WO1999055143A1

WO1999055143A1 - Strict self pollinating plants with modified flower morphology

Info

Publication number: WO1999055143A1
Application number: PCT/NL1999/000236
Authority: WO
Inventors: Eveline Johanna Van Der Zeeuw
Original assignee: Rijk Zwaan Zaadteelt En Zaadhandel B.V.
Priority date: 1998-04-29
Filing date: 1999-04-23
Publication date: 1999-11-04
Also published as: JP2002512050A; NL1009044C2; EP1075176A1; AU3445999A

Abstract

The invention relates to a method for producing fusion plants for use in obtaining plants suitable for cross-pollination, comprising of preparing protoplasts of a first parent plant and protoplasts of a second parent plant, fusing the first protoplasts with the second protoplasts so as to obtain a fusion product, and regenerating fusion plants from the fusion product, wherein at least one of the two parent plants possesses one or more traits favourable for cross-pollination, at least one of which is expressed in the fusion plant. Such traits favourable for cross-pollination comprise for instance at least a flower size and/or flower contour attractive to insects. The method according to the invention can for instance be applied for lettuce. The produced fusion plants and seed thereof can be used in the production of plants suitable for cross-pollination, hybrid plants and seed.

Description

STRICT SELF POLLINATING PLANTS WITH MODIFIED FLOWER MORPHOLOGY

The present invention relates to a method for producing plants of an originally strictly self- fertilizing species, in particular lettuce plants, which are nevertheless suitable for cross-fertilization. The invention further relates to the plants produced in this manner and seed thereof, in addition to the use of the plants in a method for obtaining hybrid plants and the hybrid plants obtained in this manner and seed derived therefrom. Hybrid varieties in virtually all cultivated crops are commercially attractive, inter alia because of the uniformity of hybrids and the possibility of utilizing heterosis. Heterosis is the phenomenon that hybrid plants which are the result of crossing (more or less) inbred parent plants have a better performance for particular traits, such as for instance yield, than either parent. It is further a great advantage of hybrid varieties that on average they perform better and more stably under changing environmental conditions. Inbred (pure line) lettuce varieties thus for instance often have a very limited cultivation environment (place, soil type, season) in which they perform well. Hybrid lettuce varieties could be suitable for larger cultivation regions and/or environments. In addition, hybrid lettuce can for instance be an interesting product because of said uniformity, increased growth vigour and stability owing to heterosis effects and combination of traits. A greater growth vigour is especially important for winter crops and for lettuce types of dark red colour.

In hybrid lettuce it would also be easier to use genes from wild species . Some interesting genes from L. virosa are for instance linked to genes which cause reduced growth when crossed into a genome of cultivated lettuce. This has been found in particular for a gene for resistance against Bremia and against the aphid Nasonovia ribisniσri . The reduced growth, which is in principle 2 undesirable, is only expressed in the homozygote and therefore, with a correct choice of the parent plants, not in the hybrid, while the resistance linked to the reduced growth is dominant and does therefore result in resistance in the hybrid. Such a gene for reduced growth in a hybrid would moreover be advantageous from the viewpoint of seed companies, because this makes illegally reproduced progeny even more unattractive.

For various reasons the ability to obtain hybrid varieties is therefore advantageous and desirable. A prerequisite for making hybrid varieties is that cross- pollination can occur within the species. However, some cultivated crops are by nature strictly self-fertilizing. Within a genus however, cross-fertilizing species can occur in addition to self-fertilizing species.

An example of a strictly self-fertilizing species is the cultivated lettuce. Lettuce belongs to the genus Lactuca, which can in turn be subdivided into a number of sections, within which subsections can again be distinguished. The Lactuca species occurring in Europe are subdivided into four sections, i.e. Lactuca, Lactucopsis , Mulcredium and Phaenixopus . Within the section Lactuca can be distinguished two subsections: Lactuca and Cyanicae . The cultivated lettuce (Lactuca sativa L.) belongs to section Lactuca, subsection

Lactuca, as do the related species which can be crossed (L.serriola, L.aculeata, L . scarioloides , L . azerbaijanica, L.georgica, L.dregeana, L.altaica, L.saliqna and L. virosa) . L . tenerrima, L.perennis and L. graeca belong to the section Lactuca, subsection Cyanicae .

