NL1009044C2 - Strict self-fertilizers with a modified flower morphology. - Google Patents

Description

STRICT SELF-RECOMMENDERS WITH A MODIFIED FLORAL morphology

The present invention relates to a method for manufacturing plants of an originally strictly self-fertilizing type, in particular lettuce plants, which are nevertheless suitable for cross-fertilization. The invention further relates to the plants and seed thereof manufactured in this way, as well as the use of the plants in a method for obtaining hybrid plants, and the hybrid plants and seed derived therefrom.

Hybrid varieties are commercially attractive in almost all cultivated crops, partly because of the uniformity of hybrids and the possibility of exploiting heterosis. Heterosis is the phenomenon that hybrid plants, which are created by crossing (more or less 15) inbred parent plants, have a better performance for certain characteristics, such as yield, than both parents. A major advantage of hybrid varieties is that they perform better and more stable on average under varying environmental conditions.

For example, seed-resistant lettuce varieties often have a very limited growing environment (place, soil type, season) under which they perform well. Hybrid lettuce varieties could be suitable for larger growing areas and / or environments. Hybrid lettuce can further be an interesting product for example because of the said uniformity, increased vigor and stability due to heterosis effects, and combination of properties. Greater vigor is especially important for winter crops and dark red colored lettuce types.

In hybrid lettuce, it would also be easier to use genes from wild species. For example, some interesting genes from L.virosa have been linked to genes that cross-breed a culture lettuce genome to cause reduced growth. This has been found in particular for a gene for resistance to Bremia and to the aphid Nasonovia ribisniori. The reduced growth, which is in principle undesirable, is only expressed in the 1009044 2 homozygous, and with the correct choice of the parent plants therefore not in the hybrid, while the resistance associated with the reduced growth is dominant, and therefore in the hybrid does lead to resistance. Moreover, in a hybrid such a gene for reduced growth would be beneficial from the perspective of seed companies, because this makes after-crop cultivation even more unattractive.

Obtaining hybrid varieties is therefore interesting and desirable for various reasons.

10 In order to make hybrid varieties, it is a requirement that cross-pollination can occur within the species. However, some crops are strictly self-fertile by nature. Within a genus, in addition to self-fertilizing species, cross-fertilizing species can occur.

15 An example of a strictly self-fertilizing species is the cultivated lettuce. Lettuce belongs to the genus Lactu-ca. which in turn can be subdivided into a number of sections, within which subsections can again be distinguished. The Lactuca species occurring in Europe are divided into four sections, namely Lactuca. Lactu copsis. Mulqedium and Phaenixopus. Two subsections can be distinguished within the Lactuca section: Lactuca and Cvanicae. The cultivated lettuce (Lactuca sativa L.) belongs, as do the crucible relatives (L. serriola. L. aculeata.

25 L. scarioloides. L.azerbaiianica, L.qeorqica. L.dreqeana. L.altaica. L.saliqna and L.virosa) to section Lactuca. subsection Lactuca.

To the Lactuca section. subsection Cvanicae include L. tenerrima. L.perennis and L.qraeca.

30 The Mulqedium section includes L. tatarica and L. sibirica. These species are taxonomically close to the genus Mulqedium. L. tatarica has been reported to have been crossed with Mulqedium bourgaei. and with the American amphidiploid (2n = 34) Lactuca species L.floridana.

3 5 L. canadensis and L. qraminifolia.

Within the genus Lactuca, both strictly self-fertilizing (autogame) and cross-fertilizing (xeno-game) species occur. The problem is that the species 1009044 3 in the Lactuca section. subsection Lactuca. to which the cultivated lettuce belongs are strict self-fertilizers. Natural cross-fertilization rates of only 1-3% were found for L. open-air cultivation with 5 different genotypes grown adjacent to each other (Thompson, RC, TW Whitaker, & GW Bohn, Proc. Natl. Soc. Horticul. Sci 36 (1958)).

It is now the object of the invention to provide the possibility of making plants, which are naturally self-fertilizing and have flowers, which are unattractive to pollinating insects, attractive to pollinators, so that these plants can be used for the production of hybrid seed in commercially required quantities.

This is accomplished according to the invention by a method of manufacturing fusion plants, for use in obtaining plants suitable for natural cross-pollination, comprising producing fusion plants by preparing protoplasts from a first parent plant and protoplasts from a second parent plant, fusing the first protoplasts with the second protoplasts to obtain a fusion product, and regenerating fusion plants from the fusion product, at least one of both parent plants having one or more properties for natural cross-pollination, at least one of which is in the fusion plant.

