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WO1992017598A1 - Production d'especes de soja transgeniques - Google Patents

Production d'especes de soja transgeniques Download PDF

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Publication number
WO1992017598A1
WO1992017598A1 PCT/US1992/002226 US9202226W WO9217598A1 WO 1992017598 A1 WO1992017598 A1 WO 1992017598A1 US 9202226 W US9202226 W US 9202226W WO 9217598 A1 WO9217598 A1 WO 9217598A1
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Prior art keywords
protoplasts
medium
plants
protoplast
calli
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PCT/US1992/002226
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English (en)
Inventor
Jack M. Widholm
Sarwan K. Dhir
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The Board Of Trustees Of The University Of Illinois
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Publication of WO1992017598A1 publication Critical patent/WO1992017598A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated

Definitions

  • the present invention relates to a process for transforming soybean plant cells and the regeneration of said cells to produce transgenic soybean plants. BACKGROUND OF THE INVENTION
  • gene transfer techniques for plant species is of great interest and value to plant breeders because it can be used for the rapid transfer of beneficial genetic traits to plants.
  • gene transfer techniques for leguminous plants is of commercial interest because they facilitate the development of new cultivars with improved disease resistance, tolerance to specific herbicides and increased nutritional value.
  • Recombinant DNA techniques are being used to transfer foreign genes into agronomically important crops and to study the expression and regulation of genes in plant systems. The meaningful application of these techniques is dependent upon efficient transfer of genes into cells capable of regenerating into fertile plants. Numerous methods have been developed for transferring genes into plant tissues including Agrobacterium-mediated transfer, direct DNA uptake, microinjec- tion, high-velocity microprojectiles and electroporation.
  • Agrobacterium-mediated gene transfers are by far the most widely used gene transfer techniques.
  • the use of Agrobacterium strains for gene transfers can be limited.
  • dicotyledonous species such as leguminous plants are susceptible to Agrobacterium infections, its use for transformation is limited due to the lack of efficient regeneration procedures for transformed tissues.
  • Patent Number 4,945,050 ('050 patent) issued July 31, 1990 to Sanford et al., which is incorporated herein by reference, provides a review of the mechanisms and limitations of direct DNA uptake, cell fusions and microinjection as methods of introducing foreign genes into cells.
  • the '050 patent relates to a method of introducing foreign substances into living cells using microprojectiles. While the '050 patent teaches a method of transforming many cells simultaneously, problems associated with microprojectile transformation include: the high mortality of transformed cells; the complexity of equipment needed; and, the difficulties in operating the equipment to efficiently perform the method.
  • the present invention relates to transgenic soybean plants.
  • Soybean species have been transformed at low frequency using the Agrobacterium or by particle bombardment method described above.
  • Electroporation one of the direct DNA transfer techniques, has been used to stably transform a number of plants. Soybean transformation using electroporation and stable integration of genes in the calli have been reported, but efforts to regenerate plants are unsuccessful.
  • Wei and Xu Plant Cell Reports 7:348-351 (1988) reported plant regeneration from soybean protoplasts. However, the efficiency reported is low and did not use commercially important strains that are transformed by electroporation.
  • the present invention relates to an improved method of producing transgenic soybean plants by transforming soybean plant cells by electroporation and regenerating the transformed cells into mature soybean plants.
  • cells of commercially important soybean varieties can be transformed by electroporation in a stable and highly efficient manner to introduce important genetic material and the resulting transformed cells can be regenerated at high efficiency to mature soybean plants.
  • many cells can be transformed simultaneously using relatively simple techniques requiring relatively simple equipment resulting in a high survivability of transformed cells which can be regenerated into mature transgenic soybean plants at high efficiencies.
  • the present invention relates to a method of generating transgenic soybean plants comprising the steps of: preparing protoplasts from soybean cotyledons; inserting foreign DNA into the prepared protoplasts by electroporation, the foreign DNA comprising a useful gene; culturing the electroplated protoplasts in medium to induce cell growth, colony formation and calli generation; and regenerating plants from calli.
  • electroporation mediated DNA transfer into protoplasts prepared from immature cotyledons is a highly efficient transformation method for soybean.
  • Previous reports on soybean transformation showed the presence of chimeric sectors in the transgenic plants produced by Agrobacterium and particle acceleration methods. This may be either due to cross protection of non-transformed tissues by transformed ones or multiple transformation events within a cell population that gives rise to callus or plants.
