CA1313153C

CA1313153C - Hybrid seed production

Info

Publication number: CA1313153C
Application number: CA000573069A
Authority: CA
Inventors: Thomas Pattinson Palmer; Anthony John Conner
Original assignee: Canada, AS REPRESENTED BY MINISTE R OF NEW ZEALAND
Current assignee: Canada, AS REPRESENTED BY MINISTE R OF NEW ZEALAND
Priority date: 1987-07-30
Filing date: 1988-07-26
Publication date: 1993-01-26
Anticipated expiration: 2010-01-26
Also published as: IT1229991B; AU615460B2; FR2618640A1; SE8802764D0; SE8802764L; AU2021088A; SE8802764A0; DK390288D0; DK390288A; GB8817191D0; NZ221375A; NL8801888A; DE3825492A1; GB2208346B; JPH02402A; GB2208346A; IT8821521A0

Abstract

ABSTRACT
The invention relates to improved methods of hybrid seed production. More particularly it relates to the use of one (or two) phytotoxic chemical resistant genes in the male (and female) parent in a hybridisation process, followed by dosing the resultant hybrids with the one (or two) phytotoxic chemicals to eliminate plants arising from unwanted contaminating seeds and thus produce pure F1 hybrids.

Description

~3~ 3~5~

IMPROVEM~TS IN OR RELATING TO HY~RID SE~D PRODUCTION

This invention relates to an improved method of hybrid seed production. More particularly it relates to the use of a phytotoxic chemical resistant gene in a male parent in a hybridisation process followed by dosing of the resultant hybrids with the phytotoxic chemical to eliminate plants arising from unwanted contaminating seeds.
For the commercial production of hybrid seed the male and female parents are generally planted in an alternating pattern and seed is harvested off only the female parent. A major problem during hybrid seed production is to ensure that all of the pollen fertilising the female parent originates from the male parent. Seed arising from any other source of pollen will contaminate the hybrid seed population. Potential sources of contaminating pollen are pollen from the female parent, causing self pollination, or pollen from a neighbouring field.
There are 6 ways of eliminating fertile pollen from the female parent (Simmonds, Principles of Crop Improvement, 1979):

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1. mechanical (e.g. tomatoes) - hand emascula-tion of the female parent, followed by pollination;

2. dioecy (e.g. spinach) - removal of the male plants of the genotype from which seed will be har-vested from a crop with separate male and femaleparents;

3. self incompatibility (e.g. brassicas) - in-terplanting of two self incompatible but cross com-patible parents and harvesting of all seed, or use of a self incompatible genotype as the female parent with a self compatible pollinator and harvesting the seed from the female parent only.

4. nuclear male sterility (many crops) segregating female parent~for male sterility (ms ms) and fertility (Ms ms); removal of male fertile plants and pollination of male sterile plants with pollen from the male parent (Ms Ms), 5. cytoplasmic male sterility (e.g. onions) -use of male sterile lines as the female parent, and 6. chemicals:
(a) male gametocides (e.g. wheat) - spraying of female parents with a chemical that induces male sterility;
(b) sex reversal (e.g. curcurbits) - spraying of female parents with plant hormones to revert male 1 3 .t ~

flowers into female flowers.
However there are several problems associated with such approaches:
(1) The physical separation of the female and male parents into alternating blocks in the field does not facilitate the efficient transfer of pollen from male to female parents and can result in less than max-imum hybrid seed production.
(2) None of these approaches can guarantee the absolute elimination of fertile pollen from the female parant (and thus self pollination of the female parent). There are 2 components in this:
(a) Human errors can occur during hand emascula-tion and removal of male fertile plants when using dioecy and nuclear male sterility. Furthermore the labour involved in these practices is very costly.
(b) When using chemical sprays to control sex ex-pression it is difficult to obtain an even application to totally prevent pollen production by female parents.