L . tatarica and L . sibirica belong to the section Mulgedium. These species are taxonomically close to the genus Mulgedium. It has been reported that L. tatarica can be crossed with Mulgedium bourgaei and with the American amphidiploid (2n=34) Lactuca species L. floridana, L.canadensis and L . raminifolia . 3

Both strictly self-fertilizing (autogamic) and cross-fertilizing (xenogamic) species occur within the genus Lactuca . The problem however is that the species in the section Lactuca, subsection Lactuca, to which the cultivated lettuce belongs, are strict self-fertilizers . Natural cross-fertilization percentages of L. sativa of only 1-3% were found in cultivation in the open air with different genotypes cultivated adjacently to each other (Thompson, R.C., T.W. Whitaker, & G. W. 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 for pollinating insects, attractive to pollinators so that these plants can be used for the production of hybrid seed in commercially required quantities .

This is achieved according to the invention by a method of producing fusion plants for use in obtaining plants which are suitable for natural cross-pollination, comprising of producing fusion plants by preparing protoplasts of a first parent plant and protoplasts of a second parent plant, fusing the first protoplasts with the second protoplasts so as to obtain a fusion product, and regenerating fusion plants from the fusion product, ' wherein at least one of the two parent plants possesses one or more traits favourable for natural cross- pollination, at least one of which is expressed in the fusion plant . The fusion plants are themselves not directly suitable for use as crossing parent in the production of hybrids. The invention therefore further relates to a method for producing plants which are suitable for natural cross-pollination, comprising of: a) producing fusion plants by preparing protoplasts of a first parent plant and protoplasts of a second parent plant, fusing the first protoplasts with the second protoplasts so as to obtain a fusion product, 4 and regenerating fusion plants from the fusion product, wherein at least one of the two parent plants possesses one or more traits favourable for natural cross- pollination, at least one of which is expressed in the fusion plant, and b) (back) crossing the fusion plants further at least once with each other and/or with a non-fusion plant with desired traits so as to obtain plants which are suitable for natural cross-pollination and possess the desired traits.

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

The desire to be able to make hybrid plants is great, particularly in the case of lettuce. Cross- fertilizing Lactuca spp . , such as .perennis , are usually pollinated by insects. Within the genus Lactuca it has been demonstrated that flower size and morphology are related to a considerable extent to the degree of cross- fertilization and therefore also to the attractiveness of the flower for pollinating insects. There is a strong correlation between xenogamy and large flower heads with a blue or bright yellow colour. It is therefore recommended to ensure that the traits in lettuce favourable for cross-pollination comprise at least a flower size and/or flower colour attractive to insects. In an embodiment of the invention both parent plants are therefore lettuce plants of the genus Lactuca and the traits favourable for cross-pollination originate from a parent plant of the species L. tatarica, and these traits consists of large and/or blue-purple or near white flowers. The plants derived from fusion plants can have yellow or blue-purple flowers. Variation in colour intensity occurs in both colours. The degree of purple- blue colouring can vary considerably. Some purple-blue 5 flowers are so pale that they appear almost white in colour.

As the other parent plant a direct choice can be made for a cultivated lettuce or for a so-called "bridge species" . In the classical meaning the term

"bridge species" designates a species which is used in crossings between species when the two species for crossing are too remote from each other for direct crossing. A bridge species must lie somewhere between the two parents for crossing and preferably be at least a little cross-breedable with both of them. In the case of fusions the term "bridge species" is used for a cross- breedable relative of the species to which it is wished to transfer genetic material but which has better or more desirable traits for the fusion process such as a better fusion capacity or a better regeneration capacity.

When a culture lettuce is chosen directly (instead of a bridge species) for the production of fusion plants, the other parent plant is for instance of the cultivated lettuce species L . sativa . In the second case, wherein use is made of a bridge species, the use of L. serriola can be envisaged.

