APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1990, p. 2404-2409 Vol. 56, No. 80099-2240/90/082404-06$02.00/0Copyright C) 1990, American Society for Microbiology

Transfer of Isolated Nuclei into Protoplasts ofTrichoderma harzianum

A. SIVAN,t G. E. HARMAN,* AND T. E. STASZDepartment of Horticultural Sciences, New York State Agricultural Experiment Station,

Cornell University, Geneva, New York 14456

Received 16 January 1990/Accepted 17 May 1990

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast,and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 ,um in diameter. Nucleiwere isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplastsobtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerousprogeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from awild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of asecond strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when theregenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating.Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-typenuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern andcolony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of thisnuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.

Fungal protoplast fusion has been established as a meansfor transfer of genetic information, and it provides an effec-tive method for genetic manipulation and strain improve-ment (20). This approach can be very powerful, especially infungi lacking a sexual stage.

Successful fusion of protoplasts in Trichoderma spp. hasbeen demonstrated (5, 20, 24). However, fusion betweendissimilar strains of Trichoderma spp. resulted in a low levelof compatibility (19, 20). This was demonstrated by a lowcomplementation frequency for fusion between complemen-tary auxotrophic parental strains and by the recovery ofprogeny which were unstable and slow-growing and whichexhibited a wide range of colony morphologies. Theseprogeny were shown to be either very imbalanced hetero-karyons or homokaryons that differed markedly from theparental strains. In imbalanced heterokaryons, nuclei of oneparental type prevailed while the nuclei of the other parentaltype became nonprevalent. Protoplast fusion in other spe-cies, e.g., Penicillium and Aspergillus spp., also has beenreported to result in limited vegetative compatibility (2, 14,17).Transfer of isolated cytoplasmic genetic elements (e.g.,

nuclei, mitochondria, or plasmids) into protoplasts mayprovide a novel means for complementation of geneticinformation. Currently, there are only a few reports on thissubject. So far as we know, none of them deals with transferof nuclei within filamentous fungi. Nuclei from Saccharomy-ces cerevisiae were transferred into protoplasts of the samespecies (3, 9). Similarly, several studies have demonstratedthat mitochondria can be isolated and transferred into pro-toplasts of S. cerevisiae (11, 27).

In Trichoderma spp., although protoplast fusion mayresult in only a low level of compatibility, nuclear transfermay allow establishment of a heterokaryon and expression

* Corresponding author.t Present address: Institutes of Applied Research, Ben Gurion

University, Beer-Sheva 84110, Israel.

of the nonprevalent nuclear types. Such a procedure mayalso provide a better understanding of the postfusion geneticevents.

Successful use of protoplast fusion requires some methodof isolating fused cells. Usually, this requires fusion of twostrains, each deficient in a complementary property, e.g.,auxotrophy. Media for regeneration of thalli following fusionwere chosen so that progeny strains which exhibited com-plementation could be identified. An example is the selectionof prototrophic strains on a minimal medium subsequent tofusion of two auxotrophic strains. Development of deficientstrains usually requires chemical mutagenesis, which maychange numerous properties other than those for which thestrain was selected. Such secondary mutations may bedeleterious. The use of nuclear transfer, however, permitsuse of any wild-type strain to serve as the nuclear donor andtherefore lessens the chance of deleterious mutations.

Finally, protoplast fusion combines the entire contents oftwo vegetative cells into one. Therefore, all cytoplasmicgenetic elements from both strains, as well as genomic DNA,are involved. Transfer of nuclei into protoplasts eliminatesrecombination of extranuclear components, and geneticanalysis of progeny resulting from nuclear transfer maytherefore be less complex.The objectives of this study were to isolate intact nuclei of

T. harzianum, insert them into protoplasts of similar anddissimilar strains, and characterize the resulting progeny.

MATERIALS AND METHODS

Media. The media used in this study included the basalmedium (BM) of Toyama et al. (24), prepared as describedby Stasz et al. (20). This medium was also amended with10% sucrose and used as a protoplast regeneration medium(PRM). BM and PRM amended with 150 ,ug of L-lysine perml, 150 ,ug of L-histidine per ml, or a combination of thetwo were designated BM+L, BM+H, BM+HL, PRM+L,PRM+H, and PRM+HL, respectively. Potato dextrosebroth (Difco Laboratories, Inc., Detroit, Mich.) amended

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with 10% sucrose served as a liquid regeneration medium forprotoplasts. Potato dextrose agar (PDA) amended with his-tidine, L-lysine, or a combination of the two was designatedPDA+H, PDA+L, or PDA+HL, respectively, and wasused for nutritional studies of progeny.

