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Plant Breeding

© 2014 Blackwell Verlag GmbH


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 Ã O 2,3
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, Brasılia, 70770-917, DF Brazil; 3Corresponding author, E-mail: francisco.

With 3 figures and 4 tables
Received October 5, 2013/Accepted March 31, 2014
Communicated by T. Debener

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
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.

GO. (2007). P.1 X BRS Pontal Embrapa 5.1 homozygous plants showed any disease symptoms (Table 3. resulting in nearisogenic lines (NILs).1 X BRS Supremo Embrapa 5. The vector was digested with the restriction enzyme FspI to interrupt the bla gene from Escherichia coli. According to Little and Hills (1978). Crosses and backcrosses: Homozygous plants of the original event were crossed to ‘Olathe Pinto’ (seed type Pinto).0 m between experimental units.067 n. M. Briefly. 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’.2% of the F1 transgenic plants challenged with BGMV displayed typical disease symptoms (Table 1). 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. A border of 5 m wide (10 rows) of common beans was sown on the same day. . and none of the BC4F2 transgenic plants showed any disease symptoms. Evaluation was done daily until all control plants had fully developed symptoms. and P = 0. Figure 1 illustrates how the disease progressed after a limited time of virus inoculation in the greenhouse at the BC4F2 for ‘BRS Pontal’. Following the fourth backcross. ‘BRS Supremo’ (seed class Black) all from the Meso-American gene pool.5 m and 1. Plant inoculation and evaluation: A BGMV viruliferous whitefly (Bemisia tabaci Genn. The v2 test was consistent with the hypothesis of a 1 : 1 ratio of resistant to susceptible plants (P = 0. 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. ‘BRS Pontal’ (seed class Carioca).0 100.01). Longitude 49°170 0000 W. ‘Perola’ were field evaluated along with the parental cultivars in the growing season of 2011. However. O. Field testing: Seventeen NILs derived from cv. Bonfim et al. Each control cultivar was represented twice in each block to increase the presence of BGMV and disease uniformity across the field. 2007). The spacing between J.1 and the proposed female parents were successfully obtained independently of seed class or germplasm origin.2 36 16 20 29 16 0 7 0 20 19. Table 2). BGMV was detected by specific PCR analysis according to Bonfim et al. Brazil (Latitude 16°280 0000 S. after confirming the presence of the transgene. both from the Andean gene pool to obtain the first generation (F1). making it non-functional mostly to avoid undesirable public perception. Common bean seedlings to be evaluated for reaction to BGMV were started in a whitefly-free greenhouse. respectively). 2013). were backcrossed to the parental commercial types up to the fourth backcross. (2007). after removing all insects and treating plants with a systemic insecticide to prevent egg development. Similarly. derived from the crosses to ‘BRS Pontal’ and ‘Perola’.1 was obtained by the introduction of the plasmid construct named pBGMVRNAiAHAS. rows was kept at 0. The commercial recurrent parent was always used as the female parent to make it easier to ensure that the actual crosses were successful. The genomic DNA was resupended in 30 ll of TE buffer or sterile milliQ water. 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.) for ‘BRS Pontal’ but not for ‘Perola’ (P = 0.1 X Dark Red Kidney 18 Embrapa 5. ‘Perola’ (Fig. A randomized complete block design was used with two blocks (replications). All F1 plants included in this study had the transgene detected by PCR analysis. Seven.0 58 37 21 36. bases 1836–2247 (DAC1hpRNA) and the Atahas gene with its own promoter and untranslated 30 region (30 UTR. ‘BRS Pontal’ and nine NILs derived from cv. In case of doubt.31 n.s. Plants were rated for the presence/absence of disease based on visual appearance of disease symptoms. which contains 410 nucleotides from the rep gene of BGMV-BR (GenBank access No.0 a All F1 plants were positive for the presence of the transgene in PCR analyses.1 X Perola Embrapa 5. ‘BRS Pontal’ and cv. PCR followed the standard protocol described by Bonfim et al. which became infected. VALDISSER. ‘Perola’ (Table 3). while the remaining plants behaved as immune.0 20 18 2 10.123 n. reared in BGMV-infected Phaseolus lunatus and Glycine max plants.s. Yates correction was used as there is only one degree of freedom. Figs 1 and 2).s. L. where the dominance for disease reaction was incomplete. A. 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.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.0 20 13 7 35. The transgene locus has been fully characterized at the molecular level (Arag~ao et al.1 (parental) Jalo Precoce (non-GM – control) Totala Resistant Susceptible % Susceptible 20 13 7 35. Plants from the F1 generation. 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’. The segregating plant population. The plants in any of the F1 generations showing susceptibility to BGMV were not used for the backcrosses. 1983). R. altitude 823 m). In addition. NOGUEIRA et al.1 X Olathe Pinto Embrapa 5.. the generations were advanced through the single seed descent methodology. for yield and BGMV resistance at the Embrapa Rice and Beans experimental station located in Santo Ant^ onio de Goias. 2). Seeds were germinated in 250-ml containers filled with fertilized soil. ‘Perola’ (seed class Carioca). FARIA. P. on all sides of the experiment. Results Analysis of F1 plants All crosses between the line Embrapa 5. M88686). 2007). conforms to the previous results observed for the F1 inoculated plants. 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. the disease symptoms progressed more quickly in cv. Statistical analysis: All plants from the segregating populations were analysed by chi-square method of goodness of fit. based on PCR analysis. ‘Jalo Precoce’ (seed class Jalo) and ‘Dark Red Kidney 18’ (seed class Red Kidney).0 20 17 3 15.) colony was maintained under greenhouse conditions. a leaf disc was taken from each plant and DNA was extracted by the Dellaporta methodology (Dellaporta et al. A proportion varying from 10 to 36. Each experimental unit consisted of four four-metre rows and 15 seeds per linear metre. It should be noticed that none of the 20 inoculated Embrapa 5.4 0. E. C. the reaction to BGMV differed.2 Material and Methods Plant material: The event Embrapa 5.

