Plant Cell Reports

Plant Cell Reports (1989) 8:313 316

© Springer-Verlag 1989

Regeneration of haploid and dihaploid plants from protoplasts of supersweet (sh2sh2) corn C.S. Sun *, L.M. Prioli, and M.R. Siindahl DNA Plant Technology Corporation, 2611 Branch Pike, Cinnaminson, NJ 08077, USA Received April 17, 1989/Revised version received July 14, 1989 - Communicated by C.T. Harms

SUMMARY. Plants were regenerated from maize ~ mays L.) protoplasts Isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures. Key words: Zea mays L - supersweet corn - protoplasts haploids - plant regeneration INTRODUCTION Plant regeneration from protoplasts offers a useful tool for a range of genetic manipulations and plant improvement. Although significant progress has been made in protoplast culture of dicotyledoneous species (Davey and Power 1988), recovery of soil-grown plants from protoplasts of Gramineae is still limited to a few species such as sugarcane (Srinivasan and Vasil 1986), rice (Abdullah et al. 1986; Yamada et al. 1986; Kyozuka et al. 1987; Ogura et al. 1987), the grasses Lolium multiflorum, FQ~;tuca arundinacea (Dalton 1988), and Dactylis glomerata (Horn et al. 1988). In maize ~ mays L.), protoplasts have been described as capable of developing into callus (Potrykus et al. 1977, 1979; Chourey and Zurawski 1981; Ludwig et al. 1985; Imbrie-Milligan et al. 1987) and somatic embryos (Vasil and Vasil 1987; Kamo et al. 1987); however, only recently plantlets (Cai et al. 1987) and plants capable of growing in soil (Rhodes et al. 1988; Shillito et al. 1989; Prioli and SSndahl 1989) were regenerated from maize protoplastderived calli. Success in maize protoplast culture appears to depend on many variables Including genotype,physiological stage of donor cells, and culture method. Maize protoplasts with regeneration capacity have been isolated from microspore-derived calli (Cal et al. 1987) and somatic cell suspension cultures (Rhodes et al. 1988; Shillito et al. 1989; Prioli and S~ndahl 1989) of field corn genotypes. The present paper reports embryogenic callus formation and plant regeneration from maize protoplasts isolated from embryogenic cell suspensions established from microsporederived callus of a commercial supersweet hybrid (sh2sh2). Haploid and dihaploid plants were observed among the

regenerated plants. MATERIALS A N D M E T H O D S Cell Suspension cultures. Embryogenic cell suspensions (H3) were established from maize calli derived from microspores of a commercial supersweet F1 hybrid which is a sh2sh2 endosperm mutant (SS7700, Abbott & Cobb, Inc., USA). The calluscultures were initiated from anthers containing uninucleate pollen. Tassels from fieldogrown donor plants were kept in a refrigerator at 6°C during 712 days. After this cold pretreatment, the tassels were sterilized for 10 rain. in a solution of 0.42% sodium hypochlorite containing 0.1% Tween-20 and then rinsed three times with sterilized water. Anthers were excised and placed on callus induction medium consisting of Ns salts (Chu et al. 1975), 30.u.Mthiamine-HCI, 15 p,M nicotinic acid, 15 p.M pyridoxine, 550 #M inositol, 500 mg/I casein hydrolysate, 10 #M 2,4-D (2,4-dichlorophenoxyaceticacid), 10 #M NAA (1naphthaleneacetic acid), 5 ,u.M 6-BA (6-benzylaminopurine), 120 g/I sucrose, 5 g/I activated charcoal, and 2.3 g/I Gelrite (Kelco Co.). Calli induced from microspores of cultured anthers were transferred to D19 medium consisting of Ns salts, 30#M thiamine-HCI, 15#M nicotinic acid, 15 #M pyridoxine, 550 #M inositol, 10 #M 2,4-D, 200 mg/I casein hydrolisate, 30 g/I sucrose, and 2.3 g/I Gelrite. The pH was adjusted to 5.8 before autoclaving. The cultures were incubated in the dark at 26+1°C. The microspore-derived calli were subcultured every 15 days. After three months of culture, a highly friable embryogenic callusline Hz was selected and used to establish cell suspensions. The H3 cell suspensions were haploid, capable of regenerating plants, and were used for protoplast isolation. Maize cell suspensions (H1) initiated from microspore-derived calli of the tropical SRRDuro supersweet (sh2sh2) variety were used as nurse cells in feeder layers. The H1 cell line was established according to the procedure described for the H3 culture. Both H3 and H1 cell suspensions were cultured in liquid medium (D17) consisting of Ns salts, 30 #M thiamine-HCI, 15 p.M nicotinic acid, 15 #M pyridoxine, 550 #M inositol, 5 mM MES (2-[N-morpholino]-ethanesulfonic acid) buffer, 200 mg/I casein hydrolysate, 10 ,u,M2,4-D, and 30 g/I sucrose. The pH was adjusted to 5.5 before autoclaving. The cultureswere incubated on a gyratory shaker (165 rpm) at 26±1 °C in the dark and subcultured every 3-4 days using a selective subculture method. Small cell aggregates were selectedevery 10-15 days by permitting the large aggregates to sediment and then subculturing the supernatant. Every 30-40 days the finely dispersed cell suspensions were filtered through a 520 p.m stainless steel filter. Plant regeneration capacityof H3 cell suspensions was evaluated at 3, 6, and 9 months after culture initiation by transferring 2.5-4.0 ml of packed fresh cells to Petri dishes containing suspension

