View of Optimizing anther culture for barley breeding

Optimizing anther ^culture for barley breeding

Outi Manninen

AgriculturalResearch Centreof^{Finland,Institute}of^Cropand Soil Science, PlantBreedingSection, FIN-31600 Jokioinen, Finland,e-mail:outi.manninen@mtt.fi

Baileyanther culture methodswereoptimizedfor theproduction of doubled haploid lines fromFinn- ishspring barley^(Hordeum vulgare) breeding material.22 F( -progeniesof two-rowed barleyculti- vars(‘Bonus’, ‘lnari’,JolölO,‘Kustaa’, ‘Kymppi’, ‘Prisma’) and six-rowedbarleycultivars (‘Arve’,

‘Botnia’, ‘LarkerMutant’, 08264, ‘Rolfi’, WW7860)were used for theexperiments. The effect of basic induction media,pretreatmentonmannitolmedium,densityofanthers,incubation temperature

and light regime were tested. Pretreatment of ^anthers for 4 days on ^medium containing 0.175 M mannitol wasbeneficial for all8genotypestestedandincreasedproduction of greenplantsper 100 anthers from 26%to74% for the best genotype(‘lnari’ x ‘Kymppi’ F(^).Alower anther density^(1.6 anthers percm²⁾wasbetter thana more ^denseone.^Amodified MS-medium with ammonium nitrate partly replaced withglutamine^(MMS-MG)wasslightly better thanamedium basedon

N 6 salts

^(N6-

MG),and addition of 100 pM silver nitrate reduced bothplantand greenplant production.Nosignif- icant differenceswere^observedbetween^the effects^ofincubationtemperatures(20°C vs.25°C) or thelight regime (darkness_vs. weaklight) duringincubation of anthers.Ineach experimentthe gen- otypic effectwasprominent and the recalcitrance ofsome genotypes was apparent. Greenplants wereproduced however from all genotypes.

Key words:^{doubled haploid,Hordeum} vulgare, lighting, mannitol,pretreatment, temperature

Introduction

Breedingnew barley varieties is basedon^creat- ingnew gene combinationsby controlledcross- es and subsequent testing and selection during the selfing generations. This takes 12-15 years using conventional methods. The early genera- tions following crossing arehighly heterozygous.

making reliable selection difficult until an ac- ceptable level of homozygosity is reached. In heterozygous plants recessive genesare not ex- pressed in the phenotype and heterosis may in- fluence performance. Such effects are lost dur- ing the later generations. To secure stable and homogeneous newbarley varieties single head selections^mustbe made during later generations when adequate homozygosity has beenreached,

©Agricultural and Food ScienceinFinland ManuscriptreceivedAugust 1997

and subsequent multiplication of seedon a com- mercial scale requires several years.

A shortcut tohomozygositycanbe achieved by producing doubled haploid lines from the microspores of the early, segregating generations (F-F3). Success in production of microspore- derived barley plants through anther culturewas first reported 25 years ago by Clapham^(1973).

Since then barley anther culture methods have been developed intoapractical breeding meth- od by modifying the donor plant growth condi- tions (Foroughi-Wehr and Mix 1979), by using a coldpretreatment of tillers_or spikes (Powell 1988^b, Huang and Sunderland 1982),ormanni- tolpretreatmentofanthers (Roberts-Oehlschlag- erand Dun well 1990),and by replacingammo- nium nitrate with glutamine in the induction medium (Olsen 1987). A major breakthrough wasachieved when alternative sugars, including maltose, were used in induction media inplace ofsucrose (Hunter 1987, Sorvari and Schieder 1987,Finnicetal. 1989).Much of the work with barley anther culturewas done with one highly responsive model cultivar, Igri. Genotypic dif- ferences in anther culture capacity complicate its practical use in breeding. To be useful for breeders, the anther culture method should be easytouse,efficient and applicabletoall breed- ing lines. In addition, the anther culture meth- ods used should^notinvoke any unconsciousse- lection of the variation existing within the bar- ley material.

The aim of this studywas ^to optimize the anther culture methodto be able toproduce an adequate number of lines from crossing combi- nations used in the Finnish spring barley breed- ing programme. In^addition,several methods for statistical analysis of datawere compared.

