CA2231423A1 - Cytoplasmic male sterile brassica oleracea plants which contain the polima cms cytoplasm and are male sterile at high and low temperatures - Google Patents
Cytoplasmic male sterile brassica oleracea plants which contain the polima cms cytoplasm and are male sterile at high and low temperatures Download PDFInfo
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- CA2231423A1 CA2231423A1 CA002231423A CA2231423A CA2231423A1 CA 2231423 A1 CA2231423 A1 CA 2231423A1 CA 002231423 A CA002231423 A CA 002231423A CA 2231423 A CA2231423 A CA 2231423A CA 2231423 A1 CA2231423 A1 CA 2231423A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/022—Genic fertility modification, e.g. apomixis
- A01H1/023—Male sterility
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
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Abstract
The present invention involves cytoplasmic male sterile Brassica oleracea plants that contain polima CMS cytoplasm. The plants remain sterile at high temperatures. The Brassica oleracea plants of the present invention can be prepared by traditional breeding techniques. Different Brassica types can then be produced using these plants by employing traditional breeding techniques or by protoplast fusion.
Description
CYTOPI~aSMIC ~Sa~E ~TERILE BhU~C~ ~rF~A~F~ Pla~r8 ~IIC~ CONTAIN THE poLI~la CM8 CYTOPI~A8M AND ARE lAI~E
5FIELD OF TB l~v~ o~l The present invention involves cytoplasmic male sterile Br~sica oleracea plants that contain polima CMS
cytoplasm, are male sterile at high and low temperatures, and exhibit good female fertility.
R~KG~u~v AND 8UMMARY OF T~E lNv~ ON
In an effort to increase the productivity of plants and food crops, plant breeders generally develop cultivars that contain certain desirable characteristics such as increased height, growth rate, higher yields, etc. One of the ways in which this may be accomplished is by infusing desirable characteristics into a plant to form a superior plant line. Superior lines are then combined to form an Fl hybrid that contains the desirable characteristics. Such superior hybrids can be developed in numerous ways.
One popular way of producing superior hybrids is by using male sterility in one of the plants for which hybridization is desired. Male sterile lines allow the breeder to produce hybrid seed more economically by controlling cross-fertilization in the flower of a plant. Cross-fertilization can be controlled by preventing the female parent from self fertilizing.
W O 97/09873 PCTrUS95/11497 Self-fertilization is eliminated by making the plant male sterile. If the plant is male sterile, then no pollen can be produced for fertilization. Once rendered male sterile, the plant may then be hybridized with a gene donor plant possessing the desired characteristics.
one way to effectuate male sterility is through the use of cytoplasmic male sterility. Present belief is that genetic factors controlling cytoplasmic male sterility (CMS) are found in the cytoplasm, particularly in the genes of the mitochondrial DNA.
Three of the most common cytoplasmic male sterilities in the Brassica species are:
1) Ogura male sterile cytoplasm of Rap*anus sati~s;
5FIELD OF TB l~v~ o~l The present invention involves cytoplasmic male sterile Br~sica oleracea plants that contain polima CMS
cytoplasm, are male sterile at high and low temperatures, and exhibit good female fertility.
R~KG~u~v AND 8UMMARY OF T~E lNv~ ON
In an effort to increase the productivity of plants and food crops, plant breeders generally develop cultivars that contain certain desirable characteristics such as increased height, growth rate, higher yields, etc. One of the ways in which this may be accomplished is by infusing desirable characteristics into a plant to form a superior plant line. Superior lines are then combined to form an Fl hybrid that contains the desirable characteristics. Such superior hybrids can be developed in numerous ways.
One popular way of producing superior hybrids is by using male sterility in one of the plants for which hybridization is desired. Male sterile lines allow the breeder to produce hybrid seed more economically by controlling cross-fertilization in the flower of a plant. Cross-fertilization can be controlled by preventing the female parent from self fertilizing.
W O 97/09873 PCTrUS95/11497 Self-fertilization is eliminated by making the plant male sterile. If the plant is male sterile, then no pollen can be produced for fertilization. Once rendered male sterile, the plant may then be hybridized with a gene donor plant possessing the desired characteristics.
one way to effectuate male sterility is through the use of cytoplasmic male sterility. Present belief is that genetic factors controlling cytoplasmic male sterility (CMS) are found in the cytoplasm, particularly in the genes of the mitochondrial DNA.
Three of the most common cytoplasmic male sterilities in the Brassica species are:
1) Ogura male sterile cytoplasm of Rap*anus sati~s;
2) Polima male sterile cytoplasm of Brassica napus; and 3) Nap male sterile cytoplasm of Brassica napus.
In Brassica, cytoplasmic male sterility can be transmitted by crossing. The female (egg) parent contributes the cytoplasm, therefore, crossing to CMS
females produces CMS progeny. The nuclear genes however are heterozygous. Therefore, six to eight generations of "backcrossing" are n~ce~c~ry to produce a CMS line breeding homozygous for nuclear characters. As an alternative, cytoplasmic male sterile lines can also be produced by protoplast fusion. In protoplast fusion, a protoplast from a plant having co~rcially desirable traits is combined with a protoplast of a C~S line. The ~ nuclear material o~ the CMS line is either removed or inactivated prior to fusion so it donates only the cytoplasm. The resulting cytoplasmic hybrid (or cybrid) possesses the CMS cytoplasm and is male sterile. For example, U.S. Patent 5,254,802 discloses B oleracea plants that contain the Ogura CMS cytoplasm. These plants were obtained by protoplast fusion.
Polima CMS cytoplasm has been used to produce CMS
in varieties such as winter-type oil seed rape (Brassica napus) ~See Barsby et al., Plant Science, 53: 243-248 (1987)). However, one significant problem with the expression of cytoplasmic male sterility by the polima CMS cytoplasm is that the polima cytoplasm is influenced by environmental conditions. Fan, Z et al. Can. J.
Plant Sci. 66:221-227 (1985). More specifically, male sterile plants cont~ini ng polima CMS cytoplasm are known to become fertile under high temperatures in the field.
Id. See also Fu, T.D., EncarDia Cruciferea Newsletter 6: 6-7 (1981).
The present invention involves Brassica oleracea plants that contain Polima CMS cytoplasm which remain male sterile at high and low t- ~atures and exhibit good W O 97/09873 PCT~US95/11497 female fertility. The Brassica oleracea plants of the present invention can be produced by traditional bree~
methods. Different Brassica types can then be developed by further crossings or backcrossings or by protoplast fusion.
To obtain the Brassica olcracca plants of this invention by traditional br~A;nq te~-hniques, an interspecific cross was made between Brassica~ cultivar 87110 and Brassica okracea cultivar 87101. The seeds resulting from the cross are collected, planted and regenerated. The resulting plants are Brassica nap~ and contain a haploid set of chromosomes. The ch~. ~mal content of said Brassica napus must be doubled by treating the plants with colchicine.
A second interspecific cross is performed by crossing Brassica napus cultivar 87118 with Brassica olera~a cultivar 87101. The seeds resulting from the cross are collected, planted and regenerated. As in the previous cross, the resulting plants are Brassica napus and contain a haploid set of chromosomes. The plants are treated with colchicine to double their chromosome content.
The Brassica nap~ plants produced as a result of the second interspecific cross are next crossed with a Brassica napYs cultivar 87102, which contains polima CMS cytoplasm and is male sterile. The seeds resulting from the cross are collected, planted and regenerated. The regenerated plants are Brassica nap~ and contained the polima CMS
cytoplasm.
The resulting plants are subsequently crossed with the Brassica nap~ plants produced as a result of the first interspecific cross. The seeds resulting from the cross are collected, planted and regenerated. The regenerated plants are Brassica nap~, contain the Polima CMS cytoplasm and are male sterile.
The resulting plants are then crossed with a normal B~ssica oleracea. AS a result of the cross, siliques are pro~llçe~, collected and examined for seeds. The seeds are collected for embryo ~e c~le, because typically, embryos produced from such interspecific hybridization abort prior to maturation. However, by employing embryo rescue techniques, interspecific hybrid plants can be produced. The resulting Fl plants contain the polima CMS
cytoplasm from the female Brassica napYs~ however; the nuclear DNA content is a combination of the Brassica napus (N=ls) and the Brassica oleracea (Nz9).
The resulting plants are then backcrossed with a Brassica okracea. Siliques are again produced, collected and W O 97/09873 PCTrUS95/11497 examined for seeds. The seeds are collected for embryo rescue. The embryos are then regenerated as in the previous cross. The resulting plants are intermediate for chromosome number and contain the polima CMS
cytoplasm. The nuclear content of the plants is a combination of Brassica napus and Brassica oleracea.
The resulting plants are then backcrossed with Brass~ca okracca. Siliques are again produced, collected and examined for seeds. The seeds are sown. The resulting plants are Brassica o~racea which are male sterile and contain the polima CMS cytoplasm.
Optionally, the male sterile Brassica olcra~a plants may be further crossed or backcrossed to produce different Brassica ~pes. Siliques will again be produced, collected and eY~;ned for seeds. The seeds are sown. The resulting plants are Brassica o~a~a which contain the polima cytoplasm and are male sterile.
Different Brassica types can also be produced by protoplast fusion. A protoplast from a male sterile 20 Brassica okra~a containing the polima CMS cytoplasm and inactivated nuclei is fused with a protoplast of a Brassica having c~: -~cially desirable characteristics. After the fusion, the allogenic cells are regenerated into CMS
Brassica plants. The resulting plants are male sterile and W O 97/09873 PCT~US95/11497 contain the polima cvtopl~m. The regenerated CMS Brassica plants contain the polima cytoplasm and can be employed in crossings with other Brassica types cont~inin~ commercially desirable characteristics.
DESCRIPTION OF THE FIGURES
Figure I shows the different hvbridization patterns of BGL I digested DNA with probe E~ in an endonuclease digest cont~ining nuclear DNA of a polima CMS donor, Brassica oleracea 904005-1, arld Brassica oleracea breeding lines used as acceptors for the CMS trait. Lane 1 shows Phage 1 Hind III
digested DNA Fra~nents (MW Standards). Lanes 2 and 3 show Polima CMS
0 Brassica oleracea 904005-1 DNA. Lanes 4 and 5 show Brassica oleracea acceptor Line K14. Lanes 6 and 7 show Brassica oleracea acceptor Line DE70.
Figure 2 shows the different hybridization patterns of BGLII digested DNA with probe CMS BRAS 4 in an en~on-l~lease digest Cn~ inin mitochondrial DNA of a polima CMS plant and mitochondrial DNA from an acceptor breeding line plant. Lanes 8 and 18 show Phage A Hind III digested DNA ~ ont~. Lanes I to 7 show Polima CMS Brassica oleracea 904005-1 DNA. Lanes 9 to 17 show Brassica oleracea acceptor Line K14. Lanes 19 to 24 show Brassica oleracea acceptor Line DE70.
DESCRIPTION OF THE INVENTION
Cytoplasmic male sterile Brassica napus plants Cont~inin~ polima cytoplasm are lcnown to ;lea-k- . Le~kinesc refers to the fact that the plants tend to lose their sterilitv under certain temperature conditions. For example, RECTIFIED SHEET (RULE 91) W O 97/09873 PCT~US9S/11497 studies have shown that CMS Brassicanap~s contA;~i~g polima cytoplasm plants are only partially sterile at high temperatures. See Fan, Z et al., Can. J. Plant Sci. 66:
221-227 (1985).