Furthermore there can often be instability associated with the expression of self-incompatibility and male sterility genes. For example, it is known that elevated temperatures or high humidity reduce self-incompatibility resulting in a high proportion of self-pollinated seed, especially in brassicas (Frankel 13 ~ rj .l and Galun, Pollination Mechanisms, Re~ gs~1s~læld Plant Breeding (1977)). This has resulted in the release of a number of "rough hybrids" containing many self-pollinated plants (Simmonds). Fertility restora-tion in certain male sterile lines of onions has madeit uneconomical to produce hybrid seed from otherwise excellent crosses (Grant, Onions, Plant Breeding in New Zealand (1983)).
3. In an attempt to circumvent foreign pollen contamination from a neighbouring field, hybrid seed blocks are grown in isolation plots. Recommended isolation distances vary from crop to crop depending on its mode of pollinations, and may range from 200 m in sorghum, corn and wheat,~up to 6.4 km in sunflowers (Wright, Commercial Hybrid Seed Production, Hybridisa-tion of Crop Plants (1980~). Attempts are made to remove all sources of contaminating pollen within these distances.
A solution to problem (1) above has been proposed in U.S. Patent Nos. 4517763, 4658084 and 4658085 (all to Beversdorf et al).
All involve creating in the same female parent a combination of cytoplasmically inherited male sterility and either cytoplasmically inherited herbicide resis-tance (4517763) or homozygosity for a dominant nuclear inherited herbicide resistant genes (46580~4). The concept allows the random mixing of the 2 parents for hybrid seed production, thereby aiding pollen transfer from the male to the female parent.
When the female parent is male sterile and her-bicide resistant and the male parent is male fertile and herbicide sensitive, the seed produced from such plants can be of 2 ~ypes:
(1) True hybrid seed from the female parent (herbicide resistant); and (2) Non-hybrid seed from self pollination of the male parent.
The non-hybrid seed resulting from the self-pollination of the male parent can be eliminated by spraying with the herbicide either after pollination but before seed harvest (thereby killing the male parent), or after sowing seed from the bulk harvest (thereby killing the non-hybrid seedlings after germination). This source of non-hybrid seed does not arise in standard hybrid seed production since in stan-dard hybrid seed production the female and male parents are physically separated into alternating blocks in the field, and seed is only harvested off the female blocks. Hence production of a non-hybrid seed com-ponent is an inherent part of these patents due to the 1313~ ~J3 mixed sowing of the two parents.
Also claimed in U.S. Patent Nos. 4,658,084 and4,658,085 is the use of resistance to two different herbicides to allow the mixed random planting of the cytoplasmic male sterile female parent, its maintainer ~ line, and the male parent. Plants from the maintainer line and non-hybrid seed from self pollination of the male parent are eliminated by their sensitivity to different herbicides.
10A possible solution to problem 2~a) above has been prepared by Wiebe (A proposal for Hybrid Barley, Agronomy Journal (1960)).
Wiebe suggests the possible finding of a close linkage between the male fertility gene and suscept-ability to a phytocide (DDT).
This allows the early identification of malefertile plants (Msms) in populations of female parents that segregate for male fertility (Msms) and male sterility (msms) when using nuclear male sterility.
Such plants can therefore be removed from the female parent population prior to flowering and possible release of pollen that may lead to selfing of the female parent.
An identical proposal was suggested by the European Patent Application to Advanced Genetic Sciences Inc. (Publication No. 198,288). However, this approach involves the random introduction of marker genes via ` ~313153 transformation into the genome of male fertile plants ~N8M8 or Msms), and many independently transformed plants are genetically analysed to find an individual with tight linkage between the marker gene and the male fertility locus. Particular emphasis is placed on the ~ use of suicide genes that will result in sensitivity to certain chemicals. The fertile plants can then be eliminated by a simple chemical spray at the seedling stage.
lOHowever the four above described patents still retain all the problems associated with contamination by foreign pollen, and contamination by self-pollination of the female parent due to a breakdown in male sterility (problems 2(b) and 3 above).
15A substantially pure Fl hybrid population of plants means a plant population which is 95~ pure, the minimum degree of purity of seed sold as hybrid seed in : the United States.
In one aspect, the present invention provides a method of forming a substantially pure Fl hybrid population of seeds, said method including:
(a) planting alternating plots of male and female parent plants, the male parent plants being resistant to a phytotoxic chemical, said resistance being attributable solely to a homozygous dominant . .
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~ 313~S3 nuclear mark~r gene, said resistance gene being absent from said female parent plants;
(b) allowing fertilization of said female plants to occur;
(c) harvesting fertilised seed from said plots of female plants only; and (d) dosing fertilised seed harvested in step (c) with said phytotoxic chemical.
step (d) may be carried out either before planting or after emergence of the seedlings. The phytotoxic chemical thus eliminates plants resulting from self-pollination of the female parent or foreign pollen sources and thereby achisves a substantially homogeneous F1 hybrid population.
The above method may be used for species of plants where both parents are self-compatibla, or where one parent only is self-incompatible.
Preferably said phytotoxic chemical is a herbicide of any class. Alternatively, said phytotoxic chemical is an antibiotic, preferably kanamycin (an antibiotic complex produced by Stre~tomYCeS
kanamvceticus).
The present invention further provides a method of forming a substantially pure Fl hybrid population of seeds in which both parents are self-incompatible or self-compatible, said method including:

