WO1986007379A1

WO1986007379A1 - Transfer of male sterility in carrots

Info

Publication number: WO1986007379A1
Application number: PCT/US1986/001110
Authority: WO
Inventors: John F. Reynolds
Original assignee: The Upjohn Company
Priority date: 1985-06-11
Filing date: 1986-05-22
Publication date: 1986-12-18
Also published as: JPS63500001A; EP0225916A1

Abstract

Process for transferring male sterility to new varieties of carrots, D. carota. Male sterile lines of carrots are used to produce functionally enucleated protoplasts which are then used to fuse with protoplasts of maintainer lines or forced to accept isolated nuclei from maintainer lines. The allogenic protoplasts are then regenerated into new carrot varieties for use in selective breeding for producing hybrid carrot seed. Products of the process, allogenic protoplasts and regenerated carrot varieties therefrom are also disclosed.

Description

TRANSFER OF MALE STERILITY IN CARROTS BACKGROUND OF THE INVENTION Field of the Invention

This invention concerns the developing of new parental lines of carrots (Daucus carota) . The parental lines are used to produce hybrid seed . Specif ically th is invent ion enables a plant breeder to incorporate the desirable quality of male cytoplasmic sterility (CMS) into a commercially desirable variety of carrot .

Male sterility is of value in carrot hybrid seed breeding because normal flowers are self- and sibling-pollinating . Male sterile carrot lines do not produce viable pollen and cannot self-poll inate . By eliminating the pollen of one parental variety in a cross, a plant breeder is assured of obtaining hybrid seed of uniform quality. At present cytoplasmic male sterility is not readily available in all carrot varieties and commercial producers of hybrid seed have to rely on traditional selective breeding to incorporate CMS into new lines of carrots.

The invent ion descri bed herein invo lves the use of modern biological techniques to hybridize protoplasts of CMS lines wi th e ither isolated nuclei or protoplasts from commercially-desirable maintainer lines. By inserting the nucleus of a male-fertile carrot cell into the cytoplasm of an enucleated CMS cell and by allowing regeneration of this allogenic cell into a mature plant, a carrot plant is obtained having both cytoplasmic male sterility and desirable commerc ial characteristics . These plants are then used as parent lines to develop new carrot hybrids using conventional selective breeding techniques .

In addition to the above process, this invention describes the product of the process , namely, novel strains of male sterile carrot protoplasts and regenerated hybrids therefrom. Information Disclosure

Previous work has established that CMS carrots can be classed into two groups, petaloid and brown-anther-type male sterility. In petaloid CMS the anthers of the affected plants are abnormal in structure and resemble petals . Thompson D.J . , Studies on the Inheritance of Male Sterility in the Carrot , Amer . Soc . Hort . Sci. , 78: 332-338 (1961 ) . In brown-anther-type male sterility the anthers are present but turn brown and fail to produce pollen. Welch, J.E. and Grimball, E.L. Male Sterility in Carrots, Science, 106:594 (1947).

The value of producing male sterile parent lines for use in production in hybrid seed is known and successful work fusing protoplasts of tobacco having CMS has been described. Zelcer A., et al., Interspecific transfer of cytoplasmic male sterility by fusion between protoplasts of normal Nictotiana sylvestris and x-ray irradiated protoplasts of male-sterile N. tabacum. Z. Pflanzenphysiol. Bd. 90.S.397-407, 1978. To the applicant's best knowledge no one has reported fusion of two carrot cells or successful insertion of allogenic nuclei between varieties of carrots. However, intergeneric fusion between the umbilliferae family (of which carrots are a member) has been reported. Dutits, D., et al., Intergeneric gene transfer mediated by plant protoplast fusion. Mole. Gen. Genet. 179:283-288, 1980. In addition, methods for producing protoplasts for carrots are also known along with methods for regenerating the protoplasts into mature plants. Stewart, F.C., et al. Observations on growth and morphogenesis in cultured cell of carrot (Daucus carota L.) Philos. Trans. R. Soc. London Ser. B 273733-53, 1980. Grambow N.J., et al., Cell division and plant development from protoplasts of carrot cell suspension cultures. Plants (Berl.) 103:348-355, 1972.

SUMMARY OF THE INVENTION This invention concerns the application of modern biology towards the development of new carrot varieties. By fusing the protoplasts of CMS carrots with desirable varieties of carrots it is possible to produce a desired parent line of male sterile carrots that when crossed with a second line will produce a hybrid carrot seed.

