Protoplast Regeneration from Normal and Bromodeoxyuridine-Resistant Sycamore Callus
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J. Cell Sci. i6, 445-463 0974) 445 Printed in Great Britain PROTOPLAST REGENERATION FROM NORMAL AND BROMODEOXYURIDINE-RESISTANT SYCAMORE CALLUS S. W. J. BRIGHT AND D. H. NORTHCOTE Department of Biochemistry, University of Cambridge, Cambridge, CBz iQW, England SUMMARY Protoplasts have been prepared from normal and mutant lines of sycamore callus, maize root and tobacco leaf. Fusion was rare in sodium nitrate solutions. A mutant tissue culture was selected for by its resistance to io/*g/ml bromodeoxyuridine. The mutant is sensitive to the Hypoxanthine-Aminopterin-Thymidine (HAT) selective medium but has normal thymidine transport and thymidine kinase activity. INTRODUCTION Tissue cultures of plant cells of many species can be induced under appropriate conditions to divide in an organized manner and to differentiate to whole plants (Steward, Mapes & Ammirato, 1969; Rao, Handro & Harada, 1973). It is now becoming experimentally possible to modify the genetic content of cultured cells and grow the tissues back to a plant. This could enable genetically useful traits, such as genes for disease resistance, to be introduced into a population of important plants where it could be propagated by conventional breeding techniques. This modification could range from the introduction of small amounts of foreign DNA to the introduction of organelles or the fusion of whole cells from genetically unrelated species. Plant cell hybridization would also be a very useful approach to studying problems of cellular organization and control. In one special case a hybrid plant has been produced from fused protoplasts and shown to be identical to the hybrid produced by conventional breeding techniques (Carlson, Smith & Dearing, 1972). We have been investigating: (1) conditions for the production of healthy protoplasts from a tissue culture of sycamore; (2) the effects of various reagents on the fusion of tobacco leaf, maize root and sycamore callus protoplasts; (3) conditions for the regeneration of walls by sycamore callus and for the resumption of cell division leading to callus formation; and (4) the production of mutant lines of tissue culture, which would be used in a system, developed for animal tissue culture cells, for selecting hybrid cells or protoplasts from a mixed population. The HAT selective system (Littlefield, 1964, 1966; Szybalski, Szybalska & Ragni, 1962) utilizes animal cells lacking in either thymidine kinase (EC. 2.7.1.21) or hypoxanthine guanosine phosphoribosyl transferase (EC. 2.4.2.8) for the selection of hybrid cells. These mutant cells are selected for by their resistance to the base 446 S. W. J. Bright and D. H. Northcote analogues bromodeoxyuridine or azaguanine (Littlefield, 1963; Kit, Dubbs, Piekarski & Hsu, 1963). Enzymic mutants of this sort would be extremely useful in plant protoplast fusion work as they would enable the selective growth of rare heterokaryons from a large number of homokaryons and unfused protoplasts. We have isolated a bromodeoxyuridine-resistant callus of sycamore and have also prepared protoplasts from it and studied their regeneration. MATERIALS AND METHODS Reagents Materials were Analytical Reagent grade or its equivalent. Aminopterin was obtained from Serva Ltd (Microbio Laboratories), thymidine phosphates from Sigma UK Ltd. [6-3H]- thymidine (2 mCi/ml) was obtained from the Radiochemical Centre (Amersham). Calcofluor white was a gift of the American Cyanamid Co. Tissue culture The tissue culture (S4) used in this study was derived from a hypocotyl explant of sycamore (Acer pseudoplatanus). The isolation and conditions for growth and differentiation of the S4 callus have been described (Wright & Northcote, 1972). The cells were grown in the PRL4 medium of Gamborg (1966) with the addition of 1 mg/1. of i-naphthyl-acetic acid and 2 % sucrose (growth medium). Agar (1 %) was used for the growth of solid calluses. Liquid suspensions of cells were grown in 500-ml conical flasks containing 100 ml of medium and an inoculum of 10-25 m' °f cells. The cells were shaken at 25 °C in the dark, and subcultured every 7-10 days. All cell cultures were maintained in a sterile condition. Media and glassware were autoclaved at 120 °C, at 103 kN m~* (15 lb in."1) for 30 min. The constituents of HAT medium (hypoxanthine io~* M, aminopterin io~6 M, thymidine io"4 M) and bromodeoxy- uridine were added to the solutions prior to autoclaving. Solutions containing high sorbitol concentrations or enzymes, were sterile-filtered by passage through a Millipore disk (045 /tm). All operations were performed on a sterile air bench. Packed cell volume was measured by centrifugation of liquid