, 13131~3 (a) planting elther alternating plot~ or a random mixture of first and second parent plants, the first parent plants being resistant to a first phytotoxic chemical and having a homozygous dominant nuclear marker gene absent from the second parent plants, the second parent plants being resistant to a second phytotoxic chemical and having a homozygous dominant marker gene absent from the first parent plants;
(b) allowing fertilization of said first and said second parent plants to occur;
(c) harvesting fertilised seed from the first parent plants and the second parent plants; and, (d) dosing the harvested fertilised seed in step (c) with both the fir~t and second phytotoxic chemicals.
Step (d) may be carried out either before planting or after emergence of the seedlings, to eliminate plants resulting from self-pollination of either said first or second parent plant or from foreign pollen sources. A substantially homogeneous Fl hybrid population is thereby achieved.
Preferably the first and second phytotoxic chemicals are herbicides of any class.

Alternatively the first and second phytotoxic chemicals are antibiotics, preferably one said antibiotic is kanamycin.

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Pre~erably, nuclear marker gene~ are inserted by a plant trans~ormation teahnlque.
Preferably the plant transformation technique used is ~robacterium-mediated transfer.
Some aspects of Aqrobacterium-mediated ~ transfer of genes into plants are disclosed in publi~hed European Patent Application No. 116718 (P. Zambryski);
or cited in published European Patent Application No.
198,288 (column 16, line 37)~
Alternatively said plant transformation technique is direct DNA uptake transformation (into protoplast or cells, plant tissues or inflorescences).
In another possible method said nuclear marker gene is selected in a somatic cell culture. The patents to Beversdorf et al refer to such a method. In a still further possible method said nuclear marker gene is selected in a transfer protoplast fusion. In yet another possible method said nuclear marker gene is selected after inducing plant mutagenesis. Alterna-tively said nuclear marker gene is present by selecting a male or first or second parent plant having the desired gene.
The present invention further provides a method of testing the purity of F1 hybrid populations of plants in which both parents are self-compatible or one parent is self-incompatible, said method including:

. ~
~, carrying out the steps (a) to ~c) o~ the first above described method; planting a small quantity as a sample of said seeds; dosing the seedlings after emergence with said phytotoxic chemical; and determining the percentage of seedlings resistant to said phytotoxic chemical.
The present invention further provides a method of testing the purity of Fl hybrid populations of plants in which both parents are self-incompatible or self-compatible, said method including: carrying out the steps (a) to (c) of the second above-described method; planting a small quantity as a sample of said seeds; dosing the seedlings after emergence with the first and second phytotoxic chemicals; and determining the percentage of seedlings resistant to the first and second phytotoxic chemicals.
In the following disclosure, reference is made to the accompanying drawings, wherein:
Fig. 1 is a diagrammatic representation of inheritance of resistance to a phytotoxic chemical in relation to hybrid seed production, according to a first preferred embodiment of the invention; and Fig. 2 is a diagrammatic representation of the use of resistance to phytotoxic chemicals for hybrid seed production, according to a second preferred embodiment of the invention.
` ~ Referring to Fig. 1, (RR) means homozygous ;~

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resistance; ~Rr) mean~ heteroZygoUs reslstance; ~rr) means homozygou~ sensitive.
The present invention includes, in a first preferred embodiment, the introduction of a dominant marker gene conferring resistance to a phytotoxic chemical into the male parent of a hybrid cultivar (RR).
Both parents are self-compatible or one parent only is self-incompatible. To maximise the efficiency of the approach the male parent should be homozygous for such a gene. In this embodiment, all the true hybrid seed harvested from the female parent (rr) would be - heterozygous for the dominant marker gene and be resistant to the corresponding phytotoxic chemical. If the hybrid seed crop is sprayed at the seedling stage , . .

