GB2429462A - Male sterile swede plants and F1 hybrids - Google Patents

Abstract

Male sterile swede (Brassica napus sppp rapifera) germplasm that is substantially homozygous is claimed. This germ line may be cytoplasmic male sterile (CMS) using the Ogura (OGU_INRA) sterility. Alternatively claimed are swede F1 hybrids which are heterozygous and substantially genetically uniform with specified characteristics.

Description

Brassicas The present invention relates to plants of the genus Brassica.

More specifically, it relates to swede, a sub-division of the amphidiploid species Brassica napus.

Considering first plants in general, typically a single plant produces both male and female gametes. Seed results from an ovule being fertilised by pollen. This pollen may be produced on the same plant as the ovule (self-pollination) or on a different plant (cross- pollination). Pollination may take place at random (pollen being transported to the ovule by wind or by insects) or by collecting pollen from the desired source and applying it to the ovule. The latter process is termed hand-crossing'. In natural conditions a collection of plants often reproduces by a mixture of self- and cross-pollination - this is referred to as open-pollination.

In modern agricultural practice, farmers prefer to grow varieties which are uniform. A field planted with a uniform variety tends to produce uniform plants, growing at the same rate and reaching maturity together, so that they can all be harvested on the same day without waste. The plants of a uniform variety have uniform phenotypic properties and disease resistance. As a result they respond well to uniform treatments with pesticides and fertilisers.

A uniform plant line may be produced by a process known as single seed descent. A plant is selected which contains desirable characteristics. This can be from a population of open- pollinated material or from a population produced from an original hand- cross between two different plants with desirable characteristics. This selected plant is then self-pollinated in the glasshouse, cross- pollination being prevented either by bagging the flowering head or by hand-crossing. The resulting seed is then sown out in trials and assessed for the desired selection characteristics or criteria. These criteria cover a wide range and differ between crops: but they normally include harvest period, quality of product, yield, ease of harvest, storage ability, disease resistance, insect resistance and resistance to lodging. Plants showing the desired characteristics are then brought into the glasshouse and once again self-pollinated. The resulting seed is again trialled out and quality plants selected and self- pollinated. This is done over at least 5 generations (it may require 6 - 7 generations, or even more). In this way seed lines with uniform genetic properties can be obtained. At this stage, for self-pollinating species the seed can be multiplied up to produce larger quantities for commercial sale. This is done by mass pollination of plants grown from seed of the uniform line.

Seeds obtained in this way are uniform as a consequence of an almost identical genetic inheritance. Being inbred, the seeds are genetically homozygous. They inherit the same set of genes (or very nearly) from both parents. The disadvantage of this is that inbred lines nearly always suffer from lack of vigour or inbreeding depression'. To obtain superior varieties, Fl hybrids may be considered. Fl hybrids are obtained by outcrossing between two different parent lines. They combine hybrid vigour' with uniformity. They are uniform because each hybrid seed has the same genetic inheritance. It receives a different genetic inheritance from each parent, so it is heterozygous: but each seed inherits the same genes from the same parent, so that the offspring, though heterozygous, are genetically uniform.

Hybrids may be produced by planting two different uniform inbred lines together in close proximity and allowing cross-pollination (e.g., with insects) to take place between the two lines. Cross-pollination between the different parent lines produces hybrid seed. However - unless special precautions are taken - many of the plants will also self-pollinate, so the seed harvested will be a mixture of hybrid and inbred seed. This seed will be non-uniform (a mixture of hybrid seed and seed of the two inbred lines) and not meet the modern farmer's needs.

In order to obtain uniform seed, there are two main ways of preventing self-pollination occurring in hybrid seed production. The first is selfincompatibility, which occurs in Brassica oleracea (cabbages and kales). Plants of this species can have various genetic traits or alleles that prevent the pollen fertilising its own flowers and any others with the same allele for this trait. To exploit this, inbred lines are produced which are homozygous for one of these self-incompatibility alleles. Fl hybrid seed can then be produced by planting together two inbred lines which contain different alleles. In this way hybrid seed is produced without any self-pollinated seed and seed can be taken from both parent plants.

When the two lines used for the hybrid production are uniform inbred homozygous lines, this produces uniform Fl hybrid seed.