Strains and markers. Strains T95 (1) (ATCC 60850) andT12 (12) (ATCC 20737) of T. harzianum Rifai were used asparental strains. We also used auxotrophic mutants derivedfrom T95 (T95 His- and T95 Lys-) (T. E. Stasz and G. E.Harman, Exp. Mycol., in press).

Preparation of protoplasts. Protoplasts were obtained from24-h cultures of prototrophic or auxotrophic strains of T.harzianum that were grown in potato dextrose broth supple-mented with appropriate amino acids for the auxotrophs andwere shaken on a rotary shaker at 25°C by a proceduresimilar to that used by Stasz et al. (20). The cultures werewashed with sterile distilled water and treated with No-vozyme 234 for 1.5 to 2.0 h at 30°C to release protoplasts.Hyphal debris was removed by filtration through four layersof cheesecloth, and protoplasts were recovered and washedtwice in 0.7 M NaCl by centrifugation in an InternationalEquipment Co. model 428 clinical centrifuge (VWR, Roch-ester, N.Y.) at 100 x g for 5 min at 4°C. Protoplasts weresuspended in a solution containing 0.6 M sorbitol, 0.01 MTris hydrochloride, and 0.01 M CaCl2 (pH 7.5) (STC) andkept on ice until used (20).

Determination of number of nuclei in protoplasts. Proto-plasts were fixed for 30 min in a solution composed of 95%ethanol, formaldehyde, acetic acid, and water (10:2:1:7) andamended with 41 mg of NaCl per ml. After fixation, proto-plasts were washed twice in 0.7 M NaCl and stained with4',6'-diamidino-2-phenylindole (DAPI) at a concentration of25 ,ug/ml for 15 min. The stained protoplasts were rinsedtwice with 0.7 M NaCl and viewed with an epifluorescencelight microscopy, as described by Stasz and Harman (inpress). Stained nuclei were counted in samples of 100protoplasts for each strain.

Isolation of nuclei. Protoplasts isolated as described abovewere concentrated by centrifugation, suspended in 0.7 MNaCl, centrifuged again, and then suspended in an extrac-tion buffer (EB) prepared by the method of Gealt et al. (10),as modified by Timberlake (23) and Taylor and Natvig (22).This solution contained 10 mM Tris hydrochloride (pH 7.0),4 mM spermidine hydrochloride (Calbiochem-Behring, LaJolla, Calif.), 1 mM spermine hydrochloride (Calbiochem),0.1 M KCl, 10 mM EDTA, 0.5 M sucrose, 1 mM phenyl-methylsulfonyl fluoride (Sigma Chemical Co., St. Louis,Mo.), and 0.1% 2-mercaptoethanol. The protoplasts werepelleted by centrifugation at 1,000 x g for 15 min at 4°C andsuspended in 0.2 ml of EB. A 50-mg portion of fine quartzsand was added, and the mixture was homogenized for 3 minin a Ten Broeck glass tissue homogenizer (Corning GlassWorks, Corning, N.Y.) to lyse the protoplasts. An additional5-ml sample of EB was added, and the number of intactprotoplasts was determined with a Petroff-Hausser bacterialcounting chamber (Thomas Scientific, Swedesboro, N.J.).The homogenized suspension was filtered sequentiallythrough 8.0-, 5.0-, 3.0-, and 2.0-,um-pore-size polycarbonatefilter membranes (Nuclepore Corp., Pleasanton, Calif.) toremove remaining protoplasts (protoplasts of Trichodermastrains are usually larger than 3 ,um in diameter). The filtratewas centrifuged at 1,500 x g for 15 min to sediment nucleibut not smaller organelles (4, 16). The supernatant wasdiscarded, and the pellet containing the nuclei was sus-pended in 1 ml of STC or EB. Numbers of intact protoplastswere assessed at various times during the isolation proce-

