WO1987001726A2 - Protoplast fusion product and process for preparing same - Google Patents

Abstract

Protoplast fusion techniques are employed to provide a progenitor plant which is cytoplasmically male sterile but which may also possess such traits as herbicide tolerance and a winter phenotype. Male sterility-conferring mitochondria of the rape plant are characterized genotypically. This allows identification of the parents of the desired fusion product and characterization of the fusion product itself. It also permits production of genotypically consistent seed by providing a means for accurately selecting the parents of a cross from which hybrid seed is produced.

Description

PROTOPLAST FUSION PRODUCT AND PROCESS FOR PREPARING SAME

This invention relates to protoplast fusion products useful in generating rape lines.

BACKGROUND OF THE INVENTION

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.

Because male sterile plants must be cross-fertilized to reproduce, they are valuable in providing hybrid plants and are often used in conventional breeding programs.

Self-fertilization is avoided in this way as is the deleterious expression of recessive genomic traits which may attend such a mating. In the case of rape, this male sterile phenotype is dictated by both nuclear genes and mitochonάrial DNA. 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.

There are other desirable traits in rape plants which are sought by breeders such as herbicide tolerance and the winter habit. 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.

Unfortunately for conventional plant breeders, cytoplasmic traits are maternally inherited meaning that seed contains only those cytoplasmic traits of the female parent. Thus, 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.

To obtain a plant having a combination of these characteristics, plant breeders are thus normally faced with the tedious task of locating them in natural environments. For example, U.S. patent 4,517,763 issued May 21, 1985 which discloses a particular breeding program using a plant whose cytoplasmic organelles confer both cytoplasmic male sterility (mitochondria) and herbicide tolerance (chloroplasts) describes a method which relies firstly on the process of natural selection to provide a population of rape plants which express herbicide tolerance. Thereafter, a comprehensive breeding program is conducted with the herbicide resistant plants to isolate one which also exhibits cytoplasmic male sterility (cms), as opposed to nuclear-conferred male sterility which can also be found in these plants. One problem faced by these breeders is the irregular spontaneity with which the desired plants arise. If not isolated and pollinated quickly, the plant will die without leaving seed and its desirable traits will be lost. Moreover, unless the pollen is derived from an appropriate maintainer genotype i.e. one which permits the cytoplasmic sterility trait to be retained in the offspring, the offspring will exhibit male fertility and will be capable of self-fertilization, and the male sterile genotype may be lost.

More recently, plant biologists have developed the technique of protoplast fusion whereby two protoplasts (plant cells devoid of cell wall) are caused to fuse to form wha.t are termed "cybrids" or somatic hybrids. Using this technique, the maternal inheritance of cytoplasmic traits is circumvented. Fusion products can be obtained which express the cytoplasm-conferred traits of both parents.

Accordingly, it is an object of the present invention to provide a means for identifying definitively cytoplasmic DNA in Brassica plants and to utilize this information in a method for developing plants and seed having a genetically consistent constitution.

It is a further object of the present invention to provide Brassica plants having desired cytoplasmically conferred traits such as male sterility and herbicide tolerance; and Brassica plants having such cytoplasmically conferred traits and, in addition, nuclear-conferred traits such as the winter habit.

SUMMARY OF THE INVENTION

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. As well, the same technique can be used to introduce both cytoplasmic traits to Brassica sp., if desired.

In one aspect, the present invention provides a process for producing a regenerable Brassica protoplast fusion product, comprising the steps of

A) providing (i) a first protoplast derived from a

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.

Preferably, 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.

To ensure that the cytoplasms of the selected protoplasts to be fused are as desired, 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.

Thus according to another aspect of the present invention, there is provided a method for characterizing cytoplasmic DNA of Brassica tissue comprising the steps of

A) extracting mitochondrion and/or chloroplast DNA from cytoplasm in an amount of Brassica tissue; B) treating said DNA with ECO RI or Mael to produce a fragmented DNA preparation; and

C) subjecting said fragmented DNA preparation to gel electrophoresis such that a restriction fragment pattern that is characteristic of said cytoplasm is generated.