The processes described herein involve the transferring of cytoplasmic male sterile characteristics into maintainer lines of carrots by either placing the intact nucleus of the maintainer variety into an enucleated protoplast of a CMS carrot variety or by direct fusion of nucleated protoplasts of maintainer varieties with functionally enucleated protoplasts having the CMS characteristic. The nucleated protoplasts may be optionally inhibited with a cytoplasmic inhibitor such as iodoacetic acid or Rhodamine 6-G. The fused protoplasts are then regenerated into plants which are ma inta ined by tradit ional breeding methods and used as parent lines for hybrid seed production.

The advantage of using allogenic nucleus insertion or hybrid cell fusion to produce new CMS parent lines of carrots is in the time saved by avoiding traditional selective breed ing techniques . Select ive breeding of CMS into a desired carrot line typically takes from 10 to 12 years. Cell fusion can achieve the same result in 4 to 5 years . In addition to time , the amount of space and manpower required to plant successive generations is also greatly reduced by these new processes . In addit ion to the processes herein described , novel products comprising allogenic protoplasts of male ster ile carrot var iet ies having nuclei from a maintainer variety are also described.

Lastly, there is described herein cells and hybr id var iet ies regenerated from the allogenic protoplasts . The term "cytoplasmic male sterility" (CMS) is meant to include both forms of sterility , i . e. , petaloid and brown-anther-type CMS.

The term "allogenic" refers to an organism having or containing the nucleus of another organism such that the phrase "allogenic protoplasts" refers to those protoplasts which are enucleated and then forced to accept the nucleus of a nonidentical plant . Allogenic protoplasts may arise by nucleus insertion or by protoplast to protoplast fusion.

The term "cell" refers to a singular unit of living plant material comprising intact cytoplasm, nucleus and cell wall . The term "enucleated" refers to a cell having a nonfunctioning exposing nucleus . This is accomplished by x-ray treatments.

The phrase "significant amount of cytoplasm" refers to that amount of cytoplasm suff icient to confer upon the allogenic cells stable CMS traits over successive generations. DETAILED DESCRIPTION

It should be understood that the detailed descript ion below contains steps that may be var ied in non-crit ical ways for the different var ieties of Daucus carota . Determinat ion of those conditions is well within the ordinary skill of a person practicing this art.

The invention disclosed herein may be preformed using any of a number of publicly available cytoplasmic male sterile carrot lines such as Michigan State University (MSU) 5986 A or MSU 1302 A . There is no preference among the known lines . In addition to selecting a suitable CMS line, the availability of a desirable maintainer line is critical. The maintainer line is a physically normal variety of carrot that when crossed with the CMS line will not restore function to the male flower parts of the second generation. Because the maintainer line supplies the nucleus for the new variety, desirable physical characteristics are also considered when choosing a suitable maintainer line. With respect to CMS lines , suitable maintainer lines are known.

The determination of alternative maintainer lines is accomplished by simple crossing of the CMS line with various male-fertile lines until a suitable maintainer is found. The following lines are examples of CMS and the ir corresponding ma intainer lines : ( 1 ) Michigan State University (MSU) Var . # Car 1209 MS and USDA 6274n; (2) MSU 1558 A and MSU 1558 B; (3 ) MSU 1302 A and MSU 1302 B; and, (4 ) MSU 5931 S and MSU 5931 M.

Alternative CMS lines having utility as starting stock for this invention are available to those familiar wi th the carrot breeding field . State and federal agricultural experiment stations have and are continuing to produce new CMS carrots . Spec if ically , CMS carrot varieties and their maintainer lines are available through the USDA Carrot and Onion Improvement Program, USDA/Hort. Dept . , University of Wisconsin, Madison, WI . Alternative sources include , for example, the Cooperative State Agricultural Experiment Stations at : the University of Florida , Gainesville , FL ; Texas A&M, College Station, Texas ; and the University of Idaho, Moscow, Idaho.

