Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
  • C12N5/12—Fused cells, e.g. hybridomas
  • C12N5/14—Plant cells

Definitions

• This invention relates to protoplast fusion products useful in generating rape lines.
• THe seed of rape (Brassica sp.) and particularly its higher quality form known as canola is recognized as a valuable source of oil and meal. Accordingly, efforts on the part of plant breeders have focused on providing improved cultivars to achieve increased crop yield and quality.
• male sterile plants must be cross-fertilized to reproduce, they are valuable in providing hybrid plants and are often used in conventional breeding programs.
• this male sterile phenotype is dictated by both nuclear genes and mitochon ⁇ rial DNA.
• the nuclear genes In order for a rape plant to exhibit the male sterile phenotype, in which anthers are incapable of dehiscing viable pollen, the nuclear genes must be homozygous recessive with respect to fertility and the mitochondria must also express the male sterile phenotype.
• rape plants which are sought by breeders
• Tolerance of the triazine herbicides is recognized as a cytoplasmically conferred trait, more specifically a trait which is expressed by DNA contained in chloroplasts of a certain variety of Brassica campestris.
• the winter phenotype, which is nuclear-conferred, is a particularly desirable trait. Winter lines may be planted in the fall and can mature earlier in the growing season than Spring lines, and provide enhanced harvest yield.
• cytoplasmic traits are maternally inherited meaning that seed contains only those cytoplasmic traits of the female parent.
• a cross between a female having mitochondria which confer male sterility and a male having chloroplasts which confer triazine tolerance will produce seed whose only cytoplasmically-determined trait is male sterility.
• the chloroplasts and therefore the triazine tolerance of the male parent will not be present in the resulting seed.
• the present invention utilizes the technique of protoplast fusion to provide a fusion product from which a plant of Brassica sp . may be regenerated.
• This technique can be used to combine a desired nucleus-conferred trait such as winter habit with any desired cytoplasm-conferred trait such as male sterility or herbicide tolerance.
• the same technique can be used to introduce both cytoplasmic traits to Brassica sp., if desired.
• the present invention provides a process for producing a regenerable Brassica protoplast fusion product, comprising the steps of
• Brassica plant containing nuclear genetic material encoding a desired nuclear-conferred trait and (ii) a second protoplast derived from a Brassica plant, said second protoplast containing, a first cytoplasmic element, the presence of which can be detected in plants carrying said element; and B) inducing fusion of said first and said second protoplast to produce a regenerable fusion product containing said nuclear genetic material and said first cytoplasmic elements.
• the fusion product contains a nucleus which is able to confer the winter habit.
• Fusion products and the plants or calluses regenerated therefrom are also within the scope of the present invention.
• the cytoplasmic traits may be either male sterility or herbicide tolerance or a combination thereof.
• the present invention also provides a method for definitively characterizing the DNA comprised within those organelles. which confer these traits. For example, it has been determined that the cytoplasmic male sterility trait (cms) is conferred by expression of mitochondrial DNA. Similarly certain types of herbicide tolerance e.g. cytoplasmic triazine tolerance are conferred by chloroplast DNA. By characterizing definitively this DNA, i.e. mitochondrial DNA and chloroplast DNA, it becomes possible to select appropriate protoplasts to be fused and to identify and select the fusion products (callus or plants) having the desired characteristics. With this knowledge, one can also identify and select appropriate maintainer and restorer lines which are necessary for the utilization of the regenerated fusion product in a hybrid seed production program.
• cms cytoplasmic male sterility trait
• chloroplast DNA By characterizing definitively this DNA, i.e. mitochondrial DNA and chloroplast DNA, it becomes possible to select appropriate protoplasts to be fused and to identify and select the fusion products (callus or
• plants of Brassica sp. possess chloroplasts whose DNA contains a fragmentation pattern which is manifest upon digestion of the chloroplast genome with either ECO RI or Mae I (a restriction enzyme recently characterized as disclosed in Nucleic Acids Research, Volume 12, Number 6, 1984, pp2619-2628).
• the triazine tolerant plant from which the desired protoplast may be extracted may be identified more clearly as compared with use of ECO RI. It has been determined that triazine tolerance results from a single point mutation in the wild-type, herbicide susceptible Brassica plant. Only if mutation of this type is present will Mae I be unable to cut the cpDNA. Thus digestion of cpDNA of a potential protoplast donor plant with Mae I followed by analysis with agarose gel electrophoresis will reveal a large segment of approximately 359 base pair (bp) size under the conditions described herein if the genome confers triazine tolerance.
