JP2002512050A - Strict self-fertilization plants with improved flower morphology - Google Patents

Abstract

  (57) [Summary] The present invention comprises the steps of preparing a first parent plant protoplast and a second parent plant protoplast, and combining the first protoplast with the second protoplast to obtain a fusion product. Fusing and regenerating the fused plant from the fusion product, wherein at least one of the two parent plants has one or more traits favorable to cross-pollination, wherein the at least one trait is The present invention relates to a method for producing a fused plant for use in obtaining a plant suitable for cross-pollination, which is characterized by being expressed in the fused plant. Such traits that are advantageous for cross-pollination include, for example, at least flower size and / or flower contours that are attractive to insects. The method according to the invention can be applied, for example, to lettuce. The produced fused plants and their seeds can be used for the production of plants, hybrid plants and seeds suitable for cross-pollination.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing a plant that is originally a strictly self-fertilized species such as a lettuce plant, but is also suitable for cross-fertilization. Moreover, the invention relates to the plants produced in this way and their seeds, as well as to the use of this plant in a method for obtaining hybrid plants and to the hybrid plants obtained in this way and to the seeds derived therefrom.

[0002] In particular, hybrid varieties are commercially attractive in virtually all cultivated crops, because the hybrids are uniform and can take advantage of heterosis. Heterosis is the phenomenon in which a hybrid plant (more or less), which is the result of crossing an inbred parent plant, has better performance than either parent, for example, for a particular trait such as yield. On average, it is a further great advantage of hybrid variants that the hybrid variants achieve more satisfactory and more stable performance under changing environmental conditions.

[0003] Inbred (pure) lettuce varieties are often very limited in cultivation environment (location, soil type, season), for example, to achieve full performance. Hybrid lettuce varieties may be suitable for larger growing areas and / or environments. In addition, hybrid lettuce can be an interesting product because, for example, as described above, it is uniform, has high growth potential, and is stable through a combination of heterosis and traits. High viability is especially important for winter crops and dark red lettuce types.

In hybrid lettuce, it may be easier to use genes from wild species. L. Some interesting genes from Virosa are linked to genes that reduce growth, for example, when crossed to the genome of cultivated lettuce. This is especially true for Bremia and aphids Nasonovia livinsn.
Genes for resistance to igri have been identified. In principle, the undesired loss of growth is only manifested in homozygotes. Thus, if the parent plant is selected correctly, rather than in a hybrid, resistance linked to reduced growth is predominant, and consequently the hybrid is resistant.
Such a gene for reduced growth in hybrids would be even more advantageous from a seed-subsidiary perspective, as it would make illegally regenerated offspring even less interesting.

[0005] It is therefore advantageous to be able to obtain hybrid variants for various reasons,
And desirable. The prerequisite for making hybrid varieties is to be able to cross-pollinate within the species. However, some cultivated crops are strictly self-fertilized species. However, within a genus, cross-fertilized species can occur in addition to self-fertilized species.

One example of a strictly self-fertilized species is cultivated lettuce. Lettuce, Lactu
It belongs to the genus ca, which can then be subdivided into a number of clauses, which can be further classified within that measure. The Lactuca species found in Europe is 4
Clauses: Lactuca, Lactucopsis, Mulqedi
um, Phenixopus. Within measures, Lactuca is 2
It can be classified into two small units (Lactuca and Cyanicae). Cultivated lettuce (Lactuca sativa L.) is a related species that can cross (L. serriola, L. aculeata, L. scariolo).
ides, L .; azerbaijanica, L .; georgica, L .; dre
jeana, L .; altica, L .; saligna, L .; Virosa), belonging to the Lactuca clause and the Lactuca bar.

L. tenerrima, L .; perennis, L .; Graeca is La
It belongs to the ctuca clause and the cyanicae measure.

L. tatarica and L. et al. sibirica belongs to the Mulgedium clause. These species are taxonomically close to the genus Mulgedium. L. data
rica has been shown to be Mulgedium burgaei and American diploid (
2n = 34) Lactuca sp. floridana, L .; canadens
is, L. It has been reported that it can cross with Graminifolia.

[0009] Both strictly self-fertilized (self-reproductive) and cross-fertilized (heterogamous) species, Lactuca
Within the genus. However, the problem is the Lactuca section to which the cultivated lettuce belongs, Lactuca
The uca bar species is a strict self-fertilization. In field cultivation, where different genotypes are cultivated adjacent to each other, L. var. sativa's natural cross-fertilization rate was only 1-3% (Thompson, RC, TW Whitaker, & GW Bohn
Natl. Soc. Horticul. Sci. 36 (1958)).