dure by using the counting chamber. Isolated nuclei wererecovered by filtration on a 0.45-p.m-pore-size polycarbonatefilter, stained with DAPI, and observed microscopically todetermine their integrity. The presence of mitochondria inthe protoplasts and in the nuclear suspension was deter-mined by staining with rhodamine 123 (Eastman Kodak Co.,Rochester, N.Y.), which specifically stains mitochondrialDNA (26).

Intrastrain and interstrain nuclear transfer. Nuclei wereisolated from 4 x 108 protoplasts of T95 His-. The nuclei,suspended in 1 ml of EB, were centrifuged at 1,500 x g, andthe pellet was mixed with 1 x 108 to 3 x 108 protoplasts ofT95 Lys- suspended in 0.5 ml of STC. The mixture ofprotoplasts and nuclei was amended with 100 p.1 of a solutioncontaining 60% (wt/vol) polyethylene glycol (PEG; molecu-lar weight, 3,350) (Sigma), 10 mM CaCl2, and 10 mM Trishydrochloride (pH 7.5). The PEG mixture was added to theprotoplast-nucleus suspension by gently rolling the tube. Asecond 250 p.1 ofPEG solution was added, the fusion mixturewas again gently mixed, and a third 250-,ul sample was addedand mixed, as described above. The fusion mixture wasincubated at 30°C for 5 min, and nuclei and protoplasts wereforced together by centrifugation for 5 min at 1,000 x g. Thispellet was incubated for an additional 5 min and thensuspended by stepwise addition of 3 portions of STC (0.55ml, 0.55 ml, and 1.1 ml). The tube was gently rolled betweensample additions. The diluted mixture was centrifuged at1,000 x g, and the supernatant was discarded and replacedwith 0.5 ml of STC. Portions of this suspension were platedon BM and BM+L.

Interstrain nuclear transfer was carried out with nucleiobtained from T12 and protoplasts of T95 Lys-. Preliminaryexperiments indicated that at least 3 x 101° nuclei wererequired to obtain progeny. In order to accumulate this highnumber, nuclei were prepared from four to five separatebatches of ca. 5 x 109 protoplasts each. The resulting nucleiwere suspended in 1 ml of EB amended with 15% glycerol,were frozen in liquid nitrogen, and were stored at -70°Cuntil use. Before fusion with protoplasts, the frozen nucleiwere thawed at room temperature, centrifuged at 1,500 x gfor 15 min, and suspended in 0.5 ml of STC containing 1 x108 to 3 x 108 protoplasts of T95 Lys-. Fusion was per-formed as described above. The fusion mixture was dilutedin STC and plated on PRM to isolate progeny and onPRM+L to test viability of protoplasts.

In preliminary tests, interstrain nuclear transfers did notresult in recovery of prototrophic progeny on PRM. There-fore, two modified procedures were utilized. The firstmethod (nuclear insertion A) included an addition of BM orBM supplemented with 1 ,ug of lysine per ml as a 10-ml agaroverlay which was poured onto the fusion mixture plates(PRM and PRM+L) 10 days after the initial plating. Theaddition of extra BM dilutes the high concentration of theosmoticant (10% sucrose), which may reduce regenerationrate and subsequent hyphal growth (unpublished data).Amendment with the low concentrations of lysine wasintended to permit initial growth of protoplasts but not topermit further hyphal growth of auxotrophic strains. In thesecond method (nuclear insertion B), samples of the fusionmixture were plated on PRM containing 1 ,ug of lysine perml. After 48 h, 10 ml of 2% water-agar or 2% water-agaramended with lysine was overlaid. All plates were incubatedat 25°C. Colonies that grew on those media were transferredto fresh BM, PDA, and PDA+L plates for further analysis.