Once selection of CMS and maintainer lines is completed, seed of the desired lines are germinated to create source mater ial for protoplas ts . Seeds are surface steril ized by immersion in a sterilizing solution, preferably dilute sodium hypochlorite, and are germinated in a dar k , mo ist env ironment at an approp riate temperature preferably at 25 °C. The seedlings are used to produce callus culture from which protoplasts are derived. Callus cultures are initiated from root segments o f the seedlings. The segments are excised a few days after germination and placed on to a basal + GR agar medium containing salts, a carbon source and growth regulators [GR] to support growth. A basal medium is a minimal medium containing a complete balance of nutrients for supporting plant cell cultures. The preferred basal medium is given in the example below; however, alternative basal media are known to those in the art. The presence of growth regulator in the basal medium is indicated by GR. The tissue is incubated in the dark at a temperature of 15-32°C, preferably from 26-28°C.

Callus culture may also be derived from CMS carrot plants having either anthocyanin-containing or white phenotypes. The roots are first surface sterilized in a sterilizing solution such as sodium hypochlorite. Small discs of approximately 1-4 cubic millimeters are excised from the cambium region and placed on basal + GR agar medium. The root segments are then incubated from 15-32°C preferably from 26-28°C. in the dark.

After about 4-8 weeks, subcultures of the callus tissue from either root segments or cambium sections are made. Small samples from 0.1 to 0.3 gm fresh weight of callus tissue are transferred to a basal agar + GR medium and incubated at similar temperatures under a cool white fluorescent light for 16 hours per day. Callus stock can be maintained by subculturing under similar conditions every two months. Callus tissue is then used to produce suspension cultures from which cell material for protoplast production is obtained.

Suspension cultures are initiated by transferring approximately 0.1 to 0.3 gm of callus tissue into 25 ml of basal + GR liquid medium in appropriate flasks. The cultures are incubated on a horizontal gyrotory shaker under cool white fluorescent lights at 25-28°C. Suspension cultures can be subcultured every 12-16 days by transferring 5 ml of the suspension to 25 ml of fresh basal + GR liquid medium in an appropriate size flask.

Protoplasts are prepared from cell suspensions by mixing the suspension with a protoplast isolation medium containing basal medium, cellulase, pectinase, and a suitable osmoticant such as mannitol. After 3-4 hours of incubation in the dark at 30°C on a rotary gyrator, released protoplasts are separated from cell clumps and other debris by filtration through screens and/or by centrifugation of the protoplasts through or onto an osmotically stabilizing dense cushion such as Lymphoprep R (Nygaard A/S Co., Norway). The technique described above is known and fully described by Larkin, P.J., Planta (Berl.) 128:213-216, 1976. In developing procedures for handling protoplasts, it is necessary to ascertain the quality and quantity of the protoplasts being formed. Such procedures permit the determination of cell viability, the determination of remaining cell wall material and the simple counting of protoplasts. The particular methods used in the disclosed process are described in the example below. Alternative methods of equal effectiveness can be found in general references on protoplast research such as in the Handbook of Plant Cell Culture. Vol. 1. Techniques for Propagation and Breeding. Ed. D.A. Evans, W.R. Sharp, P.V. Ammirato and Y. Yamada. Macmillan Pub. Co., N.Y., pp. 124-177, (1983). After determination of quality and quantity, the protoplast culture is washed three times in wash medium by repeated low speed centrifugation (700-800 g) and resuspension. Wash medium is a mixture of basal medium with a suitable osmoticant such as mannitol.

Introduction of nuclei from maintainer lines into protoplasts from CMS lines can be achieved by insertion of isolated nuclei into enucleated CMS protoplasts or by actual fusion of a maintainer protoplast to an enucleated male sterile protoplast. The preferred technique is to fuse x-ray treated protoplasts from CMS lines with protoplasts of maintainer lines after treatment of the maintainer protoplast with a cytoplasmic inhibitor.

To isolate nuclei from the maintainer variety, protoplasts are suspended in basal medium, pelleted by low speed centrifugation and resuspended in a 2% gum arabic solubilized in a nucleus stabilizing buffer. Nucleus stabilizing buffers are capable of maintaining intact nuclei and are known in the literature. The specific buffer used herein is designated RS and is fully described by Cress, D.E. et al., Planta. 143:241-253. 1978 and in the example below. Protoplasts are then loaded into a French pressure press. Protoplasts are burst by slowly increasing the pressure to approximately 1000 to 2500 psi followed by slow release and aseptic collection.

The burst protoplast solution is layered on a nucleus isolation buffer consisting of RS buffer and a substant ially greater (1 0% ) proportion of gum arable . The two solutions are then centrifuged at a force sufficient to gently pellet the nuclei yet leave other organelles in suspension. The nuclei are resuspended in 2% gum arabic in RS buffer and remaining cell debris removed by two more centrifugations and resusp ens ions . The final nucle i pellet is resuspended in a small amount of 2% gum arabic in RS buffer .

