Plant Breeding

2014 Blackwell Verlag GmbH

doi:10.1111/pbr.12189

RNAi-based Bean golden mosaic virus-resistant common bean (Embrapa 5.1)

shows simple inheritance for both transgene and disease resistance
J O S I A S C . F A R I A 1, P A U L A A . M . R . V A L D I S S E R 1, E L S A O . P . L . N O G U E I R A 2 and F R A N C I S C O J . L .
A R A G O 2,3
1
Embrapa Arroz e Feij~ao, Rodovia GO-462, km 12 Zona Rural C.P. 179, Santo Ant^
onio de Goias, 75375-000, GO Brazil; 2Embrapa
Recursos Geneticos e Biotecnologia, PqEB W5 Norte, Braslia, 70770-917, DF Brazil; 3Corresponding author, E-mail: francisco.
aragao@embrapa.br

With 3 figures and 4 tables

Received October 5, 2013/Accepted March 31, 2014
Communicated by T. Debener

Abstract
Bean golden mosaic virus (BGMV) is the causal agent of bean golden
mosaic of common beans. A transgenic bean line that has been developed based on RNA interference to silence the BGMV rep gene showed
immunity to the virus. Crosses were done between the transgenic line
and six bean cultivars followed by four backcrosses to the commercial
cultivars Perola and BRS Pontal. The transgene locus was consistently
inherited from the crosses analysed in a Mendelian fashion in the segregating populations. The disease resistance reaction co-inherited with the
transgene. Nevertheless, the expression of disease resistance displayed a
dosage effect phenomenon in the F1 generation. The analysis of the
homozygous near-isogenic lines in field conditions, under high BGMV
disease incidence, indicated that the transgenic lines were completely
resistant. These results show the strength of the disease resistance
obtained, the stability of the trait across generations and its usefulness in
the management of a disease for which there is no reported Phaseolus
germplasm with immunity.

Key words: Phaseolus common bean transformation

genetic variability transgene stability virus resistance

Bean golden mosaic virus (BGMV) is a typical circular singlestranded geminivirus (Begomovirus, family Geminiviridae), with
a narrow host range, mostly restricted to common beans and
other Phaseolus species, even though there are reports of BGMV
infecting soybeans (Fernandes et al. 2009) and Macroptillium
lathyroides (Lima et al. 2013). It is a whitefly-transmitted bipartite virus, with an A component of 2617 nucleotides and a B
component of 2580 nucleotides. Both DNA components are
needed for plant infection, but the A component is able to replicate itself in plant protoplasts. The A component harbours the
rep gene, which is responsible for the replication-associated protein, essential for the initiation of rolling circle replication,
among other functions. This gene, or part of it in a truncated
form, has been the focus in the attempt to develop pathogenderived resistance (PDR) in common beans (Arag~ao et al. 1998,
Faria et al. 2006, Bonfim et al. 2007).
Golden mosaic is considered the main viral disease of common beans in the tropics and semi-tropical regions. By itself, it
may account for yield losses of up to 100% in many bean-growing areas of Brazil, depending upon time of infection. Annual
average yield losses have been estimated at 20%. Currently, the
main measure taken to manage the disease is chemical control of
the vector, the whitefly Bemisia tabaci Genn. Finding an alternative control solution continues to be a challenge for both plant
breeders and plant pathologists, because no natural immunity or
high level of disease resistance has been identified in genotypes