[0010] It is an object of the present invention to use these plants for the production of hybrid seed in commercially required quantities, having flowers that are self-fertilized naturally and unattractive to pollinating insects. To be able to provide pollinators with a viable means of making attractive plants, if possible.

This comprises preparing, according to the invention, a first parent plant protoplast and a second parent plant protoplast, and transforming the first protoplast to obtain a fusion product. Second
Producing a fusion plant by fusing with a protoplast of the invention and regenerating the fusion plant from the fusion product, wherein at least one of the two parent plants is one or more suitable for natural cross-pollination. This is achieved by a method of producing a fused plant used to obtain a plant suitable for natural cross-pollination, wherein the plant has a plurality of traits, at least one of which is expressed in the fused plant.

[0012] Fusion plants are not directly suitable for use as parents for crosses in the production of hybrids themselves. Accordingly, the present invention further provides a) that at least one of the two parent plants has one or more traits favorable to natural cross-pollination, wherein at least one trait is expressed in the fused plant. Preparing a protoplast of a first parent plant and a protoplast of a second parent plant, wherein the first protoplast is characterized by a second protoplast to obtain a fusion product. Fusing, producing a fused plant by regenerating the fused plant from the fused product, and b) combining the fused plants with each other and / or to obtain a plant suitable for natural cross-pollination and having the desired trait.
Or further at least once (back) crossing with a non-fused plant having the desired trait.

In the present application, “natural cross-pollination” is not performed by active human intervention,
It is understood to mean any form of cross-pollination. Such natural cross-pollination is, for example, insect pollination.

The desire to be able to make hybrid plants is great, especially in the case of lettuce. L. Cross-pollinated Lactuca species, such as perennis, are usually pollinated by insects. Within the genus Lactuca, it has been demonstrated that flower size and morphology are, to a large extent, related to the degree of cross-fertilization and, therefore, to the attractiveness of the flower to pollinating insects. There is a strong correlation between cross-pollination and large flower heads with blue or light yellow. It is therefore recommended that traits in lettuce that are favorable for cross-pollination include at least insect-friendly flower size and / or flower color.

[0015] Therefore, in the embodiment of the present invention, both parent plants are Lactuca lettuce plants, and the trait convenient for cross-pollination is L. lactis. These traits are derived from a parent plant of the species tatarica, and these traits consist of large flowers or flowers having a bluish-violet or near-white flower or both. Plants derived from the fused plant can have yellow or bluish purple flowers. The change in color intensity occurs for both colors.
The degree of purple-blue tint can vary considerably. Some purple blue flowers are very thin and appear almost white.

As the other parent plant, cultivated lettuce or so-called “bridge species”
)) Can be selected directly. In the classical sense, the term "cross-linked species" refers to a species used for cross-species crossing when the two species for crossing are too far from each other to directly cross. The cross-linking species must be located somewhere between the two parents for crossing and is preferably capable of hybridizing with at least some of both. In the case of fusions, the term "cross-linked species" refers to a hybrid of species that is a subject to which genetic material is to be transferred, but that has better or more desirable traits, such as better fusion or regenerative power, etc. Used for the same system that can be formed.

When directly selecting a cultivated lettuce to produce a fused plant (instead of a cross-linked species), the other parent plants are, for example, cultivated lettuce species L. sativa. In the second case using a crosslinking species, The use of serriola is conceivable.

The plants obtained after (back) crossing of the fused plants with plants of the cultivated lettuce species can no longer be considered as fused plants and are therefore referred to herein as “fused plant derivatives”
Call. The end result of multiple (back) crossing and / or selfing is referred to herein as "plants suitable for natural cross-pollination". "Fused plants", "fused plant derivatives" and "plants suitable for natural cross-pollination" are collectively referred to as "plants according to the present invention".

Furthermore, the present invention provides fused plants, fused plant derivatives and plants suitable for natural cross-pollination, which can be obtained using the method according to the invention. In principle, all three types of plants according to the invention, adapted to natural cross-pollination, contain somewhat improved results of the fusion of randomly selected protoplasts. However, cultivated lettuce plants,
This represents a particularly advantageous embodiment.