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

The invention further provides fusion plants, fusion plant derivatives and plants suitable for natural cross-pollination which can be obtained by means of the method according to the invention. In principle these three types of plants according to the invention made suitable for natural cross-pollination all comprise the 6 more or less far advanced result of fusion of randomly chosen protoplasts. However, plants of cultivated lettuce represent a particularly advantageous embodiment hereof. A specific embodiment of plants according to the invention can be obtained by fusion of protoplasts of L. tatarica with protoplasts of L. sativa and regeneration of plants from the thus obtained fusion product. In an alternative embodiment fusion plants can be obtained by fusion of protoplasts of L. tatarica with protoplasts of L. serriola or other wild lettuce species which can be crossed with cultivated lettuce, so as to obtain a fusion product, and subsequently backcrossing plants regenerated from the obtained fusion product with L . sativa . A fusion plant derivative results herefrom. By crossing between fusion plants or fusion plant derivatives and a plant with desired culture crop traits the traits of the fusion plant or fusion plant derivative which are favourable for cross-pollination can be combined with desired culture crop traits. Via backcrossing with culture crop plants optionally combined with generations in which self-pollination is applied, this process can be repeated a number of times until the desired combination of cultivated crop traits and traits favourable for cross-pollination has been found. Of the plants obtained in this manner inbred lines are made to produce pure lines suitable as parent line for the production of hybrid seed. The final result of this process will be referred to here as "parent plants suitable for hybrid seed production" . The fusion plants as well as the fusion plant derivatives and all plants obtained by crossing of these or their next generation of progeny with other plants, including parent plants suitable for hybrid seed production, which contain the traits favourable for cross-pollination, form part of the invention.

"Hybrid plants" result from the seed of the crossing between (optionally non-hybrid) parent plants, of which at least one, in particular the mother plant, 7 possesses the traits favourable for cross-pollination. This seed is "hybrid seed" .

Also part of the invention are seeds originating from the fusion plants, fusion plant derivatives and all other plants according to the invention resulting therefrom which possess traits favourable for cross-pollination, in particular a changed flower colour and morphology, such as blue or near white flower colour and relatively large flowers. In a preferred embodiment these plants are (cultivated) lettuce plants.

The present invention will be further elucidated with reference to the accompanying examples. In the examples lettuce is used as model species. The invention is not however limited thereto.

Reference is made in the examples to the annexed figures.

Figure 1 shows the differences between a fusion plant and the two parents. Figure 2 shows the differences between leaves of a fusion plant and the two 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, the two parents of the fusion plant and a progeny of a subsequent crossing with a fusion plant .

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

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

Figure 7 shows the seed setting in a progeny of a subsequent crossing with a fusion plant with restored fertility.

Figure 8 shows insect visit to a plant derived from a fusion plant . EXAMPLES EXAMPLE 1

Establishing an in vitro stock of plants

The protoplasts are isolated from leaf material of the parent plants. In this example is described how this leaf material is obtained.

Seed is sterilized by first rinsing with 70% ethanol and thereafter with 0.7% NaOCl for 20 minutes. Rinsing is then carried out 3x with sterile demineralized water.

The seeds are sown on Murashige and Skoog (MS) nutrient medium with 2% saccharose and without hormones. To obtain green plants the seeds are first pre-germinated for 2 days at 15°C in the dark, whereafter they grow further at 25°C in the light (3000 lux, photo period of 16 hours) . When the first leaf becomes visible the tops of the shoots are cut off and transferred to MS nutrient medium with 3% saccharose, without hormones. The sterile shoots are cultured in subculture under the same conditions.

In order to obtain white tissue, for instance hypocotyls, germination is performed in the same manner but the plants are placed out of the light and are kept in the dark also at 25°C after the cold treatment at 15°C.

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

EXAMPLE 2

Isolation of protoplasts Three week-old sterile leaf material is finely chopped and preplasmolysed for 1 hour in the dark in PG solution (54.66 g/1 sorbitol and 7.35 g/1 CaCl₂.2H₂0). The PG solution is thereafter replaced by an enzyme solution 9 with 1% cellulase and 0.25% macerozyme . The material is incubated for 16 hours in the dark at 25 °C.

The suspension is subsequently filtered through a nylon filter (45 μm) and washed with a third volume of CPW16S solution (Frearson, E.M., Power, J.B. & Cocking, E.S. (1973) Developmental Biology 33: 130-137) by centrifuging at 700 rpm for 8 minutes. A band with intact protoplasts is hereby formed 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 Mes buffer) by centrifuging at 600 rpm for 5 minutes .

In the case of hypocotyls the isolation is performed in identical manner.

