and hybrids were submitted to diallel analysis. Using the grain yield data per plant, the potential of the 34 F4’s populations was predicted by the Jinks and Pooni method (1976). In the diallel analysis, BRSMG Talismã, CVIII 8511, BRS Pérola, VC 25 and VC 13 stood out in terms of the frequency of favorable alleles for FWS. Except for BRSMG Uai and IAC Formoso, these lines presented the most dominant genes associated in Fusarium wilt resistance. For YIELD, there was a predominance of dominant genes determining higher yield. The 20 F4’s populations with the highest potential included the best 12 of the 20 populations, based on diallel analysis for YIELD. Thus, the 12 populations received the addition of four that were highlighted only by the methodology of Jinks and Pooni (1976), and four based on diallel analysis, which totaled 20 populations. The use of information from more advanced inbreeding generations in complementarity with those of diallel is a promising strategy.
... The most important limitation of crop improvement using gene-editing tools is the lack of suitable protocols for plant regeneration from somatic transformed cells for many crops [32]. Antibiotic or herbicide resistance and/or marker green fluorescen protein (GFP) or β-glucuronidase (GUS) genes are included to ease the selection of transformed cells. ...
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Clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein (CRISPR/Cas) gene editing has become a powerful tool in genome manipulation for crop improvement. Advances in omics technologies, including genomics, transcriptomics, and metabolomics, allow the identification of causal genes that can be used to improve crops. However, the functional validation of these genetic components remains a challenge due to the lack of efficient protocols for crop engineering. Hairy roots gene editing using CRISPR/Cas, coupled with omics analyses, provide a platform for rapid, precise, and cost-effective functional analysis of genes. Here, we describe common requirements for efficient crop genome editing, focused on the transformation of recalcitrant legumes, and highlight the great opportunities that gene editing in hairy roots offers for future crop improvement.
... These could present major scientific opportunities for Phaseolus genetics research, as they could be used to provide direct, rather than circumstantial, evidence of gene function. The main hurdle for these approaches is the difficulty in regenerating Phaseolus through in vitro tissue culture and similar methods (Hnatuszko-Konka et al. 2014). Transgenic common bean lines have been developed through at least two main techniques: biolistics and Agrobacterium infection. ...
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Genetic vulnerability refers to (sometimes catastrophic) actual or potential losses in the production of a crop (in quantity and/or quality), attributable to spatial or temporal reduction in the crop's biodiversity. Conversely, genetic resilience refers to the natural and anthropic capabilities of this biodiversity to mitigate these reductions in crop production. Here, an assessment is provided of genetic vulnerability and resilience of Phaseolus beans, which provide an abundant and sustainable source of protein and micronutrients for populations around the world. We provide an overview of the economic, nutritional, and cultural role of Phaseolus beans and phylogenetic and diversity analyses of the genus, its five domesticated species, and seven domestications, which provide key foundational information for this appraisal. We then assess the uniformity of the crop in the United States and the main drivers of genetic erosion in the centers of origin of the genus in the Americas. Next, the current and emerging breeding constraints are discussed for biotic and abiotic stresses, morphological and phenological traits, and dietary and cooking needs. To address these vulnerabilities, several resources have been developed and, which have been applied to increase the genetic resilience of Phaseolus beans. The resilience resources include genetic resources collections such as the global collection at the Centro Internacional de Agricultura Tropical (CIAT, Colombia), national collections in the United States, Brazil, the European Union, and elsewhere, which include wild and domesticated types across the genus but focus primarily on domesticated species. Resilience resources also include genome‐wide reference DNA sequences for three of the five domesticated species, multiple diversity panels and recombinant inbred populations, and large sets of whole‐genome diversity data based on single‐nucleotide polymorphism (SNP) arrays, genotyping by sequencing, and whole‐genome sequencing of germplasm sets. Numerous marker–trait associations and genes affecting agronomic traits have also been characterized in the genus. In turn, these resources have been successfully utilized to make Phaseolus beans more resistant against biotic and abiotic stresses (including those incurred by climate change) and to improve dietary and culinary quality through significant breeding efforts in the United States, at CIAT (mainly Latin America and Africa) and in national programs in Latin America and Eastern Africa. Future challenges remain, however, which include (1) a continued need for ex situ and in situ conservation of diversity, with agroecologically informed germplasm explorations and integration of farmers into conservation and breeding activities; (2) increased pre‐breeding efforts involving gene bank curators and bean improvement scientists; (3) expansion of breeding of domesticated species other than common bean, where appropriate based on their potential adaptation to global climate change and consumer preferences; (4) an increased focus on culinary and dietary improvement; and (5) inclusion of microorganisms (both pathogenic and beneficial) in genetic conservation. We conclude that in the short term (~5 years), Phaseolus beans have limited genetic vulnerability. However, over the longer term, vulnerability due to several factors will increase, which can be addressed by a wide range of the resilience resources presented here.