The fusion plants themselves are not directly suitable for use as a cross parent in the manufacture of 30 hybrids. The invention therefore further relates to a method of manufacturing plants suitable for natural cross-pollination, comprising: a) manufacturing fusion plants by preparing protoplasts from a first parent plant and protoplasts from a second parent plant, fusing the first protoplasts with the second protoplasts to obtain a fusion product, and regenerating fusion plants from the fusion product, wherein at least 1009044 4 one of the two parent plants has one or more beneficial properties for natural cross-pollination, at least one of which is expressed in the fusion plant, and b ) further (back) crossing the fusion plants at least once with each other and / or with a non-fusion plant with desired properties to obtain plants which are suitable for natural cross-pollination and have the desired properties.

In this application, "natural cross-pollination" is understood to mean any form of cross-pollination which is not effected by active human intervention. Such a natural form of cross-pollination is, for example, pollination by insects.

The desire to be able to make hybrid plants is particularly great for lettuce. Cross-fertilizing Lactu-ca spp., Such as L. perennis, are usually pollinated by insects. Within the genus Lactuca it has been shown that flower size and morphology are strongly related to the degree of cross-fertilization, and thus also to the attractiveness of the flower to pollinating insects. Xenogamy is strongly correlated with large flower heads of a blue or bright yellow color. It is therefore preferable to ensure that the cross-pollination favorable properties in lettuce comprise at least an attractive flower size and / or flower color for insects.

In one embodiment of the invention, the two parent plants are therefore lettuce plants of the genus Lactuca and the cross-pollinating properties favorable from a parent plant of the species L. tatarica 30 and these properties consist of large and / or blue-purple or almost white flowers. The plants derived from fusion plants can have yellow or blue-purple flowers. Variation in color intensity occurs in both colors. The degree of purple-blue staining can vary widely. Some purple-blue flowers are so pale that they appear almost white in color.

As the other parent plant, a culture lettuce can be chosen immediately, or a so-called "bridge type". In 1009044 5 the classic meaning, the term "bridge species" stands for a species, which is used in species crossings when the two species to be crossed are too far apart for direct crossings. A bridge type must be between two parents to be crossed and preferably be at least slightly crossable with both. In the case of mergers, the term "bridge species" is used to mean a crossable relative of the species to which genetic material is to be transferred, but which has better or more desirable properties for the fusion process, such as better fusion capacity or better regeneration capacity.

When a cultivated lettuce (instead of a bridge species) is directly chosen for the production of fusion plants, the other parent plant is, for example, of the cultivated lettuce species L. sativa. In the second case, where a bridge type is used, one can think of L.serriola.

Plants obtained after (back) crossing a fusion plant with a plant of a cultivated lettuce species can no longer be directly considered to be fusion plants and are therefore also referred to herein as "fusion plant derivatives". The end result of multiple (back) crosses and / or self-pollinations are referred to herein as "plants suitable for natural cross-pollination". "Fusion plants", "fusion plant derivatives" and "plants suitable for natural cross-pollination" are collectively referred to as "plants of the invention".

The invention further provides fusion plants, fusion plant derivatives and plants suitable for natural cross-pollination obtainable by the method of the invention. In principle, these three types of plants made suitable for natural cross-pollination according to the invention all comprise the more or less advanced result of fusion of randomly chosen protoplasts. However, cultivated lettuce plants are a particularly advantageous embodiment of this.

1009044 6

A specific embodiment of plants according to the invention is obtainable by fusion of protoplasts of L. tatarica with protoplasts of Lsativa and regeneration of plants from the fusion product thus obtained. In an alternative embodiment, fusion plants can be obtained by fusion of L. tatarica protoplasts with L. serriola protoplasts. or other wild lettuce, which can be crossed with cultivated lettuce, to obtain a fusion product and then to backcross plants regenerated from the obtained fusion product with L. sativa. From this arises a fusion plant derivative.

By crossing between fusion plants or fusion plant derivatives and a plant with desired cultivated crop properties, the cross-pollinating properties of the fusion plant or fusion plant derivative can be combined with desired cultivated crop properties. By backcrossing with cultivated plants, possibly combined with generations in which self-pollination is applied, this process can be repeated repeatedly until the desired combination of cultivated crop properties and favorable properties for cross-pollination have been found. Inbred lines are made from the plants obtained in this way to produce pure lines suitable as parent line for the production of hybrid seed. The end result of this process will be referred to herein as "parent plants suitable for hybrid seed production".

Both the fusion plants, as well as the fusion plant-derived, and all plants obtained by crossing these or their next generation offspring with other plants, including parent plants suitable for hybrid seed production, which contain the cross-pollinating properties, are part of the invention. "Hybrid plants" arise from the seed of the cross between (optionally non-hybrid) parent plants, at least one of which, in particular the mother 1009044 7 plant, has the properties favorable for cross-pollination. This seed is "hybrid seed".

Also part of the invention are seeds from both the fusion plants, fusion plant derivatives and all other resulting plants according to the invention, which have favorable properties for cross-pollination, in particular an altered flower color and morphology, such as blue or almost white flower color and relatively large flowers. In a preferred embodiment, these plants are (cultivated) lettuce plants.