The present invention involves CMS Brassica oleracea plants that contain the polima cytoplasm. The plants are male sterile at high and low t~ reratures and exhibit good female ~ertility. Different Brassica C~S types can then be developed by employing further crossings and backcrossings or protoplast fusion.
The Breedinq Pro~am Traditional br~ g t~hn i ques were used to develop the Brassica o~racea plants of the present invention.
The inventors herewith describe the breeding program used to create the Brassica oleracca plants of this invention.
The breeding program was undertaken by making an interspecific cross between a Brassica ~ (=Brassica rapa) entry 87110 (n=10) and a Br~ssica olcracea entry 87101 (N-l9).
Entry 87110 was obtained from the Centre for Plant Research (CPR0), PØ Box 16, 6700 AA, Wageningen, The Netherlands as entry IVT 86007 paar 3MS and was genetically male sterile. 87110 has been deposited under the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852. 87110 was deposited on August 23, 1995 and received ATCC A~c~ccion Number 97246. Entry 87101 is available from the Crucifer Genetics Cooperative (CrGC) at the Department of Plant Pathology, 1630 Tlnflen Drive, University of Wisconsin, ~A~;Con~ Wisconsin as CrGC #3-3 (Ccc) and is a rapid cycling Br~sica ol~acea.
After the crossing, siliques (seeds) developed. The siliques are collected, examined for seeds and planted.
The resulting plants were haploid Br~s~ca napw ac (Nsl9) and were su~jected to colchicine treatment to double chromosomes to the diploid number aacc (2n=38). The resulting plants were "artificial" Br~sica nap~ with an aacc genome. These were labelled 87116.
A c~co~ Br~sica~ , 87118, having the same characteristics as 87110, was also crossed with 87101.
87118 was received from the CPR0 as IVT 86017 paar 3MS.
87118 has been deposited under the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852. 87118 was deposited on August 23, 1995 and received ATCC ~Cc~ccion Number 97247. As in the previous cross, the seeds were collected and planted. The resulting plants were subjected to colchicine treatment to double the W O 97/09873 PCT~US95/11497 chromosomes. The resulting plants were an "artificial"
Brassica napus with an aacc genome. The resulting plants were labeled 87119.
Plants of 87119 were then crossed with a male sterile Brassica napus (aaccj containing the polima CMS
cytoplasm (labeled 87102). 87102 is available from the Crucifer Genetics Cooperative as CrGC #5-4 (AClaacc) which is a rapid cycling Brassica nap~ with "Polima" CMS
cytoplasm. Siliques were produced, collected and examined for seeds. The seeds were planted and the resulting plants labeled 88102. These plants were Brassica napus, had an aacc genome and contained the polima CMS
cytoplasm.
A cross was then made between 87116 and 88102.
Siliques were produced, collected and ~Y~;ned for seeds. The seeds were planted. The resulting plants were Brassica napus (aacc) and were labeled 8812S. Brassica napus (aacc) is an amphidiploid species between Brassica oleracea N=9 (cc genome) and Brassica rapa N=10 (aa genome). 88125 are Brassica napus (aacc) and contains polima CMS cytoplasm and are male sterile.
An interspecific cross was made between 88125 (aacc) and a normal male fertile broccoli plant, Brassica o~acea (cc). One skilled in the art would recongize that WO 97/09873 PCT~US95/11497 any male fertile Br~sica oleracea plant could be used for this cross. Siliques developed. The siliques were then collected and ~Am; n~ for seeds. If seeds were present, then the embryo(s) is/were rescued. The embryo(s) is/were rescued because typically, embryos produced from such an interspecific hybridization abort prior to maturation. However, by employing embryo rescue, interspecific hybrids can be pro~llc~.
Embryo rescue involves removing the embryo from the sili~ue. It is preferred that the embryo rescue be conducted when the embryo is as large as possible.
Generally, 18-19 days after the cross is a good time to conduct embryo rescue. However, one skilled in the art would r~co~n;ze that the embryo rescue could take place earlier or later, so long as the seed coat has not yet hardened.
In embryo rescue, the siliques are first disinfected for about 20 minutes in a 10% hypochlorate solution (such as sodium or potassium hypochlorate) and rinsed three times in sterile water at 2.5 and lO
minutes.
Next, the siliques are cut in a longitll~;nAl direction. Young seeds are removed and embryos taken out of the seed under a microscope. The embryos are then placed on a suitable medium to facilitate growth, such as a BLKC medium which is described in Example 11.
However, any medium that facilitates growth can be used.
The embryos are grown at 20-C in 16 hours of 4500 LUX
light for at least two weeks. After the roots develop, the seedlings are placed into soil as soon as possible.
The resulting Fl plants were assigned the numbers 88132-1 through 88132-84. These plants cont~;n~ the polima CMS cytoplasm from the female Brassica napus plant, however: the nuclear DNA content was a combination of the Br~sica napus (N=19) and the Brassica oleracea (N=9) .
Essentially, the plants had mixed genomes. Because of their mixed nature, the plants were male sterile and partially female sterile. The plants were male sterile because they contained the polima CMS cytoplasm from the female (egg) parent, Brassica napus, 88125.
Phenotypical selection was used to select the plants from 88132-1 through 88132-84 that have near normal DNA content (aacc or cc). Phenotypic selection allows the breeder to select plants most similar to Brassica olera~a (cc genome). Plants with an abnormal DNA
content are visually different than plants with a normal DNA content. The plants selected were backcrossed (BCl) with a normal broccoli or cauliflower, Brassicn oleracea (cc).
However, one skilled in the art would recognize that any wos7/os873 PCT~S95111497 Brassica ol~acea could be used. After the cross, em~ryo rescue was again employed. The plants that developed were assigned the numbers 89015 through 89040 and 89046 through 89056. These plants were intermediate for chromosome number and cont~;ne~ the polima CMS
cytoplasm. Again, the nuclear DNA content was a combination of the Brassica ~us and the Brassica oleracea. The plants were male sterile (polima CMS) and partially female sterile (chL~ _ome number).
Phenotypical selection was employed to select plants from 89015 through 89040 and 89046 through 890~6 having near normal DNA content. The plants selected were backcrossed (BC2) with a normal broccoli or cauliflower, Brassica oleracea (cc). However, one skilled in the art would recognize that any Brassica oleracea could be used. Siliques again developed, however, some seeds did not require embryo rescue. These seeds were collected and planted. The plants resulting from this cross were assigned the numbers 89070 through 89141. Most of these plants had the normal Brassica oleracea set of - chromosomes (2N=18), contained the polima cytoplasm and were male sterile. The flowers of these plants had reduced anthers and petals. The plants remained sterile W O 97/09873 PCTrUS95/11497 at high and low temperatures and exhibited normal female fertility.
These resulting plants can be crossed with other Br~sica types such as broccoli, cauliflower, cabbage (red, S white, savoy), brussels sy,ouLs, kohlrabi, kale, etc. to produce CMS lines. For example, different Br~sica types can be produced by backcrossing to the CMS "polima"
plants or by protoplast fusion.
Subsequent Crosses and Selection Process for M~le Steril;tv Female FertilitY and Flower MorpholoqY
Plants of 89015 through 89640 and 89046 through 89056 and 89070 through 89141 were subsequently backcrossed with a normal broccoli and/or cauliflower and planted in the field. Siliques developed, were collected and ~A~; ne~ for seeds. The seeds were collected and then sown. Rows of polima steriles were alternated with broccoli maintainer rows. A maintainer line is understood by those of ordinary skill in the art to denote a line that is used in crosses to produce progeny that maintain the sterility of the male sterile material parent. The maint~i~er plants are fertile but otherwise genetically identical coun~erparts to the CMS
plants with which they are grown, typically in alternating strips, to produce seed from a male-sterile W O 97t09873 PCTrUS95/11497 line. After a period of growth, natural selection (by bees/flies, etc.) was employed to choose the plants exhibiting male sterility, good female fertility and the best flowers morphology, i.e. good petal size. The plants selected were assigned numbers CP0~ 2, etc.
These plants were Brassica okra~a~ cont~ the polima CMS
cytoplasm were male sterile and exhibited good female fertility.
After a period of growth, the CPO numbered plants exhibiting male sterility, good female fertility were cloned and put in a cage with a typical maintainer.
Cage production involves erecting a mesh tent (insect scr~eni~g) or a cage around the plants. This is done to prevent cross pollination by insects with other plants in the field. A beehive or other insect colony is typically put inside with the plants. Siliques that developed were collected and the seeds sown. The resulting plants were Brassica oleracea which cont~i~e~ the polima CMS cytoplasm, were male sterile and exhibited good female fertility.
ProtoPlast Fusion - Different CMS Brassica types can also be prepared by protoplast fusion. The protoplasts can be obtained from a Brassica oleracea plant that contains the CMS polima W O 97/09873 PCT~US95/11497 cytoplasm and is male sterile. For example, B ~sica oleracea plants obtained by the previously described breeding program can be used, such as plants of 89070 through 89111 and plants of the CP0 series. The protoplast fusion may be accomplished by employing polyethylene glycol (PEG) causing agglutination, in the pr~F~n~e of a fusion buffer, i.e. a high pH solution to let the membranes fuse. Such somatic hybridization may be effected under the conditions disclosed by Sundberg et al. (Plant Science 43 (1986) 155), hereby incorporated by reference, for the production of interspecific hybrids or modifications thereof. However, one skilled in the art would recognize that the protoplast fusion can be accomplished in other ways other than using polyethylene glycol (PEG). For example, the protoplasts can be fused by using electric field-induced fusion t~c-hli ques as described by Koop et al. "Electric Field-Induced Fusion and Cell Reconstruction-with Preselected Single Protoplasts and Subprotoplasts of Higher Plants"
in ~le~Lo~oration and Electrofusion in Cell BioloaY, Neuman et al. editors, pgs 355-365 (1989), herewith incorporated by reference. Additionally, protoplast fusion can be accomplished with dextran and polyvinyl alcohol as described by Hauptmann et al., "Carrot x Tobacco Somatic Cell Hybrids Selected by Amino Acid Analog Resistance Complementation", 6th International Protoplast Symposium, Basel, August 12-16, 1983, herewith incorporated by reference.
If protoplast fusion is to be accomplished with polyethylene glycol, the procedure described below can be used.
The protoplast fusion is conveniently effected in a washing solution (W5~), described below, containing an osmoticum e.g. a carbohydrate such as mannitol, sorbitol, glucose, or sucrose and potassium and calcium salts. The pH can range from 5.2 to 10 and is preferably about 5.7. The protoplasts of different origin are mixed and concentrated, conveniently to a final density of 105 and 10~ protoplasm per ml.
15The protoplast mixture should then be left undisturbed for at least 10 minutes to allow the protoplasts to settle at the bottom of the petri dish.
The mixture is then treated with polyethylene glycol tPEG)~ preferably having a molecular weight of lS00 to 6000. In general, good results are obtained when employing an aqueous solution (PFS) comprising 18.8~ by weight of PEG at a volume ratio W5' to PFS of 10:1 to - 1:1. PFS comprises conveniently an osmoticum and a calcium salt. The protoplasts are included in PFS for 15 to 20 minutes depending on the fragility of the cells.
