The present invention relates to a new method of producing double haploid plants. The invention further relates to plants thus obtained, and to progeny, cells, tissues and seeds of these plants.

Since the discovery by Guha & Maheshwari in 1964 (Nature 204: 497) that plants can be regenerated from haploid spores, a lot of research has been done to obtain similar knowledge for other species (see e.g. "In vitro Haploid production in Higher plants" Vol. 1, 2, 3, 4, 5, Eds: S. Jain, S. Sopory and R. Veilleux (1996) Kluwer

Academic Publishers) .

In modern, contemporary plant breeding the use of double haploids (DHs) has become a very valuable tool in order to speed up the creation of genetically pure lines and also to evaluate and monitor difficult traits such as those that are encoded by multiple genes/alleles .

The production and the use of DHs in breeding crop plants is well known for many species (see e.g. Thomas W. et al . (2003), In: Doubled haploid production in crop plants. A Manual. Eds. M. Maluszynski, K. Kasha, B. Forster and I. Szarejko. Kluwer Academic Publishers, pp 337-349). Thus far, DHs can be obtained from spores of the male or female organs. Spores from the male organs are called microspores and the in vitro cultures are called microspore cultures. Typical microspore cultures are well established in Brassica since a long time (see e.g. Keller et al . (1984) In: K.

Giles, S. Sen (eds.), Plant Cell Culture in Crop Improvement pp 169-183. Plenum Pub. Corp., New York) . Spores from the female organs are called megaspores, and the in vitro culture of these spores is commonly named gynogenesis.

Gynogenesis is a well established technique for e.g. sugar beet and also cucumber (see e.g. Hosemans D. and Bossoutrot, Z. Pflanzenzuchtg. 91:74-77 (1983); EP 0 374 755).

The success of both gynogenesis and microspore cultures is despite many technological advancements only limited to amenable genotypes. Not only are there plant species with low success rate for creating DHs such as watermelon (Sari N., Hort. Science 1994, vol .29 (10) , 1189- 1190) and squash (Kurtar E. S. et al . , Euphytica, Volume 127(3), 2002 , 335-344(10), some species are completely recalcitrant for induction of DHs.

This means that the enormous benefits of DHs cannot be exploited in every desired plant species.

It is therefore the object of the present invention to provide a new method for producing DHs. This object is achieved by a method for producing double haploid plants comprising the steps of:

a) allowing pollen with one functional sperm cell to fertilize an embryo sac cell which is not the central cell;

b) allowing the central cell to proliferate into endosperm;

c) and regenerating a double haploid plant from the endosperm.

The present invention does not obtain DH plants directly from using micro- or megaspores. Instead, the DH plant is regenerated from the central cell of the female gametophyte.

Sexual reproduction in Angiosperms is characterized by a unique process called double fertilization. This means that the two sperm cells from the pollen grain enter the female gametophyte. The first sperm cell will then fertilize the haploid egg cell and the second will fertilize the central cell which contains two nuclei. From the fertilized egg cell a diploid embryo will develop, and from the central cell triploid endosperm will proliferate. Without fertilization of the central cell, and/or without a trigger from the fertilized egg cell the central cell will generally not proliferate into endosperm. The only exceptions are fis and fie mutants that can give autonomous endosperm

development .

In the present invention, however, mutant pollen in which one of the sperm cells is absent or inactivated will only fertilize the egg cell. The central cell will be left unfertilized in the absence of a second sperm cell, and thus remains in the diploid stage, which is in essence a double haploid. Fertilization of the egg cell will trigger the proliferation of the unfertilized central cell into

endosperm. From there on techniques for regenerating

triploid plants out of endosperm, widely available for many plant species, can be used (see T. D. Thomas & R. Chaturvedi, Plant Cell Tissue and Organ Culture 93: 1) to regenerate double haploid plants from the unfertilized central cell.

The invention thus relates to the use of mutant pollen for the fertilization of the egg cell only, which will trigger the development of the unfertilized double haploid central cell.

In one embodiment pollen with only one functional sperm cell is created by chemical mutagenesis with EMS or chemicals like EMS such as EES, BMS, PMS, MES, or MMS.

In one embodiment pollen with only one functional sperm cell is created by mutagenesis via irradiation using e.g. UV light, X-ray, gamma ray, or ionizing radiation.

In one embodiment mutagen plants can be screened for the appropriate mutation, being inhibition of cell division in the generative cell, using eco-tilling. In one embodiment natural populations can be screened for having pollen with only one functional sperm cell, using eco-tilling.

In one embodiment, molecules inhibiting the division of the generative cell are transiently expressed during the development of the pollen, for example by a nucleic acid which is present on a plasmid. The inhibiting molecules, which can be either nucleic acid or protein, are produced in the pollen or microspores by constitutive expression from the plasmid.

In one embodiment, the molecules inhibiting the division of the generative cell are expressed from a nucleic acid that is stably incorporated in the pollen genome. The cell division inhibiting molecules, which can be either nucleic acid or protein, are produced in the pollen or microspores by constitutive expression.

According to one embodiment of the invention, pollen containing only one functional sperm cell is

obtainable by transformation with a nucleic acid. The transformation can be performed in any suitable way, such as by means of Agrobacterium tumefaciens or by means of

particle bombardment (biolistics) .

These transformation techniques are well known. Transformation of plant cells by means of Agrobacterium tumefaciens is well established and for example reviewed in De Ia Riva et al . , EJB Vol. 1(3) (1998), and Bent, Plant Physiol. 124:1540-1547 (2000).