The present invention will be further elucidated by means of the accompanying examples. In the examples, lettuce is used as a model species. However, the invention is not limited to this.

In the examples reference is made to the accompanying figures.

Figure 1 shows the differences between a fusion plant and the two parents.

Figure 2 shows the differences between leaves 20 of a fusion plant and both parents.

Figure 3 shows the differences between the flowers of a fusion plant and the two parents.

Figure 4 shows the differences between the flowers of a fusion plant, both parents of the fusion plant and an offspring of a subsequent cross with a fusion plant.

Figure 5 shows differences in PCR pattern of fusion plants, fusion plant derivatives and both parents.

Figure 6 shows the variation in flower color and flower size of a progeny of a subsequent cross with a fusion plant.

Figure 7 shows the seeding in a progeny of a subsequent cross with a fusion plant with restored fertility.

Figure 8 shows insect visits to a plant derived from a fusion plant.

1009044 8 EXAMPLES EXAMPLE 1

Setting up an in vitro stock of plants

The protoplasts are isolated from leaf material of the parent plants. This example describes how this sheet material is obtained.

Seed is sterilized by first rinsing with 70% ethanol and then with 0.7% NaOCl for 20 minutes. Then it is rinsed 3x with sterile demi 10 water.

The seeds are sown on Murashige and Skoog (MS) nutrient medium with 2% sucrose, and without hormones. To obtain green plants, the seeds are first pre-germinated for 2 days at 15 ° C in the dark, after which they continue to grow at 25 ° C in the light (3000 lux, 16-hour photoperiod). When the first leaf becomes visible, the shoot tips are cut and transferred to MS nutrient medium with 3% sucrose, without hormones. Under the same conditions, the sterile shoots are grown in subculture.

In order to obtain white tissue, for example hypocotyls, germination is done in the same way, but the plants are not put away in the light, and after the cold treatment of 15 ° C also stored in the dark at 25 ° C.

The use of green tissue from one fusion parent and white tissue from the other makes it possible to distinguish the desired fusion products microscopically from unfused cells or from autofusions (fusions between cells from the same fusion parent) in the fusion mixture.

EXAMPLE 2

Isolation of protoplasts. Three-week-old sterile leaf material is finely cut and pre-plasticized in PG solution (54.66 g / l sorbitol and 7.35 g / l CaCl2.2H2O) for 1 hour in the dark. The PG solution is then replaced with »41 1009044 9 an enzyme solution with 1% cellulase and 0.25% macerozyme. The material is incubated in the dark at 25 ° C for 16 hours.

The suspension is then filtered through a nylon filter (45 μιη) and washed with a third volume of CPW16S solution (Frearson, EM, Power, JB & Cocking, ES (1973) Developmental Biology 33: 130-137) by centrifugation at 700 rpm for 8 minutes. This creates a band with 10 intact protoplasts in the centrifuge tube. The protoplasts are collected and washed with W5 solution (9 g / 1 NaCl, 18.38 g / 1 CaCl2.2H20, 0.37 g / 1 KCl, 0.99 g / 1 glucose, and 0.1 g / 1 Knife buffer) by centrifugation at 600 rpm for 5 minutes.

In the case of hypocotyls, the isolation is carried out identically.

EXAMPLE 3

Protoplast Fusion 20 Protoplasts, isolated according to Example 2, are mixed 1: 1 to a final concentration of 6x10 5 protoplasts / ml. The protoplast suspension is mixed 1: 1 with sterile fusion solution 1 (500 g / 1 PEG 1500, 1.5 g / 1 CaCl2.2H20 and 0.1 g / 1 KH2PO4) and left at room temperature for several minutes. Then 1 ml of fusion solution 2 (50 g / 1 glucose, 7.35 g / 1 CaCl2.2H20 and 0.22 g / 1 Caps (3- (Cyclohexylamino) -1-propanesulfonic acid) buffer) is added, after which the whole Set aside for 10 to 25 minutes at room temperature. After this it is washed several times by centrifugation at 700 rpm for 5 minutes. The pellet is resuspended in B lettuce (½ B5 (Gamborg et al. (1968) Exp. Cell Res. 50: 151), 300 mg / 1 CaCl2.2H20, 14 mg / 1 Fe-330, 270 mg / 1 sodium succi -Nate, 103 g / l sucrose, 0.1 mg / l 2,4-Dichlorophenoxya-35 his acid (2,4-D) and 0,3 mg / l 6-benzylaminopurine (BAP)).

