JPH06343361A - Fungus resistant plant - Google Patents

Abstract

PURPOSE: To obtain a Lactuca sativa lettuce plant which exhibits resistance to Bremia lactucae by selecting a non-Dm gene intervened Bremia lactucae resistant plant derived from the origin different from the origin of Lactuca sativa in the stage of F2. CONSTITUTION: The wild type lettuce plant exhibiting the resistance to the Bremia lactucae and the Lactuca sativa plant having no resistance to the Bremia lactucae are mated. The mated plant is self-pollinated to an F2 generation and the plant exhibiting the resistance to Bremia lactucae is selected in the stage of cotyledon and leaves. The method is repeated until the lettuce plant having the compatibility to Lactuca sativa is obtd. The stages of an R escape test, selection of the R escape plant for mating with the Lactuca sativa plant and the self-pollination to at least the F2 generation are repeated until the useful Lactuca sativa type incorporated with the non-Dm intervened genes within the genomes is obtd. Further, the self-pollination is executed up to at least the F4 generation. The self-pollination to the female plant to be mated is required to be averted.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to resistance of Bremia lactucae to cultivated lettuce (ie, Lactuca sativa) plants.

[0002]

BACKGROUND OF THE INVENTION The fungus Bremia lactuae is a downy mildew disease that affects many lettuce crops every year.
dew) ”. Lettuce growers have been trying to grow lettuce that is resistant to fungi for many years, but the general blemia cultivated on lettuce plants of agroeconomic importance.
There has been only partial success in the transfer of Lactuca resistance.

A feature of Bremia lactuae is the ability to develop new strains at relatively short intervals. New strains of Blemia Lactucae are constantly emerging. Currently, at least 16 so-called NL varieties of Bremia lactucae are known (36 ^e Beschrivende Sassenlist Voor Groenteguewassen Voor de Terert Ondergrass (36 ^e Beschrijve
nde Rassenlijst voor Groentegewassen voor de Telt o
(Nder Glas) (1992), edited by Arlsberg Aije and Stork JH, Centrum Voor Plantenbederings en Reprodactien der Zoek, Wageningen) and other varieties of Bremia Lactucae identified. (Bonier FJM, et al. (1992) Euphytica)
a) 61: 203-211). Known commercial lettuce varieties resistant to certain Bremia lactuae strains typically contain a dominant resistance (Dm) gene in their genome, which gene is in a gene-for-gene system. Are considered to be regulated in Escherichia coli, in which the Dm gene or genes correspond to the predominant asymptomatic genes of the pathogen (Bonia et al., Supra). Dm is an abbreviation for downy mildew; the capital letter D indicates that the gene is predominant. Indicated as R unless the resistance factor proves to be due to a single gene. The Dm gene that matches most R-factors has been determined (except for R18 factor). The term Dm as used herein also includes such R-factors.

The Conduct of Tests for
Distinctness Homogenity and Stability of Lettuce (the Conduct of Tests fo
r Distinctness, Homogeneithy, Stability of Lettuce)
TG / 13/4 (revised in 1992) and TG / 13 /
7 (revised October 26, 1993) refers to useful Dm-genes and tests suitable for determining the presence of Dm-genes in lettuce. UP
Useful Dm-genes listed in OV are Dm2, D
m3, Dm4, Dm5 / 8, Dm6, Dm7, Dm1
0, Dm11, Dm16, and also includes the R18 factor. It is stated that UPOV is tested only on these routines and that the presence of Dm1, Dm4, Dm15 and Dm10 may require special testing.

Transfer of one or more Dm genes into the lettuce plant genome also does not result in a common and persistent form of lettuce that develops resistance to Bremia lecticae.
Plants carrying one or more Dm genes that provide resistance to several strains of Blemia lacquer are slightly or not resistant to other strains of B. lactus.

Therefore, lettuce-type resistance of Blemia lactuae (Bremia resistance), which is more important in agricultural economics, is not persistent over a period of time.

For all of these reasons, growers are looking for another source of resistance to Bremia, which is based on a genetic mechanism other than Dm gene-mediated resistance (such types of resistance are Hereinafter referred to as "non-Dm gene-mediated resistance").

One source of non-Dm gene-mediated resistance is the wild-type lettuce seed, Lactuca saligna.
Has been recognized for many years in (Gustavson, Ai (19
89) Euphytica 40: 227-232; Netzer, Dee et al. (1976) Hortscienc.
e) 11 (6): 612-613). Non-Dm gene resistance is effective against all known Bremia lactuae strains in at least some Lactuca saligna plant types, especially against all strains that can be regulated by the useful Dm gene and the R18 factor. It is known that (UP
OV see above).