Self-incompatibility can be a very simple and useful system to exploit for hybrid seed production. A significant problem however is the degree of incompatibility. This varies depending on the alleles contained in the parent lines. These may be strongly incompatible, weakly incompatible or even compatible. If a hybrid production is attempted using one line with an allele that is weakly incompatible or compatible then the seed can only be taken from the strongly incompatible line: only this will bear uniform hybrid seed. In the species Brassica napus (of which swede is a sub-species), we have not found self-incompatibility that occurs in a reliable form.

An alternative method of ensuring cross-pollination is to prevent one line from producing pollen by the use of cytoplasmic male sterility - a gene for which can be inherited via the cell cytoplasm, and thus only through the female line. In plants carrying this trait, the anthers in the flower either do not form at all or they are small, withered and do not produce viable pollen. A plant inbred line exhibiting this character can thus be used as the female line in an F I hybrid seed production to produce uniform hybrid seed for a crop such as globe swede where it is the vegetative plant which is harvested by the commercial grower/farmer. Male sterility is often abbreviated to ms', and cytoplasmic male sterility to cms'.

The swede (sometimes termed globe-swede', and also known in North America as rutabaga') is a sub-species of the species Brassica napus (2n38, AACC) which is an amphidiploid of Brassica oleracea (2n= 18, CC) cabbages and kales and Brassica campestris (2n= 20, AA) turnips. It has a swollen stembase (frequently referred to as a "root") with red, yellow, white or green skin and a typical globe-shaped lower stem just above the ground. Varieties of swede on the UK National List include: Brora, 1-lelenor, Magres, Laurentian, Ruby and Virtue among others. Varieties may be cooked and eaten by humans (culinary swede), or fed, uncooked, to animals as fodder.

Male sterility in swede is not known from the literature to occur in nature. To produce Fl hybrid swede one might hypothesise that laboratory' cytoplasmic male sterility could be introduced into selected, distinct, uniform breeding lines, e.g., by hand pollination using a male-fertile swede line as the pollen source and a male-sterile female line from some other Brassica species as the recipient. If male-sterile lines can be produced, after repeated back- crossing until they have become uniform, they might then be used as the female parent in a swede Fl hybrid seed production. At the outset of a back-crossing programme, it cannot be predicted whether cytoplasn-jic male sterility will successfully transfer into swede without the male- fertile nuclear DNA/genes dominating the cytoplasmic gene for male- sterility and thus producing, to a greater or lesser degree, a male- fertile plant. Neither can it be predicted that, even if such lines are established and stabilised (too high a level of fertile off-types' can readily occur), the subsequent Fl hybrid seed production will be successful at practicably viable yields of seed.

The present invention provides new swede germplasm which is substantially male-sterile and substantially homozygous. By crossing with suitable malefertile substantially homozygous swede inbred lines or varieties, it may be used to produce swede F I hybrids which are heterozygous but substantially genetically uniform. These also form part of our invention.

Male-sterile germplasm according to the invention is produced by crossing swede germplasm with cytoplasmically male-sterile oilseed rape, followed by repeated back- crossing, preferably for at least 6 generations, in order to create uniform swede lines which have stable cytoplasmic male-sterility.

Male-sterile lines do not produce viable pollen. To produce seed they must be pollinated by pollen from another plant line, thus ensuring the resulting seed is Fl hybrid. This confers a number of advantages on the resulting variety. The plants may show improvements in: evenness of emergence; initial vigour; uniformity of characteristics; and quality of product.

Certain quality characteristics from the two parent lines may be combined in the F! hybrid.

Thus we have found hybrids of the present invention to be improved in one or more of the following respects in comparison with open-pollinated varieties.

1. Evenness of emergence - In open pollinated varieties germination occurs over a number of days, even when seed of high recorded germination is precision-drilled. This results in the early germinating plants beginning to overshadow the later germinating plants. This in turn produces uneven root size in the final crop. In these hybrids germination was more uniform resulting in more uniform sized roots in the final crop.

2. Initial vigour - In some brassica species, notably the Brassica oleracea, Fl hybrids show greatly increased vigour over the uniform inbred lines and over many open-pollinated varieties. This is not so apparent in Brassica napus; the final product does not always show a marked increase in size. However there does usually appear to be an increase in initial vigour/speed of growth.

3. Unformiiy of characteristics - In any open-pollinated variety there is some degree of variation, although often this is small. This did not occur with swede hybrids which showed a very high degree of uniformity for all characteristics. Growers require a high percentage marketable pack out from their crop and hybrids are more able to provide this.

4. Quality ofproduct - Producing Fl hybrids enables combining qualities from a number of inbred lines giving the breeder a much wider pool of characteristics to draw upon in a flexible way so as to meet requirements of growers.