1009044 10 EXAMPLE 4

Selection and growth of fusion products

The protoplast suspension from Example 3 (including fusion products) is mixed 1: 1 with B-lettuce medium with agarose. This mixture is poured in a thin layer to allow it to solidify. After this it is cut into quarters and allowed to float per 2 pieces in 8 ml B-lettuce medium. The Petri dishes are taped and stored at 25 ° C. After a few days, the medium is diluted with fresh B-lettuce medium. When the calli are ± 0.5 mm in size, they are placed on callus growth medium (SH2 (Schenk, RU & Hildebrandt, AC (1972) Can. J. Bot. 50: 199) with 30 g / 1 sucrose, 5 g / 1 agarose 0.1 mg / l α-naphthalene acetic acid (NAA) and 0.1 mg / l BAP). After ± 2 weeks 15, the calli can be transferred to regeneration medium (SHreg (Schenk & Hildebrand, supra) with 15 g / 1 sucrose, 15 g / 1 glucose, 5 g / 1 agarose, 0.1 mg / 1 NAA and 0.1 mg / 1 BAP). After about 6 weeks, the first plants can be harvested and put on rooting medium (Schenk & Hildebrand with 30 g / 1 sucrose and 8 g / 1 agar). The selection of the fusion products can be done visually (based on their hybrid appearance different from both fusion parents) or by molecular biology techniques, such as PCR, AFLP, RFLP.

EXAMPLE 5

Analysis of fusion plants and their derived generations 1. Introduction

In a fusion experiment according to Examples 1-4, protoplasts of L. tatarica were fused with protoplasts of L. serriola. Fusion plants were regenerated and monitored by PCR analysis, flow cytometry, and appearance.

35 2. Appearance

The fusion plants obtained had a strong vegetative growth and large light blue colored flowers.

1 0 0 9 0 A A

11

Figures 1-4 show the differences between the parents of the mergers and the merged plants themselves.

Figure 1 shows a L. tatarica parent plant on the left, a fusion plant in the center and a L. serriola-5 parent plant on the right.

Figure 2 shows a leaf of L. tatarica at the top left, a leaf of a fusion plant in the middle and a leaf of the L.serriola fusion parent at the bottom right.

Figure 3 shows 5 flowers of L. tatarica on the left. 10 in the middle 5 flowers of a fusion plant and 5 L.serriola flowers on the right.

3. Crosses with fusion plants

Fusion plants were grown in pots in the greenhouse and blossomed. Part of the flowers were not pollinated, part of the flowers of the fusion plants were pollinated with pollen of the lettuce variety 'Norden' and part of the flowers of fusion plants was used to pollinate male sterile (MS) lettuce plants. Seeds were obtained both from self-pollination and from the indicated crosses. The populations thus created are indicated as follows.

Fusion plants: T (+) S

Self-pollinating seed on fusion plants: I1 (T (+) S)

25 Seed from crossing a fusion plant as mother with the lettuce variety 'Norden': (T (+) S) x N

Seed from crossing an MS lettuce plant (as mother) with a fusion plant: MS x (T (+) S) 30 4. Determination of the ploidization level of fusion plants

The DNA content of a number of plants of the above populations was determined by flow cytometry. Controls were the L.serriola-line. L.sativa "Norden", L.sativa "Flora", and L.tatarica.

The flow cytometry determination was performed according to standard procedure by Iribov, Laboratory for tissue culture, breeding and flow cytometry, Westeinde 149a, 1601 BM Enkhuizen. All plants were measured with a 1009044 12 internal standard. Six plants were determined in duplicate.

In the plants measured in duplicate, the largest difference between the two measured values was 1.2%. In the table below the relative DNA contents of the different types of material are given.

Table 1

Relative DNA content (L.sativa cv. "Norden" = 100) with range 10 and standard deviation of some controls, of the fusion products and of the Lactuca plants derived from the fusion products.

Population Number Relative Range Standard plants DNA content deviation L.sativa cv. Norden (N) 1 100.0 15 L. sativa cv. Flora 2 99.7 99.6-99.8 0.14 L. serriola (S) 2 99.0 98.9-99.0 0.07

Ltatarica (T) 2 156.7 156.2-157.1 0.64 T (+) S 5 257.0 254.7-259.2 2.02 I1 (T (+) S) 35 254.4 233 .6-266.9 4.79 (T (+) S) x N 4 178.0 177.6-178.5 0.44

Ms x (T (+) S) 6 177.8 177.5-182.1 2.28

The table shows that the DNA content of L. serriola (99%) is almost identical to that of L. sativa and that 25 L. tatarica has about one and a half times as much DNA (157%). The fusion products have a DNA content corresponding to the sum of the DNA content of the fusion parents: 257 is about 99 + 157. The self-pollinated seed of the fusion plants had 1 (T (+) S) plants 30 average DNA content approximately corresponding to that of the fusion plants, namely 254.4. However, these II plants did show a wider distribution (233.6-266.9), which could be explained by the occurrence of aneuploid plants in this generation. Phenotypic observations (strongly deviating, stocky growth) also suggest that some of the 35 tested plants were aneuploid.

5 The DNA content of the crossing plants was obtained by crossing the fusion plants with cv. "Norden" or with a male sterile lettuce line corresponds to a triploid level with two sets of L. serriola / L.sativa chromosomes and one set of L. tatarica level 10: 178 corresponds to approximately 100 +

Ji * (157).