• cpDNA of triazine susceptible plants will show two DNA segments of 233bp and 126bp using the same analytical procedure, revealing that protoplasts derived from such plants are unacceptable as a source of protoplasts having triazine tolerance. Fusion of the desired protoplast i.e. whose chloroplasts confer triazine tolerance, with another suitable protoplast permits generation of a Brassica plant which is tolerant to triazine.
• the desired protoplasts may be selected as described above, i.e. cms conferring protoplasts are extracted from plants having mitochondrial DNA (mtDNA) which possesses the ECO RI excisable unique DNA segments, which protoplasts are then fused, under fusion conditions, with protoplasts extracted from plants whose cpDNA is identified as possessing either ECO RI or Mae I recognizable properties.
• mtDNA mitochondrial DNA
• a plant is considered to possess cytoplasmic.
• herbicide tolerance when its ability to withstand or endure a given herbicide while carrying on its normal plant functions can be traced to the nature of the cytoplasm of the plant.
• the herbicides to which the plants acting as protoplast sources are tolerant include the s-triazines and the as-triazines which embody atrazine (2-chloro-4-ethylamino-6isopropylamino-s-triazine); cyanazine (2-[[4-chloro-6(ethylamino)-s-triazine-2-yl]amino]-2-methylpropionitrile) and metribuzin (4-amino-6-(1,1-dimethylethyl)-3-(methylthio) -1,2,4-triazine-5(4H)-one), among others.
• Figure 1 is a diagrammatic illustration of a characteristic section of agarose gel migration patterns of mitochondrial DNA from various sources after cleavage with ECO RI, and
• Figure 2 is a diagrammatic illustration of a characteristic section of agarose gel migration pattern of chloroplast DNA from various sources after cleavage with ECO RI.
• the respective parent plants Prior to extracting protoplasts to be fused, the respective parent plants are analyzed to confirm the mitochondria and chloroplast genotype. It will be appropriate to extract a tissue sample from the leaves, where mitochondria and chloroplasts are abundant. The sample may be manipulated in conventional manner to free the DNA from the organelle. In accordance with a preferred embodiment of the invention, however, novel techniques are used to isolate the organelle DNA, as disclosed in examples 1 and 2, hereinafter, which techniques require a relatively small amount of tissue to be extracted from the plant and therefore does not result in destruction of the plant. In this way, the plants can be grown to maturity and the seeds removed, if desired. After chloroplast and mitochondrial DNA is freed, it is then digested in the presence of the restriction enzyme ECO RI and analyzed for DNA segments which migrate on agarose gel under the influence of electrophoresis.
• ECO RI restriction enzyme
• plants possessing cms conferring mitochondria which are native to Brassica sp. are selected as opposed to plants possessing the desired mitochondria as a result only of cross breeding.
• Phenotypically male sterile plants whose tissue samples produce an ECO RI mitochondrial DNA restriction fragment pattern characteristic of cms lines, as shown in accompanying Figure 1, are preferably selected as protoplast donors.
• these preferred protoplast donors may be those whose tissue samples are identified, under these conditions of electrophoresis, as containing one of three distinctive markers i.e. mitochondrial DNA segments or distinctive DNA segment combinations having the following sizes in kilobases (kb):
• Phenotypically male sterile Brassica napus has been found to exhibit the desired features most often.
• the nucleotide region of the psbA gene which undergoes this single base pair change is 5'GCTAGT3' in the wild type and 5-GCTGGT3' in the mutant (triazine tolerant) type.
• the restriction enzyme Mae I isolated from Methanococcus aerolicus PL-15/H is able to cut the cpDNA of susceptible lines at 5'C TAG 3' in this region of the psbA gene.
• cpDNA of tolerant lines will not be cut by Mae I at the mutated locus.
• cpDNA of tolerant lines exhibit a Mae I band of 359 bp using agarose gel electrophoresis under the same conditions d ef ined above whereas succeptible lines f ail t o exhi bit this band but show instead two bands of DNA segments of 233 and 126 bp sizes. It has been found that cytoplasmically triazine tolerant Brassica campestris and Brassica napus exhibit the desired features of ECO RI and Mae I digestion most often.
• protoplasts may be generated from the desired tissue area of each parent. Accordingly, a first protoplast will be obtained which possesses mitochondria which confer male sterility and an ECO Rl-excisable DNA segment or combination of such segments indigenous to its mitochondrial genome of particular size and a second protoplast will be obtained which possesses chloroplasts which confer triazine tolerance and contain a DNA segment of particular size.