Although obtaining nuclei from protoplasts is preferred, nucle i can also be obtained from cells or cell suspensions rather than from protoplasts . A cell suspension in basal + GR liquid medium is centrifuged and the cells pelleted. The cells are then resuspended in 2% gum arabic in an appropriate nucle i stabilizing buffer such as RS. The cells are then placed in a French pressure cell and burst by slow release from a pressure of 2000-4000 psi . The burst cells are then screened through successively smaller Nitex or equivalent screens and the resulting organelles are layered onto a single step-gradient of 10% gum arabic and spun at an appropriate force to gently pellet the nuclei and leave the other organelles in suspension.

Protoplasts from CMS carrot lines must be enucleated pr ior to hybridization. Enucleation is achieved by exposing the protoplasts to levels of high voltage x-rays that leave the cytoplasm functioning yet effectively destroy the nucleus ' ability to divide and regulate the cell. Total dosages effective to enucleate a CMS carrot protoplast range from anywhere from 5 kiloroentgen [kr] to 100 kr depending on the var iety be ing used.

To determine the appropriate range of x-ray dosage , the minimum level of x-ray radiation to kill 100? of the protoplasts must be determined. The percent kill is determined by placing the cells in regeneration medium and counting colony formation. To obtain the most accurate x-ray dosage for 100% kill rate , it is desirable to use a cell feeder layer, conditional cell suspension medium or an appropriate low density ce ll rescue procedure. This will optimize conditions for development of low populations of unaffected celle. Upon determination of the minimum dosage required to kill 100% of the cells , protoplasts are exposed to 5 increments of x-ray levels, the minimum dosage, 10 and 20 kr above and below the minimum dosage . Protoplasts from each exposure level are used for hybridization. Cells are used immediately after exposure to x-rays.

As described above , renucleation of the functionally enucleated protoplasts can be accomplished with either isolated nuclei or intact protoplasts .

To insert isolated nucle i Into protoplasts , the functionally enucleated protoplasts are taken immediately following exposure to x-rays and mixed with a 5 to 10 fold number excess of nuclei. The mixture is centrifuged and resuspended in wash medium. To this mixture is added fusion-inducing substances that promote the uptake of nuclei by a protoplast cell membrane. Such fus ion-inducing substances are known and are generally described in the Handbook of Plant Cell Culture, supra, pp. 291-321 . The preferred buffer utilizes the polyethylene glycol (PEG) fusion procedure described by Kao & Michayluk. Planta (Berl. ) 1 15: 355-367, 1.974.

Protoplast to protoplast fusions are the preferred procedures. These procedures avoid the need to extract intact nuclei. There are two procedures that will accomplish cell fusion in a manner satisfactory for carrots. The most preferred utilizes cytoplasmic inhibitors.

In the first method, fusion of protoplasts of enucleated and nucleated protoplasts is accomplished using a PEG-containing-fusion buffer as described by Galum, E. Somatic Cell Fusion for Inducing Cytoplasmic Exchange: A New Biological System for Cytoplasmic Genetics in Higher Plants. In Plant Improv. and Somatic Cell Genetics. Ed. I.K. Vasil et al., Academic Press, pp. 205-220. Successful transfer of the CMS characteristic when using fusion between intact protoplasts depends on the complete segregation of the cytoplasmic material upon later cell divisions. Because of the possibility of inadequate segregation of cytoplasm and of unfused cells being regenerated, large numbers of regenerated plantlets must be tested in field trials to determine the success of this process.

In the most preferred method, the cytoplasm of the maintainer protoplast is inhibited by treatment with cytoplasmic inhibitors such as iodoacetic acid or Rhodamine 6-G as respectively described by Sidorov, V.A. et al., Chloroplast Transfer in Nicotiana based on Metabolic Complementation between Irradiated and Iodoacetate Treated Protoplasts . Planta 1 52 : 341 -345 , 1981 ; and Ziegler and Davidson, Illumination of Mitochondrial Elements and Improved Viability of Hybrid Cells , Somatic Cell Genetics, Vol. 7 # 1 , p. 73-88. To determine the optimal dosage of cytoplasmic inhibitor for a particular var iety, a sensitivity range must be obtained. As with x-rayed cells, a low cell density rescue procedure would be desirable to determine the optimal killing level of the cytoplasmic inhibitor .

