US 20140223611A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0223611 A1 LNDEMAN et al. (43) Pub. Date: Aug. 7, 2014

(54) METHOD FORTRANSFERRING ONE OR Publication Classification MORE GENETIC TRAITS FROMA OF THE PURPLE-FLOWERED CAPSCUM (51) Int. Cl. SPECIES TO A PLANT OF THE AOIH 5/08 (2006.01) WHITE-FLOWERED CAPSCUMI SPECIES (52) U.S. Cl. CPC ...... A0IH 5/08 (2013.01) (71) Applicant: Enza Zaden Beheer B.V., Enkhuizen USPC ...... 8OO/317.1 (NL) (57) ABSTRACT (72) Inventors: Wouter LINDEMAN, Enkhuizen (NL); Iris Alke HEIDMANN, Enkhuizen (NL) The present invention relates to a white-flowered annuum plant, comprising a gene encoding the bacterial spot (73) Assignee: Enza Zaden Beheer B.V., Enkhuizen disease resistance protein 4 (BS4), wherein the gene is derived (NL) from a purple-flowered Capsicum species plant and the gene conveys resistance to a bacterial spot pathogen disease caused (21) Appl. No.: 14/091,275 by Xanthomanas campestris pv. vesicatoria. The invention also relates to a white-flowered plant, (22) Filed: Nov. 26, 2013 comprising a gene encoding the bacterial spot disease resis tance protein 4 (BS4), wherein the plant is resistant to bacte Related U.S. Application Data rial spot pathogen disease caused by Xanthomanas campes (60) Continuation of application No. 13/232,466, filed on tris pv. vesicatoria race I, II, III, IV, and VI. The invention Sep. 14, 2011, now Pat. No. 8,618,370, which is a further relates to a seed that produces a white-flowered Cap division of application No. 11/992,503, filed on May sicum annuum plant, comprising a gene encoding BS4, and 19, 2008, now Pat. No. 8,022,278, filed as application pepper fruit produced by a white-flowered Capsicum annuum No. PCT/EP2005/054759 on Sep. 22, 2005. plant, comprising a gene encoding BS4.

Product of self pollinated C. pub, C. ex, C. frut plant combined with C. chin and C. ann. hybrid Patent Application Publication Aug. 7, 2014 Sheet 1 of 6 US 2014/0223611 A1

Patent Application Publication Aug. 7, 2014 Sheet 2 of 6 US 2014/0223611 A1

C. pubescens with desired resistance FG. 2B Patent Application Publication Aug. 7, 2014 Sheet 3 of 6 US 2014/0223611 A1

frutescens F.G. 3B

Product of step FG. 3A Patent Application Publication Aug. 7, 2014 Sheet 4 of 6 US 2014/0223611 A1

h eSe

Product of C pub, ... exi and C. fruit FG. 4A Patent Application Publication Aug. 7, 2014 Sheet 5 of 6 US 2014/0223611 A1

Product of self poll inated C pub, C. ex, C. frut plant comb ine d Wi th C. chin and C hybri FG 5 Patent Application Publication Aug. 7, 2014 Sheet 6 of 6 US 2014/0223611 A1