-- , 131 31~3 with the appropriate chemical, only seedlings arising from the true hybrid seed (Rr) will survive, and any seedlings arising from contaminating pollen will be eliminated.
The production of a true hybrid seed (Rr) is rep-resented by the path illustrated in (A) of Fig. l.
Seed produced by contaminating pollen (rr) is il-lustrated in (B) of Fig. 1. Hybrid seed is usually predominantly of true F1 origin, with varying propor-tions of contaminating seed. Contaminating seed can be eliminated on the basis of sensitivity to a phytotoxic chemical.-After harvest of seed from the female parent in ahybrid seed block, a small seed sample (e.g. lO00 seeds) can be treated with the appropriate chemical, or germinated and the seedlings sprayed with the ap-propriate chemical, to determine the percent contamina-tion. Recommendations can then be made for the re-quired increase in sowing rates to counter the propor-tion of contaminating seedlings that will be subse-quently eliminated.
Referring to Fig. 2, in a second preferred embodi-ment of the invention self-incompatibility in both parents is used for hybrid seed production and seed is harvested off both parents. Each parent must therefore t3131~3 1~

be homozygous for different chemical resistance markers. Hybrid seedlings must then be sprayed with both corresponding chemicals to eliminate seedlings arising from contaminating pollen.
The first parent (AAbb) is resistant to the chemi-cal A, the second parent (aaBB) is resistant to the chemical B. Pure hybrid seeds (AaBb) will be resistant to both chemicals A and B. Non-hybrid seed, arising from contaminating pollen (Aabb or aaBb) will carry a resistance to only chemical A or chemical B.
Because the methods of this invention include the step of eliminating non-hybrid seed during hybrid seed production, there is far less need to isolate plots of the parent plants from contaminating pollen sources.
These methods allow hybrid seed production to be more efficient. Also many excellent female parents previ-ously unable to be used for commercial hybrid seed production due to irregular reversion to male fertility can now be employed.
Provided germplasm sources are available, conven-tional plant breeding approaches can be used to trans-fer resistance to phytotoxic chemicals into male parents or hybrids. Resistance genes can be added directly to specific plant genotypes by exploiting recent advances in plant cell genetics. The applica-13131~3 tions of somatic cell selection, protoplast fusion and transformation have allowed the genetic manipulation of plants for resistance to specific chemicals tConner and Meredith, ~enetic Manipulation of Plant Cells, The Biochemistrv of Plants. A comprehensive treatise Vol.
15, Molecular BioloqY, (1988)).
In most instances such techniques result in in-dividual plants heterozygous for resistance, which must be self-pollinated, then the progeny tested, to gener-ate homozygotes. Although genes for resistance to anyphytotoxic chemicals could be used, the most useful ap-proach would involve genes for resistance to her-bicides. Aqrobacterium-mediated transformation offers an especially convenient method, since several poten-tially useful genes have been cloned and confer resis-tance to specific herbicides when integrated into plants.
The production of herbicide resistant plants via transformation may induce glyphosate resistance (Shah et al, Engineering Herbicide Tolerance in Transgenic Plants, Science, Vol. 233, (1986); Comai et al, Nature, Vol. 317 (1985); US Patent No. 4535060 (Comai); Fil-latti et al, Bio/ Technoloqv, Vol. 5 (1987~) or chlor-sulfuron resistance (Haughn et al, Molecular and 25 General Genetics, Vol. 211, (1988)); or 1 3 1 3 1 ~ ! i phosphinothriun/bialaphos resistance (De Block, The EE~
Journal~ Vol.6 (1987)).
Example 1 The use of dominant marker genes with resistance to a phytotoxic chemical for circumventing foreign pol-len contamination during the production of hybrid seedis illustrated in Nicotiana Plumbaqinifolia plants resistant to kanamycin.
A. Testinq for kanamvcin resistance Kanamycin resistance can be conveniently studied in N. ~lumbaainifolia by screening for seedlings with green cotyledons (kanamycin-resistant) versus white cotyledons (kanamycin-sensitive), growing in the presence of kanamycin. Seeds were soaked overnight in 1 mM gibberellic acid, surface sterilised with 3%
sodium hypochlorite for 5 min. and rinsed thoroughly in sterile distilled water. They were then sown on 1/2 MS
salts (Murashige and Skoog, A Revised Medium for Rapid Growth and Bioassays With Tobacco Tissue Culture, Phvsioloqia Plantarum, Vol. 15, (1~62)) plus 0.8% agar, supplemented with 300 mg/L Xanamycin.
The culture media was autoclaved for 20 min at 121 kPa, with filter sterilised kanamycin being added after autoclaving. Germinating seedlings were incubated at 26C under cool white fluorescent light (100 umol.

m 2.sec 1; 16 h light; 8 h dark daily). Green versus ~.3~31~3 white seedlings can be screened after 7 to lO days.
Using this approach a single green kanamycin resistant seedling of N. Plumbaqinifolia was observed among many thousands of white kanamycin-sensitive see-dlings. This seedling was transferred to akanamycin-free medium. After 8 weeks it was trans-ferred to soil. Controlled pollinations were made at flowering and the, resulting progeny screened for kanamycin resistance.