It is necessary to treat CMS and fertile protoplasts wi th a range of x-ray or cytoplasmic inhibitors because of the inherent variation in protoplast sens it ivity to the treatments. For any part icular protoplast preparation, it is impossible to accurately predict the minimum amount of x-ray dosage or exposure to cytoplasmic inhibitors that will achieve a 100% kill. Therefore fusions of multiple levels of x-ray treated and cytoplasmically inhibited protoplasts are conducted in an a matrix experimental design which best identifies the inhibition levels for b iochemical complementat ion. By using a matrix experimental design wherein maintainer protoplasts are exposed to varying exposures of cytoplasmic inhibitors and fused with CMS protoplasts exposed to vary ing amounts of x-ray radiation, one can select the populat ion having maximum numbers of allogenic cells. This is accomplished by sampling the chemically inhibited and x-ray irradiated cells from each trial pr ior to fus ion. Populat ions having restored viability after fusion but no viability prior to fusion are presumed to be allogenic cells.

Fusion with x-ray treated cultures is accomplished in the manner described above for non-cytoplasmic-Inhibited protoplants . This procedure is preferred because the forced selection for allogenic protoplasts avoids growth of unfused cells and the numbers of colonies and plants selected for field trials is substantially reduced.

The regeneration of allogen ic protoplas ts into rault icellular colonies takes place in a regeneration medium containing a complete balance of salts, a variety of carbon sources, osmoticants and growth stimulants . Several media are known to promote regeneration of protoplasts for a variety of plant spec ies. Each medium must be tested for its ability to regenerate the particular protoplasts used. Such experimentation is well within the ability of a person skilled within the art . The preferred regeneration medium is designated A & S and is fully described in the example below. Regeneration of allogenic cells is optionally encouraged by the use of low density cell culture methods such as feeder layers, conditional medium or alginate encapsulation. Fused protoplasts growing in a regeneration medium quickly regain cell walls and form microscopic colonies wi thin 2-4 weeks . The medium is changed weekly and after 3-4 weeks the colonies are plated out onto agar containing an embryonic basal medium. Embryonic basal media promote embryo formation and contain a complete balance of nutrients but do not contain the variety of carbon sources found in regeneration media. The preferred embryonic medium is the same basal medium used to culture callus tissue but without growth regulators. Colonies forming embryos are removed and placed in tubes containing basal agar medium. Once a suitable size is reached, the plant lets are transferred to soil and grown in a mist chamber . Established plants are vernalized to force flowering and pollinated with a maintainer line , preferably the same line that served as the source of nuclei. For the production of hybrid seed, the plants would be fertilized with pollen from a second desirable variety. Resulting seed is tested for stability of the CMS and other des ired horticultural traits using convent ional breed ing techniques known in the art.

EXAMPLES The following detailed example describes how to prepare allogenic protoplasts of D . carota and to perform the various processes of the invention. The example is to be construed as merely illustrative , and not as a limitation of the preceding disclosure in any way whatsoever. Those skilled in the art will promptly recognize appropriate variations from the procedures related to media contents , growing conditions, and other techniques.

I . Callus and Cell Culturing A. Culture Media

The basal medium used to culture callus tissue contains the salt formulat ion of Murashige and Skoog (Physiol. Plant. 15: 473-497 , 1962) and the following in mg/1 : sucrose, 20 ,000; thiamine-HCl , 0. 1 ; i- Inositol, 100; nicotinic acid, 0.5; pyridoxine -HCl , 0.1 , glycine , 3.0. Basal + GR medium contains growth regulators 2 ,4-dichlorophenoxy acetic acid (2, 4-D) , 1 .0 mg/ml ; and kinetin , 0.2 mg/ml, in addition to the components in the basal medium. Agar medium contains 7 g/1 Sigma agar. Media pH is adjusted to 5.5 prior to autoclaving or addition of agar. Liquid media is dispensed 50 ml into 125 ml delong flasks. Agar media is dispensed, after melting , into 25 x 150 mm tubes at a volume of 20 ml/tube . Tubes and flasks are capped and autoclaved for 15 min at 121 °C, 1 .05 kg/cm².

