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Rapid, Efficient and High-Performance Protocol for Agrobacterium rhizogenes-Mediated Hairy Root Transformation of the Common Bean Phaseolus vulgaris

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A rapid, efficient and high-performance transformation protocol employing Agrobacterium rhizo-genes was developed for the common bean Phaseolus vulgaris. In this study, we examined compe-tencies of various protocols to induce and explants that respond to hairy root transformation in bean plants. Utilizing young seedlings with severed radicles/hypocotyls, we developed a highly efficient procedure for achieving hairy root transformation frequencies as high as 100% as visualized by GUS reporter gene expression system. Transgenic hairy roots in these young composite plants were susceptible to nodulation by rhizobia, and form an excellent system for high throughput genomic analysis to study root biology and endosymbiosis in common bean.
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Advances in Bioscience and Biotechnology, 2014, 5, 333-339
Published Online March 2014 in SciRes. http://www.scirp.org/journal/abb
http://dx.doi.org/10.4236/abb.2014.54041
How to cite this paper: Khandual, S. and Reddy, P.M. (2014) Rapid, Efficient and High-Performance Protocol for Agrobacte-
rium rhizogenes-Mediated Hairy Root Transformation of the Common Bean Phaseolus vulgaris. Advances in Bioscience and
Biotechnology, 5, 333-339. http://dx.doi.org/10.4236/abb.2014.54041
Rapid, Efficient and High-Performance
Protocol for Agrobacterium
rhizogenes-Mediated Hairy Root
Transformation of the Common Bean
Phaseolus vulgaris
Sanghamitra Khandual1*, Pallavolu Maheswara Reddy2
1Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), Guadalajara,
México
2The Energy and Resources Institute, New Delhi, India
Email: *khandual@yahoo.com, Pallavolu.Reddy@teri.res.in
Received 9 January 2014; revised 13 February 2014; accepted 4 March 2014
Copyright © 2014 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
Abstract
A rapid, efficient and high-performance transformation protocol employing Agrobacterium rhizo-
genes was developed for the common bean Phaseolus vulgaris. In this study, we examined compe-
tencies of various protocols to induce and explants that respond to hairy root transformation in
bean plants. Utilizing young seedlings with severed radicles/hypocotyls, we developed a highly ef-
ficient procedure for achieving hairy root transformation frequencies as high as 100% as visua-
lized by GUS reporter gene expression system. Transgenic hairy roots in these young composite
plants were susceptible to nodulation by rhizobia, and form an excellent system for high throughput
genomic analysis to study root biology and endosymbiosis in common bean.
Keywords
Phaseolus vulgaris; Agrobacterium rhizogenes; Hairy Root Transformation; GUS Expression
1. Introduction
Leguminous plants are capable of entering into symbiotic association with soil bacteria commonly known as
*
Corresponding author.
S. Khandual, P. M. Reddy
334
rhizobia. A molecular dialogue between the legume host and its bacterial partner leads to the formation specia-
lized plant organ, the nodule, in which rhizobia reside and carry out nitrogen fixation. Two legumes, Lotus ja-
ponicus and Medicago truncatula, have been extensively used as model plants to advance our understanding of
the molecular dialogue between plants and rhizobia, and the signaling pathway that mediates the development of
symbiosis.
Common bean, Phaseolus vulgaris, is a very important crop legume, which originated in Latin America. This
crop plant is widely cultivated throughout the tropical regions of the world, including south Asia, as it is the
most consumed grain legume by the humankind. It is a source of more than 50% of dietary protein for poor
people living in Latin America, Africa and Asia [1]. P. vulgaris is a diploid plant with relatively a small genome
(650 Mb), and hence forms an excellent model plant among crop legumes to study nodulation and nitrogen fixa-
tion. Yet it has lagged behind the model legumes L. japonicus and M. truncatula in becoming an ideal system to
study root symbiosis because of its recalcitrance to genetic transformation. This lack of appropriate genetic
transformation technology formed a greatest impediment to the functional analysis of genes in bean. To our
knowledge, thus far, the only method for obtaining P. vulgaris transgenic plants, albeit with a very low effi-
ciency, has been reported by Aragão et al. [2]. However, Estrada-Navarrete et al. [3] developed an Agrobacte-
rium rhizogenes-mediated hairy root transformation protocol for common bean, and this paved way to further
molecular studies on nodule development in this plant species. Thus far, this is the most widely used hairy root
transformation protocol for bean. However, the efficiency of hairy root transformation by this method is highly
variable, which ranged between 20% and 80%. Hence, the present investigation was undertaken to develop a
simple, rapid and high performance alternative technique to achieve 100% hairy root transformation efficiencies
in bean. For achieving this, we examined the effectiveness of various protocols, explants and introduced appro-
priate amendments to boost hairy root transformation efficiencies to 100%, thus making it a suitable system for
high-throughput functional genomic studies in bean.
