Proc. Natl. Acad. Sci. USAVol. 83, pp. 5616-5619, August 1986Genetics

Somatic hybridization in the Gramineae: Saccharum officinarum L.(sugarcane) and Pennisetum americanum (L.) K. Schum.(pearl millet)

(protoplast fusion/grasses/cereals/amino acid analog/somatic embryo)

ZOHREH TABAEIZADEH, ROBERT J. FERL, AND INDRA K. VASIL*Department of Botany, University of Florida, Gainesville, FL 32611

Communicated by John G. Torrey, April 18, 1986

ABSTRACT Somatic hybrid cell lines with embryogeniccapacity were obtained by fusion ofprotoplasts isolated from anembryogenic cell line of sugarcane with inactivated protoplastsofan S-(2-aminoethyl)-L-cysteine (Aet-Cys)-resistant cell line ofpearl millet. Initial selection for putative hybrids was per-formed by plating protoplast-derived microcolonies onto me-dium containing 300 ,#g of Aet-Cys per ml. Calli growing in thepresence of Aet-Cys were screened with respect to theirelectrophoretic pattern of phosphogluconate dehydrogenase(6-phospho-D-gluconate; EC 1.1.1.44), a dimeric enzyme. Thesomatic hybrid calli showed both parental homodimer bandsplus an intermediate heterodimer band. Somatic hybrid lineswere characterized further by electrophoretic patterns ofshikimate dehydrogenase (shikimate; EC 1.1.1.25) andarylesterase (aryl-ester hydrolase; EC 3.1.1.2). All cell linesexpressed the nuclear genomes of both parents. Use of a maizeribosomal DNA probe allowed the identification of diagnosticribosomal DNA fragments inXba I digests of total DNA in bothparents and the somatic hybrids. The somatic hybrid linescarried both sugarcane and pearl millet ribosomal DNAfragments. The somatic hybrid calli formed somatic embryos.The banding pattern of phosphogluconate dehydrogenase fromthe somatic embryos was similar to that of the somatic hybridcalli.

Protoplast fusion has been widely used in attempts to transfernuclear and/or cytoplasmic traits from one plant species toanother (1, 2). Successful results have been obtained mostlyin the family Solanaceae and generally between sexuallycompatible species. Among other methods, selection ofsomatic hybrids has been accomplished by using aminoacid-analog-resistant cell lines in Nicotiana sylvestris (3), forwhich dominant and semidominant behavior for resistance toS-(2-aminoethyl)-L-cysteine (Aet-Cys) and 5-methyltryp-tophan, respectively, was demonstrated. The lack of efficientprotoplast regeneration systems and selectable markers haddiscouraged attempts toward somatic hybridization in impor-tant crop species such as those belonging to the familyGramineae. Recently, however, plants have been regenerat-ed from protoplasts isolated from embryogenic cell suspen-sions of Pennisetum americanum, Pennisetum purpureum,Panicum maximum, and Saccharum officinarum in thislaboratory (4-7).

Somatic hybridization in the Gramineae has proven to bevery difficult. In the only other report of somatic hybridiza-tion in this family (8), no organized development was ob-tained. In this paper we describe the selection of somatichybrid cell lines and the formation of somatic hybrid embryosof sugarcane and pearl millet, using an amino acid-analog-resistant cell line and metabolic inhibitors (9, 10).

MATERIALS AND METHODSCell Suspensions. A cell line of Pennisetum americanum

(Gahi 3, cytoplasmic male sterile) resistant to Aet-Cys wasisolated in 1980 (Z. Haydu and I.K.V., unpublished results)from an embryogenic cell suspension culture (11). Thesuspension had been maintained in Murashige and Skoog's(12) medium supplemented with 2 mg of 2,4-dichloro-phenoxyacetic acid (2,4-D) per liter, 30 g of sucrose per liter,100 mg of inositol per liter, 5% coconut milk, and 100 ,ug ofAet-Cys per ml. The amount of Aet-Cys in cell suspensionculture medium was gradually increased to 400 ,ug/ml beforefusion experiments. An embryogenic cell suspension ofSaccharum officinarum (clone B43-62) was isolated in 1984(ref. 7; see also ref. 13) and has been maintained in MSmedium containing 2.5 mg of 2,4-D per liter, 30 g of sucroseper liter, 100 mg ofinositol per liter, 5% coconut milk, and 500mg of casein hydrolysate per liter. The suspensions weresubcultured every week by adding 8 ml (for pearl millet) and2.5 ml (for sugarcane) of the cell suspension to 35 ml of freshmedium in 250-ml Erlenmeyer flasks. The cultures wereincubated in darkness at 26-28°C on a Gyratory shaker at 150rpm.