Analysis of nuclear insertion progeny. Progeny from theintrastrain and interstrain nuclear insertion experiments

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TABLE 1. Purification of nuclei from protoplastsof T. harzianum T95 His-

Purification step Protoplast yield ReleasedPurificatinstep Totalb Viablec nuclei'

Initial protoplast preparation 3.8 x 108 8.5 x 107 0

Cell disruption' 3.2 x 106 2.5 x 104 8.6 x 108

Filtration (pore size)e8,um 6.5x105 3.8x103 7.7x1085,um 3.4 x 104 2.5 x 102 5.5 x 1083 ,m 2.1 x 102 0-5 3.8 x 108

a Direct counts of nuclei trapped on a 0.45-rim-pore-size membrane filterand stained with DAPI.

b Number of protoplasts in the nuclear fraction counted in a Petroff-Hausser counting chamber.

c Number of colonies on PRM+L originating from protoplasts present inthe nuclear fraction.

d Protoplasts were ground in a Ten Broeck glass tissue grinder.e Ground nuclear fraction was filtered through polycarbonate membrane

filters. Numbers of protoplasts and nuclei were assessed after each filtrationstep.

were grown on the original BM and then transferred toadditional BM plates to confirm prototrophy. Heterokaryonswere separated into their homokaryotic constituents bysingle sporing (20). In order to evaluate the relative numbersof parental nuclei in the original heterokaryotic thallus,conidia of the initial progeny were plated on BM, BM+L,BM+H, and BM+HL.Progeny obtained from interstrain nuclear insertions (T12

nuclei x T95 Lys- protoplasts) and single-spore isolatesderived from the resulting progeny were transferred to BM,BM+L, PDA, and PDA+L to assess prototrophy and mor-phological phenotypes. The isozyme phenotype of progenywas tested by horizontal starch gel electrophoresis with theenzymes fumarase (EC 4.2.1.2), phosphoglucomutase (EC2.75.1), and ot-D-glucosidase (EC 3.2.1.20). Strains T12 andT95 differ in mobility of these three enzymes, with Rfs (forT12 and T95, respectively) of 0.58 and 0.56 for fumarase,0.66 and 0.69 for phosphoglucomutase, and 0.73 and 0.76 forglucosidase. Such differences are readily distinguishable onstarch gels (21).

RESULTS

Isolation of nuclei from protoplasts. Protoplasts of thewild-type strain T12 contained between 0 and 8 nuclei perprotoplast, with an average of 3.4. Most (81%) of thesampled protoplasts contained three to six nuclei per proto-plast. Protoplasts of T95 His- and T95 Lys- contained 2 to10 nuclei, with an average of 4.1 nuclei per protoplast.The purification procedure resulted in a nuclear suspen-

sion nearly free of protoplasts. Shortly after protoplastswere suspended in EB, significant changes in their morphol-ogy occurred. Their vacuole size increased, and most ofthem became irregular in outline. The plasmalemma sur-rounding some of them ruptured. Less than 1% of theprotoplasts remained intact after homogenization. For mostbatches of protoplasts of T95 auxotrophs, filtration of thehomogenized protoplasts through 8.0-, 5.0-, and 3.0-,um-pore-size polycarbonate filters resulted in almost no proto-plasts in the nuclear suspension (Table 1). However, in somecases, especially with protoplasts of T12, a very smallnumber of viable protoplasts (i.e., 4 x 10-5 to 1 x 10-4% ofthe initial protoplast number) were recovered after filtration.

TABLE 2. Regeneration of T. harzianum T95 Lys- protoplastsafter fusion with nuclei isolated from T. harzianum

T95 His- protoplastsa

No. of regenerating protoplasts (102)bFusion mixture

PRM PRM+L PRM+H PRM+LH

T95 Lys- protoplastsc 0 500 0 510T95 His- nuclei' ND ND 0 NDT95 His- nuclei x T95 3.5 310 ND NDLys- protoplasts

aThe complementation frequency (proportion of nuclear transfer progenygrowing on PRM compared with the number of viable protoplasts ofT95 Lys-developing on BM+L) was 0.68%.

b ND, Not done.c 4.5 x 107 protoplasts.d About 6.4 x 108 nuclei isolated from 3.5 x 108 protoplasts.