2. Materials and General Methods
2.1. Development of Agrobacterium rhizogenes Strains Carrying Binary Vectors and
Inoculum Preparation
Binary vectors used for bean transformation, pBI101.1 (control vector; Clontech), pBI101-35S:GUS or pIG121
(containing 35S:IntGUS; [4]) were introduced into Agrobacterium rhizogenes K599 by electroporation [5]. A.
rhizogenes strains harboring the transformation vectors were grown at 30˚C in Luria-Bertani (LB) plates sup-
plemented with 50 µg·ml1 kanamycin. The bacterial cells from a culture grown overnight from a single plate
were harvested and re-suspended in 5 ml sterile distilled water (OD600—0.3), and used for infecting bean seedlings/
cotyledons for the induction of genetically transformed hairy roots employing different protocols (see below).
2.2. Surface Sterilization of Bean Seeds and Germination
All operations for surface sterilization of seeds were performed at room temperature. Bean (Phaseolus vulgaris
var. Negro jamapa) seeds were washed with sterile distilled water and immersed in 70% ethanol for 1 - 2 min.
Subsequently, the seeds were immediately washed with sterile distilled water (3 × 10 min) and incubated in 10%
(v/v) commercial bleach (sodium hypochlorite) for 10 min. Following this treatment, the seeds were washed re-
peatedly in excess amounts of sterile distilled water for 6 - 8 h on a shaker before seeding them in petri dishes,
magenta boxes or steel trays containing filter paper/paper towels moistened with sterile distilled water and ger-
minated in a plant growth incubator at 28˚C. With this method, more than 90% of the seeds germinated. The
3-day to 8-day seedlings devoid of any contamination was used in the experiments.
2.3. Histochemical Visualization of GUS Expression and Microscopy
GUS staining was essentially the same as that described by Reddy et al. [6]. Briefly, hairy roots were excised
from the transformed plants, washed twice with 0.1 M potassium phosphate buffer (pH 7.0), immersed in the
GUS substrate solution containing 1 mM X-gluc (5-bromo-4-chloro-3-indolyl glucuronide, sodium salt; Bio-
synth AG, Switzerland), 10 mM EDTA, 0.1% Triton X-100, 0.5 mM potassium ferricyanide, 0.5 mM potassium
ferrocyanide, and 100 mM potassium phosphate buffer (pH 7.0), and incubated in the dark at 30˚C for 12 - 24 h.
Subsequently, stained tissues were rinsed in phosphate buffer, fixed for more than 4 h in a solution containing
S. Khandual, P. M. Reddy
335
3.7% formaldehyde, 5% acetic acid, and 50% ethanol, examined as whole specimens, and photographed under a
stereomicroscope (Carl Zeiss Stemi 2000-C, Germany).
3. Results
In this study we have used different types of plant explants, employed various methods and introduced several
modifications in order to develop a rapid and high performance protocol for hairy root transformation in com-
mon bean, P. vulgaris.
3.1. Hairy Root Transformation in P. vulgaris Using Rock Wool Support
This protocol is essentially based on the method developed by Collier et al. [7], with minor modifications to
suite bean plants. Bean seeds were surface sterilized and transferred to trays containing a wet paper towel. Trays
were covered with aluminum foil and held at 28˚C until seed germination (approximately 3 days). Then the
sprouts were transferred to small plastic pots containing sterile vermiculite, irrigated with Summerfield nutrient
solution [8] and grown in a greenhouse maintained at 28˚C under normal light for 3 weeks. The apical stem por-
tions (twigs) needed for the induction of hairy roots was derived by excising approximately 3 cm long stem sec-
tions. Subsequently, the apical meristems were severed out and discarded, leaving one or two auxiliary buds in
each stem section. The twigs thus generated were utilized as explants for the induction of composite plants from
these twigs.