It has been found that protoplasts extracted from plants of Brassica sp. possess mitochondria whose DNA contains unique, recognizable DNA segments upon digestion of the mitochondrial genome with ECO RI. Thus selection of a plant whose mitochondrial DNA possesses such unique segments as a source of protoplast for fusion with another suitable protoplast permits generation of a Brassica plant which is cytoplasmically male sterile. These unique segments are identified in accordance with the agarose gel migration patterns generated by the methods described in the examples disclosed herein.

In addition, it has been found that 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).

Where use is made of Mae I as the analytical enzyme, 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. By contrast, 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.

Therefore, to provide a cms, triazine tolerant fusion product from which a Brassica plant having these characteristics may be generated, 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.

As used herein, 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, according to a preferred embodiment of the invention, 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.

BRIEF REFERENCE TO THE DRAWINGS

The invention is further illustrated in the accompanying drawings in which:

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.

In Figure 1, only DNA segments of a size greater than 4 kilobases are shown. In Figure 2, only DNA segments of a size greater than 2.5 kilobases are shown.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

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.

In accordance with the preferred embodiment, 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. In accordance with the preferred embodiment, the agarose gel electrophoresis is conducted using a 1/10th volume of 50% glycerol containing 0.05% bromophenol blue mixed with an ECO RI digested preparation of organelle DNA. The mixture is applied to a 1% agarose horizontal gel slab containing 40mM Tris, 5mM sodium acetate, ImM EDTA pH=7.8 and electrophoresis performed in the same buffer at 2.5 v/cm for 15 hours.

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. For example, 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):

1) 13.897 + 0.044 kb; 11.415 + 0.044 kb; 9.353 + 0.044 kb; 8.271 + 0.044 kb and 7.328 + 0.044 kb;

and 2) 13.897 + 0.044 kb; 11.415 + 0.044 kb; 10.726 + 0.044 kb; 9.353 + 0.044 kb ; 8.554 + 0.044 kb; 8.271 + 0.044 kb; 6.345 + 0.044 kb, and 5.454 + 0.044 kb;

and 3) 6.096 + 0.044 kb.

Phenotypically male sterile Brassica napus has been found to exhibit the desired features most often.

By the same method the DNA of chloroplasts derived from phenotypically triazine resistant rape plants, particularly from tissue samples derived from the leaves, are analyzed to identify those which possess an ECO RI chloroplast DNA restriction fragment pattern characteristic of triazine tolerant lines, as shown in Figure 2, e.g. a segment having a size of 3.33+ 0.065kb under these electrophoresis conditions. In a similar method, but using Mae I rather than ECO RI as restriction enzyme, correct selection of herbicide resistant parent may be confirmed by digestion of chloroplast genome followed by agarose gel separation of DNA segments generated. The presence of a 359 bp segment indicates the chloroplasts of the plant confer triazine resistance, use of Mae I has the attendant advantage that there is no need to ensure that the proposed donor is phenotypically triazine tolerant as is necessary with analysis using ECO RI. It has been found that tolerance to such herbicides by rape plants occurs by virtue of a single base pair alteration in the chloroplast psbA gene. This change (an adenine residue in the wild type and a guanine residue in the resistant gene) causes an amino acid change in the protein encoded by this gene. The single amino acid substitution is sufficient to prevent the triazine herbicides from binding to the chloroplast membrane protein. 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. However, because the A residue has been substituted by a G residue in tolerant lines, 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.

Once the appropriate parents are identified, 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.

Once obtained, 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. As with the parent plants, novel techniques optimized for small sample size are used to avoid destruction of the plant.

While male sterility is desirable in that self-fertilization is precluded, it is still necessary to cross-breed the plant without losing the male sterility trait in order to multiply the plant. Breeders commonly employ maintainer lines for this purpose. These lines possess alleles which are recessive in respect of the nuclear genes which restore male fertility. Selection of an appropriate line of maintainer plants is an absolute necessity to the maintenance of the progenitor plant. Appropriate maintainers can be selected only with the knowledge of the type of 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 breeding.

Once the appropriate maintainer has been crossed with the progenitor plant provided by protoplast fusion, seed is recovered from which the maintained plants are grown for further breeding with selected restorer lines which restore male fertility to the offspring through dominant genes in the nucleus rather than in the mitochondria. Plants grown from the seed resulting from the appropriate latter crosses will exhibit hybrid vigor and can be expected to increase crop yields by as much as 40%.