* Present address. Institute of Botany-, Academia Sinica, Beijing, China Ojfprint requests 1o. M.R. SSndahl

314 regeneration medium (SRM-1) consisting of N6 salts, 30 p,M thlamineHCI, 15 pM nicotinic acid, 15 p.M pyridoxine, 550 pM inositol, 0.05 pM 2,4-D, 10 p,M zeatin, 30 g/I sucrose, 2.3 g/I Gelrite. The pH was adjusted to 5.8 before autoclaving. Protoolast isolation. Protoplasts were isolated from H3 cell suspension cultures 5-6 months after initiation. Cells harvested two days after subculture were mixed with isolation solution (ca. 0.4 g of fresh cells per 10 ml solution) Consisting of 0.62 mM KH2PO4, 1.5 mM CaCI2.2H20, 7.4 mM KNO3, 0.75 mM MgSO4.7H20, 0.45 M mannitol, 5 mM ME'S buffer, 2 mg/ml bovine serum albumin (Sigma Co. A-8022), 3% cellulase Onozuka RS (Yakult Pharmaceutical Industry Co.), 1% macerase (Calbiochem-Behring Corp.), and 1% pectolyase Y-23 (Seishin Pharmaceutical Co.). The pH was adjusted to 5.6 before filter sterilization. The protoplast-enzyme mixture was incubated at 26°C on an orbital shaker (60-70 rpm) for 5-6 h and then filtered successively through 200 pm, 75 pm, and 37 pm stainless steel mesh filters. Protoplasts were sedimented by centrifugation at 50 x g for 5 min. The pellet was resuspended in the isolation solution without enzymes and sedimented again. This washing procedure was repeated three times and then the protoplasts were resuspended in culture medium. B~fore plating, protoplast samples were counted using a hemacytometer for yield evaluations. The presence or absence of cell wall was determined by staining with 0.1% calcofluor white (Aldrich Chem. Co.). Protoplast culture. Protoplasts were cultured in either N5P or NsK media. The NaP protoplast culture medium consisted of N5 salts, N5 vitamins, 5 mM MES buffer, 5/,¢M 2,4-D, 3 mM sucrose, and 0.45 M glucose. The NsK medium consisted of N5 salts, KM-aP (Kao and Michayluk 1975) vitamins, KM-aP organic acids, KM-aP sugar and sugar alcohols, KM-aP amino acids, 20 ml/I coconut water, 5 mM MES buffer, 5 pM 2,4-D, 3 mM sucrose, 0.45 M glucose. The pH was adjusted to 5.5 before filter sterilization. Protoplasts at densities between 1 x 105 and 3 x 106 were cultured either in a thin layer of protoplastliqtiid medium(ca. 1.5 ml) in 35 x 10 mm Petri dishes (Falcon primaria 3801) or on 47 mm diameter filters placed on a feeder layer of maize cells. Four different supporting filters were tested in the feeder layer method: cellulose-nitrate filter (Whatman 7141204WCN; 0.45/zm pore size), cellulose-acatate/cellulose-nitrste filter (Millipore SCWP04700; 8.0 p,m pore size), porous polypropylene membrane (Celgard 3500; 0.04/~m pore size), and Whatman No. 1 qualitative filter paper (11 jum pore size). The supporting filter moistened with NaP or NsK medium was placed on approximately 1 g of H1 maize nurse cells previously washed with protoplast medium. The nurse cells were spread onto 20 ml of D19 medium in 100 x 15 mm Petri dishes. Protoplast suspensions (ca. 0.3 ml) were dropped on the supporting filters. As a control, protoplasta were cultured onto the supporting filters moistened with NaP or NsK medium and placed directly on D19 medium without feeder layer. Petri dishes were sealed with parafilm and cultures were incubated in a growth chamber at 26+1 °C under cool white fluorescent lights (ca. 30/~E.m" 2.s4) in a 16/8.h ligM/dark photoperiod, The osmolality of the liquid medium used in the thin layer method was reduced after 15 days of culture by adding 0.5 ml of D17 medium. After 5 days of culture, fresh protoplast medium (1 ml) was added to the nurse cells. Filters containing protoplasts were transferred 10 days after plating to fresh nurse cells moistened with D17 medium. Cell colonies derived from protoplasts cultured either on feeder layer or in liquid medium were counted under a stereomicroscope 21 days after plating. The plating efficiency was calculated as the number of cell colonies per number of plated protoplasts. Callus maintenance and plant regeneration. Cell colonies (1.0-1.5 mm dia meter) derived from protoplasts were transferred to either D 19 medium or to regeneration media in 100 x 20 mm Petri dishes. The protoplast regeneration media (PRM-1 and PRM-2) consisted of MS salts (Murashige and Skoog, 1962), 15/~M thiamine-HCI, 7.5 /~m nicotinic acid, 7.5/~M pyridoxine, 550 p.M inositol, 10/~M zeatin, 2.3