' 10 The original isolated seedling (NpKR) was ~ heterozygous at a single locus for spontaneous mutation ¦ to kanamycin resistance. This is evident from the self-pollinated and backcrossed progeny segregating 3:1 and 1:1 respectively for kanamycin resistant and sensi-tive seedlings (Table 1). From the self-pollinated progeny a single plant (NpKR B) was identified that bred true for kanamycin resistance (Table l). Clearly, this plant was homozygous at a single locus for kanamycin resistance.
B. HYbrid Seed Production Hybrid seed production was simulated using a wild-type N. plumbaqinifolia plant as the female parent and NpKR B (homozygous for kanamycin resistance) as the male parent. Three levels of control over foreign pol-len contamination were created by pollinating the emas-~`
) 13131~3 1~

culated the female parent with:
1. pollen from NpKR ~3 only (tight control), 2. pollen primarily from NpKR 8, with a small amount from a wild-type plant (relaxed control), and 3. substantial amounts of pollen from both NpKR
B and a wild-type plant (loose control).

Table 1: Inheritance of kanamycin resistance in a spontaneous kanamycin-resistant mutant of Nicotiana Plumba~inifolia (Np KR). Seedling progeny were screened for resistance to 300 mg/L kanamycin.
Cross Number of Seedibgs Ratio Chi (female x male~ Greena White tested square - Wild-type (selfed) 0 1,165 _ NpKR (selfed)1,150 -367 3:1 0:53 Wild-type x NpKR 292 266 1:1 1:21 NpKR x Wild-type 256 281 1:1 1:16 NpKR B (selfed) 8-08 0 a) kanamycin-resistant b) kanamycin-sensitive When NpKR B is used to pollinate flowers of wild-.
type plants with these varying levels of pollination20 control, green kanamycin-resistant true Fl hybrid see-dlings can be readily distinguished from the white kanamycin-sensitive seedlings arising from the "foreign" wild-type pollen (Table 2). As expected, the percent of contaminating seed increases as the control over foreign pollen is relaxed. This experiment demonstrates the principle of eliminating non-hybrid seeds when there is the threat of pollen contamination during hybrid seed production.

Example 2 The use of dominant marker genes with resistance to a phytotoxic chemical for circumventing self-pollination in the female parent during the production of hybrid seed is illustrated by Nicotiana plum-baainifolia plants genetically engineered for resis-tance to kanamycin.
A. Transformation A binary vector system involving Aarobacterium tumefaciens (strain LBA 4404), Hoekema et al., A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Tiplasmid, Nature, 303, p 179-180 (1983), harbouring the plasmid pKIWI 6 was used for transformation. This plasmid contains between the left and right borders of the T-DNA, 2 chimeric genes capable of being expressed inplant cells. One confers resistance to the antibiotic kanamycin and consists of the coding region from the neomycin phosphotransferase II gene (from the bacterial transposon Tn5) under the control of the octopine synthase promoter and poly A signal (OCS-NPTII-OCS).

13131~
19~1 The other confers chloramphenicol acetyl transferase (CAT) activity and consists of the coding region of CAT
(from bacterial transposon Tn9) under the control of the mannopine synthase promoter and octopine synthase poly A
signal.

1313~

~ The use of kanamycin re31stance to eliminate seed arising from contaminating pollen during hybrid seed production in Nicotiana plumbaqinifolia.
Female parent = wild-type kanamycin sensitive plant with varying levels of self-pollination. Male parent ~ NpKR
B (homozygote for kanamycin resistance). Seedling progeny screened for resistance to 300 mg/L kanamycin.

Cantrol over N~r Percent P~x~nt of foreign pollen of seeds true Fl cont~nating 10 ocr;l~rDtiona scn#~ed hybrid ~ selfed seedC

Tight 341 100 0 Relaxed 273 8~ 14 ~x~e 229 57 43 a see above b green surviving seedlings, heterozygous for kanamycin resistance c white dying seedlings, sensitive to kanamycin.