FG 6A FG . 6B FG

Ll CD CO O US 2014/0223611 A1 Aug. 7, 2014

METHOD FORTRANSFERRING ONE OR wild or other domesticated species of Capsicum, Such as for MORE GENETIC TRAITS FROMA PLANT example in C. pubescens which carries unique and specific OF THE PURPLE-FLOWERED CAPSCUM traits, such as the resistance against certain races of the bac SPECIES TO A PLANT OF THE terium Xanthomonas vesicatoria, that are not present in the WHITE-FLOWERED CAPSCUMI SPECIES white flowering species. It thus may be advantageous to trans fer Such favourable genetic traits, such as any resistance gene, RELATED APPLICATIONS from the distinctly related purple-flowered C. pubescens to 0001. This application is a continuation of U.S. applica the commonly used white-flowered species such as C. tion Ser. No. 13/232,466, filed Sep. 14, 2011, which is a annuum and/or C. frutescens. divisional of U.S. application Ser. No. 11/992,503, filed May 0008. It is well known, however, that strong crossing bar 19, 2008 (now U.S. Pat. No. 8,022.278), which is a U.S. riers of varying degree exist between the different species of National Phase Application of PCT/EP2005/054759, filed the Capsicum genus. Some crosses have even been proven Sep. 22, 2005. impossible, and other crosses are only possible with the help of in vitro culture techniques, such as embryo rescue. In FIELD OF THE INVENTION addition, in most cases the products derived from said crosses are completely or partially sterile which makes them not 0002 The present invention relates to a method for trans accessible for any further breeding purposes. ferring one or more genetic traits from a plant of the purple 0009. A need therefore exists for a method by which desir flowered Capsicum species comprising said genetic trait, to a able genetic traits can be transferred between the different plant of the white-flowered Capsicum species. The invention purple and white flowered Capsicum species. further relates to the plants perse, as well as to the fruits, seeds and other plant parts derived from said plants. SUMMARY OF THE INVENTION BACKGROUND OF THE INVENTION 0010. The object of the present invention is to provide a method for transferring one or more genetic traits, such as, but 0003. The genus Capsicum belongs to the large family of not limited to, resistance genes, from plants of the purple the . Several species of the Capsicum group are flowered Capsicum species to plants of the white-flowered valuable crops that are grown in open fields or underprotected Capsicum species. conditions in many countries all over the world. Capsicum species with pungent fruits are generally used as a spice, 0011. This is achieved by providing a method, comprising either fresh, dry or extracted. Species with non-pungent fruits the steps of: in a big variety of shapes and colours, commonly known as 0012 (a) preparing a first hybrid plant comprising said Sweet peppers, are widely used as Vegetable. genetic trait by crossing a first plant of the purple-flowered 0004. The genus Capsicum can be divided in two distinct Capsicum species comprising said genetic trait with a second groups based on the flower colour: the white-flowered group, plant of another purple-flowered Capsicum species, and comprising e.g. the closely related species C. annuum, C. selecting from the progeny thereof plants comprising said frutescens, C. baccatum, C. Chacoense, C. galapagoense, C. genetic trait; praetermissum and C. Chinense, and the purple-flowered 0013 (b) preparing a second hybrid plant by crossing said group, comprising e.g. C. to varii, C. pubescens, C. eximium first hybrid plant comprising said genetic trait, with a first and C. cardenasi. Of the white-flowered group Capsicum plant of the white-flowered Capsicum species: annuum, and its closely related species C. frutescens are the 0014 (c) crossing said second hybrid plant with said first best known domesticated species, which are mainly used for plant of the purple-flowered Capsicum species comprising the production of Sweet and hot peppers for processing and said genetic trait, and selecting from the progeny thereof consumption. plants comprising the genetic trait; 0005 Capsicum, like other plants, is attacked by many 00.15 (d) preparing a third hybrid plant by crossing the pests and diseases, including insects, nematodes, fungi, bac plants obtained step c) with a second plant of the white teria and viruses. One of such bacterial diseases is bacterial flowered Capsicum species, and selecting from the progeney leafspot, caused by the bacterium Xanthomonas campestris thereof plants comprising the genetic trait. pV. vesicatoria. Bacterial leafspot of Capsicum can be recog 0016. According to the present invention, for the first time nized by numerous angular spot on the leaves. Initially, the a genetic trait has been successfully transferred from a purple spots are water-soaked. Leaves infected at an early stage flowered Capsicum species to a white-flowered Capsicum become deformed. Often the margins of affected leaves are species. The method of the present invention is based on a rimmed with a narrow band of necrotic tissue. Infected Cap Suitable combination of several interspecific crosses, as well sicum leaves drop prematuraly, by which the fruit is exposed as crosses that serve as a kind of bridge to the next species to Sun which may result in Sun scald, secondary fruit rots and (FIG. 1). The desired genetic traits can for example be fol reduced yields. Capsicum fruits rarely show symptoms but lowed by molecular markers or bio-assay according to well may drop if infected early. known molecular biological techniques known to the skilled 0006 X. vesicatoria is widespread and damaging to Cap person. sicum in particular in field-grown crops in warm-temperate 0017. According to a preferred embodiment of the present and tropical countries, and thus has a large economic impact. invention, the method further comprises: Control of the disease relies principally on the production of 0018 (e) selecting plants obtained in step (d) not compris plants from healthy (treated) seeds and on preventive steps ing the genetic trait and crossing these plants with said first taken during the entire season. plant of the purple-flowered Capsicum species comprising 0007 Useful resistances to such diseases, as well as other said genetic trait, and selecting from the progeny thereof traits such as fruit quality characteristics etc., may exist in plants comprising the genetic trait; and US 2014/0223611 A1 Aug. 7, 2014