Non-Dm gene mediated Bremia resistance is also referred to in the literature as "horizontal resistance" and "immunity", which means in particular to work independently of the Dm gene mediated resistance mechanism. In accordance with the above, it is highly desirable to transfer non-Dm gene mediated resistance into commercially acceptable, ie cultivated lettuce plants. No successful case of this type of Bremia resistance transfer to Lactuca sativa has been reported. Rakutsuka Saligna and Rakutsuka
Attempts were made to sativa mating, but the result was only plants with a poor phenotype with regard to plant traits and fertility (De Vries I.M. (1990) Plant System Evolution (Pl. Syst. .Evol.) 171:
233-248).

Difficulties encountered by growers can cultivate non-Dm gene-mediated Bremia resistance derived from a source of resistance to Bremia (wild-type lettuce such as Lactuca saligna) different from that of Lactuca sativa It is therefore due to various factors, including the difficulty of fully populating agronomically (and commercially) acceptable lettuce. In fact, the non-discovered in Rakutka Saligna
Non-DM gene mediated blemia resistance as conferred by Dm gene mediated bremia resistance appears to have recessive properties. In addition, any plant suitable for use in a breeding program for the non-Dm gene mediated Bremia resistance to L. cativa allogeneic breeding is first selected as a Lactuca sativa plant, ie agroeconomics. It must be fully compatible with important lettuce plants that are commercially and commercially acceptable.

[0011]

According to the present invention, it is possible to sufficiently transfer non-Dm gene-mediated Bremia resistance derived from a source different from that of Lactuca sativa into a Lactuca sativa plant.
It has been discovered that this is possible by selecting suitable Bremia lactuae resistant plants at the F2 stage. Furthermore, it links plants that exhibit non-Dm gene-mediated resistance at the F2 stage with plant viability required for use in backcrossing commercial varieties with selected F2 plants for restoration of plant viability. It looks like The F2 population is segregated at least in terms of Bremia lactocae resistance characteristics. F2
Lettuce plants with non-Dm gene-mediated resistance selected from the population can be crossed with an agronomically important lettuce line whose product can then be used for further breeding.

[0012] The present invention provides the ability of Bremia vulgaris to be compared with that exhibited in Lactuca sativa plants, including resistance to Bremia lactuae based solely on Dm gene-mediated resistance.
The present invention relates to a Lactuca sativa lettuce plant capable of showing a broader and more stable tolerance to Lactuca tae.

The present invention provides a Lactuca sativa plant capable of displaying resistance to Bremia lactuae, and resistance to Bremia lactuae includes non-Dm gene-mediated resistance. Invention cultivated lettuce plant).

The non-Dm gene-mediated resistance is preferably derived from Lactuca saligna. For convenience, the cultivated lettuce plant of the present invention has sufficient fertility. The cultivated lettuce plant of the present invention may or may not contain one or more Dm genes in its genome.

Cultivated lettuce plants, that is, the lacquer plant of the present invention
Sativa plants are lettuce plants that are suitable for food. Virtually all seeds of the Lactuca sativa plants of the present invention are the basis for cultivated lettuce plants that express non-Dm gene-mediated resistance to Bremia lactuae. The phenotypic traits of the plant are commercially acceptable and sufficiently homogeneous to meet the criteria for obtaining a permit for commercialization.

The cultivated lettuce plant of the present invention may be tetraploid (2n = 2x = 36) or diploid (ie 2n = 2x = 18) in characteristics, but is preferably diploid. The term plant as used herein includes plant parts, cells and seeds.

Some examples of plants are suitable parts for consumption (lettuce heads, their leaves) and preferred parts for plant production,
Examples include organ tissues (eg leaves, stems, roots, shoots, etc.), protoplasts, somatic embryos, anthers, stamens, petioles and cells in culture. The cultivated lettuce plant of the present invention can be cultivated or propagated from a part of the plant using a plant tissue culture technique.
Such techniques are known in the art.

When the cultivated lettuce plant of the present invention is attacked and / or infected with a strain of Bremia lactucae, it has a wider range than that of a lettuce plant having Dm gene or Dm gene-mediated resistance itself. It is possible to show resistance to the strain. Therefore, non-D
M gene mediated resistance is observable under the test conditions and also under normal field or greenhouse conditions.