L. T. tarica protoplasts are Specific embodiments of the plant according to the present invention can be obtained by fusing with the S. protoplasm and regenerating the plant from the fusion product thus obtained. In an alternative embodiment, L. to obtain a fusion product. T. tarica protoplasts are plants fused with the protoplasts of S. seriola or other wild lettuce species capable of crossing cultivated lettuce, and then regenerated from the resulting fusion product were transformed into L. cerevisiae plants. sativa. By backcrossing with the above, a fused plant can be obtained. This gives rise to fused plant derivatives.

By crossing the fused plant or fused plant derivative with a plant having the desired cultivated agricultural product trait, the trait of the fused plant or fused plant derivative that is convenient for cross-pollination is converted to the desired cultivated agricultural product trait. Can be combined with This method is repeated many times until the backcross of the self-pollinated generation with the arbitrarily combined cultivated agricultural plant finds the desired combination of cultivated agricultural trait and cross-pollinating trait. Can be repeated. Among the plants obtained by this method, an inbred line is made to produce a suitable pure line as a parent line for producing hybrid seeds. The end result of this method is referred to herein as "parent plant suitable for hybrid seed production".

Obtained by crossing fused plants and fused plant derivatives, and their progeny or progeny of the next generation, with other plants, including parent plants, suitable for hybrid seed production containing traits favorable to cross-pollination All plants are part of the present invention.

A “hybrid plant” is obtained from the seeds of a cross between parent plants, at least one of which, in particular, the mother plant has a trait favorable for cross-pollination (optionally non-hybrid). This seed is the “hybrid seed”.

Also, a part of the present invention is obtained by the present invention having traits that are favorable for cross-pollination, especially the altered flower color and morphology, such as flower color close to blue or white and relatively large flowers, Seeds from fused plants, fused plant derivatives and all other plants according to the invention. In a preferred embodiment, these plants are lettuce plants (cultivated).

The present invention will be further described with reference to the accompanying examples. In the embodiment, lettuce is used as a model type. However, the invention is not limited to lettuce.

In the examples, reference is made to the accompanying figures.

[Examples] [Example 1] [Establishment of parent strain in vitro] A protoplast is isolated from a leaf material of a parent plant. In this embodiment, a method for obtaining the leaf material will be described.

The seeds are sterilized by first rinsing with 70% ethanol and then rinsing with 0.7% NaOCl for 20 minutes. Then rinse three times with sterile demineralized water.

Murashige and Skoog (MS) containing 2% sucrose and no hormones
) Sow the seeds on a nutrient medium. In order to obtain green plants, first at 15 ° C in the dark
The seeds are pre-germinated for 2 days at, and then grown further at 25 ° C in the light (3000 lux, 16 hour light period). When the first leaves become visible, the top of the shoot is cut off and transferred to an MS nutrient medium containing 3% sucrose and no hormones. Sterilized shoots are subcultured under the same conditions.

After cold treatment at 15 ° C. to obtain a white tissue, for example hypocotyls, germination is carried out in the same manner except that the plants are placed in the dark and kept at 25 ° C. in the dark.

Using the green tissue of the fused parent and other white tissue, non-fused cells or autologous fusion (
It is possible to microscopically discriminate the desired fusion product in the fusion mixture from the fusion between cells from the same fusion parent.

[Example 2] [Isolation of protoplasts] A 3-week-old sterilized leaf material was finely chopped, and a PG solution (54.66 g /
1 sorbitol, 7.35 g / l CaCl ₂ .2H ₂ O) for 1 hour. Thereafter, the PG solution was replaced with an enzyme solution containing 1% cellulase and 0.25% macerozyme. This material was incubated for 16 hours at 25 ° C. in the dark.

Next, the suspension was filtered through a nylon filter (45 μm),
By centrifuging at 700 rpm for 8 minutes, the CPW16S solution (Frearson,
Em, Power JB & Cocking, ES (1973) Developmental Biology 33: 130-137)
Wash with one third of the volume. This results in a band containing the intact protoplast in the centrifuge tube. The protoplasts were collected and centrifuged at 600 rpm for 5 minutes to give a W5 solution (9 g / l NaCl, 18.38 g / l CaCl ₂ .2H ₂ O, 0.37 g / l KCl, 0. 99 g / l glucose and 0.1 g /
1 Mes buffer solution).

In the case of hypocotyls, the isolation is performed in the same manner.