Leaf segments from in vitro plant~ of N.
~lumbaginifolia were dipped in a suspension of A.
tumefaciens (an overnight culture in MG/L broth - Gar-finkel and Nester, Aarobacterium tumefaciens Affected inCrown Gall Tumourigenesis and Octopine Metabolism, Journal of Bacteriology Vol. 144, (1980)), blotted dry 131315'3 and cultured on RMOP medium (Sidorov et al, Isoleucine-requiring _icotiana Plant Deficient in Threonine Deaminase, Nature, Vol. 29~ (1981)). After 2 days the leaf segments were transferred to the same medium supplemented within 500 mg/L cefotaxime (to prevent Aarobacterium overgrowth) and 300 mg/L
kanamycin (to select for transformed plant cells).
Regenerated Xanamycin-resistant shoots were rooted on MS salts (Murashige ~ Skoog, 1962) plus 3% sucrose and 0.8% agar, then transferred to soil. Controlled pol-linations were made at flowering, and the resulting progeny screened for kanamycin resistance, as described in Example 1.
B. Testina for Kanamycin Resistance Inheritance of kanamycin resistance can be con-; veniently studied in N. plumbaqinifolia by screening for seedling with green cotyledons (kanamycin-resistant) vèrsus white cotyledons (kanamycin-sensitive), 7-10 days after sowing on medium containing 20 300 mg/L kanamycin. Inheritance was studied in 10 transformed plants; results from only one of these plants (NpT 17) is reported here.
The original transformed plant of NpT 17 was heterozygous for insertion into a single locus of the kanamycin-resistant genes. This is evident from the , 1313~3 self-pollinated and backcrossed progeny segregating 3:1 and 1:1 respectively for kanamycin resistant and sensi-tive seedlings (Table 1). From the self-pollinated progeny a single plant (NpT 17 D) was identified that bred true for kanamycin resistance (Table 3). Clearly, this plant was homozygous at a single locus for kanamycin resistance.
NpT 17D was backcrossed to the wild-type in order to generate a large population of seedlings (over 5,000) heterozygous for inserted kanamycin resistant genes. Any genetic instability of kanamycin resistance would be recognised by the appearance of rare, white, kanamycin-sensitive seedlings within such a population.
All the seedlings proved~to be kanamycin resistant (Table 3~, indicating high genetic stability. There-fore genes with resistance to phytotoxic chemicals, in-troduced into plants via Aqrobacterium-mediated trans-formation, are sufficiently stable to be used as genetic markers to monitor the seed purity of crop cul-tivars.
C. Hybrid Seed Production Hybrid seed production was simulated using awild-type N. plumbaqinifolia plant as the female parent and NpT 17D (homozygous for kanamycin resistance) as the male parent. Varying levels of control over female 1313~3 pollen contamination were created by emasculating the female parent at different stages of flower develop-ment. N. plumbaqinifolia is cleistogamous and flowers self-pollinate just prior to opening of the corolla and development of corolla pigmentation. Three levels of control over selfing of the female parent were estab-lished.

Table 3: Inheritance of kanamycin resistance in a trans*ormed plant of Nicotiana plumbaqinifolia (NpT
17). Seedling progeny were screened for resistance to 300 mg/L kanamycin.

Cross Number of Seedlbngs Ratio Chi (female x male) GreenaWhite tested square _ Wild-type (selfed) 0 1,693 _ NpT 17 (selfed) 409141 3:1 0:12 Wild-type x NpT 17 586 552 1:1 1:02 NpT 17D (selfed) 1,240 0 _ NpT 17D x Wild-type 5,339 0 a) kanamycin-resistant b) kanamycin-sensitive i 1. tight control was exerted by emasculation of flower buds 2-3 cm long, well before pollen is shed, 2. relaxed control was exerted by emasculation of flower 4-5 cm long, just at the point of reaching maximum corolla length and when pollen is beginning to be shed, and 3. loose control was exerted by "emasculation"
after corolla pigment development and just as flowers were opening. Considerable self-pollination has oc-curred by this stage.
When NpT 17D is used to pollinate flowers of wild-type plants with these varying levels of pollina-tion control, green kanamycin-resistant true Fl hybrid seedlings can be readily distinguished from the white kanamycin-sensitive seedlings arising from self-pollination of the female parent (Table 4). As ex-pected, the percent of contaminating seed increases as the control over female selfing is relaxed. This ex-periment demonstrates the principle of eliminating non-hybrid seeds when there is the threat of pollen contamination from self-pollination of the female parent during hybrid seed production.
Seed of NpKR B (Example 1) and NpT 17D (Example 2), have been deposited in or are available from the Crop Germplasm Resource Centre, Crop Research Division, DSIR, Private Bag, Christchurch, New Zealand. It is available on request from the curator of the collection.