The invention is further hereinafter illustrated by way of example only.

Example 1 - Analysis of Mitochondrial DNA of Potential Parent Plants

All procedures were performed at 4°C unless otherwise stated.

Fifteen grams of leaf tissue from an individual plant of B. napus cv. Regent, a commercially available cultivar exhibiting male sterility phenotype in the absence of nuclear restorer to fertility genes, was homogenized in a Waring blender (2 x 5 sec pulses at high speed) containing 70ml of homogenization buffer (HB)(10mM TES pH 7.2, 0.5M mannitol, 1 mM EGTA, 0.2% BSA, 0.05% cysteine) .

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.

To this supernatant was added 1M Mg Cl₂ to a final concentration of 10mM and DNase (lmg/.01ml solution freshly prepared) to a final concentration of 0.01 mg/g fresh weight of starting leaf tissue. After gently mixing, the preparation was incubated on ice for 60 minutes before overlaying onto 20 ml of sucrose buffer (SB) (10mM TES pH 7.2, 20mM EDTA, 0.6M sucrose) and centrifuged at 17,000 x g for 20 minutes.

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.

All subsequent procedures were performed at room temperature unless otherwise stated.

To the lysate was added 0.3ml 2M ammonium acetate and, after gentle mixing, 3ml of redistilled phenol saturated with sterile distilled H₂O. This was gently mixed and 3ml chloroform containing 1% octanol added and also gently mixed. [Such phenol-chloroform extractions of DNA are common to those knowledgeable in the art.] The organic and aqueous phases were separated by centrifugation in an IEC HN-SII benchtop centrifuge containing a swing-out rotor at full-speed for 10 minutes. The aqueous phase was carefully removed and the phenol-chloroform extraction repeated twice more.

After the 3rd extraction, the aqueous phase was removed, 2 1/2 volumes of cold 95% ethanol were added, mixed and incubated at -20°C overnight.

The precipitated DNA was collected by centrifugation, washed twice in 70% ethanol, lyophilized, resuspended in 0.07ml sterile distilled H₂O 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.

th

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.

By the same precedure, the mitochondrial DNA of various other Brassica sp. plants were analyzed. The results appear in Figure 1 alongside the results for Brassica napus cv Regent shown in lane 1. The lanes represent leaf tissue as follows:

Lane Plant Phenotype

1 * Brassica napus cv Regent male sterile (ms)

2 Brassica campestris male fertile (mf)

3 * Brassica napus with polima cytoplasm ms

4 * Brassica napus with ogura cytoplasm ms

5 Raphanus sativus mf

6 Diplotaxis mύralis mf

7 Brassica carinata mf

8 Brassica nigra mf m size marker standards formed by digestion of lambda DNA with ECO RI * available from Crop Science Department, University of

Manitoba, Canada, all others are commercially available.

Comparison of the migration patterns permits identification of certain unique DNA segments which are unique to each cytoplasmic type i.e. markers. 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.

Using the migration pattern of the standardized DNA segments shown in lane "m" as references, 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.

In addition, the phenotypically male sterile plants represented in lanes 3 and 4 of Figure 1 also show unique restriction fragments which may act as markers to select the appropriate protoplast donor plant. 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:

Segment # size

(+ 0.044 kilobases) 20 13.897

22 11.415

24 10.726

26 9.353 28 8.554

30 8.271

32 6.345

34 5.454

The presence collectively of these segments confirms a genotype capable of conferring the cytoplasmic male sterility trait. Accordingly, phenotypically male sterile plants possessing mitochondrial DNA containing collectively ECO RI excisable fragments of these sizes, as determined according to the method described above, may be used as donors of protoplasts for the purpose of the present invention.

Further, 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.

Since specific markers can be seen to exist in each of lanes 1, 3 and 4 of Figure 1, it will be realized also that the entire migration pattern depicted in each of those lanes also 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. In accordance with the most preferred embodiment of the invention, however, 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. In the preceding description, 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.

Example 2 - Analysis of Chloroplast DNA of Potential Parent Plants

All procedures were performed at 4°C unless otherwise stated.