Therefore, the nuclear suspension obtained from T12 proto-plasts was filtered through a 2.0-,um-pore-size polycarbonatefilter after the earlier filtration steps. This completely re-moved protoplasts from the suspensions of nuclei and pro-toplasts. However, about 5 to 15% of the nuclei wereretained by the 2.0-,um-pore-size filter. After staining withrhodamine 123, no mitochondria could be detected in thepurified nuclear suspension, whereas several mitochondriawere observed in each protoplast, and free mitochondriawere observed in suspension after protoplasts were homog-enized.

Intrastrain transfer of nuclei into protoplasts of T95 auxo-trophic mutants. Only 0.11% of the protoplasts of T95 Lys-were viable following the PEG treatment. However, whenfused with nuclei isolated from T95 His-, 0.68% of viableprotoplasts regenerated on PRM, whereas no colonies de-veloped when either nuclei or protoplasts were plated on thesame medium (Table 2). These nuclear transfer progenywere balanced heterokaryons in which nuclei segregatedthrough conidiation, giving rise to both auxotrophic parentaltypes in approximately equal numbers (Table 3). Nuclei ofT95 Lys- that were stained with DAPI prior to their inser-tion into protoplasts of T95 His- could be observed in therecipient protoplasts after treatment with PEG (Fig. 1).

Similarly, nuclei could also be isolated and transferredfrom T95 Lys- protoplasts into protoplasts obtained fromT95 His- (Table 4). The frequency of complementation was0.33%, as determined from the numbers of prototrophiccolonies developed on PRM and on PRM+H. In this exper-iment, the nuclear fraction was found to contain a very smallnumber of protoplasts of T95 Lys-, as indicated by growthon PRM+L. They might have been fused with T95 His-protoplasts and thus might have given rise to prototrophicfusants. However, these surviving protoplasts could accountfor no more than 4% of the resulting prototrophic colonies.

TABLE 3. Segregation of nuclei through conidiation in progenyisolates' from the transfer of T. harzianum T95 His- nuclei

into protoplasts of T. harzianum T95 Lys-

Total no. of colonies originating from single sporesIsolate no.

BM BM+L BM+H BM+LH

1 0 7.5 x 105 5.0 x 105 2.5 x 1062 0 6.2x106 9.0x106 1.3x1073 0 7.7 x 106 6.7 x 106 1.3 x 1074 0 5.0 x 106 3.3 x 106 1.0 x 1075 0 4.2 x 105 3.0 x 105 6.6 x 105

a Sample of 5 of 350 isolates.

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FIG. 1. Fluorescence photomicrograph of isolated nuclei of T.harzianum T95 His- transferred into protoplasts of T. harzianumT95 Lys-. Nuclei were stained with DAPI before the transfer.Fused protoplasts (f.p.), free nuclei (f.n.), and nuclei incorporatedinto fused protoplasts (i.n.) are indicated. Bracket, 20 p.m.

Interstrain transfer of nuclei of T12 into protoplasts of T95Lys-. Interstrain nuclear transfers required a higher ratio ofnuclei to recipient protoplasts than did intrastrain transfers(100:1 compared with 10:1, respectively). Progeny were notobtained when smaller numbers of nuclei were utilized. Innuclear insertion A, 1.0 x 1010 nuclei isolated from T12 werefused with 1.3 x 108 protoplasts ofT95 Lys-. No protoplastscould be detected by microscopic observation of the nuclearpreparation or by observing growth of T12 after plating ofsamples on PRM. During the first 10 days after the fusionmixture was plated on PRM, no colonies developed. Afterthese 10 days, colonies were overlaid with BM plus 1 ,ug oflysine per ml, and 24 h later, two types of colonies (sparseand dense) were noted. None of these colonies grew whentransferred to PDA, BM, or BM+L, even after 60 days ofincubation. Seventeen days after the initial plating of thefusion mixture, one progeny colony (Al) was detected onone of the plates overlaid with BM. The hyphal growth ofthis colony was extremely slow: 21 days after the initialplating, its diameter was only 7 mm. Subcultures from thiscolony grew very slowly on PDA, PDA+L, BM, andBM+L. The greatest colony size was obtained on BM (9.5mm of radial growth after 7 weeks of incubation). Some ofthe colonies on PDA, PDA+L, or potato dextrose brothproduced a brown pigment which was excreted into themedium. The isozyme patterns of oa-glucosidase, phospho-glucomutase, and fumarase of these progeny were identicalto those of strain T95.