... Multiple transformation techniques have been utilized to deliver foreign DNA sequences into an ample range of plant species [25]. The combination of recombinant DNA technologies, genetic transformation, and plant tissue culture are at the core of the production of transgenic plants in a variety of crops [26][27][28][29][30][31][32][33][34][35][36]. ...
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The tomato is one of the most important vegetables in the world. The demand for tomatoes is high in virtually any country, owing to their gastronomic versatility and nutritional and aromatic value. Drought, salinity, and inadequate temperature can be major factors in diminishing yield, affecting physiological and biochemical processes and altering various metabolic pathways, from the aggregation of low molecular-weight substances to the transcription of specific genes. Various biotechnological tools can be used to alter the tomato genes so that this species can more rapidly or better adapt to abiotic stress. These approaches range from the introgression of genes coding for specific enzymes for mitigating a prevailing stress to genetic modifications that alter specific metabolic pathways to help tomato perceive environmental cues and/or withstand adverse conditions. In recent years, environmental and social concerns and the high complexity of the plant response may increase the attention of applied plant biotechnology toward biomimetic strategies, generally defined as all the approaches that seek to develop more sustainable and acceptable strategies by imitating nature's time-tested solutions. In this review, we provide an overview of some of the genetic sequences and molecules that were the objects of biotechnological intervention in tomato as examples of approaches to achieve tolerance to abiotic factors, improving existing nature-based mechanisms and solutions (biomimetic biotechnological approaches (BBA)). Finally, we discuss implications and perspectives within the GMO debate, proposing that crops modified with BBA should receive less stringent regulation.
... Additionally, the extraction and purification of therapeutic proteins from tobacco is expensive due to the presence of alkaloids (e.g., nicotine) that are toxic to humans. Crops, including soybean, rapeseed, common bean, maize, wheat, and rice, seem to be free from these drawbacks [22][23][24]. Therefore, they can be successfully used as a factory for molecular farming and as a subject of biofortification, aiming at the production of crops with increased nutritional value, which can be obtained through genetic engineering [23,25]. ...
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Tremendous advances in crop biotechnology related to the availability of molecular tools and methods developed for transformation and regeneration of specific plant species have been observed. As a consequence, the interest in plant molecular farming aimed at producing the desired therapeutic proteins has significantly increased. Since the middle of the 1980s, recombinant pharmaceuticals have transformed the treatment of many serious diseases and nowadays are used in all branches of medicine. The available systems of the synthesis include wild-type or modified mammalian cells, plants or plant cell cultures, insects, yeast, fungi, or bacteria. Undeniable benefits such as well-characterised breeding conditions, safety, and relatively low costs of production make plants an attractive yet competitive platform for biopharmaceutical production. Some of the vegetable plants that have edible tubers, fruits, leaves, or seeds may be desirable as inexpensive bioreactors because these organs can provide edible vaccines and thus omit the purification step of the final product. Some crucial facts in the development of plant-made pharmaceuticals are presented here in brief. Although crop systems do not require more strictly dedicated optimization of methodologies at any stages of the of biopharmaceutical production process, here we recall the complete framework of such a project, along with theoretical background. Thus, a brief review of the advantages and disadvantages of different systems, the principles for the selection of cis elements for the expression cassettes, and available methods of plant transformation, through to the protein recovery and purification stage, are all presented here. We also outline the achievements in the production of biopharmaceuticals in economically important crop plants and provide examples of their clinical trials and commercialization.
... Cependant, la modification d'une EBE existante ou le placement d'une EBE dans le promoteur d'un gène de résistance implique une modification du génome. Le haricot est réputé pour être récalcitrant à la transformation (Hnatuszko-Konka et al., 2014), néanmoins de nouvelles avancées récentes pourraient rendre possible la transformation de certains génotypes de haricot (Hnatuszko-Konka et al., 2019). Par ailleurs, la modification des génomes des plantes suit une législation complexe et diversifiée à travers le monde, ce qui peut compliquer la commercialisation de plantes portant ce genre de résistance. ...