The fusion is accomplished by washing the protoplasts, for example, three times, with washing solution (W5~) cont~in;ng an osmoticum (e.g. glucose) in a ~onc~ntration giving a lower osmolarity than PFS and potassium, sodium and calcium salts. The fusion procedure is carried out at a temperature in the range between 16~ and 20-C, preferably 18-C. The conc~ntration of PEG in the fusion mixture is gradually decreased with each consecutive washing step (see e.g.
Example 6). Each washing step should take at least 5 minutes to allow the protoplasts to adjust slowly to the lower osmolarity of the medium, to avoid bursting of the cells. After the washing steps have been accomplished the fused protoplasts should be in the range of 105 to 106 protoplasts per ml. The obtained fusion products may be regenerated in the presence of non-fused parental protoplasts or after optical selection from the culture.
Such optical selection may be performed by micro-manipulation of the cells, e.g. according to the procedure disclosed by Patnaik et al., Plant Science T~tters 24 (1982) 105, hereby incorporated by reference, for the r~ntl~l isolation and identification of plant heterokaryon.
W O 97/09873 PCTAUS95/11497 _ When employing the selection strategy, the parental protoplasts are for example StA; ne~ with fluorescent dyes, e.g. fluorescein diacetate. When fluorescein diacetate is used with protoplasts of hypocotyl origin it will stain yellow under a W light. Protoplasts from leaves will contain chloroplasts which give red auto-fluorescence under W light.
The obtained fusion products are cultivated in an appropriate culture medium comprising a well-balanced nutrient supply for protoplast growth. The medium contains micro- and macro-elements, vitamins, amino acids and small ~ uilLs of carbohydrates, e.g. various sugars such as glucose. Glucose serves as a carbon source as well as an osmoticum. The culture medium also comprises plant hormones (auxins and cytokine) which are able to regulate cell division and shoot regeneration.
Examples of suitable auxins include naphtyl acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). Examples of suitable cytok;nin~ include benzyl aminopyrine (BAP), zeatin (Zea) and gibberellic acid (GA3). In general NAA and 2,4-D are used in combination with BAP to initiate cell division. The ratio of auxin/cytokinin must then be high, for example greater than 1. Two or three days after the fusion treatment, the medium is largely W O 97/09873 PCTrUS9S/11497 replaced by culture medium (BP) which comprises agarose, by which the fusion products and the parental protoplasts are embedded.
After 14 days, the concentration of auxins is diluted by addition of other culture media containing no or substantially less auxins. Star-shaped micro calli will generally develop after 3 to 4 weeks. Such micro calli are then transferred to a regeneration medium to initiate shoot formation, preferably after adaption in an intermediate regeneration medium to differences in composition and physical properties between the culture medium and the regeneration medium. For shoot formation the ratio auxin/cytokinin in the regeneration medium should conveniently be low, e.g. below l:10. In general it will be preferable to employ the auxin NAA in combination with the cytoki n; n~ Zea and BAP for shoot regeneration. The regeneration media, BR and K3, are relatively poor media compared to the culture medium.
They contain less vitamins, the content of carbon source is lower, they comprise solely sucrose and xylose as carbon source and do not contain amino acids. The regeneration media also have a higher viscosity that the culture medium. The regeneration medium Br is a solid medium and contains the growth regulators 2,4-D, NAA and BAP, with the ratio of auxin to cytokinin being less than 1. Medium K3 contains Zea, GA3 and also silver nitrate to promote the shoot development.
After two weeks regeneration on Br medium, calli of approximately 3 mm in diameter are transferred to K3 regeneration medium con~; n i nq a low sucrose concentration. At this stage shoots will develop within 2 to 3 weeks. The obtained shoots are then rooted on a basic medium, such as B5, without additional hormones.
The nuclear DNA and mitochondrial DNA of the obtained plantlets may then be identified in a manner known per se, e.g. employing suitable restriction endonuclease and ~ ~ing the thus obt~;ne~ DNA digest pattern of the fusion products with that of the parental lines.
B~sica oleracea protoplasts and inactivated protoplasts of a Polima CMS Brassica oleracea plant, employed herein as starting material may be obtained in a manner known per se from the corresponding plant cells. Cell wall-free cells, i.e. protoplasts are obtained from green plant material e.g. leaf material and from white plant material e.g. etiolated seedlings, according to the method disclosed by Glimelius, Ph~sioloqia Plantarum 61 (1984) 38, hereby incorporated by reference, for the regeneration of hypocotyl protoplasts. Where optical selection of the fusion products is int~n~e~, the starting materials will be conveniently selected from a green plant or where they are from white-plant material they will advantageously be stained to facilitate selection. The inactivated protoplasts of a Polima CMS
Brassica o~racea plant are obtained in a manner ~nown per se corresponding to Polima CMS Brassica olcracea plant cells or protoplasts, for example, by irradiation.
The inactivation of the nucleus by irradiation can be effected with the aid of gamma, W or X-rays. Where irradiation is effected with an X-ray source, nucleus inactivation will in general be obtained by applying a dose of e.g. 10 krad. min for 3 to 20 minutes. The appropriate X-ray dosage may for example be established by determi n; ng the i n;~t level of X-ray irradiation killing 100% of the protoplast population: the percentage of dead cells is estimated by counting the number of formed colonies after 10 to 20 days in culture.
As described above, after the fusion, the allogenic cells are regenerated to form Brassica oleracea plants con~; n; ng the CMS cytoplasm. These plants may be subse~uently crossed with other Brassica oleracea plants.
It will be appreciated that the Brassica oleracea plant of this invention may be employed as starting material for the preparation of other Brassica oleracea varieties having W O 97/09873 PCT~US95/11497 Polima CMS by in vitro and/or crossing techniques. Such in vitro and crossing t~hn;ques are known in the art by the skilled breeder.
Solutions employed include:
A. ENZYME SOLUTION (lL) 90 g mannitol 0.0272 g KH,PO4 0.1 g KNO3 0.246 g MgS04 ~ 7H20 0.0008 g KI
0.00025 g CuSO1 ~ SH~O
1.4 g CaCI~ ~ 2H~O
1.1 gMES
6 g Cellulase R10 1 g pH 5.8 Macarozyme ¦B. WASHING SOLUrIONS (W5) (1L) 18.4 g CaCl, ~ 2H~O
In Brassica, cytoplasmic male sterility can be transmitted by crossing. The female (egg) parent contributes the cytoplasm, therefore, crossing to CMS
females produces CMS progeny. The nuclear genes however are heterozygous. Therefore, six to eight generations of "backcrossing" are n~ce~c~ry to produce a CMS line breeding homozygous for nuclear characters. As an alternative, cytoplasmic male sterile lines can also be produced by protoplast fusion. In protoplast fusion, a protoplast from a plant having co~rcially desirable traits is combined with a protoplast of a C~S line. The ~ nuclear material o~ the CMS line is either removed or inactivated prior to fusion so it donates only the cytoplasm. The resulting cytoplasmic hybrid (or cybrid) possesses the CMS cytoplasm and is male sterile. For example, U.S. Patent 5,254,802 discloses B oleracea plants that contain the Ogura CMS cytoplasm. These plants were obtained by protoplast fusion.
Polima CMS cytoplasm has been used to produce CMS
in varieties such as winter-type oil seed rape (Brassica napus) ~See Barsby et al., Plant Science, 53: 243-248 (1987)). However, one significant problem with the expression of cytoplasmic male sterility by the polima CMS cytoplasm is that the polima cytoplasm is influenced by environmental conditions. Fan, Z et al. Can. J.
Plant Sci. 66:221-227 (1985). More specifically, male sterile plants cont~ini ng polima CMS cytoplasm are known to become fertile under high temperatures in the field.
Id. See also Fu, T.D., EncarDia Cruciferea Newsletter 6: 6-7 (1981).
The present invention involves Brassica oleracea plants that contain Polima CMS cytoplasm which remain male sterile at high and low t- ~atures and exhibit good W O 97/09873 PCT~US95/11497 female fertility. The Brassica oleracea plants of the present invention can be produced by traditional bree~
methods. Different Brassica types can then be developed by further crossings or backcrossings or by protoplast fusion.
To obtain the Brassica olcracca plants of this invention by traditional br~A;nq te~-hniques, an interspecific cross was made between Brassica~ cultivar 87110 and Brassica okracea cultivar 87101. The seeds resulting from the cross are collected, planted and regenerated. The resulting plants are Brassica nap~ and contain a haploid set of chromosomes. The ch~. ~mal content of said Brassica napus must be doubled by treating the plants with colchicine.
A second interspecific cross is performed by crossing Brassica napus cultivar 87118 with Brassica olera~a cultivar 87101. The seeds resulting from the cross are collected, planted and regenerated. As in the previous cross, the resulting plants are Brassica napus and contain a haploid set of chromosomes. The plants are treated with colchicine to double their chromosome content.
The Brassica nap~ plants produced as a result of the second interspecific cross are next crossed with a Brassica napYs cultivar 87102, which contains polima CMS cytoplasm and is male sterile. The seeds resulting from the cross are collected, planted and regenerated. The regenerated plants are Brassica nap~ and contained the polima CMS
cytoplasm.
The resulting plants are subsequently crossed with the Brassica nap~ plants produced as a result of the first interspecific cross. The seeds resulting from the cross are collected, planted and regenerated. The regenerated plants are Brassica nap~, contain the Polima CMS cytoplasm and are male sterile.
The resulting plants are then crossed with a normal B~ssica oleracea. AS a result of the cross, siliques are pro~llçe~, collected and examined for seeds. The seeds are collected for embryo ~e c~le, because typically, embryos produced from such interspecific hybridization abort prior to maturation. However, by employing embryo rescue techniques, interspecific hybrid plants can be produced. The resulting Fl plants contain the polima CMS
cytoplasm from the female Brassica napYs~ however; the nuclear DNA content is a combination of the Brassica napus (N=ls) and the Brassica oleracea (Nz9).
The resulting plants are then backcrossed with a Brassica okracea. Siliques are again produced, collected and W O 97/09873 PCTrUS95/11497 examined for seeds. The seeds are collected for embryo rescue. The embryos are then regenerated as in the previous cross. The resulting plants are intermediate for chromosome number and contain the polima CMS
cytoplasm. The nuclear content of the plants is a combination of Brassica napus and Brassica oleracea.
The resulting plants are then backcrossed with Brass~ca okracca. Siliques are again produced, collected and examined for seeds. The seeds are sown. The resulting plants are Brassica o~racea which are male sterile and contain the polima CMS cytoplasm.
Optionally, the male sterile Brassica olcra~a plants may be further crossed or backcrossed to produce different Brassica ~pes. Siliques will again be produced, collected and eY~;ned for seeds. The seeds are sown. The resulting plants are Brassica o~a~a which contain the polima cytoplasm and are male sterile.
Different Brassica types can also be produced by protoplast fusion. A protoplast from a male sterile 20 Brassica okra~a containing the polima CMS cytoplasm and inactivated nuclei is fused with a protoplast of a Brassica having c~: -~cially desirable characteristics. After the fusion, the allogenic cells are regenerated into CMS
Brassica plants. The resulting plants are male sterile and W O 97/09873 PCT~US95/11497 contain the polima cvtopl~m. The regenerated CMS Brassica plants contain the polima cytoplasm and can be employed in crossings with other Brassica types cont~inin~ commercially desirable characteristics.