Protoplast Isolation, Inactivation, Fusion, and Culture.Protoplasts were isolated by incubating 1.5 ml (=0.5 g offresh weight) of a 3-day-old cell suspension in 20 ml ofenzyme solution. The enzyme solution consisted of 3.5%cellulase "Onozuka" R-10 and 0.8% pectinase (Serva) forpearl millet and of 2% cellulase "Onozuka" RS, 0.5%pectinase, and 0.25% driselase for sugarcane, dissolved in0.45M mannitol, 7 mM CaCl2 2H2O, 0.7 mM NaH2PO4 H2O,and 3 mM 2-(N-morpholino)ethanesulfonic acid (Mes) at pH5.6. After 4-5 hr of incubation at room temperature, theprotoplast/enzyme mixture was filtered through 100-, 50-,and 25-,um stainless steel filters. Protoplasts were washedonce by centrifugation (100 x g) in a solution containing thesame concentration of salts and Mes as in the enzymesolution, supplemented with 0.5 M glucose. Protoplasts ofpearl millet were inactivated by incubation in 1 mMiodoacetic acid (dissolved in wash solution) for 10 min. Theselection of this concentration of iodoacetate was based onthe results of preliminary experiments using 1-10 mM iodo-acetate with different durations of treatment (5-15 min).Treatment with 1 mM iodoacetate for 10 min did not have anyadverse effect on pearl millet protoplasts on the day of theexperiment but it successfully inhibited cell divisions. Theprotoplasts were washed two times after this treatment.Protoplasts of pearl millet and sugarcane were then mixed ina ratio of 1:2. Fusion was carried out in 6-cm Petri dishes. The

Abbreviations: Aet-Cys, S-(2-aminoethyl)-L-cysteine; 2,4-D, 2,4-dichlorophenoxyacetic acid; Mes, 2-(N-morpholino)ethanesulfonicacid; 6PGDH, phosphogluconate dehydrogenase (6-phospho-D-glu-conate).*To whom reprint requests should be addressed.

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fusion solution contained 20%o polyethylene glycol (M, 8000,Sigma), 0.2 M glucose, 10 mM CaCl2 2H2O, buffered to pH10 by 0.1 M glycine/NaOH. After 10-15 min, the fusedprotoplasts were eluted by using a solution containing 150mM CaCl2 2H2O, 3 mM Mes, and 0.2 M glucose. Protoplastswere collected after 30-40 min, washed once, and cultured inmodified Kao and Michayluk's (14) medium 8P according toVasil and Vasil (4). In each experiment unfused protoplastsas well as homologous fusion products of each species werealso cultured as controls. A total of nine fusion experimentswas performed.

Aet-Cys Selection. After 2 weeks of development, micro-calli derived from protoplasts were transferred to a selectionmedium containing MS salts and vitamins, 0.5 mg of 2,4-Dper liter, 30 g of sucrose per liter, 0.8% agar, and 300 Ag ofAet-Cys per ml. This culture was maintained for 6 weeks.Colonies that appeared on the selection medium after 2 weekswere transferred to the same medium containing 400 ug ofAet-Cys per ml (first harvest). After 2 more weeks the newcolonies, which appeared on the original Petri dish containingthe selection medium, were transferred to the fresh mediumsupplemented with 400 ktg of Aet-Cys per ml (second har-vest). This process was repeated one more time 6 weeks afterstarting the selection (third harvest).

Electrophoretic Separation of Isozymes. Approximately200-300 mg of callus tissue was ground in the extractionbuffer containing 0.2 M Tris (pH 6.8), 20%o sucrose, and 10%2-mercaptoethanol. Electrophoresis was performed undernondenaturing conditions, using 7.5% polyacrylamide gels.The gels were stained for phosphogluconate dehydrogenase(6PGDH; 6-phospho-D-gluconate; EC 1.1.1.44) and shikim-ate dehydrogenase (shikimate; EC 1.1.1.25) activity using theprocedure of Vallejos (15). Arylesterase isozymes (aryl esterhydrolase; EC 3.1.1.2) were detected according to the pro-cedure of Shaw and Prasad (16).Ribosomal DNA Analysis. DNA was isolated from 0.7-1.0