From these flow cytometry determinations it can be concluded that the fusion plants and most of the plants obtained from self-pollinating seed on the fusion plants contain the total diploid genome of the L. tatarica parent plant and the total diploid genome of the L. serrio-1a parent plant. Thus, these plants are allotetra ploid. The plants obtained by crossing fusion plants with L.sativa genotypes have a DNA content corresponding to the total of a complete haploid set of L.serriola chromosomes, a complete haploid set of L.sativa chromosomes and a complete haploid set of L. tatarica chromosomes. So these plants have a triploid DNA level.

25 EXAMPLE 6

Teruakruisina on (L.tatarica (+) L.serriola) x Norden plants

Plants obtained from crossing fusion plants as a mother with the lettuce variety 'Norden', hereinafter referred to as (T (+) S) xN for brevity, were grown in the greenhouse and blossomed. The (T (+) S) x N plants had large, very light blue-tinged flowers, which on average were slightly smaller in size than flowers of the 35 fusion plants. Figure 4 shows characteristic flowers of L. tatarica. L.serriola. a fusion plant, the lettuce variety 'Norden' (L.sativa) and a (T (+) S) x N plant (Tl). Flowers of the (T (+) S) x N plants were pollinated with pollen from plants of the lettuce variety 'Norden'.

No seeding was observed on non-pollinated flowers of (T (+) S) x N plants. After pollination with; 5 pollen from 'Norden' was obtained seed. The population obtained from this cross is referred to below as! ((T (+) S) x N) x N plants. The plants of the ((T (+) S) x N) x N population were grown in the greenhouse and flowered. The DNA content was determined by flow cytometry. Samples of T (+) S and (T (+) S): x N were included as controls.

Flow cytometry measurements were performed in the same manner as in Example 5. 11 plants were measured in duplicate. In the plants measured in duplicate, the largest difference between the two measured values was 2.2%. The table below shows the relative DNA contents of the different populations.

Table 2 20 Frequency distribution, mean and standard deviation for relative DNA content (L.sativa. Cv. Norden = 1.0) of ((T (+) S) x N) x N plants) and of some control populations

Population Relative DNA content N Average- ~ 1 deld deviation <1.0 1.0-1.3 1.3-2.0> 2.0 25 T (+) S 3 3 2.67 0.04 (T (+) S) x N 2 2 1.86 0.02 ((T (+) S) x N) 12 106 21 2 141 1.18 0.21

xN

Figure 5 shows a PCR pattern of the L.tatricica and L.serriola fusion parents. of fusion plants and of fusion plant derivatives. From left to right: 1. DNA ladder (for fragment size determination) 2. L.tatarica (T), relative DNA content: 1.61 tl 5 X »" 1009044 l Λ r 1 f 1 15 3. L.sativa "Norden" (N), relative DNA content: 1.00 4. L.serriola (S), relative DNA content: 0.99 5. fusion plant (T (+) S), relative DNA content: 2, 62 6. fusion plant (T (+) S), relative DNA content: 2.66 5 7. fusion plant (T (+) S), relative DNA content: 2.71 8. (T (+) S) x N plant, relative DNA content: 2.36 9. (T (+) S) x N plant, relative DNA content: 1.84 10. (T (+) S) x N plant, relative DNA content: 1.85 11. ((T (+) S) x N) x N plant, relative DNA content: 10 1.11, blue flowering 12. ((T (+) S) x N) x N -plant, relative DNA content: 1.04, blue flowering 13. ((T (+) S) x N) x N plant, relative DNA content: 1.44, blue flowering 15 14. ((T (+)) S) x N) x N plant, relative DNA content: 1.00, yellow flowering

The plant from lane 8 may be a Ιχ (T (+) S) plant created by the self-fertilization of a fusion plant.

Figure 5 shows that the fusion plants and the (T (+) S) x N plants obtained by crossing fusion plants with the "Norden" lettuce variety have a marker pattern consisting of the sum of the markers that were produced in both fusion parents. found. Plants obtained after crossing twice with "Norden" [((T (+) S) x N) plants] showed only the markers of L.serriola or L.sativa parents in this PCR reaction.

In the ((T (+) S) x N) x N population, created by crossing triploid (T (+) S) x N mother plants with 30 diploid N father plants, mainly diploid plants or aneuploid plants with a or expect some extra chromosomes above the diploid level. For the diploid level, a relative DNA content of 1.0 is expected if the plant does not contain L. tatarica chromosomes, and 1.3 (0.5 + 0.8) if the plant still has the total haploid genome of L.tatarica contains. A large proportion (84%) of the ((T (+) S) x N) x N plants have a DNA content in the range of 0.98 - 1.30.

1009044 16 21 ((T (+) S) χ Ν) x N plants have a relative DNA content between 1.3 and 1.8, which indicates an aneuploid chromosome number.