DESCRIPTION OF THE FIGURES
Figure I shows the different hvbridization patterns of BGL I digested DNA with probe E~ in an endonuclease digest cont~ining nuclear DNA of a polima CMS donor, Brassica oleracea 904005-1, arld Brassica oleracea breeding lines used as acceptors for the CMS trait. Lane 1 shows Phage 1 Hind III
digested DNA Fra~nents (MW Standards). Lanes 2 and 3 show Polima CMS
0 Brassica oleracea 904005-1 DNA. Lanes 4 and 5 show Brassica oleracea acceptor Line K14. Lanes 6 and 7 show Brassica oleracea acceptor Line DE70.
Figure 2 shows the different hybridization patterns of BGLII digested DNA with probe CMS BRAS 4 in an en~on-l~lease digest Cn~ inin mitochondrial DNA of a polima CMS plant and mitochondrial DNA from an acceptor breeding line plant. Lanes 8 and 18 show Phage A Hind III digested DNA ~ ont~. Lanes I to 7 show Polima CMS Brassica oleracea 904005-1 DNA. Lanes 9 to 17 show Brassica oleracea acceptor Line K14. Lanes 19 to 24 show Brassica oleracea acceptor Line DE70.
DESCRIPTION OF THE INVENTION
Cytoplasmic male sterile Brassica napus plants Cont~inin~ polima cytoplasm are lcnown to ;lea-k- . Le~kinesc refers to the fact that the plants tend to lose their sterilitv under certain temperature conditions. For example, RECTIFIED SHEET (RULE 91) W O 97/09873 PCT~US9S/11497 studies have shown that CMS Brassicanap~s contA;~i~g polima cytoplasm plants are only partially sterile at high temperatures. See Fan, Z et al., Can. J. Plant Sci. 66:
221-227 (1985).
The present invention involves CMS Brassica oleracea plants that contain the polima cytoplasm. The plants are male sterile at high and low t~ reratures and exhibit good female ~ertility. Different Brassica C~S types can then be developed by employing further crossings and backcrossings or protoplast fusion.
The Breedinq Pro~am Traditional br~ g t~hn i ques were used to develop the Brassica o~racea plants of the present invention.
The inventors herewith describe the breeding program used to create the Brassica oleracca plants of this invention.
The breeding program was undertaken by making an interspecific cross between a Brassica ~ (=Brassica rapa) entry 87110 (n=10) and a Br~ssica olcracea entry 87101 (N-l9).
Entry 87110 was obtained from the Centre for Plant Research (CPR0), PØ Box 16, 6700 AA, Wageningen, The Netherlands as entry IVT 86007 paar 3MS and was genetically male sterile. 87110 has been deposited under the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852. 87110 was deposited on August 23, 1995 and received ATCC A~c~ccion Number 97246. Entry 87101 is available from the Crucifer Genetics Cooperative (CrGC) at the Department of Plant Pathology, 1630 Tlnflen Drive, University of Wisconsin, ~A~;Con~ Wisconsin as CrGC #3-3 (Ccc) and is a rapid cycling Br~sica ol~acea.
After the crossing, siliques (seeds) developed. The siliques are collected, examined for seeds and planted.
The resulting plants were haploid Br~s~ca napw ac (Nsl9) and were su~jected to colchicine treatment to double chromosomes to the diploid number aacc (2n=38). The resulting plants were "artificial" Br~sica nap~ with an aacc genome. These were labelled 87116.
A c~co~ Br~sica~ , 87118, having the same characteristics as 87110, was also crossed with 87101.
87118 was received from the CPR0 as IVT 86017 paar 3MS.
87118 has been deposited under the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852. 87118 was deposited on August 23, 1995 and received ATCC ~Cc~ccion Number 97247. As in the previous cross, the seeds were collected and planted. The resulting plants were subjected to colchicine treatment to double the W O 97/09873 PCT~US95/11497 chromosomes. The resulting plants were an "artificial"
Brassica napus with an aacc genome. The resulting plants were labeled 87119.
Plants of 87119 were then crossed with a male sterile Brassica napus (aaccj containing the polima CMS
cytoplasm (labeled 87102). 87102 is available from the Crucifer Genetics Cooperative as CrGC #5-4 (AClaacc) which is a rapid cycling Brassica nap~ with "Polima" CMS
cytoplasm. Siliques were produced, collected and examined for seeds. The seeds were planted and the resulting plants labeled 88102. These plants were Brassica napus, had an aacc genome and contained the polima CMS
cytoplasm.
A cross was then made between 87116 and 88102.
Siliques were produced, collected and ~Y~;ned for seeds. The seeds were planted. The resulting plants were Brassica napus (aacc) and were labeled 8812S. Brassica napus (aacc) is an amphidiploid species between Brassica oleracea N=9 (cc genome) and Brassica rapa N=10 (aa genome). 88125 are Brassica napus (aacc) and contains polima CMS cytoplasm and are male sterile.
An interspecific cross was made between 88125 (aacc) and a normal male fertile broccoli plant, Brassica o~acea (cc). One skilled in the art would recongize that WO 97/09873 PCT~US95/11497 any male fertile Br~sica oleracea plant could be used for this cross. Siliques developed. The siliques were then collected and ~Am; n~ for seeds. If seeds were present, then the embryo(s) is/were rescued. The embryo(s) is/were rescued because typically, embryos produced from such an interspecific hybridization abort prior to maturation. However, by employing embryo rescue, interspecific hybrids can be pro~llc~.
Embryo rescue involves removing the embryo from the sili~ue. It is preferred that the embryo rescue be conducted when the embryo is as large as possible.
Generally, 18-19 days after the cross is a good time to conduct embryo rescue. However, one skilled in the art would r~co~n;ze that the embryo rescue could take place earlier or later, so long as the seed coat has not yet hardened.
In embryo rescue, the siliques are first disinfected for about 20 minutes in a 10% hypochlorate solution (such as sodium or potassium hypochlorate) and rinsed three times in sterile water at 2.5 and lO
minutes.
Next, the siliques are cut in a longitll~;nAl direction. Young seeds are removed and embryos taken out of the seed under a microscope. The embryos are then placed on a suitable medium to facilitate growth, such as a BLKC medium which is described in Example 11.
However, any medium that facilitates growth can be used.
The embryos are grown at 20-C in 16 hours of 4500 LUX
light for at least two weeks. After the roots develop, the seedlings are placed into soil as soon as possible.
The resulting Fl plants were assigned the numbers 88132-1 through 88132-84. These plants cont~;n~ the polima CMS cytoplasm from the female Brassica napus plant, however: the nuclear DNA content was a combination of the Br~sica napus (N=19) and the Brassica oleracea (N=9) .
Essentially, the plants had mixed genomes. Because of their mixed nature, the plants were male sterile and partially female sterile. The plants were male sterile because they contained the polima CMS cytoplasm from the female (egg) parent, Brassica napus, 88125.
Phenotypical selection was used to select the plants from 88132-1 through 88132-84 that have near normal DNA content (aacc or cc). Phenotypic selection allows the breeder to select plants most similar to Brassica olera~a (cc genome). Plants with an abnormal DNA
content are visually different than plants with a normal DNA content. The plants selected were backcrossed (BCl) with a normal broccoli or cauliflower, Brassicn oleracea (cc).
However, one skilled in the art would recognize that any wos7/os873 PCT~S95111497 Brassica ol~acea could be used. After the cross, em~ryo rescue was again employed. The plants that developed were assigned the numbers 89015 through 89040 and 89046 through 89056. These plants were intermediate for chromosome number and cont~;ne~ the polima CMS
cytoplasm. Again, the nuclear DNA content was a combination of the Brassica ~us and the Brassica oleracea. The plants were male sterile (polima CMS) and partially female sterile (chL~ _ome number).
Phenotypical selection was employed to select plants from 89015 through 89040 and 89046 through 890~6 having near normal DNA content. The plants selected were backcrossed (BC2) with a normal broccoli or cauliflower, Brassica oleracea (cc). However, one skilled in the art would recognize that any Brassica oleracea could be used. Siliques again developed, however, some seeds did not require embryo rescue. These seeds were collected and planted. The plants resulting from this cross were assigned the numbers 89070 through 89141. Most of these plants had the normal Brassica oleracea set of - chromosomes (2N=18), contained the polima cytoplasm and were male sterile. The flowers of these plants had reduced anthers and petals. The plants remained sterile W O 97/09873 PCTrUS95/11497 at high and low temperatures and exhibited normal female fertility.
These resulting plants can be crossed with other Br~sica types such as broccoli, cauliflower, cabbage (red, S white, savoy), brussels sy,ouLs, kohlrabi, kale, etc. to produce CMS lines. For example, different Br~sica types can be produced by backcrossing to the CMS "polima"
plants or by protoplast fusion.
Subsequent Crosses and Selection Process for M~le Steril;tv Female FertilitY and Flower MorpholoqY
Plants of 89015 through 89640 and 89046 through 89056 and 89070 through 89141 were subsequently backcrossed with a normal broccoli and/or cauliflower and planted in the field. Siliques developed, were collected and ~A~; ne~ for seeds. The seeds were collected and then sown. Rows of polima steriles were alternated with broccoli maintainer rows. A maintainer line is understood by those of ordinary skill in the art to denote a line that is used in crosses to produce progeny that maintain the sterility of the male sterile material parent. The maint~i~er plants are fertile but otherwise genetically identical coun~erparts to the CMS
plants with which they are grown, typically in alternating strips, to produce seed from a male-sterile W O 97t09873 PCTrUS95/11497 line. After a period of growth, natural selection (by bees/flies, etc.) was employed to choose the plants exhibiting male sterility, good female fertility and the best flowers morphology, i.e. good petal size. The plants selected were assigned numbers CP0~ 2, etc.
These plants were Brassica okra~a~ cont~ the polima CMS
cytoplasm were male sterile and exhibited good female fertility.
After a period of growth, the CPO numbered plants exhibiting male sterility, good female fertility were cloned and put in a cage with a typical maintainer.
Cage production involves erecting a mesh tent (insect scr~eni~g) or a cage around the plants. This is done to prevent cross pollination by insects with other plants in the field. A beehive or other insect colony is typically put inside with the plants. Siliques that developed were collected and the seeds sown. The resulting plants were Brassica oleracea which cont~i~e~ the polima CMS cytoplasm, were male sterile and exhibited good female fertility.