The Lactuca sativa plant of the present invention is i) one of which the genetic material of one parent is provided from a wild-type lettuce plant that exhibits resistance to non-Dm gene-mediated Bremia lactuae in cotyledons and true leaves, and Parents were obtained from Lactuca sativa, which is not resistant to Bremia lactuae, the first cross, followed by selfing to the F2 generation, and the cotyledon and leaf stages (R
Selection of plants that are resistant to Bremia lactuae in (emergence plants); and then ii) a) the above-mentioned R-emergence plants and lacquer plants that exhibit the desired phenotype.
Mating Sativa; b) self-pollination to at least F2; c) selection of R escaped plants therefrom; and d) desired steps a) to c) until a lettuce plant compatible with Lactuca sativa plants is obtained. And iii) e) crossing an R escaped plant selected from compatible lettuce plants with a different Lactuca sativa plant exhibiting the desired phenotype; f) self-pollination to at least F2; g) therefrom Selection of R escaped plants; and h) e) to h) until a Lactuca sativa plant is obtained.
Repetition of the desired steps of iv) self-pollination of the plant obtained in iii) up to at least F4 generation (however, in the above-mentioned method, the female plants to be mated do not self-pollinate ) Can be obtained.

In general, the breeding method for obtaining the plants of the present invention comprises identifying suitable wild type lettuce plants that are resistant to Bremia lacquer at the cotyledon and leaf stages. Preferably, the wild-type lettuce plant is a Lactuca saligna plant having non-Dm gene-mediated resistance to B. lactus. This involves, for example, growing populations of wild-type plants, exposing them to individual strains of Bremia lactuae by infecting them with a cocktail of Bremia lactuae, or over the cotyledon stage in development, followed by foliar stages of Bremia lactuae. It is proved by re-infection with and selecting plants that are resistant at both stages. Tolerance is demonstrated if there is no visible sporulation on the leaves. Such a method, when used during a breeding program, is referred to as the R-emergence plant test, i.e., the plant that is resistant to Bremia lactuae is an R-escape plant. Selected wild-type lettuce plants that are resistant to both stages of leaf development are then grown to mature plants and crossed with Lactuca sativa plants that are not resistant to Bremia lactucae. The preferred Lactuca sativa plant type for the first cross is conveniently selected from the Lactuca sativa plants exhibiting the desired agronomic characteristics. All plant types are selected on the basis of phenotype. The progeny of the first cross can be obtained by simply sowing the crossed plants, collecting F1 seeds, sowing the seeds to produce plants, which are then selfed to produce the F2 generation. Can be achieved.

Alternatively, an embryo rescue technique can be used, in which the product of the first cross is recovered from the cross at the embryo stage and is suitable until root and shoot formation is established. Incubate in different media. Once sufficient roots and sprouts have been established,
The resulting plantlets can be transferred to soil and grown under suitable greenhouse conditions until a plant population is established, from which the plants can then be selected for use in a breeding program.
Suitable embryo rescue protocols are known in the art, eg, Meisonove B, Agronomie (198).
7), 7 (5): 313-319. F1
The generation then self-pollinates for the F2 generation as before,
Then a suitable plant is selected based on the phenotype. Suitable F2 plants are then subjected to the R escape test as outlined herein, wherein the R escape plants are Lactuca sativa plants with agronomically desirable properties, especially in restoring viability. And are self-pollinated to the F2 generation, where the preferred plants are again selected by the R escape test. The method is repeated until a lettuce plant compatible with Ractuca sativa is obtained. Compatible plants containing non-Dm gene mediated resistance to Bremia can then be used as parent lines in any additional cultivable lettuce breeding program. Compatible lettuce plants are easy to cross with Lactuca sativa and are self-fertilizing. Since lettuce is a self-pollinating plant, it is important to establish a suitable parental line containing non-Dm gene mediated resistance to Bremia lactuae. In order to establish whether the compatibility line shows non-Dm gene mediated resistance, the Bremia lactuae strain is infected at the cotyledon stage and the first true leaf stage as described for wild type lettuce plants. Therefore, in one aspect, there is a lettuce plant compatible with Lactuca sativa, which has resistance to non-Dm gene-mediated Bremia-lactuae in the genome. Once a plant compatible with Lactuca sativa is obtained, the R escape test, the selection of the R escape plant for mating with the Lactuca sativa plant, and the process of self-pollination to at least the F2 generation are not included in the genome. -Repeat until a Lactuca sativa type of agronomic interest that incorporates Dm-mediated gene resistance is obtained. The selected plant lines containing non-Dm gene-mediated resistance are repeated until cultivated lettuce plants are obtained,
Self-pollinate. For this purpose, self-pollination is carried out for at least F4 generation, preferably F5, more preferably F6,
Non-Dm gene-mediated resistance is considered to be stably contained in the genome in that generation (s). It is believed that such plants are suitable for the production of commercial quantities of seed that can be the basis for Lactuca sativa plants containing non-Dm gene-mediated Bremia lactuae resistance in their genome.