2.5 gms of leaf tissue from an individual plant of B. napus cv 'TT' Regent (triazine tolerant), available from the Crop Science Department of the University of Manitoba, Canada, was homogenized in a Waring blender (1 x 10 sec and a 1 x 5 sec pulse at high speed) containing 70ml of isolation buffer (IB) (0.35M sorbitol, 50mM Tris-HCl pH 8.0, 5mM EDTA, 0.1% BSA, 15mM 2-mercapto-ethanol, ImM spermine, ImM spermidine) .

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.

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 H₂O.

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 Plant Tolerance of Triazine

1 Brassica napus no

2 *Brassica campestris 'TT' Candle yes

3 Brassica napus with polima cytoplasm no

4 Brassica napus with ogura cytoplasm no

5 Diplotaxis muralis no

6 Brassica carinata no

7 Brassica nigra no m size marker standaicds formed by independent digestion of lambda DNA with ECO RI and Hae III

available from Crop Science Department; University of Guelph, Canada - contains same chloroplast genome as Brassica napus cv. 'TT' Regent. 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. For example 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.

As a further check, or as an independent method, 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. Where 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.

The following examples show fusion of protoplasts which have been extracted from plants determined by the preceding methods to be suitable donors.

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

(6-benzylaminopurine). After two hours the "Soak" solution was removed and replaced with a digestion solution containing 0.1% cellulose R-10 0.01% (Kinki Yakult Mfg. Co. Nishmorniya, Japan) Macerozyme R10, and 0.35M sucrose. (Approximately lg leaves/100g enzyme). The mixture was incubated for 16 hours at 25°C with gentle agitation (50 rpm).

b) Isolation of protoplasts from Brassica napus cv. Regent

(having polima cytoplasm)

Hypocotyls were removed from sterile 4-day old seedlings growing on MS-(Murashige and Skoog, 1962 - see Physiologia Plantarum, Vol. 15, 1962 pp 473-481) medium with 1% sucrose at 25°C in the dark, chopped into 2-5 mm transverse segments and incubated in digestion solution as described above.

Treated separately, 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.

Pre-fusion Treatments

Regent - Iodoacetic acid treatment

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.

This treatment prevents division of these protoplasts. Candle

These protoplasts were not treated, as they are incapable of growth and division under the culture conditions subsequently employed.

Fusion

Samples of the two protoplast populations prepared above are mixed to 5ml total in a 1:1 ratio at a concentration of 1 x 10⁶ /ml. To this is added an equal volume (0.5ml) of PEG solution (25% polyethylene glycol, 0.12M sucrose and .01M CaCl₂.2H₂O). The mixture is gently agitated by hand and incubated for 10 minutes at room temperature. Then 0.5ml of Ca²⁺/high pH solution (.05M CaCl₂.2H₂O, 0.3M Mannitol,

.05M glycine, pH adjusted to 10.5 with KOH) was added and gently mixed. At the next two five minute intervals 1.0ml of Ca²⁺/high pH was added. After a further 5 minutes 2.0ml of

"Rinse" solution was added. The mixture was incubated for a further 5 minutes then centrifuged, (200 rpm, 5 minutes). This pellets the protoplasts. "Rinse" solution was added to the pellet and the mixture was centrifuged again (750 rpm, 10 minutes). Undamaged fused and unfused protoplasts collected at the surface.

Protoplast plating

1. Fusion Products + Nurse

Protoplasts following fusion treatment were added (at 3 x 10³/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 10⁴/ml.

2. Fusion Products

Protoplasts following fusion treatment were plated at 3.5 x 10³/ml with no nurse.