Example 3 Protoplast Fusion Protoplasts isolated as described in Example 2 were mixed 1: 1 to form 6 × 1
0 ⁵ to a final concentration of protoplasts / ml. This protoplast suspension is mixed with sterile fusion solution 1
(500 g / l PEG 1500, 1.5 g / l CaCl ₂ .2H ₂ O and 0 g
. 1g / l KH ₂ PO ₄₎ of the 1: 1 mixture, placing 2-3 minutes at room temperature. Then
1 ml of fusion solution 2 (50 g / l glucose, 7.35 g / l CaCl ₂ .2H ₂ O and 0.22 g / l Caps (3- (cyclohexylamino) -1-propanesulfonic acid) buffer solution) Addition, then the whole is left at room temperature for 10-25 minutes. Thereafter, washing is performed several times by centrifugation at 700 rpm for 5 minutes. A medium called “B-lettuce” (1/2 B5 (Gamborg et al. (1968)
.) Exp Cell Res 50:. 151), CaCl 2 · 2H 2 O of 300mg / l, 14mg /
1 Fe-330, 270 mg / l sodium succinate, 103 g / l sucrose, 0.1 mg / l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0
. Resuspend the pellet in 3 mg / l 6-benzylaminopurine (BAP)).

[Example 4] [Selection and growth of fusion product] The protoplast suspension (including the fusion product) of Example 3 was subjected to “B-
1: 1 with Lettuce Medium. The mixture is poured in a thin layer and allowed to solidify.
This is then cut into four pieces and two pieces are floated on 8 ml of "B-lettuce" medium. Seal the Petri dish with tape and place at 25 ° C. After 2-3 days, the freshly prepared “B”
Dilute the medium with "lettuce" medium. When the callus size was ± 0.5 mm, the callus was grown in a callus growth medium (30 g / l sucrose, 5 g / l agarose, 0.1 m
S containing g / l of α-naphthaleneacetic acid (NAA) and 0.1 mg / l of BAP
H2 (Schenk, RU & Hildebrandt, AC (1972) Can. J. Bot. 50: 199). After ± 2 weeks, the regeneration medium (15 g / l sucrose, 15 g / l glucose, 5 g / l agarose, 0.1 mg / l NAA, 0.1 mg / l BAP
Calli can be transferred onto SHregs containing Schenk & Hildebrand (supra). After about 6 weeks, the first plants are harvested and placed on rooting medium (Schenk & Hildebrand containing 30 g / l sucrose and 8 g / l agar). Visually (based on the appearance of two hybrids and a different hybrid) or PCR, AFLP, RFLP
Selection of fusion products can be made using molecular biology techniques such as.

[Example 5] [Analysis of fused plants and progeny derived therefrom] [1. Introduction] In the fusion experiments described in Examples 1 to 4, L. T. tarica protoplasts were obtained from
Serriola protoplasts were fused. The fused plants were regenerated and confirmed using PCR analysis, flow cytometry and based on external characteristics.

[2. Appearance] The obtained fused plant showed strong plant growth and had large pale blue flowers. 1 to 4 are diagrams showing the difference between the parent of the fused plant and the fused plant itself.

FIG. Tataica parent plant, fusion plant in the center, L on the right
. It is a figure which shows a serriola parent plant.

FIG. tatarica leaves, fusion plant leaves in the center, and L. cerevisiae in the lower right. It is a figure which shows the leaf of a serriola fusion parent plant.

FIG. 5 flowers of the fusion plant in the center and L. It is a figure which shows five flowers of serriola.

[3. Hybridization with fused plant] The fused plant was cultivated in a flowerpot in a greenhouse and was flowered. Some flowers were not pollinated, some of the flowers of the fused plants were pollinated with pollen of the lettuce variety "Norden", and the flowers of the fused plants were used to pollinate male sterile (MS) lettuce plants. Seeds were obtained by self-pollination and hybridization as described. The population obtained in this way was T (+) S for the fusion plant, I1 (T (+) S) for the self-pollinated seed of the fusion plant, and the fusion plant (as mother) with the lettuce variety “Norden”. Seeds from the cross are (T (+) S)
× N, Seeds from the cross between the MS lettuce plant (as mother) and the fused plant are MS × (
T (+) S).

[4. Determination of Ploidy Level of Fusion Plants] The DNA content of many plants of the above population was determined by flow cytometry.
The control is L. serriola system, L. et al. sativa "Norden", L.A. s
ativa "Flora", and L.A. Tatarica.