Two ((T (+) S) x N) x N plants have a relative DNA content above 2.0 (2.18 and 2.31). A plant with a relative DNA content of 2.3 can arise from fertilization of an unreduced (triploid) gamete: 2.3 = 1.8 + 0.5.

The ((T (+) S) x N) x N plants were examined for flower morphology and seeding by self-pollination.

Figure 6 shows variation for flower size and flower color in ((T (+) S) x N) x N plants (= T2 fusion). In the ((T (+) S) x N) x N population, a significant variation was found for flower size and flower color (from 15 very light yellow to very intensely yellow colored in addition to blue (to sometimes almost white) blooming flowers).

1009044 17 m__ <u ---- n ns ° H S ...

rH

p-J (N ------ I s Λ a | «- s ,,.

CD__________ 03 <- I — I § 0 n a ° H w ° o -I »,,, π 4) <N ^ yy · Η · -------- - r * * -> m

pH <0 - H

OrH H for rj - π μ α ο ίο * · u> ©

IN Η CN rH

'_________ «4-1 C

0) Q fc

I z r- 2 E

1 1 ® -S 0) Λ) x - E 0

Ü ^ VOfN ^ J · »- * X rH

ZC <D Η fN Η O, Q

a * w ------ 2.

<1> Λ r. C 8

- CN CD Φ 1J

_ Μ »Φ 0 1-4 ajOJ ^ r-HCOH 5 fl) - · rH rH rH rH c ^ 4

^ Z ______. ______ £ ID

fn HC * 0 Φ "- 'r» ιΛ Φ - - rH jj ij U fN r · MfNfD «ΛΙ —I rH« —I Η (N rH AJ g C -------- 4> c (0 v ay Ή ««

Qa CQ i i i i q, I * 2 ------ ~ 5 φ

2 CN

Ν '«0 σι Li V X Co CQiAin nr-rn -h o <N IN fN TJ s

2 H

<You

^ 7 - μ c * ^ + a - 6Γ μ

Sr1 Φ? S Η 4) Ό Ό

"r rH f — I C

~ __-_____ i s 0

W * - ï f H

cn tr g «Q Φ

—- · T ffi n «r ~ ocor * _ ® S

+ «J” "no <i Ü; - s H" 88 3_______q μ ai r, 3 ---- φ m σι c

r £ 5 - S 8 s

-Htninr * ocovo m a 'θ' rH

U fN CNN ^ Λ25 C 2 - 0) o b ^ E Ό 0 4)

aj j® cd E c λ 4J

> iJ H 4) E ®>

J aj C E Φ C

~ j = ή o o h * υ M rH 1>

CU___ _ _ _ «« ai μ Λ rH

You? A § aj M

n fi * S 3 S> ü «O 1) * * o δ u X potDt- o®« 0 rwcu W μ «H <H Ή« 4-1 t 4> (¾ 4) ^ £ ° g | > e

CD * I rH 4) AJ

öï Λ 4> aJ C

1 —-----—— .. μ Ό ay re

C m 4J Q rH

7 [~ «4» v o a <L) cd a λ μ μ c φ σ> 4). -η ·· c ϊ Τ3 I g CQ flj 4) i. 4) -0 ^, ^. > AJ T3 μ

H C 0 -H g £ * C Ή Q

rH 4) rH J4) 4) rH4Jfl {4) 0 rr yy A Ή00 4) AJrHO> (U r. c ITJ rH rH Jl Λ 5 r μφ4> τ3λλ σι ο -μ e ΓΊ I — I 3 rH τι μ < / 4J ah N m 4) 4) "'fci £ r ^ 3 rH 4) 4) Φ AJ AJ Ü Id Ö4 CD * ri lHC Ü X rH T3 AJ TJ row

nirti e 1 <0 Ό AJ C AJ C

ΜτΙφ E 4) AJ Ή Q Ιβ O ‘rH

no S o c ε o aj o 4>

S i 5 4 »μ W μ rH

(¾ H ^ tn ^ ocD'-uk: f-t an - ______ 1009044 18

On 141 ((T (+) S) x N) x N plants, 33 bloomed blue. There were also plants with clearly larger flowers than the lettuce variety "Norden".

A large part of the ((T (+) S) x N) x N plants 5 showed fully restored fertility, which made it possible to obtain large amounts of self-pollinating seed (>> 1000 per plant).

Figure 7 shows seed set on a very light blue flowering ((T (+) S) x N) x N plant with restored fertility.

EXAMPLE 7

Suitability of fusion plants for insect pollinators

Two insect-proof mesh isolation cages, each 15 with an area of about 10 m2, were placed in a greenhouse. Of eight lettuce varieties and II (T (+) S) plants, nine plants were always planted per cage (May 1997). The varieties were Camaro RZ (red batavia type), Remco RZ (green head lettuce), Kublaï RZ (red oak leaf type), Pan-20 theon RZ (green batavia type), Kristine RZ (green oak leaf type) , Roxette RZ (ice lettuce), Loretta RZ (red lollo type) and Remus RZ (green twine lettuce). At the beginning of flowering (mid-June 1997) honey bees (Apis mellife-ra) and bumblebees (Bombus terrestris) were brought into the cage.