` ~

13131~3 Table 4: The u~e oP kanamycin resistance to eliminate seed arising ~rom self-pollination of the female parent in Nicotiana plumbaginifolia. Female parent = wild-type kanamycin sensitive plant with varying levels of self-pollination. Male parent = NpT
17D (homozygote for kanamycin resistant). Seedling progeny screened for resistance to 300 mg/L kanamycin.

Control over Nmber ~ent ~x~nt of f~gn p~llen of seeds true Fl o~hr~n~ting 10 ocntaminationa hybrid seedb selfed seedC

Tight 3,110100 0 Relaxed 3,85492 8 15 La~ 6,03943 57 a see above b green surviving seedlings, heterozygous for kanamycin re~istance c white dying seedlings, sensitive to kanamycin.

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Claims

1. A method of forming a substantially pure F1 hybrid population of seeds, said method including:
(a) planting alternating plots of male and female parent plants;
(b) allowing fertilization of said female plants to occur; and (c) harvesting fertilised seed from said plots of female plants only; wherein the improvement comprises:
the male parent plants being resistant to a phytotoxic chemical, said resistance being attributable solely to a homozygous dominant nuclear marker gene, said resistant gene being absent from said female parent plants; and the method includes the step of:
(d) dosing fertilised seed harvested in step (c) with the phytotoxic chemical.

2. A method as claimed in claim 1 wherein both parents are self-compatible.

3. A method as claimed in claim 1 wherein one parent only is self-incompatible.

4. A method as claimed in claim 2 wherein said phytotoxic chemical is a herbicide.

5. A method as claimed in claim 2 wherein said phytotoxic chemical is an antibiotic.

6. A method as claimed in claim 5 wherein said antibiotic is kanamycin.

7. A method as claimed in claim 3 wherein said phytotoxic chemical is a herbicide.

8. A method as claimed in claim 3 wherein said phytotoxic chemical is an antibiotic.

9. A method as claimed in claim 8 wherein said antibiotic is kanamycin.

10. A method as claimed in either claim 1 or 4 wherein step (d) is carried out before planting said seed.

11. A method as claimed in either claim 1 or 4 wherein step (d) is carried out after the emergence of seedlings from said seed.

12. A method as claimed in either claim 3 or 6 wherein step (d) is carried out before planting said seed.

13. A method as claimed in either claim 3 or 6 wherein step (d) is carried out after the emergence of seedlings from said seed.

14. A method as claimed in claim 1 wherein said nuclear marker gene is inserted by a plant transforma-tion technique.

15. A method as claimed in claim 14 wherein said plant transformation technique is Agrobacterium-mediated transfer.

16. A method as claimed in claim 14 wherein said plant transformation technique is direct DNA uptake transformation.

17. A method as claimed in claim 1 wherein said nuclear marker gene is selected in a somatic cell cul-ture.

18. A method as claimed in claim 1 wherein said nuclear marker gene is selected in a transfer protoplast fusion.

19. A method as claimed in claim 1 wherein said nuclear marker gene is selected after inducing plant mutagenesis.

20. A method as claimed in claim 1 wherein said nuclear marker gene is selected after inducing plant mutagenesis.

21. A method of forming a substantially pure F1 hybrid population of seeds in which both parents are self-incompatible or self-compatible, said method including:
(a) planting either alternating plots or a random mixture of first and second parent plants, the first parent plants being resistant to a phytotoxic chemical A;
(b) allowing fertilization of said first and said second parent plants to occur; and (c) harvesting fertilised seed from the first parent plants and the second parent plants; wherein the improvement comprises:
the second parent plants being resistant to a phytotoxic chemical B, said resistance to said phytotoxic chemicals A or B being attributable in each plant solely to a homozygous dominant nuclear marker gene, said gene resistant to chemical A being absent from said second parent plants and said gene resistant to chemical B being absent from said first parent plants; and the method includes the step of:
(d) dosing the fertilised seed harvested in step (c) with both said phytotoxic chemicals A and B.