3. Unfused Mixed Control

Protoplasts of Candle and Regent were mixed after IOA treatment but prior to fusion and plated (as above 1.) at 3 x

10³/ml in N. tabacum (8x 10⁴/ml) . Culture Media

Cultures were initiated in the 'cell layer' medium of Shepard (see Genetic Improvement of Crops: Emergent Techniques, Rubenstein etal 1980. University of Minnesota Press at p. 196), modified by substituting the hormone concentrations of Medium A of Glimelius (see Physiol. Plant. 61:38-44, Copenhagen 1984) (0.1mg/l 1-Naphthaleneacetic acid (NAA) 0.4mg/l BAP, 1.0mg/l 2,4-D) and 0.06% agarose. The suspension was plated in plastic quandrant plates in contact with a reservoir medium (Shepard, 1980 - see Genetic Improvement of Crops: Emergent Techniques, Rubenstein etal 1980. University of Minnesota Press at p. 196) containing 0.1mg/l NAA and 1.0mg/l 2,4-D. Plated protoplasts were incubated at 25°C in the dark. Only fusion products cultured with a nurse had survived.

Seven days after plating, 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.

Twelve days later the colonies had reached 0.5-1.0/mm in size. A second transfer was then effected either by pipetting the suspension of colonies onto the surface of the proliferation medium and carefully drawing off excess dilution medium, or by individual colony transfer, onto MS (Murashige and Skoog, 1962 - see Physiologia Plantarum, Vol. 15, 1962 pp 473-481) medium with 1% sucrose, 0.5mg/l 2,4-D and 0.05mg/l kinetin.

Two weeks later, calli of 1-2mm in diameter were transferred to a similar basal medium containing 2.0mg/l kinetin, 2.0mg/l zeatin riboside, 0.1mg/l Indole-3-acetic acid (IAA) with 0.2% sucrose to induce differentiation.

Three weeks later, calli were transfered to fresh medium, composed as described above but containing 0.1% sucrose.

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.

Thirteen days thereafter, 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.

When root development was clearly evident, established plants were transfered to 'Metromix' potting compost.

When the plants attained a large enough size they were analyzed for mitochondrial and chloroplast composition. Four of these regenerants was found to have the desired cytoplasm i.e. cms conferring mitochondrial DNA having the agarose gel migration pattern shown in lane 3 of Figure 1 and triazine tolerance conferring chloroplast DNA having the agarose gel migration pattern shown in lane 2 of Figure 2.

The fusion products (as females) were crossed with the appropriate maintainer lines (as males).

Several similar crosses were made to increase the amount of seed carrying the desired cytoplasm. The progeny of these crosses were crossed (as females) with the appropriate restorers (as males) to produce triazine tolerant hybrid seed. Plant produced by these crosses were distinguished by ECO RI restriction enzyme fragment pattern analysis of chloroplast and mitochondrial DNA.

Example 4 Fusion of cms Brassica napus Regent (napus cytoplasm) with triazine tolerant Brassica napus cv. 'TT' Regent

Triazine-tolerant 'Regent'

Protoplasts were isolated from leaves as described in example 3 for Candle, except the plants were 36 days old.

cms Regent

Protoplasts were isolated from hypocotyls as described in example 3 for Regent.

Pre-fusion treatment

Regent - fluorescein diacetate (FDA) treatment

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.

'TT' Regent

No treatment.

Fusion

Fusion was carried out as described in example 3. Heterokaryon Isolation

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).

Cultures progressed essentially as described in Example 3.

Three of the regenerated plants were analyzed and found to possess ECO RI digested mitochondrial DNA whose agarose gel, migration pattern corresponded to lane 1 of Figure 1 and triazine-tolerant chloroplasts whose ECO RI digested DNA migration pattern in agarose gel corresponded to lane 2 of Figure 2. Moreover, a 359bp segment was isolated after Mae I digest ion.

These fusion products were crossed with the appropriate maintainers and restorers to produce hybrid seed essentially 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

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. Five were found to possess ECO RI digested mitochondrial and chloroplast DNA whose agarose gel migration patterns corresponded to that of Santana, and so were essentially similar to the parent plant. Four were found to possess ECO RI digested mitochondrial and chloroplast DNA whose agarose gel migration patterns corresponded to that of polima cms B. napus cv Regent. One plant possessed ECO RI digested mitochondrial DNA whose agarose gel migration pattern corresponded to that of polima cms B. napus cv Regent, and ECO RI digested chloroplast DNA whose agarose gel migration pattern corresponded, to that of Santana. This plant and one of the four with polima chloroplasts and mitochondria were compared. There, were no phenotypic differences attributable to these, chloroplast DNA patterns. These plants were crossed with the appropriate B. napus maintainer genotype (in this case, Santana).