The preferred type of male sterility used in the hybrids of our invention is cytoplasmic male sterility. The genes for this characteristic are present in the cytoplasmic DNA of the female line. Progeny receive cytoplasmic DNA only from the female parent, and so are carried unaltered in the female line through subsequent generations. Each generation must be checked for fertile pollen, to ensure the nuclear DNA does not carry genes that are dominant to the cytoplasmic male sterility genes and that would render the plant male- fertile.

Generally, male-sterile lines must be back-crossed for at least six generations to the selected uniform pollinator swede line to ensure the male sterile line and the respective pollinator line become uniform with one another, for all characteristics (other than male- sterility); the degree of uniformity (like the maintenance of the male sterility) is checked at each generation by growing in trials. After the sixth back-cross, substantially homozygous male-sterile seed may be produced in quantity, for example in a bulk/cage production system using flies for pollination. This seed can then be used for F! hybrid seed production by crossing with any male-fertile homozygous Swede line. It is preferred to select a suitable male-fertile line by doing test crosses to assess the properties of the resulting hybrids. We have found, for example, that hybrids of inbred lines from Magres when crossed with Lizzy, and from Lizzy when crossed with Helenor, have improved qualities as compared with their parents, in particular having better skin colour and shape than Magres alone and better frost tolerance than Helenor or Lizzy (see Table 1).

The following Examples illustrate the invention:

Example 1

Initially, in 1997, hand crosses were made in the glasshouse between a number of uniform globe-swede lines, as shown in Table 1, to produce small quantities of F! hybrid seed. This was done to test whether F I hybrid Swede in fact gave any improvements. The resulting materials showed an improvement in evenness of emergence, uniformity of characteristics, initial vigour and quality of product.

Table 1 - A comparison of 3 test hybrids and their parent lines as recorded from trials in 1998.

Cross Germination Uniformity Skin colour Flesh Shape colour/texture Magres x Lizzy + + + + ++ Helenor x Lizzy + + +1- - ++ B 85027 x Lizzy ++ + ++ + ++ Parent line Helenor +1- + +1- +1- + Magres +1- +1- +1- - + Lizzy - + + + +1- B 85027 - - - + +1- + A total of 14 test hybrids were assessed alongside 18 open pollinated inbred lines. This table gives an indication of the scores of three of the best crosses and the corresponding parent lines; qualities were judged by eye.

- poor +1- average + above average ++ excellent The hand-crossing procedure was carried out by opening a bud (when the stigma was receptive but the anthers had not de-hissed) on the female line, just before it flowered, using tweezers. An anther was then taken from an open flower of the male line using tweezers and the pollen was applied to the stigma of the female line. The pod was then allowed to grow and mature a process which takes about 6 weeks. The pod dried off and the seed could then be harvested. Care was taken to ensure seed from each plant was kept separate. This was done to test whether FL hybrid Swede in fact gave the hoped-for improvements. The resulting materials showed an improvement in a number of characteristics (see Table 1).

Example 2

This Example illustrates back-crossing the male sterility character, obtained from oil seed rape, into uniform (homozygous) lines of swede germplasm.

The male-sterile source was the oilseed rape variety Synergy which contains the cytoplasmic male sterility system named "OGU-INRA" (French patent no 90 11670).

Initial hand-crosses with four varieties were made in Spalding in 1997, using four commercial varieties, Acme, Magres, Merrick and Helenor. The seed obtained from these hand-crosses was sown out with the respective pollen maintainer plants, as part of the swede breeding trials, on a trial site at Spalding in 1997 and selections made for the first back- crosses to be done.

The first back-crosses were done at Spalding in spring 1998. Subsequently, hand crosses were done between each of these initial back-crossed lines and the selected breeding lines FSWO5 and FSWO99. These lines originated, respectively, from Melfort' and Devon Champion', two old fodder swede varieties.

The resulting seed from these crosses was once again sown out with the corresponding pollen maintainer lines on the trial site at Spalding in 1998.

The first backcrosses from the 1998 trials were used in Spring 1999 to make second back- crosses with Acme, Magres and Merrick. They were also crossed with breeding lines FSWO5 (derived from Melfort) and FSW99 (derived from Devon Champion). In addition hand crosses were done between male-sterile Acme back-cross I and in-house breeding lines GSW 132 (Lizzy), GSW 106 (Angela) and GSW34 (B85027), in order to introduce the cytoplasmic male sterility into further swede material.