g of callus tissue from the somatic hybrids and parental cellsuspension culture lines following the procedure ofDellaporta et al. (17). Approximately 1-2 ;Lg ofDNA isolatedfrom each source was digested with Xba I in a 20-,l reactionmixture at 37°C for 1.5 hr. The restricted DNA was electro-phoresed at 190 mA for 2-3 hr in a 1% agarose gel (0.04 MTris/0.02 M sodium acetate/1 mM EDTA, pH 8.0). Subse-quently, DNA was denatured and transferred to nitrocellu-lose (18). The blot was then dried in a vacuum oven at 80°Cfor 2 hr. A maize ribosomal DNA clone (provided by P. S.Chourey) was nick-translated with [32P]dCTP (New EnglandNuclear) and used as a probe. The Southern blot wastransferred to prehybridization solution containing 0.9 Msodium chloride/0.09 M sodium citrate, 5x concentratedDenhardt's solution (Denhardt's solution = 0.02% bovineserum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone),0.1% NaDodSO4, and 100 ug of denatured salmon spermDNA per ml at 65°C for 1 hr. Hybridization was performedat 65°C for about 14 hr after adding 4 x 10- cpm of the probeper ml of the prehybridization solution. After hybridizationthe blot was first washed at room temperature for 5 min in0.45 M sodium chloride/45 mM sodium citrate and 0.05%NaDodSO4. Another wash was carried out at 65°C for 20 min,using the same solution. Finally, after a third wash in 0.15 Msodium chloride/15 mM sodium citrate and 0.05% NaDod-S04 at 65°C, the blot was exposed to x-ray film along with anintensifying screen at -80°C for 72 hr.Somatic Embryogenesis Induction Medium. Once the so-

matic hybrid nature of the calli was confirmed, differentiationwas initiated by transfer of the calli to maintenance mediumamended to include 0.1-0.25 mg of 2,4-D per liter and 1%activated charcoal. The embryogenic calli were later trans-ferred to the same medium with reduced 2,4-D (0.005-0.05

mg/liter), with or without 6-benzylaminopurine (0.1-0.5mg/liter).

RESULTS

Selection on Aet-Cys-Containing Medium. Approximately0.008% of the total protoplasts cultured following the fusiontreatment grew on the medium containing 300 ,ug of Aet-Cysper ml (each colony growing on the selection medium wasconsidered to be derived from a single protoplast). The rateof growth of sugarcane microcalli was almost similar to therate of those recovered from the fusion treatment. Iodoace-tate-inactivated protoplasts of pearl millet and their homol-ogous fusion products died after 24 hr. The growing calli weretransferred to fresh medium and were allowed to grow furtherin order to have enough tissue for isozyme screening.Isozyme Screening. Among 15 different isozymes that were

tested in the parental lines, 6PGDH, a dimer isozyme, waschosen to screen the somatic hybrids. Sugarcane and pearlmillet contained forms of homodimeric enzyme that could beeasily distinguished from each other. Among 200 calli tested,4 were determined to be somatic hybrids, as they exhibitedboth parental bands plus a heterodimer band (Fig. la). Theheterodimer band was absent in a mixture of extracts fromboth parents. Therefore, the presence of this band indicatesthat the genes are in the same cell. The four hybrid calli-designated Hi, H2, H3, and H4-were from four differentexperiments.The hybrid lines Hi, H2, and H3 continued vigorous

growth on the selection medium and expressed the

P H3 H2 H1 SP

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S H2 HI P S

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FIG. 1. (a) 6PGDH isozyme patterns of sugarcane (S), pearlmillet (P), and three somatic hybrid lines (H1, H2, and H3). SPdenotes a mixture of extracts from two parental lines. The arrowmarks the heterodimer band. (b) Arylesterase isozyme patterns ofsugarcane (S), pearl millet (P), and four somatic hybrid lines (H1, H2,H3, and H4). The arrows mark the extra bands. (c) Autoradiogramof blot hybridization of 32P-labeled ribosomal DNA clone to Xba Idigests of total DNA from sugarcane (S), pearl millet (P), and two ofthe somatic hybrids (H1 and H2). Fragment size is shown inkilobases. (d) 6PGDH isozyme patterns of sugarcane (S), pearl millet(P), and somatic hybrid embryos (E). The arrow marks theheterodimer band.

Genetics: Tabaeizadeh et al.

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5618 Genetics: Tabaeizadeh et al.

heterodimer pattern of 6PGDH repeatedly. In contrast, lineH4 showed decreased potential for growth on the selectionmedium and also did not show any expression of 6PGDH 2months after initial screening. It could not be determinedwhether gene expression is repressed in this line or if somechromosomes were eliminated.