Seed from these crosses were planted in the greenhouse and the plants compared to those derived from repeated conventional crossing of polima cms B. napus cv Regent (as female) to Santana (as male). No differences were observed, except that the original fusion-derived plants had taken nine months to create as opposed to thirty-six months for the original conventionally produced plants. Only progeny plants derived from the single fusion product having polima cms mitochondria and the B. napus chloroplasts (from Santana) could be distinguished at any time analyzing mitochondrial and chloroplast DNA. These progeny plants were crossed with the appropriate restorer genotype to produce hybrid seed.

Example 6

An essentially similar technique was used to fuse gamma-irradiated protoplasts of polima cms B. napus cv Regent (having the mitochondrial DNA migration pattern shown in Figure

1, Lane 3, the chloroplast DNA migration pattern shown in Figure

2, Lane 3, and the spring phenotype) with IOA-treated protoplasts of the B. napus breeding line AWR 0009 (having the cam mitochondrial DNA migration pattern shown in Figure 1, Lane 2, the cam chloroplast DNA migration pattern shown in Figure 2, Lane 2, and the winter habit). Forty-two plants were regenerated from four fusion experiments. All forty-two plants had the winter phenotype (i.e. required 8 weeks at 4°C to induce flowering), cp (chloroplast) and mt (mitochondrial) DNA was analyzed from each of the plants. Fourteen were found to contain both mt and cp DNA of cam and so were essentially similar to the AWR 0009 parent plant. Twenty-three had both mt and cp DNA of polima and five had the mt of polima and the cp of cam. There were no phenotypic differences between plants having polima mt and cp and those having polima mt and cam cp. One plant of each type (having polima mt) was chosen and crossed with the appropriate B. napus maintainer genotype (in this case AWR 0009).

Seed from these crosses were planted in the greenhouse and field and the plants compared to those derived from repeated conventional crossing of polima cms B. napus cv Regent (as female) to AWR 0009 (as male). The latter plants had polima mt and cp DNA and the winter habit. No differences were observable between these plants and their progeny and those derived from fusion, except that the original fusion-derived plants had taken nine months to create as opposed to thirty-six months for the original conventionally produced plants. Progeny plants derived from fusions having both polima mt and cp were thus indistinguishable from their conventional counterparts, whereas those having polima mt and cam cp were identifiable at any time by DNA analysis alone.

These progeny plants (derived from crossing the fusion product with the maintainer) were crossed, as females, with the appropriate B. napus restorer genotypes (as males). These crosses yielded commerial hybrid seed.

International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification ~* . (11) International Publication Number: WO 87/ 017

C12N 15/00, A01H 1/02 A3 C12N 5/00, C12Q 1/68 (43) International Publication Date: 26 March 1987 (26.03.

(21) International Application Number : PCT/GB86/00565 (72) Inventors; and

(75) Inventors/Applicants (for US only) : BARSBY, Ti

(22) International Filing Date: 22 September 1986 (22.09.86) Lorraine [GB/CA]; 7 Armbro Avenue, Brampt Ontario L6Y 1W9 (CA). KEMBLE, Roger, J [GB/CA]; R.R. #1, Inglewood, Ontario LON 1

(31) Priority Application Number : 778,685 (CA).

(32) Priority Date: 23 September 1985 (23.09.85) (74) Agent: EYLES, Christopher, Thomas; Batchellor, K & Eyles, 2 Pear Tree Court, Farringdon Road, L

(33) Priority Country: US don EC 1R ODS (GB).

(60) Parent Application or Grant (81) Designated States: AT (European patent), AU, BE (

(63) Related by Continuation ropean patent), CH (European patent), DE (Eu

US 778,685 (CIP) pean patent), DK, FI, FR (European patent),

Filed on 23 September 1985 (23.09.85) (European patent), IT (European patent), JP, (European patent), NL (European patent), SE (Eu pean patent), US.

(71) Applicant (for all designated States except US): ALLE- LIX INC. [CA/CA]; 6850 Goreway Drive, Mississau- ga, Ontario L4V P1L (CA). Published

With international search report.