B . Initiation of Callus and Suspension Cultures The seed from D. carota, Michigan State University var iety #Car

1209 MS having cytoplasmic male sterile characteristics and maintainer l ine USDA 6274n are steril ized by immers ion in a 1 0% sod ium hypochlorite solution for 1 5 min and germinated in the dark on moistened sterile filter paper at 25°C . To initiate stock callus, root segments are excised after 5-7 days, and placed on basal + GR agar medium and incubated at 26-28 °C in the dark.

After 6 weeks, approximately 0.2 g fresh weight of the callus tissue is excised from root segments, and transferred to a basal + GR agar medium and incubated at 25 °C under cool white fluorescent lightfor 16 hours per day. Callus stock cultures are ma inta ined by subculturing approximately 0.2 g of tissue on basal + GR agar medium every two months.

Suspension cultures are initiated by transferring approximately 0.2 g callus tissue into 50 ml of basal + GR liquid medium in 125 ml delong flasks. Cultures are incubated on a horizontal gyrotory shaker (Model G-2 , New Brunswick Sc ientific Co. , New Brunswick, NJ) set at 150 rpm at 25 °C under cool white fluorescent light . Suspension cultures are subcultured every 14 days by transferring 5 ml of cell suspension to 50 ml of fresh basal + GR liquid medium in a 1 25 ml flask. II . Protoplast Isolation

Cell suspensions are pelleted by centrifugation and resuspended in wash medium comprising basal medium and 0.56 M mannitol. An equal amount of enzyme so lution contain ing 2.0% cellulase R-1 0 , 2.0% cellulase RS, and 1 .0% Pectolyase Y23 in basal med ium at pH 5.8 having 0.56 M mannitol as an osmoticant is added to the cell suspension in wash medium. The cell suspens ion is Incubated in the presence of the enzymes at 28-30 °C on a gyrotory shaker at 75 rpm. The f irst protoplasts are released after 1 hour and the process is completed after 3-4 hours in the enzyme solution. Some tight cell clumps will never produce protoplasts and are removed by filtering through a 25 μm mesh. Further purificat ion is done by layer ing 10 mis o f crude protoplast suspension on top of 5 mis of Lymphoprep in a 15 ml centrifuge tube and spinning at -200xg for 10 min. The purif ied protoplasts are collected from the medium-LYMPHOPREP interface with a pasteur pipette . After washing 3 times with wash medium at 100xg for 5 min , protoplasts are ready for counting , plating or other manipulation.

III . Preparative Procedures for Protoplast Isolation

A. Cell Viability Determination

Protoplast viabili ty may be determined by the fluoresce in diacetate method of Widholm (Stain Technol. 47: 189-194 , 1972) . The fluorescein diacetate staining solution (0.01 %) is prepared by diluting a 5 rag/ml acetone solubilized stock solution of the dye with protoplast culture medium. This preparation is usable for only a few hours and should be made fresh. Protoplast viability is tested by mixing equal amounts of stain solution and cell suspens ion. After 5 minutes cells are placed in a haemocytometer and examined using an Olympus Vanox fluorescent microscope equipped with exciting filter , 330-500 μm, and barrier filter , >460 nm. Living cells fluoresce yellow-green ; dead cells do not fluoresce.

B . Cell Wall Determination Liquid Calcolfluor White ST (American Cyanamid Co., Wayne, NJ, USA) is diluted 100x with protoplast culture medium and mixed with equal amounts of protoplast suspension. After several minutes, cells are examined under a fluorescent microscope at the same filter setting as for viability determination. Only cells with undigested cell walls will show fluorescence.

C. Cell Counting

Quantitative es timat ions of protoplasts are carried out by a Fuchs-Rosenthal haemocytometer. IV. Protoplast Plating and Culturing Isolated protoplasts at a concentration of 0.5-1 .0 x 10⁵ cells /ml are plated in a regeneration medium and placed in a dark growth chamber at 25°C for 5-7 days. Regeneration medium contains basal medium plus 150 mg/1 D-xylose , 150 mg/l D-arabinose, 100 rag/1 glucose, 34 g/1 sucrose, 45.5 g/1 sorbitol, 0.01 mg/1 dimethylallyladenine and 0.2 mg/1 2,4-dichlorophenoxyacetic acid (pH 5.8 ) . The cultures are then moved to indirect low light.