EXAMPLE 3

Fusion of protoplasts

Protoplasts isolated as according to example 2 are mixed 1:1 to form an end concentration of 6xl0⁵ 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 KH₂P0₄) and placed for a few minutes at room temperature. 1 ml fusion solution 2 (50 g/1 glucose, 7.35 g/1 CaCl₂.2H₂0 and 0.22 g/1 Caps (3- (Cyclohexylamino) -1-propane sulphonic acid) buffer) is then added, whereafter the whole is placed for 10 to 25 minutes at room temperature. Washing is hereafter carried out several times by centrifuging at 700 rpm for 5 minutes. The pellet is resuspended in a medium internally named "B-lettuce" GA B5 (Gamborg et al . (1968) Exp . Cell Res. 50:151), 300 mg/1 CaCl₂.2H₂0, 14 mg/1 Fe-330, 270 mg/1 sodium succinate, 103 g/1 saccharose, 0.1 mg/1 2,4- Dichlorophenoxyacetic acid (2,4-D) and 0.3 mg/1 6- benzylaminopurine (BAP) ) . 10

EXAMPLE 4

Selection and growth of fusion products

The protoplast suspension of example 3 (inclusive of fusion products) is mixed 1:1 with "B- lettuce medium" with agarose. This mixture is poured out in a thin layer to allow it to coagulate. It is then cut into four and floated per 2 pieces in 8 ml "B-lettuce" medium. The petri dishes are taped shut and placed at 25°C. After a few days the medium is diluted with fresh "B-lettuce" medium. When the calluses are ± 0.5 mm in size they are placed on callus growth medium (SH2 (Schenk, R.U. & Hildebrandt, A.C. (1972) Can. J. Bot . 50:199) with 30 g/1 saccharose, 5 g/1 agarose, 0.1 mg/1 α-naphthalene acetic acid (NAA) and 0.1 mg/1 BAP) . After ± 2 weeks the calluses can be transferred onto regeneration medium (SHreg (Schenk & Hildebrand, supra) with 15 g/1 saccharose, 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 placed on rooting medium (Schenk & Hildebrand with 30 g/1 saccharose and 8 g/1 agar) . The selection of the fusion products can take place visually (on the basis of their hybrid appearance differing from the two fusion parents) or using molecular biological techniques such as PCR, AFLP, RFLP .

EXAMPLE 5

Analysis of fusion plants and generations derived therefrom

1. Introduction In a fusion experiment as according to examples

1-4 protoplasts of L. tatarica were fused with protoplasts of L. serriola. Fusion plants were regenerated and checked using PCR analysis, flow-cytometry and on the basis of external characteristics.

2. Appearance

The obtained fusion plants had a strong vegetative growth and large light-blue coloured flowers 11

Figures 1-4 show the differences between the parents of the fusions and the fusion plants themselves.

Figure 1 shows on the left an L . tatarica parent plant, in the middle a fusion plant and on the right an L. serriola parent plant .

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

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

3. Crossings with fusion plants

Fusion plants were cultured in the greenhouse in pots and brought to flower. Some of the flowers were not pollinated, some of the flowers of the fusion plants were pollinated with pollen of the lettuce variety 'Norden' and some of the flowers of fusion plants were used to pollinate male sterile (MS) lettuce plants. Seeds were obtained from self-pollination as well as from the stated crossings. The thus resulting populations are designated as follows.

Fusion plants : T(+)S

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

4. Determining ploidy level of fusion plants

Via flow-cytometry the DNA content was determined of a number of plants of the above stated populations. Controls were the L. serriola line, L. sativa "Norden", L. sativa "Flora", and L. tatarica. The flow-cytometry assay was performed according to standard procedure by Iribov, Laboratory for tissue culture, improvement and flow-cytometry, Westeinde 12

149a, 1601 BM Enkhuizen. All plants were measured with an internal standard. Six plants were assayed in duplicate.