ProtoPlast Fusion - Different CMS Brassica types can also be prepared by protoplast fusion. The protoplasts can be obtained from a Brassica oleracea plant that contains the CMS polima W O 97/09873 PCT~US95/11497 cytoplasm and is male sterile. For example, B ~sica oleracea plants obtained by the previously described breeding program can be used, such as plants of 89070 through 89111 and plants of the CP0 series. The protoplast fusion may be accomplished by employing polyethylene glycol (PEG) causing agglutination, in the pr~F~n~e of a fusion buffer, i.e. a high pH solution to let the membranes fuse. Such somatic hybridization may be effected under the conditions disclosed by Sundberg et al. (Plant Science 43 (1986) 155), hereby incorporated by reference, for the production of interspecific hybrids or modifications thereof. However, one skilled in the art would recognize that the protoplast fusion can be accomplished in other ways other than using polyethylene glycol (PEG). For example, the protoplasts can be fused by using electric field-induced fusion t~c-hli ques as described by Koop et al. "Electric Field-Induced Fusion and Cell Reconstruction-with Preselected Single Protoplasts and Subprotoplasts of Higher Plants"
in ~le~Lo~oration and Electrofusion in Cell BioloaY, Neuman et al. editors, pgs 355-365 (1989), herewith incorporated by reference. Additionally, protoplast fusion can be accomplished with dextran and polyvinyl alcohol as described by Hauptmann et al., "Carrot x Tobacco Somatic Cell Hybrids Selected by Amino Acid Analog Resistance Complementation", 6th International Protoplast Symposium, Basel, August 12-16, 1983, herewith incorporated by reference.
If protoplast fusion is to be accomplished with polyethylene glycol, the procedure described below can be used.
The protoplast fusion is conveniently effected in a washing solution (W5~), described below, containing an osmoticum e.g. a carbohydrate such as mannitol, sorbitol, glucose, or sucrose and potassium and calcium salts. The pH can range from 5.2 to 10 and is preferably about 5.7. The protoplasts of different origin are mixed and concentrated, conveniently to a final density of 105 and 10~ protoplasm per ml.
15The protoplast mixture should then be left undisturbed for at least 10 minutes to allow the protoplasts to settle at the bottom of the petri dish.
The mixture is then treated with polyethylene glycol tPEG)~ preferably having a molecular weight of lS00 to 6000. In general, good results are obtained when employing an aqueous solution (PFS) comprising 18.8~ by weight of PEG at a volume ratio W5' to PFS of 10:1 to - 1:1. PFS comprises conveniently an osmoticum and a calcium salt. The protoplasts are included in PFS for 15 to 20 minutes depending on the fragility of the cells.
The fusion is accomplished by washing the protoplasts, for example, three times, with washing solution (W5~) cont~in;ng an osmoticum (e.g. glucose) in a ~onc~ntration giving a lower osmolarity than PFS and potassium, sodium and calcium salts. The fusion procedure is carried out at a temperature in the range between 16~ and 20-C, preferably 18-C. The conc~ntration of PEG in the fusion mixture is gradually decreased with each consecutive washing step (see e.g.
Example 6). Each washing step should take at least 5 minutes to allow the protoplasts to adjust slowly to the lower osmolarity of the medium, to avoid bursting of the cells. After the washing steps have been accomplished the fused protoplasts should be in the range of 105 to 106 protoplasts per ml. The obtained fusion products may be regenerated in the presence of non-fused parental protoplasts or after optical selection from the culture.
Such optical selection may be performed by micro-manipulation of the cells, e.g. according to the procedure disclosed by Patnaik et al., Plant Science T~tters 24 (1982) 105, hereby incorporated by reference, for the r~ntl~l isolation and identification of plant heterokaryon.
W O 97/09873 PCTAUS95/11497 _ When employing the selection strategy, the parental protoplasts are for example StA; ne~ with fluorescent dyes, e.g. fluorescein diacetate. When fluorescein diacetate is used with protoplasts of hypocotyl origin it will stain yellow under a W light. Protoplasts from leaves will contain chloroplasts which give red auto-fluorescence under W light.
The obtained fusion products are cultivated in an appropriate culture medium comprising a well-balanced nutrient supply for protoplast growth. The medium contains micro- and macro-elements, vitamins, amino acids and small ~ uilLs of carbohydrates, e.g. various sugars such as glucose. Glucose serves as a carbon source as well as an osmoticum. The culture medium also comprises plant hormones (auxins and cytokine) which are able to regulate cell division and shoot regeneration.
Examples of suitable auxins include naphtyl acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). Examples of suitable cytok;nin~ include benzyl aminopyrine (BAP), zeatin (Zea) and gibberellic acid (GA3). In general NAA and 2,4-D are used in combination with BAP to initiate cell division. The ratio of auxin/cytokinin must then be high, for example greater than 1. Two or three days after the fusion treatment, the medium is largely W O 97/09873 PCTrUS9S/11497 replaced by culture medium (BP) which comprises agarose, by which the fusion products and the parental protoplasts are embedded.
After 14 days, the concentration of auxins is diluted by addition of other culture media containing no or substantially less auxins. Star-shaped micro calli will generally develop after 3 to 4 weeks. Such micro calli are then transferred to a regeneration medium to initiate shoot formation, preferably after adaption in an intermediate regeneration medium to differences in composition and physical properties between the culture medium and the regeneration medium. For shoot formation the ratio auxin/cytokinin in the regeneration medium should conveniently be low, e.g. below l:10. In general it will be preferable to employ the auxin NAA in combination with the cytoki n; n~ Zea and BAP for shoot regeneration. The regeneration media, BR and K3, are relatively poor media compared to the culture medium.
They contain less vitamins, the content of carbon source is lower, they comprise solely sucrose and xylose as carbon source and do not contain amino acids. The regeneration media also have a higher viscosity that the culture medium. The regeneration medium Br is a solid medium and contains the growth regulators 2,4-D, NAA and BAP, with the ratio of auxin to cytokinin being less than 1. Medium K3 contains Zea, GA3 and also silver nitrate to promote the shoot development.
After two weeks regeneration on Br medium, calli of approximately 3 mm in diameter are transferred to K3 regeneration medium con~; n i nq a low sucrose concentration. At this stage shoots will develop within 2 to 3 weeks. The obtained shoots are then rooted on a basic medium, such as B5, without additional hormones.
The nuclear DNA and mitochondrial DNA of the obtained plantlets may then be identified in a manner known per se, e.g. employing suitable restriction endonuclease and ~ ~ing the thus obt~;ne~ DNA digest pattern of the fusion products with that of the parental lines.
B~sica oleracea protoplasts and inactivated protoplasts of a Polima CMS Brassica oleracea plant, employed herein as starting material may be obtained in a manner known per se from the corresponding plant cells. Cell wall-free cells, i.e. protoplasts are obtained from green plant material e.g. leaf material and from white plant material e.g. etiolated seedlings, according to the method disclosed by Glimelius, Ph~sioloqia Plantarum 61 (1984) 38, hereby incorporated by reference, for the regeneration of hypocotyl protoplasts. Where optical selection of the fusion products is int~n~e~, the starting materials will be conveniently selected from a green plant or where they are from white-plant material they will advantageously be stained to facilitate selection. The inactivated protoplasts of a Polima CMS
Brassica o~racea plant are obtained in a manner ~nown per se corresponding to Polima CMS Brassica olcracea plant cells or protoplasts, for example, by irradiation.
The inactivation of the nucleus by irradiation can be effected with the aid of gamma, W or X-rays. Where irradiation is effected with an X-ray source, nucleus inactivation will in general be obtained by applying a dose of e.g. 10 krad. min for 3 to 20 minutes. The appropriate X-ray dosage may for example be established by determi n; ng the i n;~t level of X-ray irradiation killing 100% of the protoplast population: the percentage of dead cells is estimated by counting the number of formed colonies after 10 to 20 days in culture.
As described above, after the fusion, the allogenic cells are regenerated to form Brassica oleracea plants con~; n; ng the CMS cytoplasm. These plants may be subse~uently crossed with other Brassica oleracea plants.
It will be appreciated that the Brassica oleracea plant of this invention may be employed as starting material for the preparation of other Brassica oleracea varieties having W O 97/09873 PCT~US95/11497 Polima CMS by in vitro and/or crossing techniques. Such in vitro and crossing t~hn;ques are known in the art by the skilled breeder.
Solutions employed include:
A. ENZYME SOLUTION (lL) 90 g mannitol 0.0272 g KH,PO4 0.1 g KNO3 0.246 g MgS04 ~ 7H20 0.0008 g KI
0.00025 g CuSO1 ~ SH~O
1.4 g CaCI~ ~ 2H~O
1.1 gMES
6 g Cellulase R10 1 g pH 5.8 Macarozyme ¦B. WASHING SOLUrIONS (W5) (1L) 18.4 g CaCl, ~ 2H~O
4.91 gNaCl 0.372 g KCl 0.901 g glucose pH = 5.8 C. WASHING SOLUTION (W5') (lL) 18.4 g CaCl, ~ 2H,O
4.91 g NaC 1 0.372 g KCl 0.901 g glucose 9.76 g MES
pH = 5.8 SUBSTITUTE SH~Er (RULE 26) WO 97/09873 PCI~/US95/11497 D. CPW 16S (lL) 160 g sucrose 0.0272 g K H.PO4 0.1 g K~O3 1.45 g CaCl, ~ 2H2O
0.246 g MgS04 ~ 2H2O
0.0008 g KI
0.000025 g CusO4- 5H2O
pH = 5.5-5.8 E. PEG fi~sion solution (PFS) 18.8% PEG (MW 4000) 0.06 M CaCl2 ~ 2H2O
0.1 M m~nnitol 0.025 M ~lueine 10% (v/v) DMSO
F. SPA SOLUTION (lL) 20 g SeaPlaque agarose 100 g Sucrose TABLE 1: COMPOSITION OF THE MS MEDL~ (MG/L) 8P MAC Br K 3 B 5 MS
KNO. 956 956 1556 1556 3000 1900 MgS04 ~ 2H.O 300 300 250 250 250 370 KH.PO4 164 164 -- -- -- 170 CaCI. ~ 2H~O 600 600 300 300 150 440 RECTIFIED SHEET(RULE 91) W O 97/09873 PCTrUS9~/11497 _ (NH4)2SO4 134 134 134 K 0.75 0.75 0.75 0.75 0.75 0.83 ~nSO4- 10 10 10 10 10 22.3 4H.O
H,BO3 3 3 3 3 3 6.2 znso4- 2 2 2 2 2 8.6 NaMoO4- 0.25 0.25 0.25 0.25 0.25 0.25 2H,O
0 C4S04- 0.025 0.025 0.025 0.025 0.025 0.025 4H~O
CoC l, ~ 0.025 0.025 0.025 0.025 0.025 0.025 6H,O
Fe-EDTA 40 40 40 40 40 43 Thi~mine- 10 10 10 10 10 0.1 HCL
Pyridoxine- 1 1 1 1 1 0.5 HCL
Nicotinic 1 1 1 1 1 0.5 acid Sodium 20 20 -- -- -- --pyruvate Citric Acid 40 40 -- -- -- --Maleic acid 40 40 -- -- -- --Furnaric 40 40 -- -- -- --acid Glycine -- -- -- -- -- 2 Xylose -- -- 250 -- -- --Inositol 100 -- -- -- -- 100 Sucrose 50,000 80.000 40.000 10.000 20~000 **
Glucose 40,000 -- -- -- -- --SUBSTI~UTE ~HEET IRULE 26) W O 97/09873 PCT~US95/11497 Casein 250250 -- -- -- --hydrolysate S Agarose -- -- 16,000 -- -- --Agar -- -- -- 8,000 8,000 8,000 NAA 0.1 0.1 0.1 -- -- --2,4-D 1 0.2 0.1 -- -- --Zeatin -- -- -- 0.26 -- --BAP 0.5 0.5 0.5 -- -- --GA3 0.006 -- --AgNO3 -- -- -- 5 ~~ ~~
* * see examples By way of example, and not limit~tion, examples of the present invention will now be given.