TABLE 4. Regeneration of T. harzianum T95 His- protoplastsafter fusion with nuclei isolated from T. harzianum

T95 Lys- protoplastsa

No. of regenerating protoplasts (102)bFusion mixture

PRM PRM+L PRM+H PRM+LH

T95 His- protoplastsc 0 0 300 300T95 Lys- nucleid 0.04T95 Lys- nuclei x T95 1 ND 89His- protoplastsa The complementation frequency (proportion of nuclear transfer progeny

growing on BM compared with the number of viable protoplasts of T95 His-developing on PRM+H) was 0.33%.

b ND, Not done; -, not applicable.c 6.5 x 107 protoplasts.d About 7 x 108 nuclei isolated from 3.8 x 10i protoplasts.

TABLE 5. Linear growth of selected single-spore progenyfrom nuclear insertion A'

Isolateb Colony radius (mm) Continuous sectorBM BM+L formation

AS4AC 20 3 +AS4B 25 24 +AS7 8 8 +AS7Ld 12 2.5 -AS1OA 11 32 -AS1OB 9 12 +AS17 8 0.5 -AS18 9 20 -AS24A 5 3 +AS24B 23 15 -AS25A 8 2 +AS25B 3.5 5 +AS27A 2 8 +AS27B 8 18 +AS28 25 5 -AS29 9 11 +AS31 0 9 +AS36A 7 1 +AS36B 4 5 +AS37 23 27 +AS38 15 1 -AS39 9 11 +AS41 28 13 +AS42 15 2 -Control:T95 Lys- 0 30 -T12 43 44 -a Data represent 23 of 56 single-spore isolates.b All isolates had the same isozyme phenotype as T95, determined on the

basis of a-glucosidase, fumarase, and phosphoglucomutase enzyme profiles.c Letters that follow isolate numbers represent sectors produced by the

original thallus of the single-spore isolate.d The L indicates an unusual lumpy phenotype.

Spores collected from Al gave rise to the same number ofcolonies when plated on minimal or complete medium. Thus,Al nuclei were prototrophic. The growth rates of thesesingle-spore isolates were somewhat greater than that of theoriginal Al isolate. Among single-spore isolates, 34% grewbetter on BM+L than on BM alone, 14% were not affectedby lysine, and 52% grew better on BM than on BM+L.These isolates differed in colony morphology from bothparental types, but the isozyme pattern of these single-sporeprogeny was identical to that of T95 (Table 5). Most of thesesingle-spore strains were unstable and produced one or morerapidly growing sectors.

In nuclear insertion B, 1.2 x 1010 nuclei isolated from T12were fused with 2.4 x 108 protoplasts of T95 Lys-. Nocolonies were produced when nuclei were plated on BM,indicating that the suspension of nuclei contained no viableprotoplasts. The fusion mixture was plated on BM or PRM,each amended with 1 ,ug of lysine per ml. After 48 h ofincubation, an additional water-agar overlay or a water-agaroverlay amended with lysine (1 ,ug/ml) was poured onto thefusion mixture plates. Colonies developed only on BM platesoverlaid with water-agar amended with lysine. Mycelialdisks from the colonies that were transferred to BM gave riseto slow-growing progeny that never exceeded 30 to 40 mm indiameter. Upon subculturing, some of these (45%) lost theirability to grow on any medium. Isozyme pattern analysis ofa-glucosidase, fumarase, and phosphoglucomutase of theseprogeny revealed that two of them (designated B3 and B6)were identical to the prototrophic T12 type nuclear donor,