Iribo, a laboratory for tissue culture, modification and flow cytometry
v, Flow cytometry assays were performed according to standard procedures by Westeinde 149a, 1601 BM Enkhuizen. All plants were measured using an internal standard. Six plants were assayed in duplicate.

For plants measured in duplicate, the maximum difference between the two measurements was 1.2%. The relative DNA content of the different types of material is shown in the table below.

[0046]

[Table 1]

From the table, it is apparent that L. Serriola DNA content (99%) was determined by sativ
a. is substantially the same as the DNA content of L. a. It can be seen that Tatarica has about 1.5-fold DNA (157%). The fusion product has a DNA content consistent with the sum of the DNA content of the fusion parents. 257 is about 99 + 1
57. The I1 (T (+) S) plants obtained from self-pollinated seeds of the fused plants had an average DNA content, approximately 254.4, which roughly matched the fused plants.
However, these I1 plants show a broad distribution (233.6-266.9),
This could be explained by the appearance of aneuploid plants in this generation. Phenotypic observations (large deviations, dense growth) also suggest that 2-3 of the 35 plants tested were aneuploid.

[0048] The fused plants were cv. The DNA content of the hybrid plants obtained by crossing "Norden" or male sterile lettuce lines was determined according to L. et al. serriola / L. sativ
a set of chromosomes at the level of L. a. at the level of Tatarica, which is consistent with the triploid level containing one set of chromosomes. 178 roughly matches 100 + 1/2 * (157).

Based on the three flow cytometric assays, the majority of the plants obtained from the fused plants as well as from self-pollinated seeds of the fused plants are L. cerevisiae. whole plant diploid genome and L. It can be concluded that the entire diploid genome of the Serriola parent plant is included. Thus, these plants are diploid. The fused plant was used as L. plants obtained by crossing with the C. sativa genotype are DNA
Has content. L. The complete haploid set of the Serriola chromosome, L. sa
The complete haploid set of the Tiva chromosome and the L. This is consistent with the sum of the complete haploid set of the Tataca chromosome. Therefore, these plants have a triploid DNA
Have a level.

[Example 6] [Backcrossing of (L. tatarica (+) L. seriola) x Norden plant] A plant obtained by crossing a fused plant as a mother with a lettuce variety “Norden” (briefly, For the sake of illustration, (T (+) S) × N is cultivated in a greenhouse and flowered. The (T (+) S) × N plants had large, very pale blue flowers that were, on average, somewhat smaller in size than the flowers of the fused plants. FIG. tatarica, L .; seriola, fusion plant, lettuce variety
FIG. 2 shows characteristic flowers of “Norden” (L. sativa) and (T (+) S) × N plant (T1). (T (+) S) × N plant flower with lettuce variety “Nor
The pollen of the "den" plant was pollinated.

Seed setting was not observed in the non-pollinated flowers of the (T (+) S) × N plants. However, after pollinating the pollen of "Norden", seeds were obtained. The population obtained by this cross is hereinafter referred to as ((T (+) S) × N) × N plants. ((
Plants of T (+) S) × N) × N population were cultivated in a greenhouse and flowered. DNA content was determined by flow cytometry. T (+) S and (T (+) S) × N
Samples were included as controls.

A flow cytometry assay was performed in the same manner as in Example 5. Eleven plants were measured in duplicate. For plants measured in duplicate, the maximum difference between the two measurements was 2.2%. The relative DNA content of the different populations is shown in the table below.

[0053]

[Table 2]

FIG. tatarica and L. et al. [Fig. 2] Fig. 2 shows PCR patterns of a fusion parent of seriola, a fusion plant, and a fusion plant derivative. From left to right: 1. DNA ladder (for fragment size determination); L. tatarica (
T), relative DNA content 1.61; L. sativa "Norden" (
N), relative DNA content 1.00, 4. L. seriola (S), relative DNA content 0.99,5. 1. Fusion plants (T (+) S), relative DNA content
62, 6. 6. Fusion plant (T (+) S), relative DNA content 2.66, 7. 7. fused plant (T (+) S), relative DNA content 2.71; (T (+) S) × N plants,
Relative DNA content 2.36, 9. (T (+) S) × N plants, relative DNA content 1.84,10. (T (+) S) × N plants, relative DNA content 1.85, 1
1. ((T (+) S) × N) × N plants, relative DNA content 1.11, blue flowering, 12. ((T (+) S) × N) × N plants, relative DNA content 1.04, blue flowering, ((T (+) S) × N) × N plants, relative DNA content 1.4
4. blue flowering; ((T (+) S) × N) × N plants, relative DNA content 1.00, yellow flowering.