of Phaseolus (Morales and Anderson 2001, Faria et al. 2006,

Dinon et al. 2012). Complete resistance to BGMV has, however,
been achieved by genetically engineering the common bean variety Olathe Pinto, known as Embrapa 5.1, using a construct to
express small interfering RNAs from part of the viral rep gene
(Bonfim et al. 2007, Arag~ao and Faria 2009). The complete
molecular characterization of the transgene insert indicated the
presence of a single locus in which there are two intact copies of
the RNAi cassette in opposite orientation (Arag~ao et al. 2013).
Mendelian inheritance patterns are typically observed in transgenic plants, independently of the method used for transformation (Soares et al. 2005, Anuradha et al. 2006, Faria et al. 2006,
Yao et al. 2006, Sriskandarajah et al. 2007, Terakami et al.
2007, Pinheiro et al. 2009). However, atypical non-Mendelian
segregations of transgenes have also been reported in 10 to 50%
of independent transgenic lines (Arag~ao et al. 1996, Yin et al.
2004, Romano et al. 2005, Tizaoui and Kchouk 2012). Even
though the transgene might segregate normally, gene expression
may be suppressed or partially expressed as a function of zygosity (Carvalho et al. 1992, Deng et al. 2013). The stable inheritance pattern of a transgene is an essential requirement in the
biosafety evaluation of transgenic events to be commercially
released. It is important to determine the inheritance patterns and
genotypic stability of the transgenes in different genetic backgrounds for the appropriate use of genetically modified organisms in agriculture. Several studies have been carried out to
analyse the inheritance of transgenes in primary transformants,
evaluating only the T0 and T1 generations (Butterfield et al.
2002, Rooke et al. 2003, Sriskandarajah et al. 2007, Terakami
et al. 2007, Nanasato et al. 2011). Nevertheless, to verify that
the transgene has a stable inheritance pattern, it is necessary to
evaluate its segregation in different genetic backgrounds, for at
least three generations. Schmidt et al. (2004) reported that transferring transgenes to distinct genetic backgrounds could lead to
different gene expression levels. Analysis of the progeny of
backcrosses can also be a useful tool in confirming whether the
progeny can normally recover traits of the recurrent parent and
whether the inserted genes can account for any undesirable
effects.
The objective of the present work was to study the inheritance
of disease resistance and of the transgenes inserted in the event
Embrapa 5.1 (previously Olathe 5.1) after at least ten generations
of self-pollination of the original event and after one cross and
four backcrosses to commercial cultivars. This transgenic line is
among the first applications of RNA interference technology to
generate a solution to an important plant disease.

Material and Methods

Plant material: The event Embrapa 5.1 was obtained by the
introduction of the plasmid construct named pBGMVRNAiAHAS, which
contains 410 nucleotides from the rep gene of BGMV-BR (GenBank
access No. M88686), bases 18362247 (DAC1hpRNA) and the Atahas
gene with its own promoter and untranslated 30 region (30 UTR; Bonfim
et al. 2007). The vector was digested with the restriction enzyme FspI to
interrupt the bla gene from Escherichia coli, making it non-functional
mostly to avoid undesirable public perception. The transgene locus has
been fully characterized at the molecular level (Arag~ao et al. 2013).
Crosses and backcrosses: Homozygous plants of the original event
were crossed to Olathe Pinto (seed type Pinto), BRS Pontal (seed
class Carioca), Perola (seed class Carioca), BRS Supremo (seed class
Black) all from the Meso-American gene pool, Jalo Precoce (seed class
Jalo) and Dark Red Kidney 18 (seed class Red Kidney), both from the
Andean gene pool to obtain the first generation (F1). Plants from the F1
generation, derived from the crosses to BRS Pontal and Perola, after
confirming the presence of the transgene, were backcrossed to the
parental commercial types up to the fourth backcross, resulting in nearisogenic lines (NILs). The commercial recurrent parent was always used
as the female parent to make it easier to ensure that the actual crosses
were successful. The segregation patterns for both the presence of the
transgene and resistance to BGMV were assessed in the F1 for all
crosses and in other generations for backcrosses to BRS Pontal and
Perola. Following the fourth backcross, the generations were advanced
through the single seed descent methodology. Homozygous lines were
ascertained on the basis of a progeny test of 20 plants derived from a
single plant beginning in the sixth generation of self-pollination. The
plants in any of the F1 generations showing susceptibility to BGMV
were not used for the backcrosses.
Presence of the transgene: All plants were analysed by polymerase
chain reaction (PCR) for the presence of transgene using specific primers
as previously described (Bonfim et al. 2007). Briefly, a leaf disc was
taken from each plant and DNA was extracted by the Dellaporta
methodology (Dellaporta et al. 1983). The genomic DNA was
resupended in 30 ll of TE buffer or sterile milliQ water. PCR followed
the standard protocol described by Bonfim et al. (2007).
Plant inoculation and evaluation: A BGMV viruliferous whitefly
(Bemisia tabaci Genn.) colony was maintained under greenhouse
conditions, reared in BGMV-infected Phaseolus lunatus and Glycine max
plants. Common bean seedlings to be evaluated for reaction to BGMV
were started in a whitefly-free greenhouse. Seeds were germinated in
250-ml containers filled with fertilized soil. Seven- to eight-day-old
plants were moved to the greenhouse with the whiteflies for a period of
eight days after which the plants were moved to the whitefly-free
greenhouse, after removing all insects and treating plants with a systemic
insecticide to prevent egg development. Plants were rated for the
presence/absence of disease based on visual appearance of disease
symptoms. Evaluation was done daily until all control plants had fully
developed symptoms. In case of doubt, BGMV was detected by specific
PCR analysis according to Bonfim et al. (2007).
Statistical analysis: All plants from the segregating populations were
analysed by chi-square method of goodness of fit. According to Little
and Hills (1978), Yates correction was used as there is only one degree
of freedom.
Field testing: Seventeen NILs derived from cv. BRS Pontal and nine
NILs derived from cv. Perola were field evaluated along with the
parental cultivars in the growing season of 2011, for yield and BGMV
resistance at the Embrapa Rice and Beans experimental station located in
Santo Ant^
onio de Goias, GO, Brazil (Latitude 16280 0000 S, Longitude
49170 0000 W; altitude 823 m). A randomized complete block design was
used with two blocks (replications). Each experimental unit consisted of
four four-metre rows and 15 seeds per linear metre. The spacing between