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TABLE 6. Linear growth and isozyme phenotype of selectedprogeny from nuclear insertion B'

Isoenzymeb Colony radius (mm) after 48 hIsolate phenotype PDA PDA+L BM BM+L

B3 T12 25 30 0 28B4 T95 12 29 0 29B5 T95 0 27 0 26B6 T12 22 30 0 29B9 T95 24 29 0 29B10 T95 25 30 0 29Bll T95 23 30 0 29B24 T95 24 31 0 32B47 T95 20 31 0 31B48 T95 18 29 0 29B52 T95 23 28 0 27B64 T95 24 31 0 32Control:T95 Lys- T95 25 32 0 30T12 45 44 43 44

a Of the 69 initial progeny isolated, 32 did not grow on any of the testedmedia.

b Based on a-glucosidase, fumarase, and phosphoglucomutase enzymeprofiles.

yet neither of them grew on BM (Table 6). Their colonymorphology was similar to that of T95. T12 sporulatesreadily to give a green colony, while T95 grows and sporu-lates more slowly than T12 and so gives rise to a light greencolony. Mature colonies can be readily differentiated on thisbasis.

Single-spore analysis revealed that nuclei of both parentaltypes were present in B3 and B6. From 11 to 17% of theprogeny were identical to strain T12, as determined byisozyme profiles, colony morphology, and prototrophy,while the remainder were identical in all respects to T95Lys- (Table 7).

DISCUSSION

This study demonstrated the successful isolation andtransfer of nuclei into protoplasts of T. harzianum. Isolationand transfer of nuclei into animal, plant, and yeast proto-plasts has been previously described (3, 6-8, 15, 16). How-ever, similar reports on filamentous fungi have not yet beenpublished so far as we know.

In the past, fungal nuclei have been isolated and purifiedfrom frozen mycelia (3, 9) or from protoplasts (6, 8). Isola-

TABLE 7. Isozyme pattern and colony morphology ofsingle-spore isolates of B3 and B6 progeny

Isolatea Isoenzyme phenotype Colony GrowthPGM GLU FUM morphology on BM

B6S1 T12 T12 T12 T12 +B6S2 T12 T12 T12 T12 +B6S3 T12 T12 T12 T12 +B6S4 T95 T95 T95 T95 Lys- -B6S5 T95 T95 T95 T95 Lys- -B3S1 T12 T12 T12 T12 +B3S2 T95 T95 T95 T95 Lys- -B3S3 T95 T95 T95 T95 Lys- -a All the isolates showing the T12 phenotypes are presented, but among

those with the T95 phenotype, only samples of 2 of 14 from B3 progeny and2 of 8 from B6 progeny are shown.

b PGM, Phosphoglucomutase; GLU, a-glucosidase; FUM, fumarase.

tion of nuclei from protoplasts, rather than mycelia, has theadvantage of applying lower shearing forces than thoseneeded when the cell wall is present (7). The absence of cellwalls may also result in higher nuclear yields, because cellwall fragments have been shown to trap nuclei during theisolation procedure (15).

In the present study, in order to eliminate the possibility ofprotoplast fusion occurring concomitantly with fusion ofnuclei into protoplasts, it was necessary to have no remain-ing protoplasts in the nuclear fraction. Therefore, we devel-oped an isolation technique which used consecutive filtra-tions through polycarbonate membrane filters, whichremoved intact protoplasts from the nuclear fraction. In-deed, in almost all of the tests, no remaining protoplastswere detected in the nuclear fraction. In no case were morethan 10-6 of the initial protoplasts present in final nuclearpreparations. Thus, remaining protoplasts in the nuclearfraction, which might have fused with the recipient proto-plasts, did not account for the resulting progeny. Previousreports showed that when nuclei were isolated from proto-plasts and purified by sucrose gradient centrifugation, about0.5% of the initial protoplasts were present in the finalpreparation (6, 18).PEG has been successfully used to insert nuclei into yeast

and plant protoplasts. Although the mechanism by whichPEG mediates nuclear transfer into protoplasts is unclear,Ferenczy and Pesti (9) suggested that nuclei were trappedbetween two or more fusing protoplasts in S. cerevisiae. It islikely that a similar mechanism was responsible for thetransfer of isolated mitochondria into protoplasts of thisyeast (11).