suspensions (10 ml) from replicate cultures at 50 g for 10 min in a sterile graduated centrifuge tube. The contents were then resuspended and replaced in the flask. Growth of solid tissues was measured by placing 3-5 replicate pieces of callus tissue of o-i±o-o5g on to 20 ml agar medium. The initial and later weights were measured and the mean percentage increase and standard error calculated. Microscopy Cells and protoplasts were viewed on a Zeiss Ultraphot II equipped with Nomarski optics and u.v. lamp. Petri dishes were viewed on a Union inverted microscope. Cell walls and remnants of cell wall material were detected by staining the protoplasts for 1 min with o-i % Calcofluor in growth medium and viewing under u.v. light. Protoplasts from sycamore callus Protoplasts were isolated from liquid suspension cultures of callus using a mixture of macerozyme (a pectinase) and Cellulase Onozuka SS (Unwin Ltd, Welwyn Garden City). The Onozuka was purified by passage through a Sephadex G-25 column in glass-distilled water (25 g/80 ml on 800 ml bed volume). The front running dark brown peak of material excluded from the gel was collected and freeze-dried (Hanke & Northcote, 1974). Cell suspension (20 ml) was mixed with an equal volume of 09 M sorbitol in growth medium. The cells were allowed to settle and the supernatant was drawn off down to 4 ml. To this was added 4 ml of 5 % cellulase and pectinase in 0-45 M sorbitol in growth medium. Regeneration of normal and mutant protoplasts 447 The pH of this mixture was in the range 4'8-5'4 and no adjustment was made. The cells were shaken gently on a reciprocal shaker for 4-12 h during which time protoplasts were released into the medium. The protoplasts were separated from the remaining cell clumps by a 43-/tm stainless steel mesh (R. Cadisch and Sons, Finchley), and then washed on a glass-fibre filter paper cone by dispensing 100 ml medium with 0-45 M sorbitol over them slowly with a Pasteur pipette. The last 10 ml of protoplast suspension in the filter funnel were poured off and then centrifuged at 45 g for 10 min to concentrate the protoplasts in 1-2 ml of medium. A sample was removed for counting in a haemocytometer and the rest of the protoplasts were embedded in agar medium by the method of Nagata & Takebe (1971). The final concentrations of components in the agar regeneration medium were agar o-6 %, sorbitol 0-45 M, calcium 5 mM, glucose and ribose C25 g/1. (Kao et al. 1973), zeatin 125 /tg/1., all in growth medium made up in Analar water. Leaf and root protoplasts Sterile maize (Zea mays var. Caldera 535) roots (15) each 1-3 cm long were taken from 4-day-old seedlings and chopped into 2-mm lengths before incubation in 8 ml purified cellulase (2-5 %) and pectinase (2-5 %) in 0-45 M sorbitol in growth medium. The mixture was gently shaken for 19 h or more and the protoplasts collected by passage through 63-/MT1 steel mesh and centrifugation. Mesophyll protoplasts were prepared by a modification of the method of Power & Frearson (IO73)- Mature leaves of Nicotiana Java or xanthi were surface-sterilized briefly in ethanol and then Milton solution (Richardson-Merrell Ltd., London) and washed in sterile water. The leaves were then allowed to wilt for 4 h before the lower epidermis was stripped from the leaf and the exposed mesophyll cells plasmolysed on 05 M sorbitol. The leaf pieces (0-5 g) were then laid on to 8 ml of purified cellulase (2^5 %) and pectinase (2-5 %). The flask was evacuated for 5 min to infiltrate the air spaces with enzyme solution. The leaf pieces were incubated for 12 h in enzyme solution before being strained through 43-/4111 steel mesh to separate the protoplasts. Fusion experiments were performed on freshly isolated protoplasts usually taken directly from the enzyme solution. Protoplast suspensions were spun at 11-50JJ to pellet them and were then resuspended in the test solution and spun again to bring about protoplast contacts. The pellet was then gently resuspended with a Pasteur pipette and a sample viewed for protoplast fusion and viability. Assay of thymidine kinase The assay is modified from Schwarz & Fites (1970). Suspension cells (4 days after sub- culture) were collected on 2 layers of muslin, and 5 g fresh weight were added to 5 ml of cold 04 M phosphate buffer (NaH,PO4. 2HaO, 24-3 g/1.; K2HPO4, 42-2 g/1.) pH 70. This was sonicated twice at 4 °C for 2-5 min to break the cells. The sonicate was centrifuged at 30000g for 30 min and the supernatant used as the enzyme preparation. The assay mixture consisted of enzyme extract 50 fi\; ATP 50 mM, MgCl,, 4 mM, 50 /i\; [6-'H]thymidine 0-19 mM, 325 /tCi/ml, 5 fi\; all in 0-2 M phosphate buffer pH 70. The assay mixture was incubated at 30 °C for 30 min and the reaction terminated by the addition of 03 ml cold methanol.