J. C. FARIA, P. A. M. R. VALDISSER, E. O. P. L. NOGUEIRA et al.

rows was kept at 0.5 m and 1.0 m between experimental units. A border
of 5 m wide (10 rows) of common beans was sown on the same day, on
all sides of the experiment. Each control cultivar was represented twice
in each block to increase the presence of BGMV and disease uniformity
across the field.

Results
Analysis of F1 plants
All crosses between the line Embrapa 5.1 and the proposed
female parents were successfully obtained independently of seed
class or germplasm origin.
All F1 plants included in this study had the transgene detected
by PCR analysis. A proportion varying from 10 to 36.2% of the
F1 transgenic plants challenged with BGMV displayed typical
disease symptoms (Table 1), while the remaining plants behaved
as immune. Similarly, based on PCR analysis, the generation
BC1F1 showed the expected Mendelian segregation ratio of 1 : 1
(transgenic to non-transgenic) for the two crosses analysed (to
cvs Perola and BRS Pontal; Table 2). However, the reaction
to BGMV differed. The v2 test was consistent with the hypothesis of a 1 : 1 ratio of resistant to susceptible plants (P = 0.31
n.s.) for BRS Pontal but not for Perola (P = 0.01).
Analysis of BC4F2 plants
The plants from the generation BC4F2 showed the expected
Mendelian segregation ratio of 3 : 1 (transgenic to non-transgenic) for the crosses to cv. BRS Pontal and cv. Perola
(Table 3). The v2 test also confirmed the segregation hypothesis
of a 3 : 1 ration for disease reaction (resistant to susceptible
plants) for BC4F2 to both BRS Pontal and Perola (with
P = 0.067 n.s. and P = 0.123 n.s., respectively).
Figure 1 illustrates how the disease progressed after a limited
time of virus inoculation in the greenhouse at the BC4F2 for
BRS Pontal. The segregating plant population, which became
infected, conforms to the previous results observed for the F1
inoculated plants, where the dominance for disease reaction was
incomplete. It should be noticed that none of the 20 inoculated
Embrapa 5.1 homozygous plants showed any disease symptoms
(Table 3; Figs 1 and 2). In addition, the disease symptoms progressed more quickly in cv. Perola (Fig. 2), and none of the
BC4F2 transgenic plants showed any disease symptoms.
Table 1: Response to Bean golden mosaic virus (BGMV) of F1 plants
from the crosses to different commercial bean seed types/origin
Cross/identity F1
generation
Embrapa 5.1 X Jalo
Precoce
Embrapa 5.1 X Olathe
Pinto
Embrapa 5.1 X Dark
Red Kidney 18
Embrapa 5.1 X BRS
Supremo
Embrapa 5.1 X BRS
Pontal
Embrapa 5.1 X Perola
Embrapa 5.1 (parental)
Jalo Precoce
(non-GM control)

Totala

Resistant

Susceptible

% Susceptible

20

13

35.0

20

17

15.0

20

13

35.0

20

18

10.0

58

37

21

36.2

36
16
20

29
16
0

7
0
20

19.4
0.0
100.0

a
All F1 plants were positive for the presence of the transgene in PCR
analyses.