In intrastrain transfers, protoplasts of both the nucleardonor and the recipient were derived from auxotrophicmutants. Nuclear transfer resulted in the establishment ofheterokaryons, as evidenced by data from single-spore iso-lations. The frequency of progeny that developed on theminimal medium was relatively high (0.33 to 0.68%), indi-cating that nutritional complementation must have been aresult of incorporation of nuclei of one strain into protoplastsof the other. However, in interstrain transfers, a wild-typeprototrophic strain served as the nuclear donor, and alysine-deficient mutant served as the acceptor. Normal func-tioning of the inserted nuclei should have been sufficient forgrowth, and either wild-type homokaryons or prototrophicheterokaryons were expected.Recovery of nuclear transfer progeny appeared to be

affected by the presence of low concentrations of lysine inthe overlay. The timing of application of lysine appearsimportant, since nuclear transfer progeny were obtainedonly when it was performed soon (48 h) after the initialplating but not when it was delayed several days. Thus,initiation of regeneration by minimal concentrations of thedeficient amino acid is presumably needed for growth ofheterokaryons after nuclear transfers.

In nuclear insertion A, heterokaryons were not obtained.However, nutritional complementation occurred and gaverise to a single colony. Consecutive transfers of theseisolates gave rise to unstable sectoring colonies, whichresulted in a variety of morphological phenotypes. Thesecolonies demonstrated more rapid growth on several media,including the deficient BM, than the original single-sporeisolates did. Subprogeny derived from single conidia gaverise to similar variation. Such phenomena were demon-strated with interstrain and interspecies protoplast fusionprogeny of Trichoderma spp. (20; Stasz and Harman, in

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TRANSFER OF NUCLEI INTO PROTOPLASTS OF T. HARZIANUM

press) and suggest that nuclear transfer or protoplast fusiongave rise to genetic instability.Nuclear insertion B, however, resulted in the formation of

heterokaryotic progeny which showed the isozyme patternof the donor strain (e.g., T12). Their colony morphology,however, was similar to that of T95, and none of them wasprototrophic. After segregation of nuclei through conidia-tion, 11 to 17% of the resulting subprogeny were similar toT12 in isozyme and colony morphology phenotypes, andthese subprogeny were also prototrophic. Thus, at leastsome of the initial thalli were heterokaryons containing 11 to17% prototrophic T12 nuclei and 83 to 89% T95 Lys- nuclei.These data, taken together, suggest that within heterokary-ons at different times, one of the two nuclear types wasexpressed while the other was quiescent and that thispreferential expression changed over time. The preferentialexpression of genes from one parent over genes from theother has been previously described for mammalian hybridcells (25) and for protoplast fusion progeny in Bacillussubtilis (13). In the latter case, expression of the parentaltype within heterokaryons altered over time. However, inthis work, the fact that both nuclear types were recovered insome single-spore subprogeny indicates that relatively bal-anced heterokaryons were formed. Thus, some of the vege-tative incompatibility previously reported to interfere withgrowth of heterokaryons formed by protoplast fusion wasovercome. In protoplast fusion between strains T12 and T95,greatly imbalanced heterokaryons in which T12 nuclei out-numbered T95 by 10,000:1 were formed (20).

Transfer of nuclei into Trichoderma protoplasts may leadto a better understanding of nucleus-cytoplasm interactionsin somatic hybrids produced by protoplast fusion or natu-rally occurring plasmogamy. This understanding may lead tobetter methods of transferring genetic information betweenstrains. However, further investigations on the fate of thetransferred nuclei in the recipient cell are needed, as areimprovements in the process of nuclear transfer.

ACKNOWLEDGMENTS

This research was supported in part by grants from the CornellBiotechnology Program, which is sponsored by the New YorkScience and Technology Program, and from the U.S.-Israel Bina-tional Agricultural Research and Development Fund (grant no.US-1224-86) and by the Eastman Kodak Co.

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