SUBSTITIJTE SHEET ~RULE 26) W 097/09873 PCT~US95/11497 Example 1: Seed sterilization and germination Seeds of Brassica oleracea, broccoli with Polima CMS (hereinaflcer ~$iPn~te~l B. oleracea, 904005-1) are dipped for approximately 10 seconds in 70% alcohol and stPrili7~ in a 1.5% sodium hypochlorite solution for two times for 10 minlltes at 20~C. An~,.~ds ~h-~i~e rinsing with sterile distilled water is required. The seeds are placed on the MS ml~iPnt meAillm (see Table I ) with 3% sucrose and without hormones. To obtain green sterile plants, the seeds are grown in glass jars in the light (8000 lux), for a 16 hour photoperiod with temperatures of 25~C day and 20~C night. Sterile shoots are subcultured under 10 the same conditions in plastic containers. To obtain white tissue for protoplast isolation, e.g. hypocotyls, the seeds are grown in petri dishes in the dark at 25~C.
Example 1 a: Crosses leading to 904005-1 904005-1 is the seed harvested from plant number 1 in entry number 904005.
904005 was a backcross of CMS Polima 892731-2 and a normal fertile broccoli.
SUBSTITUTE SHEEI (RULE 26) is the seed harvested from plant selection 2 in entry 892731. This was backcross of CMS Polima 89094-2 and a normal fertile broccoli.
89094-2 is the seed harvested from plant selection 2 in entry 89094. This was a backcross of CMS Polima 89022 and normal fertile broccoli.
89022-4 is the seed harvested from plant 4 in entry 89022.
Example 2 Analogous to the procedure of Example 1, seeds of Br~sica olerao~, culti~ar DE70, are sterilized and germinated. Cultivar DE70 is a fully fertile inbred cauliflower line that is used as a parental line.
However, any typical Brassica okra~a plant can be used for the protoplast fusion.
Example 3: Isolation of protoplasts Leaves of four week old shoots of plant material according to Example 1 are cut into small pieces and incubated in an enzyme solution for 16 hours at 25-C on a gyratory .ch~ke~ at 40 rpm. The suspension is filtered through a nylon mesh (40 ~m) and washed with two-thirds - of a volume of CPW 16S solution by centrifugation at 817 rpm for 5 minutes. This results in flotation of the intact protoplasts. The protoplasts are collected and W O 97/09873 PCTrUS95/11497 rinsed twice with W5 solution by centrifugation at 708 rpm for 5 minutes. The protoplasts are diluted to a density of 1 x 105 protoplasts per ml W5 solution before being used for fusion experiments.
Example 4 Six to eight day old hypocotyls of the plant material according to Example 2 are isolated according to the process of Example 3, except that during the enzyme treatment 3 ~g/ml of fluoresceinediacetate is added. Stained protoplasts for hand selection and for determination of fusion frequency are obtained.
Example 5: Irradiation of protoplast Freshly isolated protoplasts according to Example 3 are plated in a 6 cm petri dish in W5 solution (2 to 3 ml). The protoplasts are irradiated using an X-ray source (Baltograph CE100), at a dose of 3500 Gy during 20 minutes. After irradiation, the inactivated protoplasts are washed with W5 solution by centrifugation at 708 rpm for 5 minutes. The protoplasts are diluted to a density of 1 x 105 protoplasts per ml W5 solution before being used for fusion experiments.
W O 97/09873 ~CTrUS95/11497 Example 6: Fusion procedure Protoplasts according to Example 4 and 5 re mixed 1:3 in a final concentration of 1 x 105 protoplasts per ml W5 solution. Three droplets of 100 ~1 are placed in an uncoated 6 cm petri dish and the protoplasts are allowed to settle for 5 to 10 minutes. Three hundred microliters of PFS solution is added in the center of the three droplets to induce agglutination for 15 minutes. Thereafter, 300 ~1 of W5 solution is added to the mixture and again after 10 minutes and again after 5 minutes. The W5 medium is replaced by two times concentrated 8P solution and the protoplasts are cultivated for one to three days at 25-C in the dark.
Example 7 The entire fusion mixture according to Example 6 is cultured for one to three days in the dark at 25-C. The cells are collected by centrifugation at 548 for 5 minutes and diluted to a density of 1 x 105 protoplasts per ml two times concentrated 8P medium. An equal volume handwarm SPA medium is added and the cells are plated in 5 droplets of 100 ~1 in a coated petri dish.
Also 5 droplets of 100 ~1 with feeder cells are added.
After two weeks, the droplets with feeder cells are removed and 2 ml MAC medium per petri dish is added.
After two weeks the droplets are dispersed on solid Br medium. After two to three weeks the individual colonies are transferred to a petri dish with solid K3 medium. The microcalli are cultured in low light intensity (2500 lux) at 25-C with a photoperiod of 18 hours.
Example 8: Selection and growth of fusion products Fused cells, which can be recognized visually by the presence of double fluorescence, are picked up with a mic~. qnipulator. The hybrid cells are cultured in 100 ~1 agarose droplets (1% SeaPlaque) at a density of 2000 to 50,000 protoplasts per milliliter. The droplets are placed in a liquid nurse culture system (Costar-Transwell col) with feeder cells and incubated at 25-C
in the dark. The droplets are dispersed on solid Br medium after two weeks. Small calli are transferred to solid K3 medium and incubated at 25-C in low light intensity (2500 lux) with a photoperiod of 16 hours.
Example 9: Plant regeneration The calli according to Example 7 and 8, having developed to a size of 2 to 5 mm in diameter, are transferred to fresh K3 medium at normal light intensity (8000 lux) at 25 C with a photoperiod of 16 hours.
Small shoots are transferred to B5 medium with 1%
sucrose without hormones and rooted on the same medium.
.
Example 10: Molecular analysis of the fusion products a) Nuclear DNA composition Characterization of the nuclear DNA
composition of the fusion product is effected by using specific DNA probes. Probe ES, a clone containing a 820 kbp Bgl 1 fragment from cauliflower nuclei DNA
hybridizes with various ~ands in an endonuclease digest pattern of nuclear DNA of the Polima CMS donor, Br~sica ok~acea breeding lines that are used as acceptor for the CMS trait (see Figure 1).
b) Mitochondrial DNA composition Characterization is effected with CMS Bras 4 DNA, a clone cont~; n ing a 1950 bp Bgl II fragment from cauliflower mitochondrial DNA. This clone produced a band of 4 kbp with CMS polima mitochondrial DNA and a band of 6 kbp with mitochondrial DNA from the acceptor br~;~g lines e.g. DE70 (see Figure 2).
Characterization is also effected by PCR amplification of a 500 bp DNA fragment, using primers 94RS01 and 94RS02. The se~uence of the forward primer 94RS01 is 5'-GAA CCA ACT GCT TTC ACA CCG-3' and of the reverse primer 94RS02 5'-CTT GGC TCT CTG CGA ATG TC-3'. This 500 bp fir~ ntis amplified from CMS polirna mitochondrial DNA, but not from mitochondrial DNA of acceptor lines. e.g. DE70.
Example 11: Embryo Rescue Generally, an embrvo is rescued 18-19 days after a cross is made.
S In order to effectuate an embryo rescue. siliques are required. The siliques required are those having as large as possible embryos with a seed coats that have not yet hardened.
Prior tO the removal of the embryo. the siliques must first be disinfected. The siliques are disinfected by placing them in a 10% chloride 10 solution such as sodium or potassium chloride for about twenty minlltes (20 min.). The siliques are rinsed three times in sterile ~vater at 2, 5 and 10 mimltes.
The embryo is rescued by cutting the siliques open in a lonPitudinal direction. removin~ the developin~ seeds and then preparing the 15 embrvo out of the seed under a microscope.
A~er the em~ryo is cut out of the seed, it is placed on a suitable growth m~inm Any m~ m that will f~ciiit~te the growth of the embrvo can be used. For example. an MS medium with 3 & sucrose and 0.4 mg/l thi~min and 0.2 mgll IDA can be used.
RECTIFIED SHEET(RULE 91) W O 97/09873 PCT~US95/11497 Example 12: The mainten~c~ of sterility of CMS
Br~si~ oler~cea plants at high and low temperatures In order to determine if the cytoplasmic male sterile plants cont~;ni~g the polima CMS cytoplasm remained sterile at high and low temperatures, broccoli cultivars 915095, 915107, 915100 and 915144 were hybridized with red cabbage to check if 100% hybridity was achieved. Cultivars 915095-915144 were crossed with red cabbage. When the seeds of these cultivars are pollinated with a normal broccoli maintainer and are sown, the hypocotyl color and leaf color of the seedling is normally green. If these cultivars are crossed with red cabbage then the hybrid seeds are inte~ e~;~te and the seedlings show a strong authocyan (purple3 expression, while inbreds remain green. Seeds of 915095, 915107, 915100, and 915144 were sown and 20 plants of each cultivar were put together with red cabbage plants in a plastic covered cage outside. In the summer, the temperature of the cage varied from 15-40-C during the day. The temperatures in the cage were recorded once a week. A sample of the seed harvested from 915095, 915107, 915100 and 915144 were sown and all resulting plants were hybrid indicating no presence of selfing and no leaking of the polima CMS cytoplasm. At W097/09873 PCT~S95/11497 no time did any of the plants resuit in partial male sterility.
~he crosses which lead to 915095, 915107, 915100 and 915144 are shown below.
88125 x broccoli (=bc (broccoli pollinator line)) 88132 x bc 89022 x bc 89036 x bc 89046 x bc 89094 x bc 89102 x bc 89083 x bc 39133 x bc 892731 x bc 892733 x bc 892736 x bc 892737 x bc 904005 x bc 904006 x bc 904008 x bc 904045 x bc 915095 9151~0 915107 91~144 When two or more arrows go from one cross (i.e.
88132 x bc), then each arrow represents a cross with a di f f erent broccoli genotype.
ZO
Example 13: Female Fertility Listed below is the seed yield data obtained after crossing by hand CMS "Polima" types with broccoli. The data shows that the seed yield of CMS "polima" is higher than those of broccoli inbreds. This is because W O 97/09873 PCT~US95/11497 replicated inbreeding affects the fertility. A better comparison is made between the broccoli Fl's and the CMS
Ogura crosses. The results show that the seed yield of the CMS polima is in the same range as the other two types of crosses.
Broccoli 8eed Yield~ 1991-1994 (Hand pollination) Year Type of CrossNumber of Seeds ~er Cross 1991 (CMS Polima x Broccoli)F1114 (n=4) (Broccoli inbred x Broccoli)Fl 64 (ns23) (Broccoli F1 x Broccoli)F1116 (n~45) 1992 (CMS Polima x Broccoli)Fl146 (ns66) (Broccoli inbred x Broccoli)Fl 100 (n=76) (Broccoli Fl x Broccoli)Fl135 (n=28) (CMS Ogura x 8roccoli)F1140 (n=32) 1993 (CMS Polima x Broccoli)F179 (n=41) (Broccoli in~red x Broccoli)Fl 53 (n=70) (Broccoli Fl x Broccoli)Fl80 (n=35) (CMS ogura x Broccoli)Fl144 (n=5) 1994 (CMS Polima x Broccoli) Fl93 (n=9) (Broccoli inbred x Broccoli)Fl 47 (n=50) (Broccoli Fl x Broccoli)Fl84 (n=35) (CMS ogura x Broccoli)Fl74 (n=9) Weighted means (CNS Polima x Broccoli)Fl115 (n=120) (Broccoli inbred x Broccoli)F1 60 (n=227) (Broccoli F1 x Broccoli)F1103 (n=143) (CMS Ogura x Broccoli)Fl(127)((n=40)) Although the invention has been described primarily in connection with the special and preferred W O 97/09873 PCTrUS95/11497 embodiments, it will be understood that it is capable of modification without departing from the scope of the invention. The following claims are intended to cover all variations, uses or adaptions of the invention, following, in general, the principles thereof and including such departures from the presented disclosure as come within known or customary practice in the field to which the invention pertains, or as are obvious to persons skilled in the field.