Crossing of self-pollinated plants is necessary to prevent self-propagation of the plants used as female parents. This can be done by manually emaculation of the male parts of the genitals, which requires their physical removal, or emasculation by chemical means and / or using water on the flowers. Can be done. All such methods of emasculation are known in the art.

Those skilled in the art will find that the above outlined breeding program takes many years, and the above outlined breeding program is suitable for breeding all types of cultivable lettuce, including indoor, greenhouse, and outdoor lettuce. You will recognize that.

The following examples are provided to further illustrate the present invention. It should be understood that the examples should not be viewed as limiting the scope of the invention in any way.

[0025]

【Example】

Example 1 In S3113 and S3114, the Bremia lactucae strains Nl1 to Nl16, and further isolated strains of the Bremia fungus, given internal reference TV, Bremia lactuae resistance to ITAl and GER, of the Bremia lactuae strains. The inoculum containing the cocktail is administered by cotyledon 6 days after sowing and is tested by observing cotyledons and first leaves aged 13 and 18 days after sowing, respectively. S3113 and S3114 are resistant to all Bremia lactuae strains tested as shown in Table 1, while the resistance produced by the Dm gene itself is sensitive to at least one strain of Bremia lactuae. Is.

In Table 1, − and (−) represent bremia.
It is shown to be resistant or partially resistant to Lactucaae, respectively, and (+) indicates to be sensitive to Blemia lacquee.

[0027]

[Table 1]

Example 2 S3113 and S3114 were isolated from individual Bremia lactuae strains and NL16, GER and TV Bremia.
Cocktails of the Lactuca strains were tested for resistance to Bremia lactucae and compared to the resistance of different commercial lettuce varieties containing only Dm gene-mediated resistance to B. lactuae. As can be seen in Table 2, S3113 and S3114 are resistant to all Bremia lacquer strains tested, whereas the lettuce type containing only Dm gene-mediated resistance was sensitive to strains with B. lactus. is there.

[0029]

[Table 2] Variant Dm gene NL16 Ita 1 TV, mixture of NL16 and GER Dandie Dm3 + + + UCDm2 Dm2 + + + Judy (Judy) Dm3, Dm11 + + + m + D11, Ramona (Ramona) Safier Dm3 + Dm7 + Dm16 + − + Capitan Dm11 + + + Mariska R factor 18 − + + S3113 − − − S3114 − − − Inoculants: NL16, Ita1, NL16 + GER +
Mixture of TV Inoculation at cotyledon stage: 0 + 6 days after sowing Seed cotyledons: 0 + 13 days after sowing Leaves observation: 0 + 18 days after sowing-resistance + sensitivity

S3113 and S3114 are Scotland's International Depositary Authorities, National Collections of Industrial and Marine Bacteria.
Limited (National Collections of Industrial and
Marine Bacteria Ltd.) deposited on May 19, 1993 with accession numbers NCIMB 40556 and NCIMB4.
0557 respectively.

[0031]

[Explanation of breeding schedule]

Breeding Plan Table 1: Shows an example of a breeding program that initiates insertion into non-Dm gene mediated Bremia lactuae resistant Lactuca sativa. The plant obtained, S3113, can be further subjected to self-pollination. S3113 contains in the genome the non-Dm gene-mediated Bremia resistance gene and the Dm gene group, Dm3 and Dm11.