The number of bees and bumblebees observed on flowers of the varieties or the II (T (+) S) plants was then recorded on 6 dates during flowering.

j i! 1 0 Ü 9 0 4 4 19 <υ

Ti

rH

<υ

Ti

Ti Ή ε <υ Q) Ti tn £

<D

4JO

ai c Λ <L)

Cn c fÖ <u nj Ό m c ...........

cd T3> g rH ij 0) C § e <y 3.

H £ vntnt'fNmr ^ r'OO

H, -HrooofsmHvor * · vU 0) «......

J> Q prlf * lrHmOC> r-i

rH

• Λ ---------

Ö Q) OU> OOOOrH

(DQ ^ Dooooooo 4-J O ¢) 0000000 P * flj Λ --------- rH U - ft -H u

, .. UHOOOOOfN

IN OlfHOOOOOO

you ......

OOOOOOOO

CO ft 3

Ή., I · - ... - - I ---— I

+ 4-)

v CQ S

Hrr- <4JpH ^ * OOOVOO

^ 0) Η ^ ΟΟΟΟιΗ

- TH X

,, ο00®0000 ι — I Ή (2 Μ YY __________ ρ ν s Q) XJ · Η

^ Η yJtT'J'OOOOiH

ι — I n ^ -coooom ö d) vJhoooooo <ΰ £ «

Cfl Jh --------- ca <υ ft t> c U o VJ, 4) dj JJ j ^ fHrN ^ JOooo

_I fli U «-4 <NOOOOO

r-i QJ c ......

0} ^ A50000000 ft ft ---------- 0 0 njoo * noou> ®

i-HOlT | rHOOO (N

C 4J Λ .......

<1) 03 | rwoooooo TH ___________

H

Λ - frj ij or-r-tooooin ë c g -; ^ 00000 _1 W 4) 0000000 Ή (y Qj C rH _________ O ft

rC

Li. O

. Γ] ._, MCO * HVÖVOOVD * i C d) Tj m ^ r-rHOOCOm fÖ ft C <Öflj0r- * 00000> 0 κ u ü> --------- d) d) ooooood) TH ^ Ό oonomo O · Η · η (Λ co <τ> cn σ \ ο

Nil) Η Η ^ 0 -Η ---------

Α Ή CQ

ι — ι £ cd w Λ \ Λΐ. 1 Λ \ C C0 00 CO C0 00 00 0) (D4 -> <iJ 3 ο ο ο ο ο ο _η Ο d η ^.

-j. d Γ »* Γ- cn -q · in (Ν σ> 4) (ΰ rH Π3 UQ Η -> Η Ρ1 (Ί Ο Η ffl πί nJ II— ... ------- - 1009044 20 α > τ3 φ -η η φ ε Ό Φ Ό Ο • Η Ö ε <υ φ Cη σι φ 4J τ3 φ Λ G Π3 α> (0 ΓÖ Ö I,., -U Φ CQ 0 c

α) S

τ3 ο ϊ

(- I - I

S'r-for-ojr-im ^ C -HyjLnvor ~ rHrnrr

L «Q. W

rl M4 τοΟΟΟΟΟΟ fl) Ο tu> ^ ---------- • Π ηοοοοοοο -i 30000000 ^ 0) 0000000 + J * α a --------- <Τ5 · Η * υ

rH 4-J jJOOOOOVOrH

ρ | φ 0) 0000000 I Τ3 οοοοοοοο - · η 13 C0 -Η ----------

- 4J

+

iJOVOOVOOOCN

ν— "QJ 0) 0000000 Ε- * ΟΟΟΟΟΟΟΟ ^ rH« Η (ϋ --------- Μ £ Μ Η £ Ijvohhoooui Φ S <noHi- »oooo οοοοοοοο £ XJ * 0) (L) - ------- CQ ^ β w 8

(Τ5 Q-I. COVOOOOH

^ Q JJOOOOOOO

teooooooo ι — I 4-) co as ---------- f), fOOVOOOOHrn

0 i-HOOOOOHO

xi ......

j \ 30000000

Cfl G ___ * ________

r — I aS

(U rH ΟΟΟΟΟΟΟΟ g οοοοοοοοο 0 Οοοοοοοο OM * ^ ω --------- ft ο

£ j ^ WOOHOOOCN

it η c „ι ° οΗ. ° ° ° °>> __ l ο re οοοοοοοο ΓΠ Ir 'οί υ Φ> λ; ε φ --------- Φ ε 1-0 ο ο ο ο ο ο QQ Ό ο ο η ο η ο