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La pathogénie des bactéries du genre Xanthomonas repose en partie sur leur capacité à injecter des effecteurs TAL (transcription activator-like) dans la cellule végétale. Ces effecteurs agissent comme des facteurs de transcription eucaryotes en induisant des gènes de sensibilité chez la plante hôte afin de favoriser l’infection. L’objectif de cette thèse est de mieux comprendre l’interaction Xanthomonas-haricot, et notamment le rôle des effecteurs TAL dans la graisse commune causée par Xanthomonas phaseoli pv. phaseoli et X. citri pv. fuscans. Pour cela, une étude transcriptomique a été menée pour évaluer l’impact de Xanthomonas sur le transcriptome du haricot en contextes résistant et sensible. Cette étude a permis de révéler des gènes et des voies métaboliques en lien avec la résistance ou la sensibilité de la plante.A l’aide d’une combinaison d’approches transcriptomiques et de tests de pouvoir pathogène, les cibles potentielles de deux effecteurs TAL ont été étudiées. Ces approches ont montré que XfuTAL1 et XfuTAL2 étaient importants pour l’agressivité de Xanthomonas et que XfuTAL1 induisait le gène de sensibilité PvAIL1 codant un facteur de transcription. Cette étude a aussi révélé que la structure atypique de XfuTAL2 augmentait sa capacité d’accroche à l’ADN et jouerait potentiellement un rôle dans la pathogénie. Par ailleurs, une nouvelle méthode de phénotypage a été mise au point pour évaluer l’agressivité de Xanthomonas sur haricot. Ensemble, ces découvertes permettent d’apporter de nouvelles perspectives pour le développement de résistances à Xanthomonas chez le haricot et soulignent l'importance des effecteurs TAL dans ce pathosystème.
... An important reason for this gap is the declining genetic diversity of cultivated legumes, which reduces their tolerances to biotic and abiotic stresses. There are also apparent technical limitations in legumes breeding, such as the lack of effective haploid doubling technology, anatomical constraints for making hybrids, and low transformation efficiency that impedes gene modification (Hnatuszko-Konka et al. 2014). All these limitations have imposed difficulties in expanding the genetic diversity of cultivated legume crops as well as deep investigation of the functions of important genes. ...
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The Leguminosae constitutes the second largest family of plants in the world. In the face of global climate change and reduction of cultivated land area, legumes have been attached more importance to feed the future in light of their nutritional values and environmental benefits. However, the yields of legume crops have thus far been far below their theoretical potentials. Despite that hybrid breeding and gene editing technologies have hold great promises to the improvement of crops, they are generally difficult to apply in legumes because of the anatomical constraints for making hybrids in these species and the low transformation efficiency impeding gene modifications. Mutation technologies, as an alternative, can speed up breeding and facilitate functional genomic studies in legume species, yet mutant screening technique remains a major challenge in this field. In this review, we detail the currently available mutant library resources in major legume crops following a brief introduction of the methods of mutation induction. We then revisit the current mainstream technologies for mutant screening, with focus on the new emerging next-generation sequencing-based approaches. Overall, this review provides a collection of information that dedicates to assisting legume researchers and breeders to more easily find and better utilize mutant resources for the improvement of these crops.
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Micronutrient deficiencies (hidden hunger), particularly in iron (Fe) and zinc (Zn), remain one of the most serious public health challenges, affecting more than three billion people globally. A number of strategies are used to ameliorate the problem of micronutrient deficiencies and to improve the nutritional profile of food products. These include (i) dietary diversification, (ii) industrial food fortification and supplements, (iii) agronomic approaches including soil mineral fertilisation, bioinoculants and crop rotations, and (iv) biofortification through the implementation of biotechnology including gene editing and plant breeding. These efforts must consider the dietary patterns and culinary preferences of the consumer and stakeholder acceptance of new biofortified varieties. Deficiencies in Zn and Fe are often linked to the poor nutritional status of agricultural soils, resulting in low amounts and/or poor availability of these nutrients in staple food crops such as common bean. This review describes the genes and processes associated with Fe and Zn accumulation in common bean, a significant food source in Africa that plays an important role in nutritional security. We discuss the conventional plant breeding, transgenic and gene editing approaches that are being deployed to improve Fe and Zn accumulation in beans. We also consider the requirements of successful bean biofortification programmes, highlighting gaps in current knowledge, possible solutions and future perspectives.