expected.s. E. considering that the two transgene copies are located in a single locus (Arag~ao et al.43 n. all PCR-positive plants remained symptomless.5 339. The observed segregation patterns are consistent with the concept of gene dosage effect.s. 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. R.31 n. .5 0. the transgene inheritance fitted the Mendelian segregation of 1 : 1 for the transgene. As it is not possible to completely control the number of whiteflies per plant during inoculation. S. O. On the other hand. probability. 2007).54 n.Transgene inheritance in BGMV-resistant bean 3 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 E v2 P BC to Pontal PCR + 336 PCR  443 Total 79 339. While the transgene was present in all F1 plants. 37b 19 2. for the detection and expression of dose effect phenomenon (as in Table 3). as expected.62 0. PCR+/ refers to polymerase chain reaction results as positive/negative.s. S.175 n.) for ‘BRS Pontal’ but not for ‘Perola’ (P = 0. A previous example of this phenomenon was the expression of b-1. 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’. susceptible.06 0.5 BC to Perola PCR + PCR  Total 37 19 56 42 14 Parentals BRS Pontal Perola Embrapa 5. the gene’s full expression was determined to be strictly associated with the homozygous state of the plant. Furthermore.s. 29 51b 6. in fact.1 10 20 20 2.s. observed. there were 10 transgenic plants with BGMV symptoms. BC to Perola PCR + 36 PCR  44 Total 80 40 40 O 0. but not for the reaction to BGMV. 0 0 20 10 20 0 0.01** O(R) 0. there were seven PCR-positive plants with BGMV symptoms and no escapes among the PCR-negative plants. 2009).83 0. an incomplete dominance for the resistance to BGMV was observed (Table 1). 1992) and concluded that there was a phenomenon of dosage-dependent suppression of gene expression.5 52. resistant.03 0. Table 4 and Fig. This type of transgene segregation was expected. R. b For the Perola backcross population.31 n. P. resistant. observed.149 n. 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). For the BC1F1 generation (Table 2).38 0. and 35 PCR-negative plants remained symptomless. a For the BRS Pontal backcross population.01**). O. but not in hemizygous plants. Regarding the cross to ‘Perola’. Discussion The proposed plant population generations reported in the current study were successfully obtained. have been previously published (Bonfim et al. the near-isogenic lines had higher yield than the conventional parentals. in part. The control plots showed a significant amount of disease. while the homozygous backcrossed isolines showed no disease incidence. and there were no escapes among the PCR-negative plants. a For the BRS Pontal backcross population. there was one transgenic plant with BGMV symptoms and nine PCR-negative plants remained symptomless. probability. our results expand these findings.3-glucanase. 2013). P. 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.81 O(S) v2 P 44a 35 1. These results are important in the process of conversion of elite bean cultivars. The authors extensively reviewed the subject (Carvalho et al. Thus. reinforcing the need for absolute certainty that the newer breeding lines are homozygous for the transgene when being evaluated for disease resistance.s PCR +/ refers to polymerase chain reaction results as positive/negative. susceptible. Field testing The results from the field plot. expected.123 n. followed by selfing. BC to Pontal PCR + PCR  Total 149 61 210 157. **Significantly different from the expected ration of 1 : 1. E. It has been observed that plants engineered to express foreign genes presented the phenomenon of gene suppression in homozygous plants.38 O(R) O(S) v2 P 169a 41 1.123 n. In spite of the effect of zygosity on resistance expression.s. Detection of the transgene O Reaction to BGMV E v2 P 2. where no corresponding mRNA could be detected. b For the Perola backcross population. The Mendelian transgene inheritance studies in the T1 progeny from the original event. the high population of viruliferous whiteflies helped to minimize escapes upon inoculation of segregating lines. confirm the stability of disease resistance in homozygous near-isogenic lines (NILs). the high number of whiteflies used might have accounted. 3.s.07 0. It was correlated with the transgene dose in the plant genome.