The plants in lane 8 show the resulting I ₁ (T (+) S
) Considered a plant.

From FIG. 5, it can be seen that the fused plant obtained by crossing the fused plant with the lettuce variety “Norden” and the (T (+) S) × N plant show a marker pattern consisting of the sum of the markers found in both fused parents. It can be seen that The plant ((T (+) S) × N) × N plant obtained after crossing twice with “Norden” was obtained by the PCR reaction.
L. serriola parent or L. Only the markers of the sativa parent are shown.

In a ((T (+) S) × N) × N population obtained as a result of crossing a triploid (T (+) S) × N mother plant with a diploid N father plant, it is mainly doubled. Above the somatic or diploid level, aneuploid plants with one or several additional chromosomes are expected. At the diploid level, the plant is If no T. tarica chromosome were included, a relative DNA content of 1.0 would be expected, and plants would still be L. data
If the entire haploid genome of ica is included, a content of 1.3 (0.5 + 0.8) is expected. ((T (+) S) × N) × N Most of the plants (84%)
. It has a DNA content ranging from 98 to 1.30.

Twenty-one ((T (+) S) × N) × N plants have a relative DN between 1.3 and 1.8.
It has an A content, which indicates the chromosome number of the aneuploid.

Two ((T (+) S) × N) × N plants have a relative DNA content of more than 2.0 (2.18 and 2.31). Plants with a relative DNA content of 2.3 are obtained as a result of fertilization of non-reduced (triploid) germs: 2.3 = 1.8 +
0.5.

(For flower morphology and seed growth in case of self-pollination, (T (+) S)
XN) xN plants were tested.

FIG. 6 is a diagram showing changes in flower size and flower color of ((T (+) S) × N) × N plants (= T2 fusion). In the ((T (+) S) × N) × N population,
Significant changes in flower size and flower color were observed (flower bloom from very pale yellow to very dark yellow in addition to blue (sometimes close to white)).

[0062]

[Table 3]

Out of 141 ((T (+) S) × N) × N plants, 33 had blue flowers.
Some plants had flowers that were significantly larger than the lettuce variety "Norden".

Most of the ((T (+) S) × N) × N plants have fully restored fertility, and therefore obtain large amounts of self-pollinated seeds (>> 1000 per plant). Can be.

FIG. 7 shows restored fertility with very pale blue flowers ((T (+) S
It is a figure which shows the growth of the seed of a)) * N) * N plant.

Example 7 Suitability of Insect Pollination of Fusion Plants Two insect cages (each with a tight mesh with a surface area of about 10 m ² ) were placed in a greenhouse. Nine species were planted per cage, in each case eight lettuce varieties and I1 (T (+) S) plants (May 1997). Variants are Camaro RZ (red Batavia type), Remco RZ (green butterhead lettuce), Kublai RZ (red oak leaf type), Panth
eon RZ (green Batavia type), Kristine RZ (green oak leaf type), Roxette RZ (ice burger lettuce), Loletta
RZ (red laro type) and Remus RZ (green cosme lettuce). When flowering began (mid-May 1997), bees (Apis mel)
lifeera) and bumblebees (Bombus terrestris) were introduced into cages. Next, the number of bees and bumblebees observed on the flowers of the varieties or I1 (T (+) S) plants was recorded on day 6 during flowering.

[0067]

[Table 4]

[0068]

[Table 5]

From the above results, it can be seen that both honeybees and bumblebees visit the I1 (T (+) S) plant more frequently than lettuce varieties. Honeybees were confirmed to visit flowers of the I1 (T (+) S) plant on average 12 times more than all test lettuce varieties. , The most frequently visited lettuce variety (Cam)
Compared to aro RZ), the I1 (T (+) S) plant was 5-fold. Bumblebees visit flowers on I1 (T (+) S) plants on average 28 times more than lettuce varieties, and bumblebees visit the most frequent lettuce varieties (Kristine R)
Z) was 10 times. This shows that the I1 (T (+) S) plants are clearly more suitable for natural crossing (pollination by insects) than the lettuce varieties tested.

FIG. 8 shows the flowers of the I1 (T (+) S) plant visited by the bees.