• These two types of protoplasts are generated separately according techniques standard in the art which involve, in general, removal of the cell wall under conditions carefully controlled to regulate osmotic pressure, pH and the like.
• first and second protoplasts are fused using standard procedures.
• the fusion products are grown to culture, ultimately to mature plants when phenotypic traits can be observed and the genotype confirmed by the procedure described, to confirm the desired cybrid plant has been generated.
• novel techniques optimized for small sample size are used to avoid destruction of the plant.
• cytoplasmic male sterility (cms) system in the progenitor plant since maintainer lines are very specific and do not necessarily "maintain" plants having other than one particular cms system. The identity afforded using the present invention therefore provides valuable information for purposes of subsequent
• the homogenate was filtered through 4 layers of cheesecloth and 1 layer of Miracloth (both presoaked in HB) prior to centrif ugation at 1,000 x g for 10 minutes in a Sorval RC-5B centrifuge containing a SS-34 rotor. [All subsequent centrifugation steps employ this centrifuge and rotor unless otherwise stated.]
• the supernatant was then centrif uged at 17,000 x g for 10 minutes.
• the resultant pellet was resuspended in 10ml HB and recentrifuged at 1,000 x g for 10 minutes. All pellets were resuspended with a small (#4) artists brush.
• the pellet was resuspended in 10ml SB and recentrifuged at 17,000 x g for 10 minutes.
• the resulting mitochondrial pellet was resuspended in lysis buffer (2ml 50 mM Tris HCl pH 8.0, 10 mM EDTA + 0.5ml 10% sarkosyl + 0.03 ml autodigested pronase at 10mg/ml concentration) and incubated, with gentle agitation, at 37°C for 60 minutes.
• the precipitated DNA was collected by centrifugation, washed twice in 70% ethanol, lyophilized, resuspended in 0.07ml sterile distilled H 2 O and stored at -20°C. A portion of the mtDNA was digested with a specific activity excess of ECO RI (Boehringer-Mannheim, Canada) at 37°C according to the manufacturers specifications.
• a 1/10 volume of 50% glycerol containing 0.05% bromophenol blue was added, mixed and the preparation applied to a 1% agarose horizontal gel slab containing 40mM Tris, 5mM sodium acetate, ImM EDTA, pH 7.8. Electrophoresis was performed in the same buffer at 2.5 V/cm for 15 hours.
• the agarose gel was stained in an aqueous solution of 500ng/ml ethidium bromide for 30 minutes, destained in water for 30 minutes, illuminated with 302nm ultra-violet light and photographed.
• Lane 1 of Fig. 1 shows a diagrammatic representation of the resultant unique and characteristic ECO RI restriction fragment pattern of B. napus cv. Regent mtDNA.
• Lane 1 shows five segments denoted by reference numerals 10, 12, 14, 16 and 18 respectively, which are unique collectively relative to the other lanes shown.
• the sizes of the unique segment 10 can be estimated to be 13.897 + 0.044 kilobases (kb); the size of the segment 12 can be estimated to be 11.415 + 0.044 kb; segment 14 estimated at 9.353 ⁇ 0.044 kb; segment 16 estimated at 8.271 + 0.044 kb and segment 18 estimated at 7.328 + 0.044 kb. Plants possessing both male sterile phenotype and the combination of segments 10 through 18 upon analysis as described above are suitable as donors of protoplasts for fusion according to the present invention.
• Lane 3 which represents the migration pattern of phenotypically male sterile Brassica napus with polima cytoplasm DNA segments, exhibits segments 20, 22, 24, 26, 28, 30, 32 and 34 which are unique collectively among the migration patterns disclosed in Figure 1. These segments are estimated to be of the following sizes:
• phenotypically male sterile plants possessing mitochondrial DNA containing collectively ECO RI excisable fragments of these sizes may be used as donors of protoplasts for the purpose of the present invention.
• lane 4 which shows the migration pattern of mtDNA of phenotypically male sterile Brassica napus with ogura cytoplasm exhibits one unique segment i.e. 36, the size of which can be estimated to be 6.096 + 0.044kb. Segment 36 represents a marker, the presence in mtDNA of which indicates that the mitochondria is capable of conferring cms.
• each of lanes 1, 3 and 4 of Figure 1 may serve as an identification of the desired mitochondria i.e. the plant from which appropriate protoplasts may be extracted for use in fusion.
• These appropriate protoplasts, containing mitochondrial DNA as described above may be fused with a second protoplast extracted from Brassica sp. providing that both which do not contain dominant fertility restorer genes in order to obtain a cybrid protoplast which expresses cytoplasmic male sterility.