Supporting medium/material for growing the twigs was prepared out of rock wool (Grodan, Denmark;
http://www.rockwool.com). Rockwool plugs were cut into sections of approximately 3 cm3 cubes, a hole was
poked on top of each plug (approximately 3/4th of the thickness of the plug) with a pipette tip, and autoclaved.
Two plug sections were placed into a Petri dish bottom and one apical stem section, prepared as described above,
was inserted into the hole of each rock wool plug (Figure 1). Subsequently, 5 ml of re-suspended A. rhizogenes
culture carrying pBI101.1 or pIG121 (prepared as above) was inoculated carefully with a pipette tip on to the cut
end of the twig in each plug placed in open petri dishes within plant growth trays, covered with plastic domes
and incubated in an incubator at 28˚C in dark. After 24 h the trays were opened and the stem sections were al-
lowed to dry for a few hours, until the leaves are not turgid. Later, the plugs were saturated with Summerfield.
Nutrient solution, covered with clear plastic domes and returned to the growth room maintained at 28˚C and
14/10 h light/dark cycle. Plugs were checked periodically and watered when necessary for the remainder of the
induction period.
Approximately two weeks later, the first hairy roots emerged, and within another 15 - 20 d a composite plant
with fully developed root system was established (Figures 1(a) and (a’)). Under N-free nutrient medium regime,
these composite plants, when inoculated with Rhizobium tropici CIAT899, showed normal nodulation (Figures
1(a) and (a’)). With this optimized protocol [9], modified from Collier et al. [7], even though approximately 70%
of explants produced transgenic roots, with an average of 2 - 4 transgenic roots per composite plant, only
about 30% of emerging roots showed GUS expression (Figure 1(b), Table 1). Another major drawback of this
Figure 1. A. rhizogenes-mediated hairy root transformation of the twigs of P. vulgaris inserted in rockwool
plugs. (a) Nodulating hairy roots; (a’) Enlarged picture of (a); (b) Transgenic hairy roots expressing GUS.
Note that only some roots are stained blue, indicating that only a limited number of the roots are trans-
formed with 35S-IntgusA gene.
S. Khandual, P. M. Reddy
336
Table 1. Efficiency of various methods/types of explants used in promoting hairy root transformation of the common bean
Phaseolus vulgaris plants.
Explant/method used for hairy root transformation
by A. rhizogenes K599 % of explants with
transformed roots % of GUS
expressing roots
A. Infection of the twigs on rockwool support 60 - 70 30
B. Infection through cotyledonary node injection in intact seedlings 60 - 80 20 - 60
C. Infection at the cut end of the radicle severed, cotyledon-bearing seedling 100 100
protocol is that due to high humid conditions, fungal contamination frequently occurred on the rock wool plugs.
3.2. Hairy Root Transformation in P. vulgaris through Cotyledonary Node Injection
This procedure is fundamentally the one developed by Estrada-Navarrete et al. [3], with slight variations [10].
Seeds of P. vulgaris were surface sterilized and transferred small plastic pots containing sterile vermiculite im-
pregnated with Summerfield nutrient solution [8] supplemented with 5 mM KNO3, covered with clear plastic
bags, and incubated in a growth room (28˚C; 14/10 h light/dark regime).
Five days after planting, plantlets with newly unfolded cotyledons were infected by injection directly into the
cotyledonary nodes (4 - 5 times at different positions around the node) with a syringe equipped with 21 G needle
delivering approximately 10 - 20 μl of the suspension of A. rhizogenes K599 cells transformed with pBI101.1 or
pBI101-35S:GUS vectors (prepared as described above) into the wound. After injection, the plants were securely
covered with clear plastic bags (to maintain high internal humidity) and returned to the growth room. Normally,
bean plants infected by A. rhizogenes started to show tumors approximately a week after inoculation (Figure
2(a)). Ten to twelve days after infection, plantlets showing profuse hairy root differentiation at the site of infec-
tion were selected, primary root removed by severing approximately 1 cm below the cotyledonary node and
replanted in fresh pots containing sterile vermiculite. Immediately after transferring to new pots, each of the A.