0019 (f) preparing a fourth hybrid plant by crossing the hybrids thereof, more preferably the second plant of the plants obtained step (e) with a second plant of the white white-flowered Capsicum species is C. Chinense, C. annuum flowered Capsicum species, and selecting from the prog and/or a hybrid thereof. eny thereof plants comprising the genetic trait. In this 0030. According to the present invention a hybrid is a way, plants that are obtained in step (d) of the method of plant obtained from a cross between two different species of the invention that do not comprise the genetic trait, still the Capsicum genus, e.g. a cross between a plant of the can be used further in the method of the invention. species C. Chinense and a plant of the species C. annuum 0020. According to a further preferred embodiment, the (interspecific cross), or a plant obtained from a cross between method further comprises: populations or cultivars of a single species (intraspecific 0021 g) preparing a fifth hybrid plant by crossing the cross). fourth hybrid plant comprising the genetic trait with a second 0031. According to a further preferred embodiment of the plant of the white-flowered Capsicum species, and selecting invention, the genetic trait is a resistance gene against a spe from the progeny thereof plants comprising the genetic trait. cific Capsicum disease attacking all cultivated species of Thus, a stable white-flowered plant is obtained comprising Capsicum. By transferring one or more resistance genes from the genetic trait of interest derived from the purple-flowered distinctly related species of the Capsicum genus to commonly species but with all the quality aspects of the white-flowered used species not naturally having Such resistance genes, new species. resistant plants are provided. As a consequence, the use of 0022 Preferably, after step (b) the second hybrid plant is environmental undesirable chemical treatments may for self-pollinated and the progeny thereof is used in step (c) in example be reduced or even abandoned. However, also other order to fix the resistance, to improve fertility aspects and to genetic traits, such as other resistance genes, fruit quality obtain homozygous, genetically stable plants carrying the characteristics such as shape, taste and colour, improved desired trait(s). yield, fruit set, and resistance againstabiotic stress (e.g. cold/ 0023. According to another preferred embodiment, the salt) may be transferred using the method of the present plants obtained in step (c) are self-pollinated and the progeny invention. thereof is used in step (d), again in order to fix the resistance, 0032. In a particular preferred embodiment, the resistance to improve fertility aspects and to obtain homozygous, gene is the BS4 gene, conveying resistance to the bacterial genetically stable plants carrying the desired trait(s). disease Xanthonanas campestris pv. vesicatoria race I, II, IV 0024. In addition, according to other preferred embodi and VI. ments, the plants obtained in step (d) are self-pollinated and 0033. The present invention further relates to plants, the progeny thereof is used in Step (e), and/or the plants obtainable by the method as described above. obtained in step (e) are self-pollinated and the progeny 0034. In particular, the invention relates to plants of the thereof is used in step (f). white-flowered Capsicum species, comprising a genetic trait 0025. According to the present invention embryo rescue derived from a plant of the purple-flowered Capsicum spe techniques are applied whenever it is beneficial to the process cies, said plants comprising a genetic trait which normally is interms of obtaining hybrid plants or speeding up the process. not present in the white-flowered species. 