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

Table 1

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

Population Number Relative Range Standard of plants DNA content deviation

L. sativa cv. Norden (N) 1 100.0 -- -

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

L. tatarica (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 virtually identical to that of L . sativa and that L. tatarica has about one and a half times as much DNA (157%) . The fusion products have a DNA content corresponding with the sum of the DNA content of the fusion parents: 257 is about 99 + 157. The I1(T(+)S) plants obtained from self -pollinated seed of the fusion plants had an average DNA content roughly corresponding with that of the fusion plants, i.e. 254.4. These II plants did however display a greater distribution (233.6- 266.9), which could be explained by the occurrence of aneuploid plants in this generation. Phenotypical observations (greatly deviating, compact growth) also make it likely that a few of the 35 tested plants were aneuploid. The DNA content of the crossings plants obtained by crossing the fusion plants with cv. "Norden" or with a male sterile lettuce line corresponds with a triploid level with two sets of chromosomes of the level of L. serriola/L. sativa and one set of the level of L. tatarica: 178 roughly corresponds with 100 + A* (157) . On the basis of these flow-cytometry assays it can be concluded that the fusion plants and most of the plants obtained from self-pollination seed on the fusion plants contain the total diploid genome of the L. tatarica parent plant and the total diploid genome of the

L. serriola parent plant. These plants are therefore allotetraploid. The plants obtained by crossing fusion plants with L. sativa genotypes have a DNA content corresponding with 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. These plants therefore have a triploid DNA level .

EXAMPLE 6

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

Plants obtained from crossing fusion plants as mother with the lettuce variety 'Norden', designated below for the sake of brevity as (T(+)S) x N, were cultured in the greenhouse and brought to flower. The (T(+)S) x N plants had large, very light-blue tinted flowers, which were on average somewhat smaller in size than flowers of the fusion plants. Figure 4 shows characteristic flowers of L. tatarica, L. serriola, a fusion plant, the lettuce variety 'Norden' (L . sativa) and of a (T(+)S) x N plant (Tl). Flowers of the (T(+)S) x N 14 plants were pollinated with pollen of plants of the lettuce variety 'Norden'.

No seed- setting was observed on non-pollinated flowers of (T(+)S) x N plants. Seed was however obtained after pollination with pollen of 'Norden'. The population obtained from this crossing is designated below as ((T(+)S) x N) x N plants. The plants of the ((T(+)S) x N) x N population were cultured in the greenhouse and brought to flower. The DNA content was determined via flow-cytometry . Samples of T(+)S and (T(+)S) x N were included as controls.

The flow-cytometry assays were performed in the same manner as in example 5. 11 plants were measured in duplicate. In the case of the plants measured in duplicate, the greatest difference between the two measured values was 2.2%. The relative DNA contents of the different populations are given in the table below.

Table 2

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 several control populations

Population Relative DNA content N Mean Standard deviation

< 1.0 1.0-1.3 1.3-2.0 >2.0

T(+)S 3 3 2.67 0.04

(T(+)S) x N 2 2 1.86 0.02

((T(+)S) x N) x N 12 106 21 2 141 1.18 0.21

Figure 5 shows a PCR pattern of the L. tatarica and L. serriola fusion parents, of fusion plants and of fusion plant derivatives. From left to right:

1. DNA ladder (for determining fragment size)

2. L. tatarica (T) , relative DNA content: 1.61

3. L. sativa "Norden" (N) , relative DNA content: 1.00 15

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

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: 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

14. ( (T (+) S) x N) x N plant, relative DNA content: 1.00, yellow flowering

The plant from lane 8 is possibly an I_χ (T (+) S) plant resulting from 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 lettuce variety "Norden" have a marker pattern which consists of the sum of the markers which were found in both fusion parents. Plants obtained after crossing twice with "Norden" [ ( (T (+) S) x N) 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 resulting from crossing triploid (T(+)S) x N mother plants with diploid N father plants, mainly diploid plants or aneuploid plants are expected with one or several additional chromosomes above the diploid level . For the diploid level a relative DNA content of 1.0 is anticipated if the plant contains no L. tatarica chromosomes and a content of 1.3 (0.5 + 0.8) if the plant still contains the total haploid genome of L . tatarica . A large part (84%) of the ((T(+)S) x N) x N plants has a DNA content in the range of 0.98 - 1.30. 16

21 ((T(+)S) x N) 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 result through 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 for seed setting in the case of self-pollination.

Figure 6 shows variation of flower size and flower colour in ((T(+)S) x N) x N plants (= T2 fusion) . In the ((T(+)S) x N) x N population a considerable variation was found in flower size and flower colour

(flowers blooming from very light-yellow to very intense yellow colour in addition to blue (to sometimes near- white) ) .