The bar gene from Streptomyces hygroscopicus (Jansen) Labeda & I-Nons encodes phosphinothricin acetyl transferase (PAT), which confers tolerance to the herbicide glufosinate ammonium. In Brazil, the productivity of Phaseolus vulgaris L. (dry bean) has been declining in some regions. One of most limiting factors is weed competition. This study was conducted to evaluate the possibility of obtaining transgenic bean plants resistant to glufosinate ammonium, which would facilitate weed control during the summer season. The biolistic process was used to insert this gene into dry bean, and the integration was confirmed by Southern blot analysis. Two transgenic events, PHV119 and PHV122 were tolerant to 500 g ha(-1) of glufosinate ammonium under green house conditions, with no visible symptoms and developmental growth comparable to nontransgenic P. vulgaris. Field evaluation carried out with the PHV119 event has shown that the plants tolerated up to 400 g GA ha(-1), with no visible symptoms. Inheritance studies showed that the transgenes segregated in a Mendelian fashion.
Common bean breeding requires novel techniques to incorporate genes that may not be available in bean genetic diversity. The objective of the present study was to analyze the main variables involved in Agrobacterium tumefaciens-mediated transformation of common bean towards providing an efficient system to introduce new agronomic traits. Bean cultivars Flor de Mayo Anita and Pinto Saltillo were inoculated with different A. tumefaciens strains, constructs and selection was done using two selection agents (kanamycin or glufosinate-ammonium herbicide). Five-day old hypocotyls regenerated organogenic buds (cell clusters) 5 d after inoculation. A randomized complete blocks experimental design was used and the experimental unit was a petri dish with at least 10 hypocotyls. Analysis of variance and Tukey test (p <= 0.05) were performed on data. Transformation efficiency was 10-28 cYo with small variations due to the genetic background of each cultivar, the Agrobacterium strain and selection agent used. Transformation efficiency was higher using kanamycin (28.6 %) in relation to glufosinate-ammonium (10.2 %). Similarly, Pinto Saltillo cultivar showed better regeneration response (21-28 %) than that of Flor de Mayo Anita cultivar (10-17 %) and this was consistent when selection agents were compared within each cultivar. Molecular analysis for detection and gene expression quantification by using end point PCR and q-PCR showed evidence of presence and activity at different expression levels through several generations (T0 to T3). Transformed lines of common bean with potential for fungal resistance and drought tolerance were successfully obtained via Agrobacterium tumefaciens with evidence of genetic stability.
The large-seeded legume crops, including the common bean (Phaseolus vulgaris L.), are second to cereals with regard to their role in human and animal nutrition, and they are a cheap source of dietary protein, calories, fiber, vitamins, and minerals. This nutritional value is particularly important for the poorer parts of populations in developing countries in Africa, Asia, and Latin America. In the common bean, conventional breeding methods have achieved remarkable progress using sexual recombination (including successful wide crosses, Mejia-Jimenez et al., 1994). However, the lack of genetic variation for certain resistance traits and the difficulties with further interspecific hybridizations, especially with species in the quaternary gene pools and beyond (see Chapter 2 by Debouck), still make breeding a rather slow process, even when marker-assisted breeding is being used (as discussed in Chapter 4 by Kelly & Miklas). In the past, plant geneticists tried to make use of radiation- or chemically-induced mutations, but the respective low frequencies and occurrence of many recessive and loss-of-function mutations failed to convince breeders of the value of these techniques. In addition, in the era of mutation breeding, major emphasis was on yield-related parameters and not on resistance to biotic and abiotic production constraints.
For shoot induction and plant regeneration in bean we used MS media+BA and NAA. Multiple shoot induction was obtained in case of P. vulgaris cv. Fonix and Maxidor. The efficiency of regeneration from intact seedling (IS) and cotyledonary node (CN) explants was compared. The optimum treatment for the induction of multiple shoot formation was the culturing of (IS) on MS-based media+BA and NAA. Multiple shoot induction on dry bean (CN) cultured on full MS medium+1mg/l BA and 0.1 mg/l NAA was feasible. Shoots that were 2cm in length or longer and having 2 trifoliate leaves were responsible for rooting ability, and root development. The method can be applied in transformation experiment.