Further confirmation of the hybrid nature of these calli wasobtained from the electrophoretic pattern of arylesterase. Inlines Hi, H2, and H3 both parental bands as well as an extraband could be detected. Line H4 contained all of the pearlmillet bands with three of the sugarcane bands. Three extrabands were also seen in this line (Fig. lb).The electrophoretic pattern of shikimate dehydrogenase

was another indication of hybridization between sugarcaneand pearl millet. Each ofthe parents showed one band. LinesHi, H2, and H3 contained both parental bands (data notshown). No activity of this isozyme could be detected in lineH4.Ribosomal DNA Analysis. The maize ribosomal DNA probe

was hybridized to Xba I digests oftotalDNA from sugarcane,pearl millet, and somatic hybrids Hi and H2 (Fig. ic). Thehybridization with the parental DNA yielded two fragments(7.9 and 5.5 kilobases, respectively) in sugarcane and threefragments (5.5, 4.2, and 1.6 kilobases, respectively) in pearlmillet; only one fragment was similar in both parents. Hi andH2 contained all parental fragments.Morphogenesis in Somatic Hybrid Calli. The hybrid calli

maintained continuously on the selection medium werecomposed of densely cytoplasmic embryogenic cells thatcontained many starch grains. Some of these calli formedglobular proembryoids and leaf-like structures 3-5 weeksafter transfer to the somatic embryogenesis induction medi-um. A number of somatic embryos were later formed in threeof the somatic hybrid calli-Hi, H2, and H3 (Fig. 2). Theactivity of6PGDH in the somatic embryos was similar to thatof the somatic hybrid calli (Fig. ld).

DISCUSSIONAmino acid-analog-resistant cell lines have been used byseveral investigators for somatic hybridization since the firstreport by White and Vasil (3). The work of Cella et al. (19)is the most similar to the present study. They fused theinactivated azetidine-2-carboxylic acid-resistant protoplastsof Daucus carota to the sensitive protoplasts of the samespecies; however, the only evidence for somatic hybridiza-tion in their studies, aside from the chromosome number, wasgrowth of cells on the selection medium (containing the

FIG. 2. Somatic hybrid embryo of sugarcane and pearl millet. ci,

coleoptile; sc, scutellum. (x60.)

inhibitory amounts of azetidine-2-carboxylic acid) only afterheterofusion. Our results, and those of Ozias-Akins et al. (8),show that growth on the selection medium alone is notsufficient for confirmation of somatic hybridization, since ofthe large number of calli that grew on the selection mediumonly four were confirmed to be true somatic hybrids. Theremaining were sugarcane calli that were able to grow on theselection medium. The growth rate of the sugarcane "es-capes" was even faster than that ofthe somatic hybrids, sincethree of the hybrids (H1, H2, and H3) were recovered fromthe second harvest and H4 was from the third harvest. Nosomatic hybrids could be selected from the first harvest.The use of a multimer isozyme for the second screening

was thus necessary in order to differentiate a chimeric callusfrom a somatic hybrid. Therefore, 6PGDH was chosen forscreening the somatic hybrids since it is a dimer enzyme thatshowed distinct differences between the parental lines. Al-cohol dehydrogenase and phosphoglucoisomerase wereamong the dimer enzymes that were tested but their electro-phoretic patterns were not sufficiently different between thetwo parents. Among 200 calli tested for 6PGDH, 4 proved tobe somatic hybrids. Gleddie et al. (20) have also used thisisozyme for characterization of Nicotiana rustica andNicotiana sylvestris somatic hybrids, but they did not detectany intermediate band in their somatic hybrids.Although all of the somatic hybrid lines exhibited both

parental bands for arylesterase activity, their electrophoreticpatterns varied from each other. The somatic hybrid lines H1,H2, and H3 showed one extra band, whereas H4 showedthree extra bands. Gleba et al. (21) showed a similar result foresterase activity in somatic hybrid cell lines of Atropabelladona and Nicotiana chinensis. The hybrid lines hadmore bands than the sum of parental bands and they weredifferent in their electrophoretic patterns from each other.The number of somatic hybrids that could be identified

after screening with 6PGDH does not necessarily reflect thefrequency of hybridization. The calli were screened only byone isozyme after growth on the selection medium. It is likelythat some of the somatic hybrids could not be detected dueto the sorting out of chromosomes, a frequent event inprotoplast fusion between sexually incompatible species (22,23).The fact that some of the hybrid calli formed somatic

embryos is encouraging. This shows that embryogenic ca-pacity is retained in the somatic hybrid lines. Since plantshave been regenerated from protoplasts ofthe cell suspensionculture of sugarcane used in this study (7), and becauseregeneration capacity has been shown to be a dominant trait(24, 25), plant regeneration from these somatic hybrid em-bryos may be feasible.

Technical help of Mrs. Beth Laughner in ribosomal DNA analysisis gratefully acknowledged. This work was partly supported by fundsfrom Monsanto Company, St. Louis. This paper is Florida Agricul-ture Experiment Station Journal Series no. 7270.

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