0026. Preferably, the first plant of the purple-flowered 0035. In a preferred embodiment, the plant is selected Capsicum species is selected from the group consisting of C. from the group consisting of C. baccatum, C. frutescens, C. pubescens, C. eximium, and C. Cardenasi, more preferably chinense C. annuum, and hybrids thereof, preferably, the the first plant of the purple-flowered Capsicum species is C. plant is C. chinense, C. annuum and/or hybrids thereof. pubescens. The greatest genetic distance in the genus Capsi 0036. According to a preferred embodiment of the inven cum is between the species C. pubescens and C. annuum/ tion, the genetic trait is a resistance gene against a . Direct crosses between these species have been disease, in particular the resistance gene is the BS4 gene, proven impossible until now, even with the help of embryo conveying resistance to the bacterial disease Xanthomanas rescue. By using the method of the present invention, how campestris pv. vesicatoria race I, II, IV and VI. ever, a totally new gene pool becomes accessible to the spe 0037. Furthermore, the present invention relates to fruits, cies of C. annuum and/or C. frutescens. seeds and other plant parts derived from the plants described 0027. According to the invention, the second plant of the above, wherein the other plant parts may be selected from the purple-flowered Capsicum species is selected from the group group consisting of seeds, cuttings, runners, and meristem. consisting of C. pubescens, C. eximium, and C. cardenasii, preferably the second plant of the purple-flowered Capsicum BRIEF DESCRIPTION OF THE DRAWINGS species is C. eximium or C. cardenasii, preferably C. exi 0038. The present invention is illustrated by the following mium. Preferably, the first plant is selected from another Example and Figures, which are not intended to limit the species than the second plant of the purple-flowered Capsi Scope of the invention in any way. cum species. 0039 FIG. 1 schematically shows the steps of the method 0028. In a preferred embodiment of the invention, the first of the present invention; plant of the white-flowered Capsicum species is selected from 0040 FIG. 2A and FIG. 2B are photographs of a C. exi the group consisting of C. baccatum, C. frutescens, C. chin mium plant (FIG.2A)anda C. pubescens PI235047 (FIG.2B) ense, C. annuum, and hybrids thereof. Preferably, the first plant with the desired resistance gene used in a first step of a plant of the white-flowered Capsicum species is C. frute preferred embodiment of the method of the invention; SCa2S. 0041 FIG.3A shows an example of a first hybrid plant, i.e. 0029. The second plant of the white-flowered Capsicum the product obtained in the first step of the method of the species is preferably selected from the group consisting of C. invention: FIG. 3B is a photograph of C. frutescens baccatum, C. frutescens, C. Chinense and C. annuum, and PI238059; US 2014/0223611 A1 Aug. 7, 2014