[Brief description of the drawings]

FIG. 1 shows the difference between a fused plant and two parents.

FIG. 2 shows the difference between the leaves of the fused plant and the leaves of the two parents.

FIG. 3 shows the difference between the flowers of the fused plant and the flowers of the two parents.

FIG. 4 shows the difference between the flowers of the fused plant, the flowers of the two parents of the fused plant and the flowers of the progeny of the next cross with the fused plant.

FIG. 5 shows the differences between the PCR patterns of the fused plant, the fused plant derivative and the two parents.

FIG. 6 is a diagram showing changes in flower color and flower size of the progeny of the next cross with the fused plant.

FIG. 7 is a diagram showing the growth of seeds in the progeny of the next cross with a fused plant whose fertility has been restored.

FIG. 8 shows insect visits to plants derived from fused plants.

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Claims (20)

[Claims] 1. preparing a protoplast of a first parent plant and a protoplast of a second parent plant; and transforming the first protoplast into the second protoplast to obtain a fusion product. Producing a fused plant by fusing with a trait and regenerating the fused plant from the fusion product.
One having one or more traits favorable to natural cross-pollination, wherein at least one trait is expressed in the fusion plant. Of producing a fusion plant for use in a plant. 2. a) At least one of the two parent plants has one or more traits favorable to natural cross-pollination, wherein at least one trait is expressed in the fusion plant. Preparing a protoplast of the first parent plant and a protoplast of the second parent plant, and fusing the first protoplast with the second protoplast to obtain a fusion product. Producing a fused plant by regenerating the fused plant from the fusion product; b) combining the fused plants with each other and / or to obtain a plant suitable for natural cross-pollination and having the desired trait. Further comprising a step of crossing at least once (back) with a non-fused plant having the following traits. 3. The trait favorable to cross-pollination comprises at least the size and / or color of a flower attractive to insects.
Or the method of 2. 4. The method according to claim 2, wherein the desired trait is an agricultural trait to be cultivated. 5. The method according to claim 5, wherein the two parent plants are lettuce plants of the genus Lactuca; The method according to claim 3 or 4, wherein the method is derived from a parent plant of the species tarataca and consists of a large flower having a blue-violet or white color. 6. The other parent plant is L. lettuce, which is a cultivated lettuce. The method according to claim 5, wherein the plant is a plant of the species sativa. 7. The other parent plant is L. lettuce being cultivated. The method according to claim 5, characterized in that the plant is a Lactuca plant capable of substantially completely crossing a plant of the Sativa species. 8. The cultivated lettuce L. Another parent plant, a plant of the species Lactuca, which is capable of substantially completely crossing a plant of the species sativa, is L.
. The method according to claim 6, which is a plant of the species serriola. 9. A fused plant obtained by using the method according to claim 1 or 3 to 8. 10. L. tatarica protoplasts and L. 10. A fused plant according to claim 9, characterized in that it is obtained by fusion with a protoplast of sativa and regeneration of the plant from the fusion product thus obtained. 11. L. tatarica protoplasts and L. seriola
A fused plant according to claim 9, characterized in that it is obtained by fusion with a protoplast of E. coli and regeneration of the plant from the fusion product thus obtained. 12. The fused plant according to claim 11, which is a L. plant. A fused plant derivative obtained by crossing with C. sativa. 13. A plant suitable for natural cross-pollination, which is obtained using the method according to any one of claims 1 to 8. 14. The plant according to claim 9, which is used for producing a hybrid plant by crossing said plant with a crossing partner having a desired trait. 15. The plant according to claim 14, wherein the desired trait is an agricultural trait to be cultivated. 16. The plant seed according to claim 9, which is obtained by self-pollinating such a plant. 17. A pure line is produced from a plant according to any of claims 9 to 15 or a plant obtained by one or more crossings of such a plant with a plant having the desired cultivated agricultural trait. And hybridizing a pure line plant as a first parent plant with a second parent plant from the pure line, and collecting seeds obtained from the cross. 18. A method for obtaining homozygous lines by inbringing plants suitable for natural cross-fertilization and having essential cultivated traits, or by producing doubled haploids, or 2. A pure homozygous line produced by any of the other methods known in the art.
7. The method according to 7. 19. Hybrid seed obtained using the method according to claim 17 or 18. 20. A method for producing a hybrid plant, comprising the steps of germinating a hybrid seed obtained using the method of claim 17 or 18, and cultivating the hybrid plant therefrom.