  • the protoplast-electroporation system allows for the selection of the transformed single cells which can divide and give rise to regenerable callus forming transgenic shoots. Based on the total number of microcalli formed with and without selection, a relative transformation frequency of 21.4-23.0% can be obtained, corresponding to an absolute transformation frequency of 5.7-6.8 x 10 .
  • the transformation frequency achieved for the soybean variety Glycine max is higher than that reported earlier for other protoplast culture systems. Only with two other plant systems, N. tabacum and Oryza sativa have higher transformation frequencies (up to 1 x 10 ]) been achieved by using a combination of PEG and electroporation or by electroporation only.
  • Plating and selection methods along with several other factors are important criteria in the development of a stable transformation and regeneration system.
  • an agarose-embedding system is employed to immobilize the transformed cells and therefore essentially eliminate the possibility of multiple recovery of single events or recovery of 'false positives' due to inadequate selection pressure.
  • Another element of the present invention is use of a selection marker which confers sufficient sensitivity to transformed cells carrying the chimeric gene construction.
  • hygromycin is employed as the selection agent.
  • Hygromycin as a selection agent has been used in other systems, i.e. Arbidopsis, orchardgrase and rise.
  • a transgene construct according to the present invention comprises a desired gene which confers a desired trait on the transgenic soybean plants which contain it.
  • the present invention embodies the successful transformation of soybean protoplasts via electroporation and recovery of transgenic shoots. Additionally, a plant regeneration system from protoplast derived transformed calli is disclosed.
  • plasmids which contain the genes to be introduced are constructed. Plasmids must contain the necessary genetic elements for expression in plant cells. These elements include an operably linked promoter and polyadenylation addition signal.
  • the construct optionally contains a gene which encodes a selectable marker.
  • Immature pods are collected from 60-80 day old soybean plants and surface sterilized. Cotyledons, 2-7 x 2-3 mm size are cut transversely into 0.5-2 mm thick sections, preferably 1-2 mm thick sections, and preplasmolyzed in CPW 13M. Preplasmolysis of the tissue in CPW 13M makes the plasma membrane shrink away from the cell wall, thus leaving space for enzyme to enter and act; the enzymatic treatment thereby becomes more effective. Washing the tissue helps in removing the damaged tissue thus the ultimate protoplast preparation is cleaner. The tissue is incubated in enzyme solution for about 4-6 hr in the dark.
  • the enzyme solution consists of 1.5% (w/v) Cellulase "Onozuka” RIO, 0.2% Pectolyase Y23 dissolved in CPW 9M (pH 5.8).
  • CPW 9M CPW 9M
  • the combination of Cellulase "Onozuka” R10 (1.5%) and Pectolyase Y23 (0.2%) has been found to be the optimum, because the viability of protoplasts was highest (about 85- 93%), and the yield adequate (5-6 x 10 6 per gm fresh wt.).
  • Use of Pectolyase Y23 seems to be necessary to obtain high yields of viable protoplasts.
  • Protoplasts vary in size from 20-35 ⁇ m in diameter. Protoplasts are rarely released within the first two hours and incubation longer than six hours reduces the viability of protoplasts greatly. The most suitable duration of incubation is 4-6 hr. Enzyme combination used for preparing protoplasts is somewhat critical. Higher concentration of enzyme and longer incubation period are usually deleterious to protoplast preparations and may affect plating efficiency and plant regeneration. A much lower concentration of cellulose (1.5%) and use of Pectolyase Y23, in particular seems to be helpful in isolating protoplasts and in a relatively much shorter 4-6 hr compared to 18 hr time period. Cotyledon age and size are important factors influencing protoplast yield and viability. Optimum protoplast yield and viability is obtained from cotyledons of 3-4 x 2 mm in size. Protoplast release is poor from cotyledons of very large size (6-7 x 3 mm).
  • the released protoplasts are filtered through sieves and pelleted by centrifugation. At least 43 ⁇ m sieve is used. Preferably an additional step is to filter with a 74 ⁇ m sieve also because it makes the process of removing debris more efficient.
  • the purpose of centrifugation is to get a pellet of living, uniform protoplasts. Protoplasts are washed twice by resuspending in either CPW 9M or KP8 medium. The washed protoplasts are then purified by floating over 23% (w/v) sucrose overlaid with 2.0 ml of KP8 medium and centrifuged. Protoplasts can be gently removed with a Pasteur pipette from the interface.
  • KP8 medium for washing and purification.
  • protoplasts are floated on sucrose (23%) there is no deleterious effect on viability.
  • Ficoll (13%) is used, 100% of the protoplasts burst within a few hours.
  • Sucrose concentration can be varied from 21-23%, without any major effect, washing after flotation is necessary to remove sucrose otherwise it can change the osmoticum and have deleterious effect on protoplast preparation.
  • Protoplasts are resuspended at the density of 1-2 x 10 6 /ml in electroporation buffer (10 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM CaCl 2 2H 2 O and 0.2 M nannitol).
  • electroporation buffer 10 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM CaCl 2 2H 2 O and 0.2 M nannitol.
  • HBM and HBS which are the same as the buffer described above except with 1 mM Hepes and 29 mM Hepes, respectively.
  • KP8 and 14 mM CaCl 2 7H 2 O and sodium phosphate buffer also work.
  • the samples are transferred to precooled, presterilized electroporation vessels fitted with electrodes.
  • An electric field of 500 V/cm is applied by a single discharge of a 1000 ⁇ F capacitor that is precharged with an electrophoresis power supply.
  • Aliquots electroporated without DNA and aliqouts treated with plasmid DNA without electroporation serve as controls.
  • Heat shock treatment given to freshly isolated protoplasts prior to electroporation increases the percentage of dividing protoplasts observed by day 14. The stimulatory effect of heat shock on protoplast division is reflected in the increased number of colonies in control cultures and of the resistant colonies which develop following exposure of protoplast derived cells to selection medium.
  • protoplasts are cultured for about 2 weeks without selection if selection is to be performed.
  • Protoplast density is adjusted to 2.5 x 10 5 protoplasts/ml in KP8 medium containing 2% Ficoll (Type 400-DL), 40 mM MES.
  • Use of 2% ficoll (type 400 DL) in the liquid culture medium prevents the protoplasts from adhering to the surface of the petri dish. Otherwise along with the dead protoplasts the dividing ones also settle down and the toxic substances released by the dead ones may affect the growth of dividing colonies.
  • Use of 40 mM MES buffer in the liquid medium helps in stabilizing the pH of the liquid medium (which usually drops down from 5.8 to 3.9) in the first 7 days of culture.
  • selection medium refers to medium supplemented with compounds that are lethal to all cells except those having the protein produced by the expression of the selection marker.
  • the protoplast derived transformed cells are selected by applying either liquid K8/selection medium, i.e., K8 medium supplemented with compounds which are lethal to all cells except those transformed with the selection marker, or the cells are resuspended in an equal volume of 1.2% Sea-plaque agarose (LMT, FMC Corp., ME, USA) gelled K8/selection medium. Solidified agarose cultures are cut into small beads and submerged in liquid K8/selection medium. In liquid medium, the influence of initial protoplast density reveals that at densities lower than 2.5 x lO ⁇ ml, cell budding can be frequently observed. Plating efficiencies of 38-50% can be observed after 7 days in culture.
  • liquid K8/selection medium i.e., K8 medium supplemented with compounds which are lethal to all cells except those transformed with the selection marker
  • the resistant colonies continue to grow and reach a size of 100- 200 ⁇ m or more after 4 weeks in culture.
  • visible colonies develop in agarose beads and also in the surrounding liquid medium (released from the beads).
  • Transformed colonies are also recovered when selection medium is used only in the liquid medium indicating that both the selection methods can yield large numbers of resistant colonies.
  • the advantage of embedding the protoplasts in an agarose bead over the liquid selection method is that the culture medium can be replaced without disturbing the developing colonies.
  • the resistant colonies are easily distinguishable microscopically after about 4 weeks of selection (6 weeks after electroporation) and can be scored visually after about 6 weeks.
  • Gradual dilution of KP8 medium with K8 using this time table is important, as it helps in gradually lowering the osmoticum.
  • agarose bead technique which immobilizes the transformants, replacement of liquid culture medium at regular intervals not only provides fresh culture medium (changing liquid is relatively easy) but also helps in removing the phenolic substances released by the dead cells.
  • the use of 1.2% agarose instead of 0.6% agarose gives slightly higher plating efficiency, but the main purpose of agarose immobilization is to prevent protoplast lysis during the initial culture period.
  • Transformation frequency can determined by counting the number of colonies in the agarose blocks.
  • the -native transformation frequency (RTF) is described as the ratio between the number of resistant colonies in the selected cultures and the developing colonies in the unselected cultures.
  • the absolute transformation frequency (ATF) is calculated on the basis of number of resistant colonies produced after 8 weeks from the initial number of protoplasts plated after electroporation.
  • microcalli about 1-2 mm in size can be observed.
  • the microcalli are transferred onto MSB medium supplemented with 0.5 mg mg/1 each of 2,4- D, BA, Kn, 500 mg/1 CH, 3% sucrose and 0.6% agar (with or without millipore 0.45 mm pore size filters). Where the colonies grow (to 3-5 mm) within 3-4 weeks and proliferate as light green calli. When grown on millipore filter paper the calli can grow faster and are more compact than the calli that are grown directly on culture medium. When the fast growing green calli are selectively transferred onto fresh medium of the same composition, green compact calli form after 3-4 subcultures.
  • MSB + BA + Kn + Z + NAA CA 3 + CH medium containing asparagine and glutamine. After repeated subculture, small shoots are formed but these shoots do not elongate on this medium.
  • MSB medium containing 3% sucrose and 0.5 mg/1 each of BA, Kn, ZT, 0.1 mg/1 NAA and 500 mg/1 CH and incubated under high light intensity (2,000 bu) dark green nodular structures appear on the surface and periphery of the calli.
  • shoots are transferred in 1/2 strength MS minerals containing 1.0 gm/1 KNO 3 , 0.01 mg/1 TH with or without 0.5-1.0 mg/1 GA 3 .
  • Shoots (0.5-1.0 cm) are transferred to several different media for elongation.
  • Elongation rate is significantly affected by growing the shoots on 1/2 strength MS minerals containing 1.0 gm/1 KNO3, 0.01 mg/1 TH and 1.0 mg/1 GA 3 . In two or three weeks approximately 60% of the cultures produce 2-3 cm long shoots.
  • Protoplasts are isolated several times from the immature cotyledons obtained from greenhouse as well as field grown plants and from each experiment plating efficiencies in excess of 60% are obtained. Regenerable callus with multiple shoots and plants are recovered from most of the experiments. The calli seem to retain regeneration potential even after several months. In general, it took 4-5 months to regenerate plants from isolated protoplasts including rooting of the shoots.
  • Protoplast derived resistant calli are usually subcultured once every 2 weeks on MSB medium containing 0.5 mg/1 each of 2,4-D, BA, Kn and 500 mg/1 CH. After 4 subcultures, calli started becoming nodular. Selective subculturing of the nodular calli on MSB medium supplemented with 0.5 mg "1 each of BA, KN, ZT, 0.1 mg/1 of NAA, 0.2 mg/1 of GA 3 and 50 mg/1 of asparagine and glutamine, triggered the differentiation of green buds and leafy shoots after 4-6 or more subcultures.
  • a reproducible plant regeneration method from immature cotyledon protoplasts of a commercial Glycine max genotype has been established. It is used in combination with high efficiency stable transformation of protoplast derived soybean cells via electroporation using a chimeric gene encoding hygromycin resistance and /3-glucuronidase and subsequent recovery of transformed shoots to generate transgenic soybean plants.
  • Stable transformation of soybean immature cotyledon protoplasts is achieved following electroporation with plasmid DNA carrying a chimeric gene for j8-glucuronidase (GUS) and hygromycin phosphotransferase (Hpt) under the control of the cauliflower mosaic virus (CaMV) 35S promoter.
  • GUS j8-glucuronidase
  • Hpt hygromycin phosphotransferase
  • Transformed colonies are stringently selected by growing 15-day old protoplast derived cells in the presence of 40 ⁇ g/ml of hygromycin-B for 6 weeks. Over 93% of the resistant cells and colonies exhibited GUS activity, indicating that the two marker genes borne on a single plasmid are co-introduced and co-expressed at a very high frequency.
  • Cotyledons 3-4 x 2 mm size are cut transversely into 1-2 mm thick sections and plasmolyzed for 1 hr in CPW 13M. After two washings with CPW 13M approximately 1 gm of tissue is incubated in 15 ml of enzyme solution in two 60 x 15 mm plastic petri dishes for 4-6 hr in the dark with shaking at 50 rpm.
  • the enzyme solution consisted of 1.5% (w/v) Cellulase "Onozuka" R10, 0.2% Pectolyase Y23 dissolved in CPW 9M (pH 5.8) which is filter sterilized with a 0.2 ⁇ m pore size Nalgene filter unit.
  • Protoplasts varied in size from 20-35 ⁇ m in diameter.
  • the released protoplasts are filtered through 74 and 43 ⁇ m sieves and pelleted by centrifugation at 100 x g for 10 min.
  • Protoplasts are washed twice by resuspending in KP8 medium and then purified by floating over 23% (w/v) sucrose or 13% Ficoll in CPW salts (pH 5.8) overlaid with 2.0 ml of KP8 medium and centrifuged at 80 x g for 10 min.
  • Protoplasts are gently removed with a Pasteur pipette from the interface and washed once again with KP8 medium.
  • Protoplast viability is determined with fluorescein diacetate (FDA; 24) test. When protoplasts are floated on sucrose (23%) there is no deleterious effect on viability but when Ficoll (13%) is used, 100% of the protoplasts burst within a few hours.
  • Plasmid construct
  • the plasmid pZA300 contains a hygromycin phosphotransferase gene, known to confer hygromycin ⁇ resistance to plant cells together with the ⁇ -glucuronidase gene from pBI121 (Clonetech Laboratory, CA, USA). This chimeric gene is driven by the CaMV 35S promoter and has the nopaline synthase polyadenylation signal of pCaMVNeo. The plasmid is multiplied in the E. coli vector pTZlSR. Electroporation
  • Protoplasts are resuspended at the density of 1-2 x 10 6 /ml in electroporation buffer (10 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM CaCl 2 2H 2 O and 0.2 M nannitol).
  • electroporation buffer 10 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM CaCl 2 2H 2 O and 0.2 M nannitol.
  • One ml of protoplast suspension is chilled briefly in ice water, then heat-shocked at 45°C for 5 min prior to addition of 20 ⁇ g/ml supercoiled plasmid DNA.
  • polyethylene glycol (PEG, 6000 MW; 200 ⁇ l of 28% stock) dissolved in KP8 medium containing 120 mM MgCl 2 is mixed with the protoplast:DNA suspension.
  • the samples are transferred to precooled, presterilized 0.4 cm plastic cuvettes fitted with aluminum foil electrodes.
  • An electric field of 500 V/cm is applied by a single discharge of a 1000 ⁇ F capacitor that has been previously charged with an electrophoresis power supply.
  • Four aliquots are treated with plasmid DNA and other aliquots are either electroporated without DNA or treated with plasmid DNA without electroporation to serve as controls.
  • Hygromycin sensitivity is determined by adding varying concentrations of hygromycin at different time intervals, starting 2 hr after culture initiation to 20 days after culture. The selection pressure is maintained for 4 weeks by replacing the liquid medium containing the antibiotic periodically (after every 7 days).
  • Protoplast density is adjusted to 2.5 x 10 5 protoplasts/ml in KP8 medium containing 2% Ficoll (Type 400-DL), 40 mM MES and 2.5 ml aliquots are dispensed in each 60 x 15 mm plastic petri dish.
  • protoplasts are cultured in 25 ⁇ l drops (10 drops/petri dish).
  • the osmolarity of the medium is progressively reduced by addition of 0.25 ml or 10 ⁇ l drops of a 1:1 mixture of KP8 : K8 medium to each dish at day 7 and addition of an equal volume of a 1:2 mixture of KP8 : K8 at day 10.
  • Protoplasts are resuspended in molten KP8 medium containing 0.6 or 1.2% (w/v) Sea Plaque agarose (at a final density of 2 x 10 5 protoplasts/ml and 4 ml is plated in each 60 x 15 mm petri dish or dispensed as 25 ⁇ l droplets on die bottom of petri-dishes (10 drops/dish). After 6-8 hr each layer is cut into 4 sectors which are transferred to 90 x 15 mm petri dishes. The agarose sectors and droplets are bathed in 5.0 ml of KP8 liquid medium in each dish.
  • the developing protoplasts are allowed to grow for 2 weeks without selection.
  • the protoplast derived cells at the 2-4 cell stage, are washed with K8 medium and the selection is applied either in liquid K8 medium containing 40 ⁇ g/ml of hygromycin or the cells are resuspended in an equal volume of 1.2% Sea-plaque agarose (LMT, FMC Corp., ME, USA) gelled K8 medium containing 50 ⁇ g/ml hygromycin.
  • the solidified agarose cultures cut into small beads are submerged in 10 ml of K8 liquid medium containing 40 ⁇ g/ml hygromycin and kept on a gyratory shaker at 40 rpm.
  • the liquid selection medium is replaced every 7 days over a period of 6 weeks with K8 and MSB medium (MS basal salts + B 6 organics, using different volumes of each containing 40 ⁇ g/ml hygromycin.
  • the resistant colonies continued to grow and reached a size of 100-200 ⁇ m or more after 4 weeks in culture.
  • visible colonies developed in agarose beads and also in the surrounding liquid medium (released from the beads).
  • Transformed colonies are also recovered when hygromycin is applied only in the liquid medium indicating that both the selection methods can yield large number of resistant colonies.
  • the advantage of embedding the protoplasts in an agarose bead over the liquid selection method is that the culture medium can be replaced without disturbing the developing colonies.
  • the resistant colonies are easily distinguishable microscopically after 4 weeks of selection (6 weeks after electroporation) and could be scored visually after 6 weeks.
  • the maximum number of hygromycin resistant colonies (570-685) are recovered from 1-2 x 10 5 protoplasts electroporated with plasmid DNA.
  • Hygromycin resistant is maintained for at least four subcultures on selective medium and is not lost after growing on hygromycin lacking medium for two subcultures (14 days each) and then replating on hygromycin-containing medium. Regeneration
  • the resistant calli grew to 1-2 mm in size they are transferred to different media for callus reformation and shoot regeneration as follows.
  • the microcalli grew to 1-2 mm in size, they are transferred onto MSB medium supplemented with 0.5 mg/1 each of 2,4-D, BA, Kn, 500 mg/1 CH, 3% sucrose and 0.6% agar (with or without millipore 0.45 mm pore size filters).
  • MSB medium supplemented with 0.5 mg/1 each of 2,4-D, BA, Kn, 500 mg/1 CH, 3% sucrose and 0.6% agar (with or without millipore 0.45 mm pore size filters).
  • the dark green callus pieces with nodular structures are selectively transferred to MSB medium with various combinations of auxins and cytokinins for shoot formation.
  • Protoplasts are isolated several times from the immature cotyledons obtained from greenhouse as well as field grown plants and from each experiment plating efficiencies in excess of 60% are obtained. Regenerable callus with multiple shoots and plants are recovered from most of the experiments. The calli seem to retain regeneration potential even after several months. In general, it took us 4-5 months to regenerate plants from isolated protoplasts including rooting of the shoots.
  • the protoplast development reported here is similar to that reported earlier with other Glycine max genotypes where protoplast division occurred after 3-5 days with plating efficiencies of 37-41 % recorded after 7 days of culture.
  • the reasons for the enhanced protoplast development in the present communication with another commercial genotype remains unclear, but may be caused by the combined effects of modifications in protoplast culture methods as described in this report: (i) use of 40 mM MES buffer in the culture medium to stabilize the pH in the initial stages (the pH normally dropped from 5.8 to 3.9), (ii) use of 2% Ficoll (type 400 DL) in the culture medium to prevent protoplasts from adhering to the surface of petri dishes, (iii) immobilization of the protoplasts in 1.2% agarose and initial incubation in the dark, since protoplasts placed immediately after isolation in the light bleached and many burst after 3-5 days, (iv) plating small colonies on agar solidified MSB medium with millipore filter paper to possibly adsorb phenolic substances and to facilitate gas
  • GUS activity is measured by in situ staining of electroporated and non-electroporated protoplast derived cells or calli.
  • the GUS assay buffer solution contained 100 mM sodium phosphate (pH 7.0), 0.4% X-gluc, 5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide and 0.1-1.0% triton X-100. Incubation is carried out at room temperature for 12- 24 hr.
  • DNA is prepared from twelve hygromycin resistant, GUS positive callus lines and from transgenic shoots from four of these clones by a hot phenol-lithium chloride procedure. At the LiCl precipitation step, the soluble fraction is saved and precipitated twice with ethanol. The DNA pellet is resuspended in distilled water and treated with 10 ⁇ g of RNAse to remove RNA from the sample.
  • DNA is digested with Hindlll or EcoRI, fractionated on 0.8% agarose and transferred to nitrocellulose filters.
  • the filters are hybridized with 32 P labelled 2 kb HindDI or 3 kb EcoRI fragment from pZA300 containing Hpt or GUS genes.
  • the filters are washed twice at room temperature in 2 x SSPE, 0.2% (w/v) SDS, once at 65°C and subsequently with 0.3 x SSPE, 0.1% (w/v) SDS for 1 hr at 65°C.
  • Hybridization is visualized by exposure of the membranes to Kodak XAR-5 film at -70°C with intensifying screens.
  • Protoplasts were isolated from immature cotyledons of Glycine max (L.) Merr. cv. Clark 63 and purified as described in Example 1. Protoplast viability was determined by staining with fluorescein diacetate (FDA; 29). Electroporation
  • the BAMHI - Kpnl T R fragment from pSaK5 (pSa4 vector containing Kpnl fragment 5 from pTil5955, S.K. Farrand, unpublished data) was cloned between the unique BAMHI - Kpnl sites of pUCD2001, resulting in the T-DNA vector pMAS2.
  • the insert includes T R , R c and part of the T L - region of pTil5955 (Baker, R.F. et al., (1983) Plant Mol. Biol. 2:335-350).
  • the T R -region contains ORFs 24, 25 and 26 corresponding to transcripts 2', 1' and 0', which are necessary for the biosynthesis of the mannityl opines (Ellis, J.G. et al., (1984) Mol. Gen. Genet. 195:466-473).
  • An NPTII gene fused to the 24S promoter of the CaMV was cloned into the Xbal site located in T R ORF 21 of pMAS2 (Baker, et al. supra).
  • Plasmid pUSD2001 also contains an Xbal site located in the pTAR par region (Galli, D.R.
  • Electroporated protoplasts were resuspended in Kp8 medium at densities from 2-4 x 10 5 /ml and were allowed to grow for two weeks without selection.
  • protoplast-derived cells On day 15 protoplast-derived cells, at the 2-4 cell stage, were washed with K8 medium and resuspended in an equal volume of 1.2% agarose medium (5 ml) containing 50 ⁇ g/ml kanamycin sulfate (Sigma).
  • the agarose cultures were cut into slices and allowed to grow for 6 weeks under the antibiotic selection in K8 and MSB medium as described in Example 1.
  • Neomycin phosphotransferase II (MPTII. assay
  • NPTII enzymatic activity was qualitatively detected in callus and leaves from individual transformed clones by a dot blot procedure. Tissue (100 mg fresh weight) extracts were made in 50 ⁇ l of extraction buffer. (See McDonnell, R.E. et al., (1987) Plant Mol. Biol.
  • mannityl opines For the detection of mannityl opines, 30-50 mg fresh weight of kanamycin-resistant callus, leaf or root tissue from individual plants was homogenized in 50 ⁇ l of 70% ethanol containing 5 ⁇ l of electrophoresis running buffer (formic acid:acetic acid: water, 3:6:91, v/v/v, pH 1.9). Tissue extract was spotted onto Whatman No. 3 filter paper and opine were separated and visualized as described by Savka et al., (1990) Phytopathology 80:503-508.
  • DNA was extracted from callus or leaf tissue essentially as described by Dellaporta et al., (1983) Plant Mol. Biol. Rep. 1:19-21. Ten ⁇ g of DNA digested with Xbal was electrophoresed in a 1.0% w/v agarose gel. The DNA was blotted onto a nitrocellulose filter and hybridized with the 32 P-labelled 1.7 kb Xbal fragment containing the NP ⁇ I gene from pMAS4. The filter was washed twice with a solution of 2X SSPE and 0.5% w/v SDS for 15 min at room temperature followed by 15 min at 68°C.
  • the filter was washed with 0.2X SSPE and 0.2% SDS for 15 min at 68°C with agitation, dried and exposed to X-ray film using an intensifying screen for 24 to 72 hr at -70°C.
  • the viability and division frequency of protoplasts subjected to electroporation was generally lower (55-60%) than that of untreated control protoplasts (10).
  • Electroporated protoplasts plated in agarose readily formed microcolonies within 8-10 days after transfer of the agarose slices to liquid medium containing 50 ⁇ g/ml kanamycin. On selection medium, the resistant colonies continued to proliferate and were easily distinguishable visually from the non-transformed cells after 6 weeks.
  • the maxiumum number of kanamycin resistant colonies recovered varied from 370 to 460 from 1 x 10 ⁇ protoplasts/ml in four experiments with each two individual treatments.
  • the concentration of selectable marker pCaMVNeo was increased from lO ⁇ g to 50 ⁇ g/ml in combination with lO ⁇ g/ml pMAS2 (in 50 ⁇ l TE buffer) this resulted in a slightly increased number of transformed colonies (185 to 245 recovered from 10 6 protoplasts upon selection on kanamycin medium).
  • the number of NPTII expressing colonies also increased (in one assay 18 of 20 colonies were NP ⁇ I positive showing 90% expression) but the fraction of opine expressing colonies did not change, remain nearly 65% of the selected cell clones.
  • NA molecule leads to a higher transformation frequency (85%) in comparison to co- transformation of two unlinked genes on separate plasmids (65% efficiency).
  • the co- transformation frequency reported here is higher than the frequencies of 25-59% described with other genes in Arabidopsis, maize and rice using PEG mediated transformation.
  • Example 1 the expression of linked chimeric genes (hygromycin phosphotransferase and jS-glucuronidase from the 7.7 kb plasmid pZA300, using 20 ⁇ g/ml) at the rate of more than 93%, by selecting the transformants with 40 g/ml of hygromycin in soybean is reported. However, using a larger plasmid (1" .
  • Kanamycin resistant plants were regenerated from Glycine max protoplasts electroporated with a plasmid vector containing linked chimeric genes with 85% co-expression. However, if the genes are on separate plasmids the efficiency of co-expression is reduced to about 65%. In selection of microcalli, we found that 75 ⁇ g/ml kanamycin should be used to prevent escapes. Both NPTII and all the genes from mannityl opine biosynthetic region of pMAS4 plasmids were present in different organs of transgenic plants as shown by enzyme and opine assays and by Southern hybridization indicating that nonchimerial plants are produced. EXAMPLE 3 Plant Material
  • Plants of fourteen cultivated Glycine max genotypes were grown in the field or under greenhouse conditions (16 hr supplemented light per day, 26+2°C).
  • Protoplasts were readily isolated from all fourteen soybean genotypes included in this study using 1.5% Cellulase and 0.2% Pectolyase within 4-6 hr of incubation. Usually about 5 x 10 6 protoplasts were released from one gm fresh weight of immature cotyledon tissue. However, the yield varied with the genotype and ranged from 1-8 x 10 6 protoplasts per gm fresh weight of tissue (Table 1). Viability of isolated protoplasts varied among experiments and genotypes, but was generally over 70% as indicated by FDA staining and protoplast morphology, except in Burlison and XP 3015 where the viability was 60-70% (Table 1).
  • the cultures were diluted with the D1-D9 medium, optimal for each genotype and K8 medium at a 1:1 ratio on day 8 and 14, 1:2 on days 21, and 28 and then K8 medium alone was added after 5 weeks as reported in Example 1.
  • K8 medium alone was added after 5 weeks as reported in Example 1.
  • cells of the genotype Jack divided faster than other genotypes and grew to 64 or more cells in 3 weeks of culture. Colonies that formed in agarose from all genotypes were organized and compact, whereas those formed in liquid medium were loose, unorganized and slow growing. After 5-6 weeks of culture, green microcalli (1-2 mm in size) could be observed.
  • Protoplasts of all the fourteen genotypes were resuspended at 1-5 x 10 5 protoplasts per ml and cultured in D1-D9 medium using liquid medium or embedding the protoplasts in 1.2% L P agarose. Plating efficiency was determined as the % o dividing protoplasts after 7 days of culture.

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Abstract

Un procédé de production d'espèces de soja transgéniques comprend les étapes suivantes: préparation de protoplastes à partir de cotylédons de soja; insertion par électroporation d'un ADN étranger dans les protoplastes préparés, ledit ADN comprenant un gène utile; culture des protoplastes électroplaqués dans un milieu pour induire la croissance des cellules, la formation de colonies et la génération de cal; et regénération de plantes à partir dudit cal.
PCT/US1992/002226 1991-03-29 1992-03-26 Production d'especes de soja transgeniques WO1992017598A1 (fr)

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