4.91 g NaC 1 0.372 g KCl 0.901 g glucose 9.76 g MES
pH = 5.8 SUBSTITUTE SH~Er (RULE 26) WO 97/09873 PCI~/US95/11497 D. CPW 16S (lL) 160 g sucrose 0.0272 g K H.PO4 0.1 g K~O3 1.45 g CaCl, ~ 2H2O
0.246 g MgS04 ~ 2H2O
0.0008 g KI
0.000025 g CusO4- 5H2O
pH = 5.5-5.8 E. PEG fi~sion solution (PFS) 18.8% PEG (MW 4000) 0.06 M CaCl2 ~ 2H2O
0.1 M m~nnitol 0.025 M ~lueine 10% (v/v) DMSO
F. SPA SOLUTION (lL) 20 g SeaPlaque agarose 100 g Sucrose TABLE 1: COMPOSITION OF THE MS MEDL~ (MG/L) 8P MAC Br K 3 B 5 MS
KNO. 956 956 1556 1556 3000 1900 MgS04 ~ 2H.O 300 300 250 250 250 370 KH.PO4 164 164 -- -- -- 170 CaCI. ~ 2H~O 600 600 300 300 150 440 RECTIFIED SHEET(RULE 91) W O 97/09873 PCTrUS9~/11497 _ (NH4)2SO4 134 134 134 K 0.75 0.75 0.75 0.75 0.75 0.83 ~nSO4- 10 10 10 10 10 22.3 4H.O
H,BO3 3 3 3 3 3 6.2 znso4- 2 2 2 2 2 8.6 NaMoO4- 0.25 0.25 0.25 0.25 0.25 0.25 2H,O
0 C4S04- 0.025 0.025 0.025 0.025 0.025 0.025 4H~O
CoC l, ~ 0.025 0.025 0.025 0.025 0.025 0.025 6H,O
Fe-EDTA 40 40 40 40 40 43 Thi~mine- 10 10 10 10 10 0.1 HCL
Pyridoxine- 1 1 1 1 1 0.5 HCL
Nicotinic 1 1 1 1 1 0.5 acid Sodium 20 20 -- -- -- --pyruvate Citric Acid 40 40 -- -- -- --Maleic acid 40 40 -- -- -- --Furnaric 40 40 -- -- -- --acid Glycine -- -- -- -- -- 2 Xylose -- -- 250 -- -- --Inositol 100 -- -- -- -- 100 Sucrose 50,000 80.000 40.000 10.000 20~000 **
Glucose 40,000 -- -- -- -- --SUBSTI~UTE ~HEET IRULE 26) W O 97/09873 PCT~US95/11497 Casein 250250 -- -- -- --hydrolysate S Agarose -- -- 16,000 -- -- --Agar -- -- -- 8,000 8,000 8,000 NAA 0.1 0.1 0.1 -- -- --2,4-D 1 0.2 0.1 -- -- --Zeatin -- -- -- 0.26 -- --BAP 0.5 0.5 0.5 -- -- --GA3 0.006 -- --AgNO3 -- -- -- 5 ~~ ~~
* * see examples By way of example, and not limit~tion, examples of the present invention will now be given.
SUBSTITIJTE SHEET ~RULE 26) W 097/09873 PCT~US95/11497 Example 1: Seed sterilization and germination Seeds of Brassica oleracea, broccoli with Polima CMS (hereinaflcer ~$iPn~te~l B. oleracea, 904005-1) are dipped for approximately 10 seconds in 70% alcohol and stPrili7~ in a 1.5% sodium hypochlorite solution for two times for 10 minlltes at 20~C. An~,.~ds ~h-~i~e rinsing with sterile distilled water is required. The seeds are placed on the MS ml~iPnt meAillm (see Table I ) with 3% sucrose and without hormones. To obtain green sterile plants, the seeds are grown in glass jars in the light (8000 lux), for a 16 hour photoperiod with temperatures of 25~C day and 20~C night. Sterile shoots are subcultured under 10 the same conditions in plastic containers. To obtain white tissue for protoplast isolation, e.g. hypocotyls, the seeds are grown in petri dishes in the dark at 25~C.
Example 1 a: Crosses leading to 904005-1 904005-1 is the seed harvested from plant number 1 in entry number 904005.
904005 was a backcross of CMS Polima 892731-2 and a normal fertile broccoli.
SUBSTITUTE SHEEI (RULE 26) is the seed harvested from plant selection 2 in entry 892731. This was backcross of CMS Polima 89094-2 and a normal fertile broccoli.
89094-2 is the seed harvested from plant selection 2 in entry 89094. This was a backcross of CMS Polima 89022 and normal fertile broccoli.
89022-4 is the seed harvested from plant 4 in entry 89022.
Example 2 Analogous to the procedure of Example 1, seeds of Br~sica olerao~, culti~ar DE70, are sterilized and germinated. Cultivar DE70 is a fully fertile inbred cauliflower line that is used as a parental line.
However, any typical Brassica okra~a plant can be used for the protoplast fusion.
Example 3: Isolation of protoplasts Leaves of four week old shoots of plant material according to Example 1 are cut into small pieces and incubated in an enzyme solution for 16 hours at 25-C on a gyratory .ch~ke~ at 40 rpm. The suspension is filtered through a nylon mesh (40 ~m) and washed with two-thirds - of a volume of CPW 16S solution by centrifugation at 817 rpm for 5 minutes. This results in flotation of the intact protoplasts. The protoplasts are collected and W O 97/09873 PCTrUS95/11497 rinsed twice with W5 solution by centrifugation at 708 rpm for 5 minutes. The protoplasts are diluted to a density of 1 x 105 protoplasts per ml W5 solution before being used for fusion experiments.
Example 4 Six to eight day old hypocotyls of the plant material according to Example 2 are isolated according to the process of Example 3, except that during the enzyme treatment 3 ~g/ml of fluoresceinediacetate is added. Stained protoplasts for hand selection and for determination of fusion frequency are obtained.
Example 5: Irradiation of protoplast Freshly isolated protoplasts according to Example 3 are plated in a 6 cm petri dish in W5 solution (2 to 3 ml). The protoplasts are irradiated using an X-ray source (Baltograph CE100), at a dose of 3500 Gy during 20 minutes. After irradiation, the inactivated protoplasts are washed with W5 solution by centrifugation at 708 rpm for 5 minutes. The protoplasts are diluted to a density of 1 x 105 protoplasts per ml W5 solution before being used for fusion experiments.
W O 97/09873 ~CTrUS95/11497 Example 6: Fusion procedure Protoplasts according to Example 4 and 5 re mixed 1:3 in a final concentration of 1 x 105 protoplasts per ml W5 solution. Three droplets of 100 ~1 are placed in an uncoated 6 cm petri dish and the protoplasts are allowed to settle for 5 to 10 minutes. Three hundred microliters of PFS solution is added in the center of the three droplets to induce agglutination for 15 minutes. Thereafter, 300 ~1 of W5 solution is added to the mixture and again after 10 minutes and again after 5 minutes. The W5 medium is replaced by two times concentrated 8P solution and the protoplasts are cultivated for one to three days at 25-C in the dark.
Example 7 The entire fusion mixture according to Example 6 is cultured for one to three days in the dark at 25-C. The cells are collected by centrifugation at 548 for 5 minutes and diluted to a density of 1 x 105 protoplasts per ml two times concentrated 8P medium. An equal volume handwarm SPA medium is added and the cells are plated in 5 droplets of 100 ~1 in a coated petri dish.
Also 5 droplets of 100 ~1 with feeder cells are added.
After two weeks, the droplets with feeder cells are removed and 2 ml MAC medium per petri dish is added.
After two weeks the droplets are dispersed on solid Br medium. After two to three weeks the individual colonies are transferred to a petri dish with solid K3 medium. The microcalli are cultured in low light intensity (2500 lux) at 25-C with a photoperiod of 18 hours.
Example 8: Selection and growth of fusion products Fused cells, which can be recognized visually by the presence of double fluorescence, are picked up with a mic~. qnipulator. The hybrid cells are cultured in 100 ~1 agarose droplets (1% SeaPlaque) at a density of 2000 to 50,000 protoplasts per milliliter. The droplets are placed in a liquid nurse culture system (Costar-Transwell col) with feeder cells and incubated at 25-C
in the dark. The droplets are dispersed on solid Br medium after two weeks. Small calli are transferred to solid K3 medium and incubated at 25-C in low light intensity (2500 lux) with a photoperiod of 16 hours.
Example 9: Plant regeneration The calli according to Example 7 and 8, having developed to a size of 2 to 5 mm in diameter, are transferred to fresh K3 medium at normal light intensity (8000 lux) at 25 C with a photoperiod of 16 hours.
Small shoots are transferred to B5 medium with 1%
sucrose without hormones and rooted on the same medium.
.
Example 10: Molecular analysis of the fusion products a) Nuclear DNA composition Characterization of the nuclear DNA
composition of the fusion product is effected by using specific DNA probes. Probe ES, a clone containing a 820 kbp Bgl 1 fragment from cauliflower nuclei DNA
hybridizes with various ~ands in an endonuclease digest pattern of nuclear DNA of the Polima CMS donor, Br~sica ok~acea breeding lines that are used as acceptor for the CMS trait (see Figure 1).
b) Mitochondrial DNA composition Characterization is effected with CMS Bras 4 DNA, a clone cont~; n ing a 1950 bp Bgl II fragment from cauliflower mitochondrial DNA. This clone produced a band of 4 kbp with CMS polima mitochondrial DNA and a band of 6 kbp with mitochondrial DNA from the acceptor br~;~g lines e.g. DE70 (see Figure 2).
Characterization is also effected by PCR amplification of a 500 bp DNA fragment, using primers 94RS01 and 94RS02. The se~uence of the forward primer 94RS01 is 5'-GAA CCA ACT GCT TTC ACA CCG-3' and of the reverse primer 94RS02 5'-CTT GGC TCT CTG CGA ATG TC-3'. This 500 bp fir~ ntis amplified from CMS polirna mitochondrial DNA, but not from mitochondrial DNA of acceptor lines. e.g. DE70.
Example 11: Embryo Rescue Generally, an embrvo is rescued 18-19 days after a cross is made.
S In order to effectuate an embryo rescue. siliques are required. The siliques required are those having as large as possible embryos with a seed coats that have not yet hardened.