g/I Gelrite, and either 30 g/I sucrose (PRM-1) or 60 g/I sucrose (PRM-2). Somatic embryos and plantlats were transferred to 150 ml glass jars containing PRM-1 medium without zeatin. Plantlets were further developed in 350 ml jars containing growth medium (GR-1) consisting of half-strength MS salts, 15/~M thiamine-HC[, 7.5 ,uM nicotinic acid, 7.5 #M pyridoxine, 20 g/I sucrose, and 2.3 g/I Gelrite. Cultures were incubated at 26~:1°C with a 16/8 h light/dark photoperiod (125/LE.m'2.S'I). Rooted plantlets were potted in a peat moss/perlite mixture (Sunshine Mix, Groff Inc.) and maintained for 8-10 days in greenhouse under a fog system. Plants were grown to maturity under greenhouse conditions. During short-day seasons, supplementary photosynthetic lights (High Pressure Sodium 400W) were provided to maintain a 16/8 h light/dark photoperiod. Chromosome counts. Cell samples from suspension cultures and root tips of plants regenerated from protoplasts were treated for 2 h in 0.025% colchicine, fixed in Carnoy's solution for 24 h at room temperature, hydrolyzed in 1 N HCI at 60°C for 17 min., and then stained in Schiff's reagent for 2 h at 5-8°C. Cells and root tips were macerated in an enzyme solution 0.1% ceUulasa Onozuka RS and 0.5% pectolyase Y-23 for 10 min. Squashed cells were stained with 1% acetic-carmine solution containing 45% acetic acid. RESULTS AND DISCUSSION The H3 cell suspensions used for protoplast Isolation doubled in cell mass every 3-4 days. These suspension cultures consisted mostly of fast-growing small aggregates of cytoplasmically dense cells (Fig. 1A). Among 240 cells cytologically examined, 194 (80.8%) were haploid (n=10), 45 (18.8%) were aneuploid (n= 10+1 or n= 10+2) and only one cell (0.4%) was dihaplold (2n=20). Therefore, the haploid cell population predominated in the H 3 cell suspension cultures. Variation In chromosome number has been previously reported in maize callus induced from anthers (Gu et ah 1982) and from immature embryos (Edallo et ah 1981; McCoy and Phillips 1982). The H 3 cell suspensions were capable of regenerating plants after 3, 6, and 9 months of culture. In these experiments, 1012 plants were regenerated per ml of packed cells (ca. 0.4 g fresh weight) plated on SRM-1 medium. Plants were grown under greenhouse conditions. Approximately 1 x 106 protoplasts were isolated per ml of packed fresh cells of H3 suspension cultures. Most protoplasts ranged in size from 15/~m to 30'/~m in diameter (Fig. 1B); however, a few large (ca. 501j,m ) protoplasts were also observed. Cell wall material was not present in freshly isolated protoplasts, as determined by calcofluor white staining. The first cell divisions occurred after 6-8 days and colonies were