V . Isolation of the Nantes Nucle i

A. Protoplasts as Source Material

Isolated protoplasts are incubated at high density overnight in protoplast culture media. Protoplasts are spun at 700 xg for 5 min and resuβpended in 2% gum arabic in RS buffer containing 0.4 M sucrose , 5 mM 2 ,N-morpholinoethane sulfonic acid, 50 μg/ml DTT DL-dithiothreitol, .15% 2-ethylhexanol, 6 mM magnesium acetate, 25 mM potassium chlor ide ( pH 6.4 ) and 0.5% dimethyl sulfoxide are incubated at room temperature for 2 hours. Protoplasts are loaded into a French pressure cell press (Amer ican Instrument Co. , Inc . , Silver Springs, MD) . Protoplasts need not be adjusted to any spec if ic dens ity. A steril ized 50 ml plast ic centrifuge tube with a rubber stopper is inserted over the release nozzle to catch the burst protoplasts aseptically. To burst the protoplasts , the pressure is slowly increased to 2000 psi followed by release into the receiving tube. Samples of the burst protoplast containing intact nuclei are inspected under a microscope to determine the optimal bursting conditions.

Five ml of the burst protoplast solution are layered onto 10 ml o f 10% gum arabic in RS buffer in a 15 ml Falcon plast ic centrifuge tube and centrifuged at 700 xg for 20 min . The pellet is resuspended in 2% gum arabic in RS buffer and centrifuged at 700 xg for 5 min to wash cytoplasmic material free from the nucle i. This last step is repeated three times. The final nuclei pellet is resuspended in 2 ml of RS buffer containing 2% gum arabic . A 100 ul sample of nuclei is stained with 50 ul Hoechst #33342 (1 .0 g/1 stock solut ion in water ) , and counted under a Fuchs-Rosenthal haemocytometer. Pr ior to use for fusion, the nuclei are pelleted and resuspended in wash medium.

B. Cell Suspensions As Source Material Established cell suspensions are concentrated by centrifugation.

Culture medium are discarded and cells resuspended in 2% gum arabic in RS buffer. Cells are placed in the French pressure cell and burst at 2-4000 psi . Lysed cells are screened through 117 , 61 , 43 , 25 , 15 , and 10μ Nitex screens and layered on a 11 , 15 , and 20% gum arabic viscosity step gradient and centrifuged at 900 x g for 12 min . The pellets are examined for purity of nucle i by sta ining in Hoechst #33342 as previously described.

VI . Preparation of CMS Cytoplasm by Protoplast X-Ray Irradiation Protoplasts are isolated from established CMS callus cultures of

MSU 1209MS as descr ibed above and suspended in wash medium. After cell counting and viability testing, a number of protoplasts are irrad iated by a MG-301 High Voltage X-ray machine made by Philips Elektronik

Industrie GMBH, Hamburg, Germany. Each time before using, the x-ray machine is standardized with a Victoreen probe, ( She ller -Globe Corp. ,

Victoreen Instrument Div . , Cleveland, OH) . The protoplasts are exposed to increments of 10, 13, 16 , and 19 kiloroentgens . Protoplasts from each exposure are used in the hybridization procedures described below.

VII . Hybridization

A . Uptake of Isolated Nucle i by CMS Protoplasts The polyethylene glycol (PEG) fusion procedure used was modified from that descr ibed by Kao and Michayluk. Planta (Berl. ) . 115: 355-367, 1974. X-ray treated protoplasts and isolated nuclei in wash medium are mixed at a number ratio of 1 : 5 or 1 : 10 to give a final protoplast concentration of 2 x 10⁶ cells in 0.5 ml suspension. Two ml of the PEG solution consisting of 22.5% PEG 6000 (Sigma) , 10 mM CaCl₂ and 4% sucrose (pH 5.8-6.0) are added to the protoplast mixture, followed by gentle mixing and incubation for 10 min at room temperature . The PEG solution is diluted out by gradually adding aliquot port ions of a washing medium comprised of basal medium and 0.56 M mannitol. The washing medium (12.5 ml) is added successively in 0.5 , 1 .0 , 2.0 , 2.0 , 3.0 and 4.0 ml amounts at 5 min intervals. The protoplasts are mixed by gently rolling after each dilution. The PEG is removed in a final wash by pelleting and resuspension In regeneration medium.