rhizogenes-transformed composite plants were inoculated with 1 - 2 ml R. tropici CIAT899 culture (rhizobial
inoculum was prepared by growing CIAT899 strain to a density of 106 cells·ml1 in peptone-yeast extract (PY)
medium supplemented with nalidixic acid, 20 μg·ml1), and irrigated with Summerfield nutrient solution [8]
containing a minimal level (0.385 mM) of KNO3. Subsequent to this, the plants were covered with clear plastic
bags to maintain humid conditions, and returned to the plant growth room. After 4 - 5 d of incubation, the plastic
bags were perforated to enable the gradual acclimation of transformed plants to the ambient environment for a
few days, before they were transferred to the greenhouse maintained at 25˚C. The plants were harvested 3 - 4
weeks after rhizobia inoculation, and roots were analyzed for GUS activity.
With this protocol, even though about 70% of plants produced hairy roots, the transgenic roots expressing
GUS ranged generally between 20% - 60% per plant (Figure 2(b), Table 1). In spite of several trials, we could
never achieve more than 70% transformation efficiency.
3.3. Hairy Root Transformation of Radicle Severed, Cotyledon-Bearing Common Bean
(Miniature) Seedling/Plumule Explants by A. rhizogenes
Surface sterilized P. vulgaris seeds were germinated in dark on moistened paper towels in a try under sterile
conditions in an incubator maintained at 28˚C. At day 3 after germination, when the seedlings were just emerg-
ing, seed coats were imbibed by spraying sterile water and carefully removed without injuring the cotyledons.
Subsequently, seedlings having a radicle length of about 1 cm were aseptically transferred under the laminar
flow hood, to a sterile glass Petri dish containing water to avoid desiccation, and the hypocotyl was severed,
with a horizontal cut, precisely at the lower side of the cotyledonary node (shown by an arrow in Figure 3(a))
with a sterile scalpel holding the root tip with the forceps. After removing the radicle the seedling was held by
cotyledons and the sectioned surface (at the node) was coated with A. rhizogenes (transformed with pBI101.1 or
pIG121 vectors) cells freshly scrapped from the surface of the culture plate, with the help of a sterile scalpel.
The plumule explant with intact cotyledons was then transferred to a magenta box, with sterile Whatman num-
ber 1 filter paper with nutrient medium to form a moist surface. The plants were incubated in dark at 25˚C for 3
d. Subsequently, excess agrobacterial growth was avoided with washings with antibiotic (kanamycin, 50
S. Khandual, P. M. Reddy
337
Figure 2. A. rhizogenes-mediated hairy root transformation through
infection by injection at the cotyledonary nodes in the seedlings of P.
vulgaris. (a) Development of callus and emergence of hairy roots at the
infected site; (b) Transgenic hairy roots and nodules (arrowheads)
showing GUS expression. Note that several roots are not stained blue
indicating that they are not transformed with 35S-gusA gene.
µg·ml1) solution, and the seedlings were transferred to fresh magenta boxes containing filter paper impregnated
with the nutrient solution supplemented with the kanamycin, within a laminar hood. The infected seedlings were
allowed to grow in the incubator maintained at a temperature of 28˚C with 14 h light/10 h dark regime for the
development of hairy roots (Figure 3(b)).
With this method transformed roots were rapidly induced, normally within 8 - 10 days after infection, and
proliferated profusely. The plants were harvested 2 weeks after rhizobia inoculation, and roots were analyzed for
GUS activity. With this protocol, 100% of plants produced transgenic roots with 100% of the transgenic roots
expressing GUS (Figure 3(c), Table 1). The same protocol was also tested on soybean seedlings with 100%
transformation efficiencies (Figures 3(d) and (e)).
Comparison of hairy root transformation methods using various plant parts, the radicle severed (miniature),
cotyledon-bearing plumule explants gave 100% transformation efficiency. With this method, composite plants
showed good growth with a high degree of root proliferation. This is a less laborious and least time consuming
(rapid) method, which results in highest transformation efficiency. The “miniature” plants are also easy to han-
dle/maintain under sterile conditions, and suitable for the labs where space is a constraint. The composite plants
generated using this method can also be transferred and grown in vermiculite, and can be used for nodulation
studies.