Recently, it was discovered that genetic transformation of plants is not solely restricted to

Agrobacterium, but that other bacteria too have the capacity to transform plants (Broothaerts et al . , Nature 433, 629-633 (2005) , incorporated herein by reference) . These

plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. Such transformation systems are also suitable for use in the invention.

Biolistic transformation is also well known to the person skilled in the art and tools for such applications are commercial available since several years (Ralph Bock, In: QiagenNews, Issue No. 5, 1997). Suitable techniques for use in the invention are for example also described by

Barinova et al . (J Exp Bot. 53 (371) : 1119-29 (2002)), in which delivery of DNA at the level of microspores and transient expression thereof in Antirrhinum majus is shown, or by Ramaiah et al . (Current Science 73:674-682 (1997)) for alfalfa (Medicago sativa L.). Methodology for microspore or pollen transformation with biolistic bombardment in tobacco can be found in Baubak Bajoghli, (Matrikel number: 9802743, University of Vienna, Experimentelle Genetic III. Plant Biotechnology by Alisher Touraev, July 2001). Van der Leede- Plegt, et al . , Transgenic Research 4(2):77-86 (1995)

describe direct delivery of DNA into pollen of tobacco

(Nicotiana glutinosa) by means of microprojectile

bombardment. These and other techniques can be used for the transformation of pollen or microspores for use in the invention .

In one embodiment, the pollen and microspores thus comprise the cell division inhibiting molecules by virtue of the presence of a nucleic acid. The nucleic acid that is introduced can be the cell division inhibiting molecule itself, or can encode the cell division inhibiting molecule. In the latter case the inhibiting molecule is a protein or a peptide. In the first case the inhibiting molecule is a nucleic acid. The nucleic acid can be inhibiting in itself or it can block other nucleic acids from being expressed. For example, the nucleic acid can be or code for a RNAi against members of the CDK protein family or the KRP family.

The invention is based on the principle that only one sperm cell is delivered to the embryo sac or egg cell by means of transformed or natural mutant pollen. Gene

constructs or molecules that are capable of inhibiting cell division in the generative cell are in itself known and can be used in the new method of the invention.

In one embodiment pollen grains are subsequently transferred onto the pistils of plants from the same species or a species in which pollen discharge of the said

pollen/microspore cells can occur. The latter is called heterologous pollination. An example of heterologous

pollination is the use of a species belonging to the

Solanaceae family as a pollen donor and tomato as an

acceptor. Other examples are described in de Martinis, D et al. Planta 214 (5) : 806-812 (2002) and Dore C et al . , Plant Cell Reports 15:758-761 (1996). In general, species that are suitable for heterologous pollination belong to the same plant family.

The invention further relates to a plant producing pollen with only one functional sperm cell, and microspores, egg cells, seeds, cells, or tissue from such a plant or progeny thereof.

Finally the invention relates to doubled haploid endosperm, obtainable by means of the method of the

invention, as well as to plants regenerated from such double haploid endosperm, progeny of such plants, and to seeds, cells, tissues, microspores and egg cell from such a plant or progeny thereof.

In all embodiments the pollen contains one

functional sperm cell or generative cell which is capable of successfully fertilizing the egg cell. The Figure illustrates the method of the present invention. An embryo sac cell 1 contains three antipodal cells 2, a dinuclei central cell 3 and a haploid egg cell 4 flanked by two synergids 5 and 6. When fertilization 7 takes place with wild type pollen 8 with two functional sperm cells 9 and 10 a fertilized triploid central egg cell 11 and a fertilized diploid egg cell 12 are formed in the embryo sac cell 1. Upon germination a diploid plant 13 is formed from the embryo. After fertilization 16 with mutant pollen 14, which contains only one functional sperm cell 15, no fertilization of the central cell 17 takes place. The unfertilized central cell 17 is double haploid. The egg cell 18 is diploid after fertilization. A double haploid plant 19 can subsequently be regenerated from the central cell 17.

The present invention will be further elucidated in the examples that follow. These examples are for

illustration purpose only and are not to be construed as limiting this invention in any way. EXAMPLE

Pollination with mutant pollen and endosperm culture

The CDC2A gene plays a central role in the mitotic cell cycle of plants. A negative mutation in the CDC2A region results in pollen in which mitotic division of the generative cell fails, resulting in pollen with only one sperm cell (Nowack et al, Nature genetics 38: 63 (2006)).

Tomato flowers were emasculated and pollinated with the transformed mutant pollen obtained from tomato plants in Example 1. After pollination, the ovaries expanded and formed fruit-like bodies. The young fruit-like structures were kept on the plants for 2-4 weeks. Plants were grown under climatized conditions (22 ⁰ C day, 18 ⁰ C night) . Fruits were harvested and the endosperm was separated from the rest of the embryo cells. The endosperms cells were then incubated on a medium commonly used for endosperm regeneration (see T. D. Thomas & R. Chaturvedi, Plant Cell Tissue and Organ Culture 93: 1 (2008) and

references therein) . Leaf material of successfully generated plants was used to determine the ploidy of the plant by way of flow cytometry (K. E. Arumuganathan & E. D. Earle Plant Molecular Biology Reporter 9: 229) . The majority of the plantlets regenerated from endosperm had a nuclear DNA content similar to that of a diploid tomato plant, inferring that these plants are in fact double haploids and that fertilization of the central cell had not taken place.