The seed produced from these crosses was sown on the trial site in 1999 along with the pollen maintainer lines. Selections for closeness/similarity to the globe- swede pollinator- line were made for the next back-cross generation and brought into glasshouses.

The seed resulting from this back-cross generation was sown out along with the swede breeding trials in 2000 near Berwick, a swede growing area in Scotland. The swede trials were grown near Berwick in 2000 and for subsequent years, up to 2004 and are planned to continue.

Single-plant back-crosses have been made over subsequent generations up to back-cross 5 and are planned to continue to back-cross 7. At every generation the uniformity of the male-sterile line with the uniform selected male-fertile line used as the pollen source is checked and the male sterility of the line is checked. At back-cross 5 test crosses are done with established breeding lines from the breeding programme.

The recent and planned future situation is as follows: 2004 - fixed lines of Acme, Magres and FSW 05 (Melfort selection): ready for hybrid test crosses. A sample of this lot of Acme male-sterile seed has been deposited with the is National Collections of Industrial, Marine and Food Bacteria (NCIMB Ltd), Craibstone Estate, Bucksburn, Aberdeen on 8 August 2005 under reference No NCIMB 41340 2005 - fixed lines of 1-lelenor and FSW 99 (Devon Champion selection): ready for hybrid test crosses.

2006 (planned) - Angela: ready for hybrid test crosses.

2007 (planned) - ELSW74 (Lizzy selection): ready for hybrid test crosses.

Using this method of back-crossing over 6 generations a number of distinct, uniform and stable fixed lines of male-sterile Swede (Brassica napus) have been produced. The following Tables of Descent (Table 2 and Table 3) lay out the time-scale and method of production for 1) the swede male-sterile/recurrent lines and 2) the fertile/pollinator lines.

TABLE 2

Table of descent (single seed descent) for male-sterile/recurrent lines This shows the method used to transfer male sterility from the oil seed rape variety Synergy, which contains the cytoplasmic male sterility system "OGU-INRA", into two uniform swede lines (which were used as the recurrent pollinator lines).

Se1f-pjinatedcted lines from Magres and Acme were used as below: ________ _____ Year/ + MS Magres-origin Field MS Acme-origin Field Back line code line code Cross 1997 Synergy x Magres line Synergy x Acme line

Field code ESW39 Field code ESWI9

FSW 30, FSW29 1998 (Synergy x Magres) x Magres line (Synergy x Acme) x Acme line (Back Cross FSW3O F5W35 FSW29 FSW37 BC1) GSW163 GSWI61 PS Magres BCI x Magres line MS Acme BC1 x Acme line G5W163 GSW129 GSW161 GSW135 HSW 193 HSW192 2000 Sowings lost, resown Sowings lost, resown net year nexyear MS Magres BC2 x Magres line MS Acme BC2 x Acme line GSWI62 GSW129 GSWI61 GSW135 KSW68,, KSW87 MS Magres BC3 x Magres line] MS Acme BC3 x Acme line 2002 KSW68 KSW66 I KSW87 KSW85 (BC4) I LSW77 LSW92 2003 MS Magres BC4 x Magres line MS Acme BC4 x Acme line (BC5) L5W77 LSW73 LSW92 LSW88 MSW1O5 MSW121

TABLE 2 (cont.) "P "P

MS Magres BC5 x Magres line MS Acme BC5 x Acme line (BC6) MSWIO5 MSW1O2 MSWI21 MSWI19 NSW1O7 NSW127 Trials demonstrate seed to be Trials demonstrate seed to be uniform and effectively identical to uniform and effectively identical recurrent Magres pollinator line to recurrent Acme pollinator line ______ or 2005 Bulk Production 10 x 10 Bulk Production lOx 10 MS Magres line Magres line MS Acme line Acme line NSW 107 NSW 100 NSW 127 NSW 122 Use for commercial seed production of F 1 hybrids

TABLE 3

Table of descent for fertile/pollinator lines - selections made in Scotland for frost resistance P NSW 110 NSW 91 NSW 139 Year Lizzy-origin line Devon Champion-origin Bizzy line

Field code ____________ Field code Field code

1997 Commercial stock grown in Commercial stock grown breeding trial. Best in breeding trial. Best individual single swedes individual single swedes selected. selected.