Before the expiration of the time limit for amending t claims and to be republished in the event of the receipt amendments.

(88) Date of publication of the international search report:

23 April 1987 (23.04.8

(54) Title: PROTOPLAST FUSION PRODUCT AND PROCESS FOR PREPARING SAME

(57) Abstract

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.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify Statesparty to the PCT on the frontpages ofpamphlets publishing international applications under the PCT.

AT Austria FR France ML Mali

AU Australia GA Gabon MR Mauritania

BB Barbados GB United Kingdom MW Malawi

BE Belgium HU Hungary NL Netherlands

BG Bulgaria IT Italy NO Norway

BJ Benin JP Japan RO Romania

BR Brazil KP Democratic People's Republic SD Sudan

CF Central African Republic of Korea SE Sweden

CG Congo KR Republic of Korea SN Senegal

CH Switzerland LI Liechtenstein SU Soviet Union

CM Cameroon LK Sri Lanka TD Chad

DE Germany, Federal Republic of LU Luxembourg TG Togo

DK Denmark MC Monaco US United States of America

ΪT Finland MG Madagascar

Claims

CLAIMS :

1. A process for producing a regenerable Brassica protoplast fusion product, comprising the steps of

A) providing (i) a first protoplast derived from a 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

3) inducing fusion of said first and said second protoplast to produce a regenerable fusion product containing said nuclear genetic material and said first cytoplasmic element.

2. The process according to claim 1 wherein the nuclear genetic material of said first protoplast encodes the winter, habit.

3. The process according to claim 1 or claim 2 wherein said first cytoplasmic element is a genetic determinant of male sterility.

4. The process according to claim 3 wherein said determinant is a mitochondrion determinant.

5. The process according to claim 1 or claim 2 wherein said first cytoplasmic element is a genetic determinant of herbicide tolerance.

6. The process according to claim 5 wherein said determinant is a chloroplast determinant.

7. A process according to claim 1 or claim 2 wherein said first protoplast or said second protoplast contains a second cytoplasmic element, the presence of which can be detected in plants carrying said second element, and said regenerable fusion product contains said genetic material, said first cytoplasmic el'ement and said second cytoplasmic element.

8. The process according to claim 7 wherein said first cytoplasmic element is a mitochondrion genetic determinant of male sterility and said second cytoplasmic element is a chloroplast genetic determinant of herbicide resistance.

9. A regenerable fusion product produced by the process defined in any one of claims 2-8.

10. A regenerable fusion product from which may be grown a Brassica plant having the winter habit.

11. The fusion product according to claim 10 having, in addition, the cytoplasmically conferred herbicide tolerance trait.

12. The fusion product according to claim 10 or 11 having the cytoplasmically-conferred male sterility trait.

13. A regenerable fusion product from which may be grown a Brassica plant having the winter habit, the cytoplasmic male sterility trait and the herbicide tolerance trait said trait being derived from different sources.

14. A method for characterizing cytoplasmic DNA of Brassica tissue comprising the steps of

A) extracting mitochondrion and/or chloroplast DNA from cytoplasm in an amount of Brassica tissue;

B) treating said DNA with ECO RI or Mael to produce a fragmented DNA preparation; and

C) subjecting said fragmented DNA preparation to gel electrophoresis such that a restriction fragment pattern that is characteristic of said cytoplasm is generated.

15. A method according to claim 14 wherein said cytoplasm is selected from the group consisting of B. napus cytoplasm, B. campestris cytoplasm, polima cytoplasm, ogura cytoplasm, R. sativus cytoplasm, D. muralis cytoplasm, B. carinata cytoplasm and B. nigra cytoplasm.

16. A method according to claim 14 wherein said DNA is chloroplast DNA and Mae I is used in step (B).

17. A method according to claim 14 wherein said DNA is mitochondrial DNA and ECO RI is used in step (B).

18. A method according to claim 16 wherein said restriction fragment pattern comprises a band corresponding to 359 base pair segment of chloroplast DNA characteristic of herbicide tolerance-conferring chloroplast DNA.

19. The method according to claim 14 wherein said tissue is extracted from Brassica plant or tissue thereof in an amount sufficiently small to permit continued growth of remaining tissue after tissue extraction.