2011.4 J. this should be an important consideration also from the safety point of view. P. FARIA. the transgene must be transmitted from parent to offspring in a predictable and stable way. Fig. L. 2: Disease incidence progress in plants from the BC4F2 generation using Perola as recurrent parent. which were resistant to BGMV.9%) were infected by BGMV. except for the new trait. 3) are from plants in the BC4F8 and BC4F9 generations and clearly demonstrated stability for both transgene maintenance and . The transgene segregation results demonstrate the normal development of zygote and embryo without abortions or any phenotypically undesirable Fig. 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. and all non-transgenic segregants (33. Li et al.7 The same phenomenon was observed for all of the F1 plants from the crosses reported above. 3: Bean golden mosaic virus (BGMV) incidence progress for parental lines and the pooled data for near-isogenic lines. 1: Disease incidence progress in plants from the BC4F2 generation using BRS Pontal as recurrent parent. P. line F2BC4 Total represents the nontransgenic segregants. BC4F2 Total represents both the nontransgenic segregants and the transgenic (probably hemizygous plants) that ended up showing disease symptoms. already of the commercial type. 2005. (1997). 2007) have indicated that particle bombardment may lead to the integration of a high number of copies and possible rearrangements of DNA. The results shown here (Table 4. M. Fig. Even though several researchers (Travella et al. represents all transgenic plants. O. effects. R. C. As indicated by Heeres et al. the transgenereceiving cultivar. NOGUEIRA et al. in the current case. A. line F2BC4 Transg. E.4 29. All transgenic plants showed resistance to Bean golden mosaic virus (BGMV). and above all for growers. Fig. The commercial common bean varieties that received the transgene are currently under evaluation for their possible release to the growers. VALDISSER. For the conversion of a transgenic event into useful technology for society. Five percentage of the PCRpositive (BC4F2Transg) transgenic plants showed Bean golden mosaic virus (BGMV) symptoms. the evidence is of normal inheritance both for the transgene and for disease reaction associated with its expression. should behave exactly like the non-transgenic lines.

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