• the second protoplast, to be fused with the cms protoplast described above possesses cytoplasmically conferred i.e.
• chloroplast conferred tolerance of triazine herbicide The technique by which a suitable donor of the second protoplast may be identified is exemplified in Example 2.
• three distinct mitochondrial genomes are defined as being male sterility-conferring. It should be appreciated that other such mitochondria will be discovered.
• the method of the present invention is equally applicable in that event, provided that the DNA of the newly discovered mitochondrial variety exhibits a unique marker when electrophoresed as described. The importance of the present method resides in the ability to mate rape plants precisely.
• the homogenate was filtered through 4 layers of cheesecloth and 1 layer of Miracloth (both pre soaked in IB) prior to centrifugation at 1,000 x g for 10 minutes in a Sorval RC-5B centrifuge containing a SS-34 rotor. [All subsequent centrifugation steps employ this centrifuge and rotor unless otherwise stated].
• the pellet was resuspended in 10ml wash buffer (WB) (0.35M sorbitol, 50mM Tris-HCl pH 8.0, 25mM EDTA, ImM spermine, ImM spermidine) and recent rifuged at 1,000 x g for 10 minutes. All pellets were resuspended with a small (#4) artists brush.
• WB wash buffer
• the resultant pellet was resuspended in 9.5 ml of WB and layered onto 7ml of buffer A (30% sucrose in WB) which. immediately prior, had been layered onto 18ml of buffer B (60% sucrose in WB) in a Beckman 38.5ml centrifuge tube.
• the gradient was centrifuged in a Beckman L8 ultracentrif uge using a SW28 rotor at 25,000 rpm for 40 minutes.
• Chloroplasts collected at the buffer Arbuffer B interface were removed, diluted with 30ml WB, and centrifuged at 1,500 x g for 15 minutes.
• the pellet was incubated in lysis buffer and all subsequent steps were identical to those described in example 1 except that the lyophilized cpDNA sample was resuspended in 0.2ml sterile distilled H 2 O.
• Lane 2 of Figure 2 shows the migration pattern of DNA segments generated by the above technique i.e. using leaf extract of Brassica napus cv. 'TT' Regent.
• Lanes.1 and 3-7 represent the migration patterns for cpDNA of triazine succeptible plants, when analyzed by the same method, as follows:
• Lane 2 of Figure 2 which represents the migration pattern of the only cytoplasmically triazine tolerant donor plant analyzed in this experiment, exhibits a unique DNA segment pattern.
• the segment denoted by reference numeral 38 is unique by comparison with the other genomes analysed. This segment is estimated to be of 3.33 + 0.065kb. The unique pattern indicates that the plant having this cpDNA characteristic is suitable as a source for the protoplasts.
• the chloroplast genome of a plant proposed. as a source of protoplast having cytoplasmically conferred triazine tolerance may be analyzed by the procedure described above but by using Mae I in place of ECO RI.
• the Mae I enzyme is unable to cut further a 359bp segment resulting from scission of the chloroplast genome using this enzyme in the case where the chloroplast genome is capable of conferring triazine tolerance.
• triazine succeptibility is coded by the cpDNA, however, the Mae I enzyme will cleave the 359bp segment into two fragments of 233bp and 126bp sizes. Accordingly, this 359bp segment indicates that the plant possesses a chloroplast genome which confers triazine tolerance and the plant may therefore serve as a source of useful protoplasts.
• Example 3 Fusion of triazine tolerant Brassica campestris Candle with polima cms Brassica napus cv. Regent a) Isolation of protoplasts from triazine tolerant Brassica campestris Candle
• Leaves were removed from 3 week old plants growing in a growth chamber (12h photoperiod, 23°C 10,000 lux) and surface sterilized by dipping in ethanol. The lower epidermis was brushed, the leaves chopped into 1cm pieces and incubated for 2 hours in "Soak" solution comprising the major salts and organic additives of medium A of Shepard and Totten (1977) (see Plant Physiology (1977) 60, pp 313-316) and lmg/1 2,4D (2,4 dichlorophenoxy acetic acid) and 0.5 mg/1 BAP
• the digestion mixtures were filtered through two layers of cheesecloth, and centrifuged (750 rpm, 10 min). A band which contained protoplasts collected at the surface. Following a second centrifugation in a "Rinse” solution (0.35M sucrose and salts as in the "Soak” solution), the surface band contained mostly protoplasts.