0042 FIG.4A-FIG. 4C show examples of a second hybrid Two resistant plants were obtained. These plants were fertile plant (FIG.4A), and photographs of C. annuum (FIG.4B) and and accordingly F2 seeds were obtained. C. chinense (FIG. 4C); 0052. In addition, hybrids of C. annuum (FIG. 4B) and C. 0043 FIG. 5 is a photograph of the product of the self chinense (FIG. 4C) were made by crossing individuals of C. pollinated ((C. pubescensxC. eximium)xC. frutecens)x(C. chinense PBC306 (Asian Vegetable Research and Develop ment Center) with individuals of a C. annuum plant. The chinensex.C. annuum); and product of this cross was again crossed with C. annuum. 0044 FIG. 6A-FIG 6.D show C. annuum plants carrying Subsequently F2 seeds were obtained. the resistance genes derived from C. pubescens. 0053 Subsequently, a third hybrid plant (FIG.5) was pre pared by crossing the resistant plants derived from the cross DETAILED DESCRIPTION OF THE INVENTION of the second hybrid plant with C. pubescens or plants derived Example from the F2 seeds thereof, with plants derived from the F2 seeds of the C. annuum/C. chinense hybrid. The resistant 0045 Methods: products from this cross were further crossed with pure C. 0046) Seeds were sown on vermiculate, potted into rock annuum lines. wool, blocks, and fertilised and propagated until flowering. 0054. In addition, plants derived from said cross that were Flowerbuds were emasculated and pollinated with the chosen Susceptible for Xcv6, were crossed again with C. pubescens fatherlines. Between 24-28 days after pollination fruits were PI235047. The resistant plants derived from that cross were harvested, Surface sterilised and opened under sterile condi then selfed and F2 seeds were obtained. Resistant plants tions. Embryos from all seeds were isolated aseptically and derived from that cross were Subsequently again crossed with transferred to embryo rescue (ER) medium as described by a line derived from the hybrid Abdera AMAl2. This hybrid is Sibi et al., (Ann. Amelior. Plantes, 29:583-606, 1979). After a cross between C. Chinense and C. annuum. The resistant 1-2 weeks well developed plants were transferred to MS plants resulting from said last cross were selfed and F2 seeds medium (Murashige T. and Skoog F., Physiol. Plant 15:473 were obtained. In addition, the plants derived from the selfing 497, 1962), supplemented with 0.8% agar and 20 g/l sucrose. of the last mentioned cross with PI235047 were further Plants with a well developed root system were subsequently crossed with several C. annuum parental lines. transferred to the greenhouse and adapted carefully to its 0055 As a result, C. annuum plants were obtained carry conditions. ing the BS4 resistance gene derived from C. pubescens (FIG. 0047 Leaves of 4-6 weeks old plants were detached and 6). This result clearly shows that it is possible to introduce inoculated with Xanthomonas campestris vesicatoria race VI traits from the purple-flowered Capsicum species into the to select the resistant plants. The bacterial clone is stored in white-flowered Capsicum species, using the method of the the freezer and grown on agar medium one week before present invention. inoculation at 22°C. The Suspension for inoculation is pre 1. A white-flowered Capsicum annuum plant, comprising a pared by resuspending the bacteria in demineralised water. gene encoding the bacterial spot disease resistance protein 4 The bacteria Suspension was applied to the leaves with a (BS4); Syringe. Inoculated leaves are kept in boxes covered with wherein the gene is derived from a purple-flowered Cap glass in a climate room at 22°C. for two days. Leaves of sicum species plant and said gene conveys resistance to resistant plants show a light brown and dry spot at the site of a bacterial spot pathogen disease caused by Xanth inoculation caused by a hypersensitivity reaction (HR). Omanas Campestris pv. vesicatoria. Leaves of Susceptible plants show no reaction or a water 2. The plant of claim 1, wherein the plant is resistant to soaked lesion at the site of inoculation depending on the time bacterial spot pathogen disease caused by Xanthomanas of incubation. Absence of a hypersensitivity reaction thus is campestris pv. vesicatoria race I. indicative of the absence of the BS4 gene. Resistant control is 3. The plant of claim 1, wherein the plant is resistant to C. pubescens PI235047, susceptible control is a susceptible bacterial spot pathogen disease caused by Xanthomanas C. pubescens accession. campestris pv. vesicatoria race II. 0048 Crosses: 4. The plant of claim 1, wherein the plant is resistant to 0049. A first hybrid plant was prepared by crossing Cap bacterial spot pathogen disease caused by Xanthomanas sicum pubescens PI235047 (male, FIG. 2B) (United States campestris pv. vesicatoria race III. Department of Agriculture), comprising the BS4 resistance 5. The plant of claim 1, wherein the plant is resistant to gene, with (female, FIG. 2A). 37 hybrid bacterial spot pathogen disease caused by Xanthomanas plants (FIG. 3A), comprising the resistance gene, were campestris pv. vesicatoria race IV. obtained. 6. The plant of claim 1, wherein the plant is resistant to 0050. A second hybrid plant was subsequently prepared bacterial spot pathogen disease caused by Xanthomanas by crossing C. frutescens PI 238059 (male, FIG.3B) with the campestris pv. vesicatoria race VI. first hybrid plants comprising the resistance gene (female). 7. A seed that produces the plant of claim 1. None of the second hyrid plants plants derived from this cross 8. A pepper fruit produced by the plant of claim 1. expressed the resistance. 9. The pepper fruit of claim 8, wherein the pepper fruit is a 0051. Subsequently, the second hybrid plant (female, FIG. green pepper fruit. 4A) was crossed with PI235047 (male).