In the diagram, the female plants produced in the breeding program are on the left and the male plants are on the right. Lactuca saligna is internally named as "B28", and this plant is a common non-Dm against Bremia lactucae.
It is implied to include gene-mediated resistance. B28-60 was the first in-house designation for the lettuce line of agronomic importance, which included non-Dm gene-mediated Bremia-lactuae resistance, which was compatible with Lactuca sativa (ie self-combined). Sex, sufficient fertility and easy mating with Lactuca sativa), and can be used as parent line in all continuous breeding programs. B28-60
Is F3 self-pollination from a cross of F2 × Palmyran. The name "Novir" is Bremia.
We show that it is the first available lettuce from INRA that is also not resistant to strains of Lactobacillus. Other names “Palmyran”, “Jessy”, “Ramona”
"na", "Judy", "Thirana" and "Penny" are S.H.
Besloten Fennaught Shap (S & G Seeds BV)
, A commercial variant of Dm3, which contains the Dm gene cluster, Dm3 and Dm11, in its genome. "E-207",
"E-99" relates to an in-house produced S-en-Hay-Says experimental system. Ramsal-19 is an in-house designation for an even more agronomically important lettuce line that can grow in short day conditions and contains B28 resistance. Ramsar-
19 is used as the parent of all greenhouse lettuce types, F2x
It is an F3 self-pollination from a Ramona cross. All of the above variants are at least non-D against Bremia lactuae
It is selected from F2 having m gene-mediated resistance. All F2 plants used throughout the breeding chart for mating and / or self-pollination are all R escaped plants. Matings of F2x Sirana and F4x Judy were made to insert an improved commercial phenotype of commercial interest, which was also due to the early insertion of B28 resistance. Stable inclusion of B2
Including 8 resistance. F6, that is, self-pollination to S3113,
B26 resistance is considered to be a stable inclusion in the genome.

Breeding Plan Table 2: Other than F2 × E-207 Essentially according to the breeding selection mode of Breeding Plan Table 1, the selected (self-pollinating) F2 plants derived from the crosses were also S. en. Mating with a different type "E-99", which is an experimental variant of Sez Besloten Fennaught Shap,
F2R escaped The resulting F4 plants derived from self-pollination were selected F2R escaped plants by crossing with a further commercial type "Penny" which is a commercial type of S.en.Hay. From S to F6, self-pollination is performed to obtain S3114.

[0034]

[Table 3]

[0035]

[Table 4]

Claims (11)

[Claims] Claim: 1. Bremia lactuca
e) A cultivated lettuce plant resistant to Bremia lactucae, which comprises a non-Dm-gene that mediates resistance in its genome. 2. The non-Dm gene has Dutch Bremia varieties NL1-7, NL9-16, TV and ITA.
The cultivated lettuce plant according to claim 1, which imparts resistance to 1. 3. The non-Dm gene is Lactuca saligna (L
The cultivated lettuce plant according to claim 1 or 2, which is obtained from actuca saligna). 4. The cultivated lettuce plant according to claim 1, wherein the cultivable lettuce plant has sufficient fertility. 5. A non-Dm gene and 1 in its genome.
5. One to four containing one or more Dm genes.
The cultivated lettuce plant according to any one of 1. 6. The genetic material of one parent is provided from a wild-type lettuce plant that exhibits resistance to non-Dm gene-mediated Bremia lactuae in cotyledons and true leaves, and the other parent is of Bremia. First cross obtained from a Lactuca sativa plant that is not resistant to Lactuca, followed by self-pollination to the F2 generation (sel
fing), and selection of plants that are resistant to Bremia lactuae at the cotyledon stage and leaf stage (R escaped plant); and then ii) a) the R escaped plant and a lacquer that exhibits the desired phenotype.
Mating Sativa; b) self-pollination to at least F2; c) selection of R escaped plants therefrom; and d) desired steps a) to c) until a lettuce plant compatible with Lactuca sativa plants is obtained. And iii) e) crossing an R escaped plant selected from compatible lettuce plants with a different Lactuca sativa plant exhibiting the desired phenotype; f) self-pollination to at least F2; g) therefrom Selection of R escaped plants; and h) e) to h) until a Lactuca sativa plant is obtained.
And iv) self-pollination of the plant obtained in iii) up to at least F4 generation (however, in the above-mentioned method, the female plants to be mated do not self-pollinate) The method for obtaining the cultivated lettuce plant according to claim 1, which comprises: 7. The method of claim 6, wherein the male parent of the first cross is a Lactuca sativa plant and the female parent is a Lactuca saligna plant. 8. A male parent used for mating is a Lactuca sativa plant, and a female parent is a non-human against Bremia lactuae.
The method according to claim 6 or 7, wherein the Dm gene-mediated resistance is transferred from Ractuca saligna. 9. A cultivated lettuce plant obtainable by the method according to any one of claims 6 to 8. 10. A head or leaf of the cultivated lettuce plant according to any one of claims 1 to 5 and 9. 11. A seed or other propagation material of a cultivated lettuce plant according to any of claims 1 to 5 and 9.