Table 3

Flower colour and size of ( (T ( + ) S ) x N) x N plants and several control populations 5i tΛ

4-. t-J

Characteristic Population ( (T (+) S) x N) x N relative DNA content

"Norden" "L tatarica" T ( +) N (T ( +) N) x N < 1 1-1 3 1 3-2 0 >2 0

Flower colour G B B B B G B G B G B G

Number of plants 5 5 4 5 - 12 12 94 20 1 1 1

Average flower size 17 4 27 4 27 2 25 4 - 17 3 14 2 16 4 16 4 16 0 29 0 24 0

(standard deviation) 0 80 0 80 0 43 1 62 - 2 56 1 83 2 87 4 61 - - -

Largest flower 18 28 28 27 - 22 18 24 26 - - -

Smallest flower 16 26 27 23 - 13 11 11 10 - - -

G yellow, B purplish or blueish size of the flower head in mm, measured as maximum spread of the petals of a flower head

Per plant the most frequent flower size was determined aavveerraaggee ssiizzee ooff tthhee fflloowweerr hheeaadd,, mmeeaassuurreedd aass under , for the plant with the largest flowers idem for the plant with the smallest flowers

n r yo o

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Of 141 ( (T (+) S) x N) x N plants, 33 flowered 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 exhibited a fully restored fertility, this making it possible to obtain large quantities of self-pollination seed (>> 1000 per plant) .

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

EXAMPLE 7

Suitability of fusion plants for insect pollination

Two isolation cages of insect-tight netting, each with a surface area of about 10 m², were placed in a greenhouse. Nine plants were planted per cage of in each case eight lettuce varieties and I1(T(+)S) plants (May 1997) . The varieties were Camaro RZ (red batavia type) , Remco RZ (green butterhead lettuce) , Kublaϊ RZ (red oakleaf type) , Pantheon RZ (green batavia type) , Kristine RZ (green oakleaf type) , Roxette RZ (iceberg lettuce) , Loretta RZ (red lollo type) and Remus RZ (green cos lettuce) . When flowering started (mid-June 1997) , honeybees (Apis mellifera) and bumblebees (Bombus terrestris) were introduced into the cage. On 6 dates during flowering the number of bees and bumblebees observed on flowers of the varieties or the I1(T(+)S) plants was then recorded.

Table 4

Flower visit of honeybees on lettuce varieties and I1(T(+)S) plants. Given are the average number of bees per plant located at the stated time on flowers of said accession. t*)

Date Time Vaπetv

Camaro Remco ublai Pantheon Knstine Roxette Loretta Remus Fusion plants

708 900 078 017 100 011 144 on 011 000 139

1208 800 111 011 050 022 044 0 4 000 006 389

1408 930 006 000 011 006 000 000 000 000 122

1508 900 006 000 000 000 000 000 000 000 333

2208 930 000 000 000 000 000 000 000 000 017

2908 1000 006 000 006 000 000 006 000 000 067

Average 034 005 028 006 031 010 002 001 178

O

:H zr VO

Table 5 O v©

Flower visit of bumblebees on lettuce varieties and I1(T(+)S) plants. Given are the average yo δnϊ number of bumblebees per plant located at the stated time on flowers of said accession. w

Date Time Variety

Camaro Remco Kublai Pantheon Kristine Roxette Loretta Remus Fusion plants

7 08 900 000 000 000 000 006 000 000 000 061

12 08 8 00 0 00 000 006 000 0 11 006 000 000 0 50

14 08 9 30 0 11 000 000 006 0 11 000 000 000 0 67

15 08 9 00 000 0 00 000 000 000 006 000 0 00 0 72

22 08 9 30 000 0 00 0 00 000 000 000 000 000 0 11 O

29 08 10 00 000 000 o n 000 000 000 006 000 033

Average 002 000 003 001 005 002 0 01 000 049

~0 o r

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21

The results show that the I1(T(+)S) plants are visited much more frequently by both honeybees and bumblebees than the lettuce varieties. Bee visits to flowers of the I1(T(+)S) plants were observed on average to be 12 times more frequent compared to all tested lettuce varieties. Compared to the lettuce variety (Camaro RZ) visited most frequently by honeybees the I1(T(+)S) plants had five times as many bee visits. Bumblebee visits to flowers of I1(T(+)S) plants were on average 28 times higher than to the lettuce varieties, and about 10 times higher than to the lettuce variety (Kristine RZ) visited most frequently by bumblebees. This shows that the I1(T(+)S) plants are clearly more suitable for natural cross-pollination by insects than the tested lettuce varieties.