3.4. Rapid Development of Axenic Hairy Root Cultures from Bean Cotyledons
During the course of our studies, we have also developed an easy and rapid protocol for generating hairy root
cultures from isolated cotyledons. This procedure is essentially based on the protocol used for soybean by Sub-
ramanian et al. [11], with minor modifications. Briefly, surface sterilized common bean seeds were germinated
on moistened paper towels in steel trays under sterile conditions as described above. On day 3, when the seedl-
ings were just emerging, seed coats were removed after moistening them by spraying sterile distilled water, and
the seedlings were allowed to grow for further 3 days. Cotyledons were harvested from the seedlings at day 6 by
gently twisting them off the hypocotyls. It is very important that the cotyledons be at the correct developmental
stage, neither very young and dense nor old and flaccid. When bent, the ideal cotyledons bow only marginally
before breaking into two. Individual cotyledons were surface sterilized again by wiping with an alcohol swab
soaked in 70% ethanol. The alcohol swab was rung out slightly before use, so that it was wet but not dripping.
The surface sterilized cotyledon was briefly dried, then etched by making shallow cuts/incisions about 2 - 6 mm
from the petiole end of the cotyledon. Cuts were made with a razor blade through a 10 - 20 µl droplet of A. rhi-
zogenes K599 (carrying the control vector or pIG121) inoculum such that the bacteria were directly introduced
into the wound site. The infected cotyledons were then transferred on to a nutrient solution-impregnated what man
number 1 filter paper placed in a Petri plate and covered with a top lid having a layer of filter paper moistened
with nutrient solution (i.e., above the cotyledons) to maintain humid conditions. The Petri dishes containing
S. Khandual, P. M. Reddy
338
Figure 3. A. rhizogenes-mediated hairy root transformation in the radicle severed, cotyledon-bearing (b)
and (c) common bean and (d) and (e) soybean seedling/plumule explants, and (f) and (g) generation of
hairy roots from cotyledonary explants. (a) Schematic representation of the bean/ soybean seedling
showing the site of cut (arrow, left), and the plumule explant used for transformation (right); (b) Bean
explant with emerging hairy roots at the cut site; (c) Hairy roots from bean showing GUS expression; (d)
Soybean explant showing hairy root emergence at the cut site; (e) Soybean hairy roots expressing GUS;
(f) Bean cotyledonary explant exhibiting calli formation on the surface and initiation of hairy root
differentiation (arrowheads, root primordia); (g) Hairy roots developed from the cotyledons showing
GUS expression. Note that 100% of the hairy roots that emerged from cotyledon-bearing plumule or
cotyledonary explants are expressing GUS, demonstrating that 35S-IntgusA is transferred with ex-
tremely high efficiency in these explants.
the infected cotyledons were incubated at 25˚C in dark. After 3 days of infection, the cotyledons were rinsed
with kanamycin (50 µg·ml1) solution to wash off bacterial cells, and then transferred to a new Petri dish with
filter papers moistened with nutrient solution (as described above) supplemented with kanamycin (50 µg·ml1).
The plates were incubated in a culture room maintained at a temperature of 28˚C and 14 h/10 h light/dark cycle,
and examined for hairy root formation at regular intervals.
With the above described method, profuse calli developed at the sites of infection (incisions) in cotyledons
within 7 - 8 d (Figure 3(f)), and hairy roots started appearing by 12 - 15 d after infection. Histochemical staining
of the hairy roots by x-glu revealed that 100% of the hairy roots generated from cotyledons were positive for
GUS (Figure 3(g)), indicating very high transformation efficiency. The isolated transgenic hairy roots could be
grown and maintained for extended periods on hormone-free MS salts medium supplemented with vitamins.
4. Discussion
The common bean P. vulgaris is a most widely cultivated and consumed crop legume in the developing world. It
is a diploid plant with relatively a small genome (650 Mb), and consumed crop legume in the developing world.
It is a diploid plant with relatively a small genome (650 Mb), hence potentially an ideal model plant from among
crop legumes to study genetic basis of plant development including root nodule symbiosis. However, its recalci-
trance to genetic transformation has formed a great stumbling block to undertake extensive molecular analysis of
cellular processes in this crop legume plant. Thus far, the only method for obtaining P. vulgaris transgenic
plants, albeit with a very low efficiency, has been reported by Aragão et al. [2] using biolistic approach. Never-
theless, in several recalcitrant plant species including common bean, Agrobacterium rhizogenes-mediated trans-
formation was used as an alternative method to obtain transgenic roots to study root biology. By the method of A.
rhizogenes-mediated transformation, “composite plants” can be produced having transgenic hairy root system
attached to non-transformed shoots and leaves. Recently, Estrada-Navarrete et al. [3] developed an A. rhizo-
genes-mediated hairy root transformation protocol for P. vulgaris plants, which paved way at least to take for-
ward the molecular studies of root biology, particularly the processes involved in nodule development and func-
tion in this plant species. However, the efficiency of hairy root transformation by this method is highly variable.