• Ν Λ Φ ΙήΙ I σ \ I col σ \! O 'I ^ I ο I II

ΙΓι φ -Η Η w 4 rH w --------- Η Ε It ΙΟ φφ4-ϊφ 3 ο ο ο ο σ ο · Λ Ο Ö Ο Α γν <r in <ν cn υ Π5 rI Π3 U ο ^ Hr-iMiso Η PQ Π3 (Ο Η- 1009044 21

The results show that the I1 (T (+) S) plants are much more frequented by both honey bees and bumble bees than the lettuce varieties. An average of 12 times higher bee visits were observed on flowers of the II (T (+) S) plants compared to all tested lettuce varieties. Compared to the most frequently visited slaras (Camaro RZ) by honey bees, the II (T (+) S) plants had a five-fold higher bee visit. The bumblebee visit was on flowers of II (T (+) S) plants averaged 28 times higher than on the lettuce varieties, and about 10 times higher than on the most frequently visited bumblebee lettuce variety (Kristine RZ). This shows that the I1 (T (+) S) plants have a markedly better suitability for natural insect cross-pollination than the tested-15 lettuce varieties.

Figure 8 shows a flower of an I1 (T (+) S) plant visited by a honey bee.

1009044

Claims (20)

A method of manufacturing fusion plants, for use in obtaining plants suitable for natural cross-pollination, comprising manufacturing fusion plants by preparing protoplasts from a first parent plant and protoplasts from a second parent plant, fusing the first protoplasts with the second protoplasts to obtain a fusion product, and regenerating fusion plants from the fusion product, characterized in that at least one of the two parent plants has one or more properties for natural cross-pollination, of which there are at least one is expressed in the fusion plant. ~ 2. A method of manufacturing plants suitable for natural cross-pollination, comprising: a) manufacturing fusion plants by preparing protoplasts from a first parent plant and protoplasts from a second parent plant, fusing the first protoplasts with the second protoplasts to obtain a fusion product, and regenerate fusion plants from the fusion product, at least one of the two parent plants having one or more properties for natural cross-pollination, at least one of which is expressed in the fusion plant, and b) further (back) crossing the fusion plants with each other and / or with a non-fusion plant with desired properties to obtain plants which are suitable for natural cross-pollination and have the desired properties. Method according to claim 1 or 2, characterized in that the properties favorable for cross-pollination comprise at least one flower size and / or flower color which is attractive to insects. 4. Method according to claim 2 or 3, characterized in that the desired properties are cultivated crop properties. jT "1 0 0 9 0 4 4 Method according to claim 3 or 4, characterized in that the two parent plants are lettuce plants of the genus Lactuca and the cross-pollinating properties are derived from a parent plant of the species L. tatarica and consist of large flowers with a blue purple or white color. Method according to claim 5, characterized in that the other parent plant is a plant of the cultivated lettuce species L. sativa. A method according to claim 5, characterized in that the other parent plant is a plant of a Lactuca species which is substantially fully crossable with a plant of the cultivated lettuce species L. sativa. 8. A method according to claim 6, characterized in that the other parent plant, which is a plant of a Lactuca species, which is substantially fully crossable with a plant of the cultivated lettuce species L. sativa. is a plant of the species L. serriola. 9. Fusion plant obtainable by the method according to claims 1 and 3-8. Fusion plant according to claim 9, obtainable by fusion of protoplasts of L. tatarica with protoplasts of L. sativa and regeneration of plants from the fusion product thus obtained. Fusion plant according to claim 9, obtainable by fusion of protoplasts of L. tatarica with protoplasts of L. serriola and regeneration of plants from the fusion product thus obtained. 12. Obtain fusion plant derivatives by crossing a fusion plant according to claim 11 with L. sativa. 13. Plant suitable for natural cross-pollination obtainable by the method according to claims 1-8. Plant according to claims 9-13 for use in the production of hybrid plants by crossing the plant with a crossing partner, which has desirable properties. 1 o 0 9 0 4 4 Plant according to claim 14, characterized in that the desired properties are cultivated crop properties. Seed of a plant according to any one of claims 9-15, obtainable by self-pollinating such a plant. 17. Method for manufacturing hybrid seed, comprising manufacturing a pure line from a plant according to any one of claims 9-15 or a plant obtained by one or more crosses of such a plant with a plant with desired cultivated crop properties, crossing a plant of the pure line as a first parent plant with a second parent plant from a pure line, and collecting seed obtained from this crossing. 18. A method according to claim 17, characterized in that the production of a pure, homozygous line is by inbreeding the plant suitable for natural cross-fertilization with the required cultural properties, by production of doubled haploids, or on any other in the Technique known for obtaining homozygous lines. Hybrid seed obtainable by a method according to claim 17 or 18. A method of manufacturing hybrid plants, comprising germinating hybrid seed obtained by the method of claim 17 or 18, and cultivating hybrid plants therefrom. 1009044