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

Claims

22CLAIMS

1. Method for producing fusion plants for use in obtaining plants suitable for natural cross- pollination, comprising of producing fusion plants by preparing protoplasts of a first parent plant and protoplasts of a second parent plant, fusing the first protoplasts with the second protoplasts so as to obtain a fusion product, and regenerating fusion plants from the fusion product, characterized in that at least one of the two parent plants possesses one or more traits favourable for natural cross-pollination, at least one of which is expressed in the fusion plant .

2. Method for producing plants suitable for natural cross-pollination, comprising of: a) producing of fusion plants by preparing protoplasts of a first parent plant and protoplasts of a second parent plant, fusing the first protoplasts with the second protoplasts so as to obtain a fusion product, and regenerating fusion plants from the fusion product, wherein at least one of the two parent plants possesses one or more traits favourable for natural cross- pollination, at least one of which is expressed in the fusion plant, and b) (back) crossing the fusion plants further at least once with each other and/or with a non-fusion plant with desired traits so as to obtain plants which are suitable for natural cross-pollination and possess the desired traits.

3. Method as claimed in claim 1 or 2 , characterized in that the traits favourable for cross- pollination comprise at least a flower size and/or flower colour attractive to insects.

4. Method as claimed in claim 2 or 3 , characterized in that the desired traits are cultivated crop traits.

5. Method as claimed in claim 3 or 4 , characterized in that the two parent plants are lettuce 23 plants of the genus Lactuca and the traits favourable for cross-pollination originate from a parent plant of the species L. tatarica and consist of large flowers with a blue-purple or white colour.

6. Method as claimed in claim 5, characterized in that the other parent plant is a plant of the cultivated lettuce species L. sativa .

7. Method as claimed in claim 5, characterized in that the other parent plant is a plant of a Lactuca species which can be substantially fully crossed with a plant of the cultivated lettuce species L. sativa.

8. Method as claimed in claim 6, characterized in that the other parent plant, which is a plant of a Lactuca species which can be substantially fully crossed with a plant of the cultivated lettuce species L. sativa, is a plant of the species L. serriola .

9. Fusion plant to be obtained by means of the method as claimed in claims 1 and 3-8.

10. Fusion plant as claimed in claim 9, to be obtained by fusion of protoplasts of L. tatarica with protoplasts of L . sativa and regeneration of plants from the thus obtained fusion product .

11. Fusion plant as claimed in claim 9, to be obtained by fusion of protoplasts of L . tatarica with protoplasts of L. serriola and regeneration of plants from the thus obtained fusion product .

12. Fusion plant derivatives to be obtained by crossing a fusion plant as claimed in claim 11 with

L. sativa .

13. Plant suitable for natural cross- pollination, to be obtained by means of the method as claimed in claims 1-8.

14. Plant as claimed in claims 9-13 for use in the production of hybrid plants by crossing the plant with a crossing partner possessing desired traits.

15. Plant as claimed in claim 14, characterized in that the desired traits are cultivated crop traits. 24

16. Seed of a plant as claimed in any of the claims 9-15, to be obtained by self-pollination of such a plant .

17. Method for producing hybrid seed, comprising of producing a pure line from a plant as claimed in any of the claims 9-15 or a plant obtained by one or more crossings of such a plant with a plant with desired cultivated crop traits, crossing a plant of the pure line as first parent plant with a second parent plant from a pure line, and collecting seed obtained from this crossing.

18. Method as claimed in claim 17, characterized in that production of a pure, homozygote line takes place by inbreeding the plant suitable for natural cross-fertilization and having the required cultivated traits, by producing doubled haploids, or in any other manner known in the art in order to obtain homozygote lines.

19. Hybrid seed, to be obtained by means of a method as claimed in claim 17 or 18.

20. Method for producing hybrid plants, comprising of causing germination of hybrid seed obtained using the method as claimed in claim 17 or 18 and cultivating hybrid plants therefrom.