(b) (c)
(f) (g)
(d) (e)
(a)
Cotyledons
Radicle
Plumule
Seedling
Cotyledon-bearing
Plumule (explant)
S. Khandual, P. M. Reddy
339
Using the conventional procedure developed by Estrada-Navarrete et al. [3], we could never improve transfor-
mation efficiencies beyond 70% (see above). Hence, the present investigation was undertaken to develop a rapid
and high performance alternative technique to achieve high hairy root transformation efficiency in bean. For
achieving this we examined the effectiveness of various protocols, explants and introduced appropriate amend-
ments to boost hairy root transformation efficiencies to 100%. By adopting a new protocol we were able to de-
velop a more efficient A. rhizogenes-mediated transformation procedure for bean plants. In the new method, we
have utilized radicle severed, cotyledon-bearing common bean (miniature) young seedlings as explants for A.
rhizogenes-mediated transformation. With these young explants we were able to achieve 100% efficiency con-
sistently (Figures 3(a)-(c)), thus making this procedure a suitable system for high-throughput functional ge-
nomic studies in bean. The major advantage of this new approach is the rapidity and efficiency of A. rhizo-
genes-mediated transformation of common bean plants. Indeed, the same protocol was also found to be equally
effective in soybean as well (Figures 3(a), (d) and (e)).
Acknowledgements
We deeply acknowledge Centro de Ciencias de Genomicas, Universidad Autonoma de Mexico, Cuernavaca,
Morelos, Mexico for financial and lab support.
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the plants tolerated up to 400 g GA ha 1 , with no visible symptoms. Inheritance studies showed that the transgenes segregated in a Mende- Transgenic P. vulgaris (cultivars Olathe and Carioca) were lian fashion. produced as described by Aragao et al. (1996), from a total of 11 607 embryonic axes (5708 cv. Olathe and 5899 cv. Cari- oca) in batches of about 250. Briefly, mature seeds were sur- face sterilized and soaked in distilled water for 16 to 18 h. I n Brazil,dry bean is nutritionally important because Then, the embryonic axes were excised from the seeds and it is part of the staple diet, but productivity of this the apical meristems were exposed by removing their primary crop has been declining in some regions, with the aver- leaves. The embryonic axes were placed with the apical region age Brazilian yield being 600 kg ha 1 . This crop has the directed upward in Petri dishes containing basal MS medium potential to yield over 4000 kg ha 1 , with the West Asian (Murashige and Skoog, 1962) immediately before the bom- and North American yields being 1100 to 1500 kg ha 1 , bardment. The bombardment was conducted with a high-pres- while yields in Latin American and African countries sure helium-driven particle acceleration device built in our are between 500 to 600 kg ha 1 . In low-yield regions, laboratory. The embryonic axes were cultivated on MS me- the main limiting factors are poor agronomic practices, dium containing 44.3 M 6-benzylaminopurine (BAP) to in- duce multiple shoot development. The vector used was pB5/ diseases, insects, nutritional deficiencies, soil-type, cli-
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This transformation procedure generates, with high efficiency (70–90%), hairy roots in cultivars, landraces and accessions of Phaseolus vulgaris (common bean) and other Phaseolus spp. Hairy roots rapidly develop after wounding young plantlets with Agrobacterium rhizogenes, at the cotyledon node, and keeping the plants in high-humidity conditions. Callogenesis always precedes hairy-root formation, and after 15 days, when roots develop at wounded sites, the stem with the normal root is cleaved below the hairy root zone. Transgenic roots and nodules co-transformed with a binary vector can be easily identified using a reporter gene. This procedure, in addition to inducing robust transgenic hairy roots that are susceptible to being nodulated by rhizobia and to fixing nitrogen efficiently, sets the foundation for a high-throughput functional genomics approach on the study of root biology and root–microbe interactions. This protocol can be completed within 30 days.
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