Single plants self-pollinated in glasshouse Field plots (of seed from Field plots (of seed from 1998 each self-pollinated plant) each self-pollinated plant) trialled in breeding trial. trialled in breeding trial. B85027 x Lizzy Fl test Single best plants selected. Single best plants selected. cross in breeding trial GSW126 GSW14O GSW25 Single plants self-pollinated in glassho use ________________________ Field Plots for F3 Field plots for F3 Field plots F2 for assessment assessment assessment 1999 best plants selected best plants selected best plants selected HSW29 HSW35 HSWO9 ft,

TABLE 3 (cont.) + + +

Single plants self-pollinated in glassho use Field Plots for F4 Field Plots for F4 Field Plots for F3 assessment assessment assessment 12000 best plants selected best plants selected best plants selected JSW51 JSW41 4, JSW92 Single plants self-pollinated in glassho use Field Plots for F5 Field Plots for F5 Field Plots for F4 assessment assessment assessment 2001 best plants selected plants selected best plants selected JSW51, KSW57 KSW113 _________ Single plants self-pollinated in glasshouse Field Plots for F6 Field Plots for F6 Field Plots for F5 2002 assessment assessment assessment best plants selected best plants selected best plants selected LSW8O LSW68 LSW1O4

TABLE 3 (cont.)

I V

+ Single plants self-pollinated in glasshouse + Field Plots for F7 Field Plots for F7 Field Plots for F6 assessment. assessment. assessment.

2003 Plots uniform - no further Plots uniform - no further Plots uniform no further self-pollination required self-pollination required selfpollination required MSW1O7 MSW92 MSW144 F] Test Hybrid. Produced by crossing with male-sterile line Bulk production for stock Bulk production for stock Bulk production for stock seed as a pollinator line for seed as a pollinator line for seed as a pollinator line for Fl production. Fl production. Fl production.

NSW11O NSW91 NSW 139 [yale-Sterile Lines. Bulk seed production of Fl hybrids

Claims (9)

1. Example 3

   A number of test hybrids of swede were produced for the culinary swede market. Swedes for human consumption desirably have two or more, preferably as many as possible of the following characteristics: a globeshaped root with a tapering base which can be cut off square;.

   a narrow neck at the leaf base, which leaves only a small cut surface when harvested and the leaves are cut off; skin colour of root rich red/purple (top half) and cream (bottom half); flesh cream or yellow; leaves mildew-resistant; root frost-resistant; good flavour.

   The breeding programme to date has produced four hybrids which show commercial potential. Their different characteristics are as follows: ELSW 230 Magres-origin ms line x Lizzy-origin pollinator line. Good globe shape, skin colour red/cream, firm crisp flesh, with even colour. Frost and mildew resistant. Mild flavour.

   ELSW 237 Acme-origin ms line x Devon Champion-origin pollinator line. Good globe shape, skin colour red/cream, hard crisp flesh, with even colour. Frost and mildew resistant. Distinct swede flavour.

   ELSW 239 Acme-origin ms line x Lizzy-origin pollinator line. Very good globe shape, skin colour red/cream, flesh medium/soft - creamy/yellow. Distinct swede flavour.

   ELSW 240 Acme-origin ms line x Bizzy-origin pollinator line. Good globe shape, skin colour red/cream, flesh good texture, even colour (mid yellow) . Mild flavour.

   All these hybrids are very uniform, germinating more evenly and slightly ahead of similar open-pollinated lines, and producing an even and vigorous crop of roots of similar size and shape.

   We claim: 1. Swede germplasm which is male-sterile and substantially homozygous.
2. 2. Germplasm as claimed in claim I in which the male-sterile character is conferred by cytoplasmic DNA
3. 3. Germplasm as claimed in claim 2 in which the male-sterile character is OGU-INRA'.
4. 4. Swede Fl hybrids which are heterozygous and substantially genetically uniform.
5. 5. Hybrids as claimed in claim 4 which show initial growth hybrid vigour as compared with their parents.
6. 6. Hybrids as claimed in 4 which have higher final globe yield as compared with their parents.
7. 7. Hybrids as claimed in any of claims 4 - 6 which show characters from both parents.
8. 8. Hybrids as claimed in any of claims 4-7 combining at least two of the following characteristics: a globe-shaped tapering stem-base which can be cut off square;.

   a narrow neck' at the leaf base/globe-top, which leaves only a small cut surface when harvested; skin colour of root rich red/purple (top half) and cream (bottom half); flesh cream or yellow; leaves mildew-resistant; root frost-resistant; good flavour.
9. 9. Seed of hybrids claimed in any of claims 4-8.

   -