Transgene inheritance in BGMV-resistant bean

Table 2: Analysis of generation BC1F1 for the presence of the transgene

and reaction to Bean golden mosaic virus (BGMV) infection
Detection of the transgene

Reaction to BGMV

v2

BC to Pontal
PCR + 336
PCR  443
Total
79

339.5
339.5

0.62

0.43 n.s.

BC to Perola
PCR +
36
PCR 
44
Total
80

40
40

0.81

O(S)

v2

44a

35

1.03

0.31 n.s.

29

51b

6.06

0.01**

O(R)

0.54 n.s

PCR +/ refers to polymerase chain reaction results as positive/negative;

O, observed; E, expected; R, resistant; S, susceptible; P, probability.
**Significantly different from the expected ration of 1 : 1.
a
For the BRS Pontal backcross population, there was one transgenic
plant with BGMV symptoms and nine PCR-negative plants remained
symptomless.
b
For the Perola backcross population, there were seven PCR-positive
plants with BGMV symptoms and no escapes among the PCR-negative
plants.

Field testing
The results from the field plot, Table 4 and Fig. 3, confirm the
stability of disease resistance in homozygous near-isogenic lines
(NILs). The control plots showed a significant amount of disease, while the homozygous backcrossed isolines showed no disease incidence. Furthermore, the near-isogenic lines had higher
yield than the conventional parentals.

Discussion
The proposed plant population generations reported in the current study were successfully obtained. The Mendelian transgene
inheritance studies in the T1 progeny from the original event,
followed by selfing, have been previously published (Bonfim
et al. 2007). While the transgene was present in all F1 plants, as
expected, an incomplete dominance for the resistance to BGMV
was observed (Table 1). For the BC1F1 generation (Table 2), the

Table 3: Analysis of generation

BC4F2 for the presence of the transgene and reaction to Bean golden
mosaic virus (BGMV) infection

v2 test was consistent with the hypothesis of a 1 : 1 ratio of

resistant to susceptible plants (P = 0.31 n.s.) for BRS Pontal
but not for Perola (P = 0.01**). The observed segregation patterns are consistent with the concept of gene dosage effect,
which is the quantitative differential action of the alleles of a
gene on the phenotypic expression of the corresponding character as described by Rieger et al. (1976). It has been observed
that plants engineered to express foreign genes presented the
phenomenon of gene suppression in homozygous plants, where
no corresponding mRNA could be detected, but not in hemizygous plants. A previous example of this phenomenon was the
expression of b-1,3-glucanase. It was correlated with the transgene dose in the plant genome; in fact, the genes full expression
was determined to be strictly associated with the homozygous
state of the plant. The authors extensively reviewed the subject
(Carvalho et al. 1992) and concluded that there was a phenomenon of dosage-dependent suppression of gene expression. A
report with RNAi-mediated cassava resistant to geminiviruses
indicated a correlation between expression of the small RNA
dosage dependence and the resistance of the transgenic lines
(Vanderschuren et al. 2009).
Thus, our results expand these findings. These results are
important in the process of conversion of elite bean cultivars,
reinforcing the need for absolute certainty that the newer breeding lines are homozygous for the transgene when being evaluated for disease resistance. As it is not possible to completely
control the number of whiteflies per plant during inoculation, the
high number of whiteflies used might have accounted, in part,
for the detection and expression of dose effect phenomenon (as
in Table 3). On the other hand, the high population of viruliferous whiteflies helped to minimize escapes upon inoculation of
segregating lines. In spite of the effect of zygosity on resistance
expression, the generation BC1F1 showed the expected Mendelian segregation of 1 : 1 for both transgenic to non-transgenic
and for disease resistance to susceptibility ratios for the cross to
BRS Pontal. This type of transgene segregation was expected,
considering that the two transgene copies are located in a single
locus (Arag~ao et al. 2013). Regarding the cross to Perola, the
transgene inheritance fitted the Mendelian segregation of 1 : 1
for the transgene, but not for the reaction to BGMV.