observed after 12-15 days of culture. In three different experiments, protoplasts formed cell colonies in frequencies between zero and 0.002% In NeP medium and between zero and 0.04% in NsK medium using the thin-layer method (Fig. 1-C). In contrast, cell colonies were formed in frequencies between 4.6% and 6.1% from protoplasts cultured on feeder layers using the cellulose-nitrate or celluloseacetate/cellulose-nitratefilters (Fig. l-D). Therefore, the feeder layer method had a clearly positive effect on the formation of cell colonies from maize protoplasts as compared to the thin layer method. The frequency of protoplast-derived cell colonies was very low (0.04% or less) when polypropylene membranes or Whatman No. 1 filter papers were used as supporting filters on feeder layers. These results show an effect of the different types of supporting membranes in cell colony formation. The low efficiency of protoplasts to develop

315

Fig. 1 A-I. (A) Cell aggregates of maize suspension cultures (H3) established from microspore-derived callus of a sh2sh2 hybrid. Bar=50 /zm. (B) Protoplasts isolated from haploid H3 celt suspensions, Bar=20 /an. (c) Cell colonies formed from H3 protoptasts after 21 days of culture in thin layer of liquid medium. Bar=2.0 mm. (O) Numerous cell colonies formed from H3 protoptasts 21 days after plating on cellulose/nitrate f i l t e r placed on a feeder layer of maize cells, Bar=5.6 ram. (E) Embryogenic H3 maize protoplast-derived c a l l i 20 days after plating on PRM-1 medium. Bar=l mm. (F) Maize ptantlets regenerated from H3 protoptast-darived c a t l i . Bar=l.0 cm. (G) Protoplsst-derived H3 soil-gromn plants under greenhouse conditions. Bar=3.3 cm. (11) Chromosome set from root t i p of a haploid H3 protoplast-derived plant (n=lO). Bar=2 /Lm. ( I ) Chromosome set from root t i p of s dihaploid plant derived from H3 protoplssts (2n=20). Bar=l.8 /ua.

cell colonies on prolypropylene membrane and Whatman No. 1 filter paper could be attributed to the membrane material, presence of toxic substances in the membranes, or to differences in membrane pore size which could affect the exchange rate of substances and/or protoplast aeration. In the case of Whatman No. 1 filter paper, the excess of liquid medium on the filter may have had an additional negative effect on protoplast divisions due to more limited aeration. Cell colonies were not obtained from protoplasts cultured on supporting filters placed directly on D19 medium without a feeder layer. The negative results of this control demonstrate that the presence of nurse cells was essential to induce divisions in the haploid H3 protoplasts; however, the inadequate osmolality of the D19 medium and/or other factors