B . Protoplast to Protoplast Fusion The preferred protoplast to protoplast fusion procedure utilizes cell inhibitors to optimize maintainer cell to CMS cell fusions. Cytoplasmic inhibitors are added to aliquot portions of maintainer protoplast suspensions. Varying exposures are tested. Iodoacetic acid is tested at a final concentration of 0.1 mM and the cells incubated at 0°C for 5, 10, 20 and 30 minutes. The preferred cytoplasmic inhibitor is Rhodamine 6-G which is tested at 1 μg/ml at 0°C for 20, 40, 60 and 80 minutes. After exposure is completed, the cells are pelleted by centrifugation and resuspended in wash medium. -

All four levels of x-ray treated CMS protoplasts and four levels of cytoplasmically- inhibited fertile maintainer protoplasts are fused in a matrix experimental design (16 treatments for this example) to cover all sensitivity variations. The inhibited maintainer protoplasts and samples of x-ray treated CMS protoplasts are mixed at 1:1 cell ratios to a final density of 2 x 10⁶ cells in 0.5 ml wash medium. Populations having viable cells after fusion and no viability prior to fusion are presumed to be allogenic populations.

Fusion is achieved by the addition of 2 mis of 22.5% PEG 6000 (Sigma), 10mM CaCl₂ and 4% sucrose at pH 5.8-6.0 in wash medium. The cell3 are incubated for 10 min at room temperature and then washed free of the fusion buffer by diluting with 12.5 ml of wash medium added in 0.5, 1.0, 2.0, 3.0, 4.0 ml portions at 5 minute intervals. The fusion buffer is removed in a final wash by pelleting and suspension in regeneration medium. VIII. Protoplast Culture Plating and Callus Regeneration

Fused protoplasts are cultured at an initial density of 1 x 105 protoplasts /ml in regeneration medium. Regeneration medium contains basal medium plus 150 mg/1 D-xylose, 150 mg/1 D-arabinose, 100 mg/1 glucose, 34 g/1 sucrose, 45.5 g/1 sorbitol, 0.01 mg/1 dime thy la lly laden ine and 0.2 mg/1 2,4-dichlorophenoxyacetic acid (pH 5.8). The protoplasts are incubated in dark growth chambers at 25 °C for 5-7 days then moved to low intensity fluorescent light at approximately 26 °C. After 3-4 weeks, protoplasts will divide and form small microscopic colonies. The colonies are placed in basal medium. The basal medium is changed twice over a 2 week period. After 2 weeks the colonies are plated out on basal agar medium. After 1-2 weeks developing embryos and 3hoot3 are isolated and cultured in tubes with basal agar medium having 0.3 mg/1 naphthylene acetic acid for root developraent.

IX. Development of Differentiated Plantlets

Mature plants are transferred out of the tubes to soil into a greenhouse misting chamber . Established plants are vernalized by plac ing in a cold environment (40°C) for 6-8 weeks. Plants that bolt and flower after vernalization are examined for male sterility and the male sterile plants are crossed with maintainer lines in a screened cage . Pollination is accomplished by insects. Seed is harvested and planted to determine the stability of the newly developed CMS line.

Claims

1. A process for transferring male sterile characteristics into maintainer varieties of Daucus carota comprising: the combining of a nucleus of a maintainer variety of D. carota with the enucleated protoplast of a cytoplasmic male sterile variety of D. carota.

2. The process of claim 1 wherein an isolated nucleus of a maintainer variety of D. carota is inserted into the enucleated protoplast of a cytoplasmic male sterile variety of D. carota.

3. The process of claim 1 wherein a nucleated protoplast of a maintainer D. carota variety is fused with a enucleated protoplast of a cytoplasmic male sterile variety of D. carota.

4. The process of claim 3 wherein the nucleated protoplast of a maintainer D. carota variety has been treated with a cytoplasmic inhibitor prior to fusion.

5. The process of claim 4 wherein the cytoplasmic inhibitor is selected from the group comprising iodoacetic acid and Rhodamine 6-G.

6. A strain of allogenic carrot protoplasts having a significant amount of cytoplasm from a cytoplasmic male sterile variety and the nucleus from a maintainer variety.

7. The strain of carrot protoplasts of claim 6 wherein the nucleus is selected from the Nantes variety of carrots.

8. The strain of cells regenerated from protoplasts of claim 6.

9. The variety of allogenic carrot regenerated from the strain of claim 8.

10. A variety of allogenic carrot consisting of cells having the cytoplasm of a cytoplasmic male sterile variety and the nucleus of a maintainer variety.

11. The variety of allogenic carrot seed consisting of cells as described in claim 10.

12. The hybrid of carrot originating from one or more crosses involving a parent line produced by a process according to claim 1.