Prior tO the removal of the embryo. the siliques must first be disinfected. The siliques are disinfected by placing them in a 10% chloride 10 solution such as sodium or potassium chloride for about twenty minlltes (20 min.). The siliques are rinsed three times in sterile ~vater at 2, 5 and 10 mimltes.
The embryo is rescued by cutting the siliques open in a lonPitudinal direction. removin~ the developin~ seeds and then preparing the 15 embrvo out of the seed under a microscope.
A~er the em~ryo is cut out of the seed, it is placed on a suitable growth m~inm Any m~ m that will f~ciiit~te the growth of the embrvo can be used. For example. an MS medium with 3 & sucrose and 0.4 mg/l thi~min and 0.2 mgll IDA can be used.
RECTIFIED SHEET(RULE 91) W O 97/09873 PCT~US95/11497 Example 12: The mainten~c~ of sterility of CMS
Br~si~ oler~cea plants at high and low temperatures In order to determine if the cytoplasmic male sterile plants cont~;ni~g the polima CMS cytoplasm remained sterile at high and low temperatures, broccoli cultivars 915095, 915107, 915100 and 915144 were hybridized with red cabbage to check if 100% hybridity was achieved. Cultivars 915095-915144 were crossed with red cabbage. When the seeds of these cultivars are pollinated with a normal broccoli maintainer and are sown, the hypocotyl color and leaf color of the seedling is normally green. If these cultivars are crossed with red cabbage then the hybrid seeds are inte~ e~;~te and the seedlings show a strong authocyan (purple3 expression, while inbreds remain green. Seeds of 915095, 915107, 915100, and 915144 were sown and 20 plants of each cultivar were put together with red cabbage plants in a plastic covered cage outside. In the summer, the temperature of the cage varied from 15-40-C during the day. The temperatures in the cage were recorded once a week. A sample of the seed harvested from 915095, 915107, 915100 and 915144 were sown and all resulting plants were hybrid indicating no presence of selfing and no leaking of the polima CMS cytoplasm. At W097/09873 PCT~S95/11497 no time did any of the plants resuit in partial male sterility.
~he crosses which lead to 915095, 915107, 915100 and 915144 are shown below.
88125 x broccoli (=bc (broccoli pollinator line)) 88132 x bc 89022 x bc 89036 x bc 89046 x bc 89094 x bc 89102 x bc 89083 x bc 39133 x bc 892731 x bc 892733 x bc 892736 x bc 892737 x bc 904005 x bc 904006 x bc 904008 x bc 904045 x bc 915095 9151~0 915107 91~144 When two or more arrows go from one cross (i.e.
88132 x bc), then each arrow represents a cross with a di f f erent broccoli genotype.
ZO
Example 13: Female Fertility Listed below is the seed yield data obtained after crossing by hand CMS "Polima" types with broccoli. The data shows that the seed yield of CMS "polima" is higher than those of broccoli inbreds. This is because W O 97/09873 PCT~US95/11497 replicated inbreeding affects the fertility. A better comparison is made between the broccoli Fl's and the CMS
Ogura crosses. The results show that the seed yield of the CMS polima is in the same range as the other two types of crosses.
Broccoli 8eed Yield~ 1991-1994 (Hand pollination) Year Type of CrossNumber of Seeds ~er Cross 1991 (CMS Polima x Broccoli)F1114 (n=4) (Broccoli inbred x Broccoli)Fl 64 (ns23) (Broccoli F1 x Broccoli)F1116 (n~45) 1992 (CMS Polima x Broccoli)Fl146 (ns66) (Broccoli inbred x Broccoli)Fl 100 (n=76) (Broccoli Fl x Broccoli)Fl135 (n=28) (CMS Ogura x 8roccoli)F1140 (n=32) 1993 (CMS Polima x Broccoli)F179 (n=41) (Broccoli in~red x Broccoli)Fl 53 (n=70) (Broccoli Fl x Broccoli)Fl80 (n=35) (CMS ogura x Broccoli)Fl144 (n=5) 1994 (CMS Polima x Broccoli) Fl93 (n=9) (Broccoli inbred x Broccoli)Fl 47 (n=50) (Broccoli Fl x Broccoli)Fl84 (n=35) (CMS ogura x Broccoli)Fl74 (n=9) Weighted means (CNS Polima x Broccoli)Fl115 (n=120) (Broccoli inbred x Broccoli)F1 60 (n=227) (Broccoli F1 x Broccoli)F1103 (n=143) (CMS Ogura x Broccoli)Fl(127)((n=40)) Although the invention has been described primarily in connection with the special and preferred W O 97/09873 PCTrUS95/11497 embodiments, it will be understood that it is capable of modification without departing from the scope of the invention. The following claims are intended to cover all variations, uses or adaptions of the invention, following, in general, the principles thereof and including such departures from the presented disclosure as come within known or customary practice in the field to which the invention pertains, or as are obvious to persons skilled in the field.
Claims (9)
1. A method for producing cytoplasmic male sterile Brassica oleracea plants which are sterile at high and low temperature and which contain Polima CMS cytoplasm, the method comprising the steps of:
a) crossing Brassica campestris cultivar 87110 with Brassica oleracea cultivar 87101 and collecting the seeds resulting from said cross;
b) regenerating said seeds into Brassica napus plants which contain a haploid set of chromosomes;
c) doubling the chromosomes of the plants of step b to provide Brassica napus plants containing a diploid set of chromosomes;
d) crossing Brassica campestris cultivar 87118 with Brassica oleracea cultivar 87101 and collecting the seeds resulting from said crops;
e) regenerating said seeds into Brassica napus plants which contain a haploid set of chromosomes;
f) doubling the chromosomes of the plants of set e to provide Brassica napus plants containing a diploid set of chromosomes;
g) crossing the plants of step f with Brassica napus cultivar 87102 which is male sterile and contains polima CMS cytoplams and collecting the seeds resulting from said cross;
h) regenerating said plants into Brassica napus plants containing the polima CMS cytoplasm;
i) crossing the plants of step c with the plants of step h and collecting the seeds resulting from said cross;
j) regenerating said plants into Brassica napus plants which are male sterile and contain the polima CMS
cytoplasm;
k) crossing the plants of step j with a Brassica oleracea cultivar and collecting the seeds resulting from said cross;
l) removing the embryos from the seeds and regenerating said embryos into F1 plants which are male sterile and contain the polima CMS cytoplasm;
m) backcrossing the plants from step l with a Brassica oleracea cultivar and collecting the seeds resulting from said backcrossing;
n) removing the embryos from the seeds and regenerating said embryos into plants which are male sterile and contain the polima CMS cytoplasm;
p) backcrossing the plants from step n with Brassica oleracea cultivar and collecting the seeds resulting from said backcrossing; and q) regenerating said seeds into Brassica oleracea plants which are male sterile and contain the polima CMS
cytoplasm.
a) crossing Brassica campestris cultivar 87110 with Brassica oleracea cultivar 87101 and collecting the seeds resulting from said cross;
b) regenerating said seeds into Brassica napus plants which contain a haploid set of chromosomes;
c) doubling the chromosomes of the plants of step b to provide Brassica napus plants containing a diploid set of chromosomes;
d) crossing Brassica campestris cultivar 87118 with Brassica oleracea cultivar 87101 and collecting the seeds resulting from said crops;
e) regenerating said seeds into Brassica napus plants which contain a haploid set of chromosomes;
f) doubling the chromosomes of the plants of set e to provide Brassica napus plants containing a diploid set of chromosomes;
g) crossing the plants of step f with Brassica napus cultivar 87102 which is male sterile and contains polima CMS cytoplams and collecting the seeds resulting from said cross;
h) regenerating said plants into Brassica napus plants containing the polima CMS cytoplasm;
i) crossing the plants of step c with the plants of step h and collecting the seeds resulting from said cross;
j) regenerating said plants into Brassica napus plants which are male sterile and contain the polima CMS
cytoplasm;
k) crossing the plants of step j with a Brassica oleracea cultivar and collecting the seeds resulting from said cross;
l) removing the embryos from the seeds and regenerating said embryos into F1 plants which are male sterile and contain the polima CMS cytoplasm;
m) backcrossing the plants from step l with a Brassica oleracea cultivar and collecting the seeds resulting from said backcrossing;
n) removing the embryos from the seeds and regenerating said embryos into plants which are male sterile and contain the polima CMS cytoplasm;
p) backcrossing the plants from step n with Brassica oleracea cultivar and collecting the seeds resulting from said backcrossing; and q) regenerating said seeds into Brassica oleracea plants which are male sterile and contain the polima CMS
cytoplasm.
2. The method of claim 1 further comprising the steps of crossing the plants from step q with a Brassica oleracea; collecting seeds resulting from the cross; and regenerating said seeds into Brassica oleracea plants which are male sterile and contain the polima CMS cytoplasm.
3. The method of claim 1 wherein the plants are selected from the group consisting of: cauliflower, brussels sprouts, cabbage, kale, kohlrabi, and broccoli.
4. A method for producing cytoplasmic male sterile Brassica oleracea plants which are sterile at high and low temperatures and which contain Polima CMS cytoplasm, the method comprising the steps of:
a) fusing a protoplast from a male sterile Brassica oleracea produced according to the method of claim 1 which contains the polima CMS cytoplasm and which remains sterile at high and low temperatures; and b) regenerating the obtained allogenic cells into CMS Brassica oleracea plants containing polima cytoplasm.
a) fusing a protoplast from a male sterile Brassica oleracea produced according to the method of claim 1 which contains the polima CMS cytoplasm and which remains sterile at high and low temperatures; and b) regenerating the obtained allogenic cells into CMS Brassica oleracea plants containing polima cytoplasm.
5. The method of claim 4 wherein the Brassica oleracea is selected from the group consisting of: cauliflower, brussels sprouts, cabbage, kale, kohlrabi and broccoli.
6. The method of claim 4 further comprising the steps of crossing the regenerated CMS Brassica oleracea containing polima cytoplasm with Brassica oleracea plants containing commercially desirable characteristics.
7. Cytoplasmic male sterile Brassica oleracea plants produced by the method of claim 1 which are sterile at high and low temperatures and contain Polima CMS
cytoplasm.
cytoplasm.
8. Cytoplasmic male sterile Brassica oleracea plants produced by the method of claim 4 which are sterile at high and low temperatures and contain Polima CMS
cytoplasm.
cytoplasm.
9. The plants of claims 7 or 8 wherein the plants are selected from the group consisting of: cauliflower, brussels sprouts, cabbage, kale, kohlrabi,and broccoli.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002231423A CA2231423A1 (en) | 1995-09-11 | 1995-09-11 | Cytoplasmic male sterile brassica oleracea plants which contain the polima cms cytoplasm and are male sterile at high and low temperatures |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002231423A CA2231423A1 (en) | 1995-09-11 | 1995-09-11 | Cytoplasmic male sterile brassica oleracea plants which contain the polima cms cytoplasm and are male sterile at high and low temperatures |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2231423A1 true CA2231423A1 (en) | 1997-03-20 |
Family
ID=4162188
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002231423A Abandoned CA2231423A1 (en) | 1995-09-11 | 1995-09-11 | Cytoplasmic male sterile brassica oleracea plants which contain the polima cms cytoplasm and are male sterile at high and low temperatures |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2231423A1 (en) |
-
1995
- 1995-09-11 CA CA002231423A patent/CA2231423A1/en not_active Abandoned
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| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |