A PROCEDURE

FOR THE MICROINJECTION OF PLANT C E L L S AND P R O T O P L A S T S

Brian Miki, Bin Huang, Sharon Bird, Roger Kemble, Daina Simmonds, and Wilfred Keller

Plant Research Center, Agriculture Canada, Ottawa, Ontario, Canada K1A 0C6 (B. M., D. S., IV. K.) and Allelix Crop Technologies, 6850 Goreway Drive, Mississauga, Ontario, Canada L4V1P1 (B. H., S. B., R. K.)

SUMMARY: This paper describes a general method suitable for the microinjection of Brassica napus protoplasts, unicellular microspores, and muhicellular microspores. By incorporating components taken from other methods, manual operations frequently involved in the microinjection of plant cells have been simplified and microinjection rates increased. The embedding of cells in agarose provides a simple alternative to the variety of sophisticated immobilization strategies devised for different plant cell types thereby reducing the manipulations often involved in the culture of microinjected cells. Use of an automatic microinjector eliminated the operation of fine control systems on manual injectors; however, precision in sample delivery was reduced. Analyses indicate that transformed tissues can be recovered from microinjected protoplasts and microspores at high frequencies.

Key words: microinjection; genetic transformation; protoplasts; microspores; Brassica napus.

I.

INTRODUCTION

formats. The method described below is acceptable for the genetic transformation of at least three different cell types of Brassica napus: protoplasts, unicellular microspores, and multicellular microspores. References will be given for alternative approaches that may enhance the efficiency at certain steps in the method.

It is possible to identify specific cell types in culture, to introduce a variety of molecules or organelles into a small number of them {1-4,6-8,10-18k in particular specific intracellular locations (1,8,13,14,16,18k and to examine the immediate biological consequences of that action. Efficient cell culture methodologies allow analysis of the stable genetic and physiologic alterations incurred by the cell (1,10,14,16). These experimental capabilities are widely applicable in plant research; however, the main advances in the technology have emerged from a desire for genetic transformation systems that are extremely precise and independent of selection systems, species barriers or ceil-type barriers (7). Although several approaches to plant cell mieroinjection have been reported {1-4,6-8,10-18), all are limited by the small number of cells that can be injected and recovered in an experiment. Governed by the experimental objectives of the research and the cell types under examination, compromises must be made in the rate of sample delivery, precision in the volume and location of the sample delivered, and extent of damage to the cells. Several separate components must be carefully integrated. These include conditions for efficient culture of individual ceils; methods for immobilizing and orienting ceils; specific microscope formats and optics; systems for controlling the microenvironment; microinjeetors and micropipette Journal of TissueCultureMethods Vol.12, No. 4, 1989

II.

MATERIALS A. Plant materials 1. Plants, Brassica napus cv Topas, Svalof, Sweden 2. Suspension culture, B. napus cv Jet neuf, W. A. Keller, Agriculture Canada, Ottawa, Ont., Canada B. Media and buffers Formula and references and provided in Procedures tIII~ C. Equipment 1. Autoclave 2. Laminar air flow cabinet, H4-MW 97T Canadian Cabinets t 3. Refrigerator, household, at 4 ° C 4. Growth room, El5 Conviron2 5. Incubators at 25 ° and 32° C, I23 Conviron z 6. Water bath at 40° C 7. Gyratory shaker, G10 or G33, New Brunswick ~ 8. Reciprocal shaker, R23 9. Low speed centriguge, IEC Centra-4, FisheP 10. Pipet-aid, filler, and dispensor, Drummond s 11. Gilson Pipetman pipetter, 1-20 gl, MandeP

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@)1989TissueCultureAssociation,Inc.

MIKI ET AL. 3. Immobilize microspores in nylon-ring units (Fig. 1 a,b) a. Cut nylon tubing into 1.0 to 1.5-mm thick rings with a razor blade and sterilize the rings in an autoclave. b. Wash 18 X 18 mm cover slips in acetic acid followed by 10 or more washes with double distilled sterile water and store in 95% ethanol. Air dry just before use. c. Place one ring onto each cover slip. d. Using a micropipetter with sterile, wide-mouth, plastic tips prepare the primary nurse culture in each ring by adding 20 pl of fresh mierospore culture in NLN-13 medium with 0.8% agarose kept at 40 ° C, at a density of 30 000 cells/ml (Fig. 1 a). This culture is prepared by centrifugation of fresh microspore culture at 100 Xg, 3 min, 25 ° C and resuspension in NLN-13 medium with 0.8% agarose adjusted to 40o C in a water bath. e. Allow the primary nurse culture to solidify at 4 ° C, 15 min. f. Cover the primary nurse culture with a guard layer (Fig. I a) by adding 60/~t NLN-13 medium with 0.8% agarose, 40° C, and allow solidification at 4 ° C, 15 rain. g. Place 1 to 2 ~1 of fresh microspore culture, containing about 10 microspores, onto the guard layer of each ring to form the sample layer (Fig.

12. 13. 14. 15. 16. 17.

Gilson Pipetman pipetter, 1-200 ~16 Pipette puller, DK1700C David KopP Micromanipulator, right hand, 2520137 Leitz 8 Pipette holder, 2520145 Leitz a Large base plate, 2520145 Leitzs Microscope, inverted fluorescent research microscope equipped with tong working distance lens objectives, 8X, 16X, 40X, IM-35 Zeiss 9 18. Automatic microinjector, 5242 Eppendorf, equipped with a nitrogen cylinder and gas regulator 9 D. Chemicals and enzymes 1. Agarose, Sea Plaque, FMC l° 2. Lucifer Yellow, L0259, Sigma" 3. MES {2-[N-morpholino]ethanesulfonic acid), M8652" 4. Acetic acid, glacial, 99.7% 4 5. Ethanol, 95%, Consolidated AlcohoP ~ 6. Celluiase, Onozuka RI0, Yakult Honshu 13 7. Macerozyme ~3 E. Glassware and plastics 1. Glass capillaries with filaments, BF 100-58-10, Sutter Instruments" 2. Glass capillaries without filaments, 2520119 Leitzs 3. Cover slips, 18 X 18 mm, Canlab 's 4. Flasks, 125-ml Erlenmeyer, Pyrex brand 4 5. Centrifuge tubes 15 ml, 30 ml Corex brand 4 6. Petri dishes, 1001 Falcon {100 X 15 mm), 1005 Falcon {100 X 20 mm), 1007 Falcon {60 X 15

la).

m i l l )4

h. Place each ring unit and cover slip into a 60 X 15mm petri dish and seal the dish with parafilm. i. For unicellular microspores, incubate at 32 ° C in darkness, for 8 to 16 h before microinjection. j. For multicellular microspores, prepare rings with 80 gl of solid NLN-13 medium with 0.8% agarose alone and 1 to 2 gl of microspore culture after 2 to 4 d at 32 ° C in darkness {Fig. 1 b). Note: The nurse layer is not required for these samples {Fig. 1).

7. Pipettes, serological, plastic, disposable, 10 ml, 5 mP 8. Hemacytometer, American OpticaP F. Miscellaneous 1. Parafilm 4 2. Nylon mesh screen, 44/~m, Fyntex is 3. Nylon tubing, 8-10 mm diameter 's 4. Measuring calipers, Canadian Tire 17 5. Razor blades 6. Aluminum foil 7. Sterile filtration units, 0.2 ~m, disposable, SM 165 34 Sartorius MinisarP 8. Sterile double distilled water

a III.

A. Preparation and culture of microspores 1. Prepare culture media for Brassica napus microspore isolation and culture as described by Huang and Keller (5). 2. Prepare microspore cultures for microinjection a. Grow B. napus cv Topas plants according to Huang and Keller{5). b. Prepare and culture microspores from about 10 individual flower buds measuring 3.5 to 4.0 mm in length from the lateral racemes as described by Huang and Keller (5). c. Utilize the cultures in NLN-13 medium [see Table 1 in (5)] for microinjection and nurse cultures, as described below {III.A.3; III.A.4). 140

b

c

PROCEDURE

sampte~

~ ; 4 sample

i

°,.,r.

I guard

==i sample guard

I

I

nurse

I nurse

FIG. 1. Use of nylon ring units for the immobilization and culture

of a, 8- to 16-h unicellular microspores; b, 2- to 4-d multicellular microspores; and c, protoplasts. In each case the microinjected samples are separated from the nurse cultures by a guard layer. In addition to the main nurse culture a separate nurse culture in the ring unit (a) is used to obtain the embryogenic 8- to 16-h unicellular microspores. Journal of Tissue Culture Methods Vol. 12, No. 4, 1989

MIKI ET AL. 4. Prepare microspore nurse cultures for the ring units (Fig. 1 a, b) a. SolMity 2.0 ml NLN-13 medium with 0.4% agarose in a 60 X 15-ram petri dish. b. Over the surface of the solid medium place a 1.2ml layer of liquid fresh microspore culture in NLN-13 medium at a cell density of 30 000/ml. B. Preparation of protoplasts 1. Prepare protoplasts for microinjeetion a. Subculture B. napus cv Jet netd cell suspension cultures weekly by a fivefold dilution with 2MS2D medium [MS medium (9) with 2% sucrose, 2 mg/liter ~2,4-D~] and culture at 25 ° C with rotary shaking at 150 rpm under 16 h fluorescent light. b. After 2 to 4 d, allow 200 to 500 mg fresh weight of cells to settle to the bottom of a 100 X 20-mm petri dish, and replace the medium with 15 to 20 ml 0.4 M mannitol, 5 mM MES, pH 5.8, 1% cellulase Onozuka R10, and 2% Macerozyme. c. Allow enzymic digestion of the cell walls to proceed overnight in darkness, 25° C, with gentle reciprocal shaking at 50 rpm. d. Filter the protoplast suspension through a 44-~m nylon mesh screen. e. Collect the protoplasts by gentle centrifugation at 100 Xg, 3 min, room temperature in 15-ml centrifuge tubes with round bottoms. f. Wash the protoplasm 3 times with 0.4 M mannitol, 5 m M M E S , pH 5.8, 1 mM CaCI2. g. Resuspend protoplasts in NLN medium with 6% sucrose, 3% mannitol, 0.5 mg/liter NAA, 0.5 rag/liter 2,4-D, 0.5 rag/liter BA at a cell density of 104 protoplasts/ml. h. Dilute protoplasts further for microinjection and nurse culture as described in III.B2 and III.B.3. 2. Immobilize protoplasts in nylon rings (Fig. 1 c) a. To nylon rings on cover slips, add 80 pl of NLN medium with 0.8°'/0 agarose, 40 ° C and allow solidification at 4° C, 15 rain. b. Dilute fresh protoplast cultures to a cell density of 1 )< 104 protoplasts/mi with NLN medium with agarose and add 2 to 5 ~1, which contains about 20 to 50 protoplasts and 0.4% agarose, to the rings. 3. Prepare protoplast nurse cultures for the ring unit (Fig. 1 c) a. Solidify 2.0 ml fresh protoplast culture with 0.4% agarose in a 60 )< 15-ram petri dish at a celt density of 5 )< 104 protoplasts/ml. Note: For microinjection, other protoplast systems with high plating efficiency, such as those described in other papers of this issue, may be used; however, appropriate substitution of media and culture conditions must be made. The protoplasts described above are used immediately after isolation because cell wall resynthesis and cell division are extremely rapid. This can interfere with the micromanipulation Journal of Tissue Culture Methods Vol. 12, No. 4, 1989

steps described later (III.E.5-7t. With other systems, the initial stages of protoplast regeneration may be slower and may occur within a predictable time, thereby permitting partial cell wall resynthesis in culture before micromanipulation and microinjection. C. Assembly of equipment (Fig. 2 a) 1. Assemble equipment in a laboratory area that is both vibration-free and clean. Note: A preferred location would be a basement floor, in a separate clean room, with filtered air. 2. If possible, locate a laminar flow hood close to the equipment for preparing samples and components such as the micropipettes. 3. Assemble the equipment in a laminar flow unit or an environmental chamber designed for micromanipulation (15). 4. Select a fluorescent microspore with a fixed stage such as an inverted microscope (e.g., Zeiss IM 35~ or a fixed stage conventional microscope designed for microinjection (e.g., Leitz Laborlux FS 12). Long working distance lens objectives may be necessary. 5. Select micromanipulators that are compatible with the microscope system. Note: The major microscope manufacturers have assembled systems with all of the necessary components. If the components are mixed, the services of a workshop will probably be required to assemble the system properly and securely. An example is illustrated in Fig. 2a. 6. Before a microinjection experiment, make sure that the equipment is aseptic by cleaning the stage with ethanol and leaving the laminar flow on until experimentation. D. Preparation of mieroinjectors I. Prepare plasmid DNA solutions for microinjection. a. Prepare plasmid DNA using standard procedures and include two CsCI gradient steps. b. Store DNA in 10 mM tris, pH 8.0, 0.1 rmM EDTA at 1 mg/ml. c. For microinjection, dilute to 10 ~g/ml with 48 mM K2HPO,, 14 mM NaH2PO4, 4.5 mM KH~PO~. Note: Filter all buffers before use through a 0.2/~m filter to ensure removal of particles that may plug the pipette tips. To aid in visualizing the location of the injected samples, 2% Lucifer Yellow may be included in the samples (12,17 ~. 2. Prepare and load the glass micropipettes a. Sterilize the glass capillaries in an autoclave. b. Mount the capillaries onto a commercial pipette puller and adjust the heating coils according to the manufacturer's instructions (Fig. 2 b). c. Determine the settings that produce fine but quickly tapered tips visible under a microscope (Fig. 2 cl. Note: The opening may be <1~ diameter and therefore it will not be visible under a light microscope. The passage of air or liquid will 141

M I K I E T AL.

FIG. 2. Illustrations of (a) microinjection apparatus, (b) vertical pipette puller, and (c) micropipette made from a glass capillary using the puller illustratedin (b). Microlnjection apparatus in (a) consists of an automatic microinjector (A), a Zeiss IM 35 inverted microscope ~B) mounted on a Leitz baseplate IC) together with Leitz micromanipulators ~D), and custom made metal blocks (E) to adjust the height of the manipulator. A Leitz micropipette holder (F) is fixed on the manipulator and connected to the N2 gas supply through the microinjector. Position of the micropipette (G) can be adjusted by four controls (H) and a joystick (I). 142

Journal o! Tissue Culture Methods Vol. 12, No. 4, 1989

MIKIET AL. indicate for practical purposes if the tip is sealed or open. The rapid taper provides rigidity during lateral movements and penetration of cells. The David Kopf Instruments model DK1 700 C provides sufficient control to pull micropipettes for the experiments described in this paper {Fig. 2 b~. d. For glass capillaries containing an inner filament, load the DNA solution by immersing the back end into the solution, taking care to avoid air bubbles. Once filled, connect to the pipette holders according to the mamffacturer's instructions. e. For glass capillaries without an inner filament, connect to the pipette holder first and draw the DNA solution into the pipette through the tip with the aid of vacuum. Note: It is only necessary to draw a small volume {Ca 0.5 ~1) into the pipette because the volumes to be injected are so small that a change in the total volume of solution in the pipette will barely be observed after the experiment {Fig. 2 c}. 3. Prepare the microinjector for delivery of the smallest volumes possible a. Following the manufacturer's instructions make sure that all connections to the Eppendorf model 5242 microinjector are secure. b. Set the cleaning, injection, and holding pressures at 5000, 500, and 100 hPa, respectively, and the injection time at 0.5 to 1.0 s. Note: For most routine applications, having the injector in automatic mode simplifies the steps in the microinjection operation, thus increasing the number of cells that can be injected in an experiment. For finer control of injected volumes a manual microinjector is recommended (15t. E. Microinjection 1. Place a drop of culture medium over the ring unit to keep the culture from drying. 2. Position the cover slips with ring units on the microscope stage. Do not use laminar flow. 3. With brighffield optics, focus sharply on the cells to be injected under low magnification {e.g., 8X objective). 4. Position the micromanipulators so that the horizontal plane of the micropipette is at a 45 ° angle to the plane of the cover slip and move the tip of the micropipette into the center of the field. 5. Slowly lower and adjust the position until the tip just touches the cell surface when viewed under high magnification {e.g., 16X, 40X objective}. 6. Use the horizontal controls of the micromanipulators to position the tip inside the cell, preferably the nucleus if visible and inject D N A by activating the footswitch of the microinjector. Note: Combine brightfield and ultraviolet illumination to observe the fluorescence of Lucifer Yellow which is coinjected with the DNA. Journal of TissueCulture Methods Vol. 12, No. 4, 1989

7.

8.

9.

10

11. IV.

Note: A blunt holding pipette may be moved into position on the opposite side to the injection pipette if problems with cell movement during penetration are experienced. Withdraw the pipette and position another cell under the tip using the microscope stage. Note: With plant cells the micropipette may become plugged during microinjection. The plug may be removed by pressing the orange light on the automatic microinjector to activate the cleaning pressure; however, the efficiency of this operation is variable and it may be necessary to change the micropipette. For cultured microspores, select those that have enlarged or have initiated equal cell divisions. Those that remain triangular after 16 h will not develop into embryos. Try to inject each of the cells in the multicellular microspores. After all the viable cells in the ring have been injected, remove the ring unit from the cover slip and place on the appropriate nurse culture {Fig. 1 L Note: This entire operation requires about 1 to 2 h. Losses due to damage to the cells and protoplasts should not occur with these methods. For microspores, complete the 4-d culture period at 32 ° C to induce embryogenesis (5), and culture at 25 ° C thereafter. For protoplasts, culture at 25 ° C.

DISCUSSION A number of different procedures have been developed for the microinjection of plant cells in culture {1-4,6-8,10,11,13-18L Efficient genetic transformation has been reported for tobacco protoptasts (1), alfalfa protoplasts (14), and B. napus embryogenic multicellutar microspores {10}. The protocols presented in this paper integrate components from the other microinjection systems to provide a general and simplified approach that can be used with different cell types. For instance, the use of the ring units for immobilization and culture of the microinjected cells i6) eliminates the complicated and time-consuming approaches used in many studies. Due to the variety of morphologic and physiologic properties of plant cells it is unlikely that a single procedure will be optimal for all plant cells considered. Control of the injected volumes and precision in the location of intracellular delivery is lower than with other systems (1,8,15,16,18}; however, the rate of microinjection of single cells and multicellular structures is relatively high. Approximately 100 protoplasts or 20 microspores may be injected within 1 h. For genetic transformation and recovery of transgenic plants, it is essential to microinject large numbers of cells to compensate for subsequent losses in culture and for ceils that do not functionally integrate the injected DNA. It is also important to utilize cell culture systems having high plating efficiency and plant regeneration ability. For physiologic studies, which do not require regeneration 143

MIKIET AL.

from the cells, precision may be a greater consideration, and both technologies and optical systems for achieving this exist {1,8,13,15,16,18t. The microinjection protocols for plant cells are generally at an early stage of development and will improve with experience. At this time, analyses indicate that the procedures described here may generate transformation frequencies comparable to those reported earlier (1,10,14,16). V.

REFERENCES

1. Crossway, A.; Oakes, J. V.; Irvine, J. M., et at. Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts. Mol. Gen. Genet. 202:179-185; 1986. 2. de Laat, A. M. M.; Biaas, J. An improved method for protoplast microinjection suitable for transfer of entire plant chromosomes. Plant Sci. 50:161-169; 1987. 3. Griesbach, R. J. Protoplast microinjection. Plant Mol. Biol. Rep. 1:32-37; 1983. 4. Griesbach, R. J. Chromosome-mediated transformation via microinjeetion. Plant Sci. 50:69-77; 1987. 5. Huang, B.; Keller, W. A. Microspere culture technology. J. Tissue Cuh. Methods 12:171-178; t989. 6. Lawrence , W. A.; Davies, D. R. A method for the microinjection and culture of protopiasts at very low densities. Plant Cell Rep. 4:33-35; 1985. 7. Miki, B. L.; Reich, J. J.; Iyer, V. N. Microinjcction: an experimental tool for studying and modifying plant cells. In: Hohn, T.; Schell, J., eds. Plant DNA injections agents. Vienna: Springer-Verlag; 1987:249-265.

Canadian Cabinets, Ottawa, Canada 2 Conviron, Winnipeg, Canada New Brunswick Scientific, Edison, NJ 4 Fisher Scientific, Ottawa, Canada s Drummond Scientific Co., Broomall, PA 6 Mandel Scientific, Guelph, Canada David Kopf Instruments, Tujunga, CA 8 Wild Leitz {Canada~ Ltd., Toronto, Canada 9 Carl Zeiss, Toronto, Canada

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8. Morikawa, H.; Yamada, Y. Capillary mieroinjecfion into protoplasts and intranuctear localization of injected materials. Plant Cell Physiol. 26:229-236; 1985. 9. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassay with tobacco tissue cuhure. Physiol. Plant 15:473-497; 1962. 10. Neuhaus, G.; Spangenberg, G.; Mittehten Scheid, O., et al. Transgenic rapeseed plants obtained by the microinjection of DNA into microsporederived embryoids. Theor. Appl. Genet. 75:30--36; 1987. 11. Nomura, K.; Komamine, A. Embryogenesis from mieroinjected single cells in a carrot cell suspension culture. Plant Sci. 44:53-58; 1986. 12. Palevitz, B. A.; Hepler, P. K. Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by mieroinjection of Lucifer Yellow. Planta 164:473-479; 1985. 13. Reich, T. J.; Iyer, V. N.; Haffner, M., et al. The use of fluorescent dyes in the microinjection of alfalfa protopiasts. Can. J. Bot. 64:12,59-1267; 1986. 14. Reich, T. J.; Iyer, V. N.; Miki, B. L. Efficient transformation of alfalfa protopiasts by the intranuclear microinjection of Ti plasmids. Bio/Technology 4:1001-1004; 1986. 15. Reich, T. J.; Iyer, V. N.; Scobie, B., et al. A detailed procedure for the intranuclear microinjection of plant protopiasts. Can. J. Bot. 64:1255-1258; 1986. 16. Spangenberg, G.; Neuhaus, G.; Schweiger, H-G. Expression of foreign genes in a higher plant cell after electrofusion-mediated cell reconstitution of a microinjected karyopiast and a cytoplast. Eur. J. Cell Biol. 42:236-238; 1986. 17. Steinbiss, H-H.; Stabel, P. Protoplast derived tobacco cells can survive capillary microinjection of the fluorescent dye Lucifer Yellow. Protopiasma 116:223-227; 1983. 18. Toyoda, H.; Matsuda, Y.; Utsumi, R., et al. Intranuclear microinjection for transformation of tomato callus cells. Plant Cell Rep. 7:293-296; 1988.

,o FMC Corp., Rockland, ME " Sigma Chemical Co., St. Louis, MO ~2Consolidated Alcohol Ltd., Toronto, Canada ,3 Yakult Honshu Co. Ltd., Japan ,4 Sutter Instrument Co., San Rafael, CA ,s Caniab, Montreal, Canada ~ Fyntex, Switzerland " Canadian Tire Corp., Ottawa, Canada

Journal of Tissue Culture Methods Vol. 12, No. 4, 1989