• Isolated protoplasts were incubated for 10 minutes at room temperatuare in a solution containing 2mM iodoacetic acid (IOA), 0.35M sucrose and salts as in the rinse solution. The mixture was then centrifuged (750 rpm, 10 minutes). Protoplasts collected at the surface were resuspended in "Rinse” solution and again collected by centrifugation.
• IOA 2mM iodoacetic acid
• Samples of the two protoplast populations prepared above are mixed to 5ml total in a 1:1 ratio at a concentration of 1 x 10 6 /ml. To this is added an equal volume (0.5ml) of PEG solution (25% polyethylene glycol, 0.12M sucrose and .01M CaCl 2 .2H 2 O). The mixture is gently agitated by hand and incubated for 10 minutes at room temperature. Then 0.5ml of Ca 2+ /high pH solution (.05M CaCl 2 .2H 2 O, 0.3M Mannitol,
• Protoplasts following fusion treatment were added (at 3 x 10 3 /ml) to a culture of N. tabacum protoplasts (previously prepared as for Candle above and irradiated with 20 kr gamma irradiation to prevent growth) at 8 x 10 4 /ml.
• Protoplasts following fusion treatment were plated at 3.5 x 10 3 /ml with no nurse.
• the suspension of dividing cells was transferred to a 10 cm petri plate and diluted with an equal volume of a medium similar to the reservoir but modified by the replacement of the hormones specified with 1.0mg/l 2,4-D and 0.1mg/l Kinetin.
• the agarose concentration was also altered to 0.06%. All subsequent procedures were carried out at 25°C with a 16h photoperiod of 4000 lux.
• Colonies with primordial shoots appearing 9 days later were tranfered to B5 basal medium (Gamborg et al, 1968 - see Exp. Cell. Res. 50:151-158) containing 0.2% sucrose and 0.03mg/l GA3, to induce grow-out of the shoots. After growing for one week, a second transfer was made to the B5-basal medium described above.
• shoots with visibly differentiated meristems were transferred to sterile 'Jiffy 7' peat pellets to induce root formation. These were placed within sterile jars to maintain a relatively high humidity, under 10h photoperiod.
• Protoplasts were isolated from leaves as described in example 3 for Candle, except the plants were 36 days old.
• Protoplasts were isolated from hypocotyls as described in example 3 for Regent.
• Isoated protoplasts were mixed with an equal volume of FDA solution, (0.1 mg/ml FDA, salts and sucrose as in rinse solution). The mixture was incubated at room temperature for 5 minutes, then centrifuged (740 rpm, 5 mins.). Stained protoplasts collected at the surface.
• FDA solution 0.1 mg/ml FDA, salts and sucrose as in rinse solution.
• the fusion mixture was observed the following day using UV-light microscopy.
• FDA-stained Regent hypocotyl protoplasts were recognized by their green fluorescence, and TT Regent mesophyll protoplasts by their red autofluorescence (of chlorophyll) .
• Protoplasts with both red and green fluorescence were recognized as fusion products (heterokaryons), and physically isolated from the fusion mixture using a micromanipulator. Isolated heterokaryons were placed into a culture of gamma irradiated N. tabacum cells (as described in Example 3).
• Example 5 Obtaining cms Parent Lines for Hybrid Seed Production by Fusing Cytoplasm Containing cms Mitochondria with a Desired Nucleus
• Example 3 The method according to Example 3 may be followed to establish fusion of protoplasts of polima cms B. napus cv. Regent (having the mitochondrial DNA migration pattern shown in Figure 1, Lane 3, and the chloroplast DNA migration pattern shown in Figure 2, Lane 3) was treated with 30 krad gamma irradiation to prevent division, with protoplasts of B. napus cv. Santana (having the mitochondrial DNA migration pattern shown in Figure 2, Lane 1) was treated with IOA as for cv. Regent as in Example 3 was used. Ten plants were regenerated from two fusion experiments. All ten plants had the winter phenotype (i.e. required 8 weeks at 4°C to induce flowering). Chloroplast and mitochondrial DNA was analyzed from each of the plants.
• progeny plants derived from crossing the fusion product with the maintainer
• progeny plants were crossed, as females, with the appropriate B. napus restorer genotypes (as males). These crosses yielded commerial hybrid seed.
• Protoplast fusion techniques are employed to provide a progenitor plant which is cytoplasmically male sterile b which may also possess such traits as herbicide tolerance and a winter phenotype. Male sterility-conferring mitochond of the rape plant are characterized genotypically. This allows identification of the parents of the desired fusion prod and characterization of the fusion product itself. It also permits production of genotypically consistent seed by providin means for accurately selecting the parents of a cross from which hybrid seed is produced.

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