may have also Influenced the protoplast response. Improvement of the frequency of cell colony formation from maize diploid protoplasts by using maize cells In feeder layers has been previously reported (Ludwig et al. 1985; Kamo et al. 1987; Rhodes et al. 1988; Shillito et al. 1989). The results obtained in the present work for H~ haploid protoplasts confirmed that feeder layer methods can improve the frequency of cell colony formation from maize protoplasts. The maize protoplast-derived calli cultured on D19 medium were friable and doubled in cell mass every 3-4 days. Upon transferring callus samples to PRM-1 and PRM-2 media, a large number of globular somatic embryos were formed within 2 weeks. Most embryos turned green in the presence of light (Fig. l-E). A noticeably higher number of somatic embryos

316 germinated on PRM-1 medium as compared with the PRM-2 medium. Plantlets were regenerated from callt derived from protoplasts (Fig. l-F). T'nese plantlets developed new leaves and a strong root system after transferring to GR-1 medium. The protoplast-derived Calli have maintained the regeneration capacity for 8-10 months as determined by periodic transfer of callus samples to PRM-1 medium. A total of 31 plants regenerated from the initial calli derived from Ha protoplasts were grown under greenhouse conditions (Fig. l-G). In maize, plant regeneration from protoplasts Isolated from embryogenic cell suspensions was recently achieved for the inbred lines A188 and B73 (Rhodes et al. 1988), B73-derivattve (Shillito et aL 1989), and Cat100-1 (Prioli and S~ndahl 1989). Previous reports had suggested that the regeneration capacity of donor suspension cultures can be partially or completely lost in protoplast cultures (Vasil and Vasii 1987; Kamo et ai. 1987). The results obtained for the supersweet haploid protoplast cultures confirmed that maize cell cultures capable of regenerating plants retain this ability after protoplast isolation. in addition, it was demonstrated that haploid maize protoplasts can regenerate plants capable of growing in soil. Maize haploid suspensions and protoplasts capable of regenerating plants could be useful for selection of mutants, as well for colchicine treatments in experiments aimed at chromosome doubling. Cytological analysis of eleven protoplast-derived plants revealed that ten were haploid (Fig. l-H) and one was dihapioid (Fig. 1-1). These results Indicated that the plants recovered from protoplasts tended to maintain the haploid chromosome number observed in the majority of donor suspension cells. The dihaploid plant may have resulted from a dihaploid cell present in the donor cultures, from spontaneous protoplast fusion, or from spontaneous chromosome doubling in protoplast-derived callus. Regeneration of haploid and dihaploid plants from protoplasts isolated from cell suspensions derived from anther callus have been reported in rice (Torlyama et ah 1986). Morphological abnormalities, such as low vigor, reduced number of nodes, short internodes, terminal ear and absence of tassel and/or ear were observed among the regenerants. The identified dihapioid plant derived from protoplast was small (ca. 40 cm tall), had a reduced number of nodes, short internodes, and developed a terminal ear with a few Silks and no male flowers. Similar morphological abnormalities were previously observed among maize plants regenerated from both protoplasts (Rhodes et al. 1988; ShillitO et al. 1989; Prioli and S~ndahl 1989) and embryo-derived calli (Earle and Gracen 1985). A total of 4 plants developed ears and tassels but did not produce pollen. As revealed by the cytological analysis, those 4 protoplast-derived plants were haploids. Both sterile plants (Rhodes et al. 1988) and fertile plants (Shillito et aL 1989; Prioli and SSndahl 1989) have been regenerated from maize protoplasts. The male sterility of the plants regenerated from H3 protoplasts was possibly due to haploidy. Pollen abortion is a well-known and expected phenomenon in haploid plants. The present work demonstrates regeneration of soil-grown plants from a haploid supersweet (sh2) genotype unrelated to the field corn inbred lines previously used for protoplast culture. R was also demonstrated that maize dihaploid plants can be regenerated from protoplasts isolated from haploid cell culture. Dihaploid plants are extremely valuable in developing

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