22. A method as claimed in claim 21 wherein said phytotoxic chemicals A and B are herbicides.

23. A method as claimed in claim 21 wherein said phytotoxic chemicals A and B are antibiotics.

24. A method as claimed in claim 23 wherein one said antibiotic is kanamycin.

25. A method as claimed in either claim 21 or 22 wherein step (d) is carried out before planting said seed.

26. A method as claimed in either claim 21 or 22 wherein step (d) is carried out after the emergence of seedlings from said seed.

27. A method as claimed in claim 24 wherein step (d) is carried out before planting said seed.

28. A method as claimed in claim 24 wherein step (d) is carried out after the emergence of seedlings from said seed.

29. A method as claimed in claim 21 wherein said nuclear marker gene is inserted by a plant transforma-tion technique.

30. A method as claimed in claim 29 wherein said plant transformation technique is Agrobacterium-mediated transfer.

31. A method as claimed in claim 29 wherein said plant transformation technique is direct DNA uptake transformation.

32. A method as claimed in claim 21 wherein said

33 nuclear marker gene is selected in a somatic cell culture.
33. A method as claimed in claim 21 wherein said nuclear marker gene is selected in a transfer protoplast fusion.

34. A method as claimed in claim 21 wherein said nuclear marker gene is selected after inducing plant mutagenesis.

35. A method as claimed in claim 21 wherein said nuclear marker gene is present by selecting a parent plant having the desired gene.

36. A method of testing the purity of F1 hybrid populations of plants, said method including:
(a) planting alternating plots of male and female plants;
(b) allowing fertilization of said female plants to occur;

(c) harvesting fertilised seed from said plots of female plants only; and (d) planting a small quantity as a sample of said seeds; wherein the improvement comprises:
the male parent plants being resistant to a phytotoxic chemical, said resistance being attributable solely to a homozygous dominant nuclear marker gene, said resistance gene being absent from said female parent plants; and the method includes the steps of:
(e) dosing the seedlings after emergence with said phytotoxic chemical; and then (f) determining the percentage of seedlings resistant to said phytotoxic chemical.

37. A method as claimed in claim 36 wherein said nuclear marker gene is inserted by a plant transforma-tion technique.

38. A method as claimed in claim 37 wherein said plant transformation technique is Agrobacterium-mediated transfer.

39. A method as claimed in claim 37 wherein said plant transformation technique is direct DNA uptake transformation.

40. A method as claimed in claim 36 wherein said nuclear marker gene is selected in a somatic cell cul-ture.

41. A method as claimed in claim 36 wherein said nuclear marker gene is selected in a transfer protoplast fusion.

42. A method as claimed in claim 36 wherein said nuclear marker gene is selected after inducing plant mutagenesis.

43. A method as claimed in claim 36 wherein said nuclear marker gene is present by selecting a parent plant having the desired gene.

44. A method of testing the purity of F1 hybrid populations of plants in which both parents are self incompatible or self-compatible, said method including:
(a) planting either alternating plots or a random mixture of first and second parent plants;
(b) allowing fertilization of said first and said second parent plants to occur;
(c) harvesting fertilised seed from the first parent plants and the second parent plants; and (d) planting a small quantity as a sample of said seeds; wherein the improvement comprises:
the first parent plants being resistant to a phytotoxic chemical A, the second parent plants being resistant to a phytotoxic chemical B, said resistance to said phytotoxic chemicals A or B being attributable in each plant solely to a homozygous dominant nuclear marker gene, said gene resistant to chemical A being absent from said second parent plants and said gene resistant to chemical B being absent from said first parent plants; and the method includes the steps of:
(e) dosing the seedlings after emergence with said phytotoxic chemicals A and B; and then (f) determining the percentage of seedlings resistant to said phytotoxic chemicals A and B.

45. A method as claimed in claim 44 wherein said nuclear marker gene is inserted by a plant transforma-tion technique.

46. A method as claimed in claim 45 wherein said plant transformation technique is Agrobacterium-mediated transfer.

47. A method as claimed in claim 45 wherein said plant transformation technique is direct DNA uptake transformation.

48. A method as claimed in claim 44 wherein said nuclear marker gene is selected in a somatic cell cul-ture.

49. A method as claimed in claim 44 wherein said nuclear marker gene is selected in a transfer protoplast fusion.

50. A method as claimed in claim 44 wherein said nuclear marker gene is selected after inducing plant mutagenesis.

51. A method as claimed in claims 44 wherein said nuclear marker gene is present by selecting a parent plant having the desired gene.