Detection of the transgene

O

Reaction to BGMV

v2

2.07

0.149 n.s.

BC to Pontal
PCR +
PCR 
Total

149
61
210

157.5
52.5

BC to Perola
PCR +
PCR 
Total

37
19
56

42
14

Parentals
BRS Pontal
Perola
Embrapa 5.1

10
20
20

2.38

O(R)

O(S)

v2

169a

41

1.83

0.175 n.s.

37b

19

2.38

0.123 n.s.

0
0
20

10
20
0

0.123 n.s.

PCR+/ refers to polymerase chain reaction results as positive/negative; O, observed; E, expected; R, resistant; S, susceptible; P, probability.
a
For the BRS Pontal backcross population, there were 10 transgenic plants with BGMV symptoms, and 35
PCR-negative plants remained symptomless.
b
For the Perola backcross population, all PCR-positive plants remained symptomless, and there were no
escapes among the PCR-negative plants.

J. C. FARIA, P. A. M. R. VALDISSER, E. O. P. L. NOGUEIRA et al.

Fig. 1: Disease incidence progress

in plants from the BC4F2 generation
using BRS Pontal as recurrent
parent. Five percentage of the PCRpositive (BC4F2Transg) transgenic
plants showed Bean golden mosaic
virus (BGMV) symptoms; BC4F2
Total represents both the nontransgenic segregants and the
transgenic (probably hemizygous
plants) that ended up showing
disease symptoms.

Fig. 2: Disease incidence progress in plants from the BC4F2 generation

using Perola as recurrent parent. All transgenic plants showed resistance
to Bean golden mosaic virus (BGMV), and all non-transgenic segregants
(33.9%) were infected by BGMV; line F2BC4 Total represents the nontransgenic segregants; line F2BC4 Transg. represents all transgenic plants,
which were resistant to BGMV.

Table 4: Yield and Bean golden mosaic virus (BGMV) incidence for
top near-isogenic lines (NIL) derived from cvs BRS Pontal and Perola
Isoline/Bean cultivar
NIL Pontal 138
NIL Pontal 097
NIL Perola 1-8-4-3-4
NIL Perola 1-15-7-1-4
Perola
BRS Pontal

Yield (kg/ha)

BGMV
incidence (%)

2050
1950
1750
1650
720
700

0
0
0
0
64.4
29.7

The same phenomenon was observed for all of the F1 plants

from the crosses reported above. The transgene segregation
results demonstrate the normal development of zygote and
embryo without abortions or any phenotypically undesirable

Fig. 3: Bean golden mosaic virus (BGMV) incidence progress for parental lines and the pooled data for near-isogenic lines, 2011.

effects. For the conversion of a transgenic event into useful technology for society, and above all for growers, the transgene must
be transmitted from parent to offspring in a predictable and stable way. As indicated by Heeres et al. (1997), the transgenereceiving cultivar, already of the commercial type, should behave
exactly like the non-transgenic lines, except for the new trait;
this should be an important consideration also from the safety
point of view.
Even though several researchers (Travella et al. 2005, Li et al.
2007) have indicated that particle bombardment may lead to the
integration of a high number of copies and possible rearrangements of DNA, in the current case, the evidence is of normal
inheritance both for the transgene and for disease reaction associated with its expression.
The commercial common bean varieties that received the
transgene are currently under evaluation for their possible release
to the growers. The results shown here (Table 4, Fig. 3) are
from plants in the BC4F8 and BC4F9 generations and clearly
demonstrated stability for both transgene maintenance and

Transgene inheritance in BGMV-resistant bean

disease resistance. Transgenic isolines were immune to BGMV

and yielded up to three times as much as the recurrent parents.
In conclusion, achievement of common bean lines with commercial types containing agronomically useful traits is feasible,
and it is a good example of a non-commodity crop benefiting
from the use of biotechnology. The Embrapa 5.1 event shows
normal transgene segregation both in the original line and when
used as a transgene donor in a backcross breeding programme.
Acknowledgments
We are grateful to Vanderlino Moreira de Santana for help in conducting
experiments under both greenhouse and field conditions and to Gesimaria
Ribeiro Costa Coelho for laboratory assistance.

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