Material and methods

22F

t

-progenies of two-rowed barley cultivars (‘Bonus’, ‘lnari’, JolölO, ‘Kustaa’, ‘Kymppi’,

‘Prisma’) and six-rowed barley cultivars(‘Arve’,

‘Botnia’, ‘Barker Mutant’, 08264, ‘Rolfi’, WW7860)from the Finnish barley breeding pro- grammewere used for the experiments. Differ- ent genotypes were used in each of the experi- ments.Plants were grown in the greenhouse in pots withpeat soil mix and fertilized regularly (11% N, 4%P, 25%K). Natural lightwas sup- plemented with high pressure sodium lamps (Sylvania SHP-T-400) for 16 h per day and the day/night temperatures were approximately 18°C/12°C. The first lot of plantswas planted in August and the second in December. Plants from both lots _wereused in each experiment.

Donor spikes were collected when the dis-

tancebetween the flag leaf ligule and the penul- timate leafwas3-7cmand the microsporeswere atthe mid- orlate uninucleatestage. Ifnototh- erwisestated,freshly collected spikeswereused for anther isolation. Usually MMS-MG medium wasused for induction. Thiswas modified MS- medium (Murashige and Skoog 1962) witham- monium nitrate partly replaced with glutamine asproposed by Olsen (1987).All induction me- dia contained 175 mM maltoseastheir carbohy- dratesource, 4,4 pM 6-benzylaminopurine and 5,7 pM indolate-3-aseticacid, and were solidi- fied with2 g/1 Gelrite. All mediawere sterilizer by autoclaving 20 min. at 120°C. If otherwise stated,the disheswerefirst kept in darkness for four weeks and then in light, thetemperaturebe- ing +25°C.

Induction media

Anthers from the

F,

generation of_twosix-rowed and four two-rowed barley crosses were used.

Two differentbasic mediawereused: MMS-MG and N.-MG. The latter contained the_o macro-and micro-salts and vitamins of theN 6 medium^(Chu

etal. 1975)inplace of those of the MS- medi- um.The third medium testedwasMMS-MG sup- plemented with 100 mM silver nitrate. Anthers from each spike were distributed ^to all three media, 50 anthers per Petri dish⁽⁵ cm0). A total of700 anthers per genotype ^x medium combi- nationwereused.

Light and temperature conditions during embryoid ^induction

Anthers from the

F,

generation offour six-rowed and four two-rowed barley crosses were used.

Anthers from several spikeswererandomly dis- tributed to four 5 cm diameter Petri dishes, 50 anthers perdish, containing the MMS-MG_me- dium. One of the four dishes was incubated in darkness at +2O°C, the second in darkness at +25°C, the third in dim lightat +2O°C, and the fourth in dim light ^at+25°C. A total of 600an- thers pergenotypex inductiontreatmentcombi- nationwere used.

Pretreatment

Anthers from the

F,

generation offour six-rowed and four two-rowed barley crosses were used.

Anthers from several spikeswererandomly dis- tributed tofourtreatments: 1) MMS-MG medi- um, 5 cm dishes, 2) MMS-MG medium, 9cm dishes 3) MMS-MAN^medium, 5 cm dishes,4) MMS-MAN medium, 9 cmdishes. 100 anthers were isolated per plate. MMS-MAN was the same mediumas MMS-MGexcept that maltose wasreplaced by 175 mM mannitol.Anthers from the mannitol disheswere transferredto normal MMS-MG medium after four dayspretreatment.

A total of 600 anthers per genotype xpretreat- mentcombinationwere used.

The number of green and albino plantlets in- duced during 8 weeks after anther isolation were counted in each experiment. The efficiency of three transformations to homogenize variances wastested with oneof the datasetsand variance analysis results were compared between differ- ent transformations. All datasets were analysed by variance analysis(ANOVA)using the

arcsinV

transformed numbers of plants per 100 cultured anthers (PPA), greenplants per 100 culturedan- thers(GPA)and proportion of green plants^(PGP).

Whenneeded,normalized indiceswereproduced by dividing all data by the maximum value in the experiment. The model included the main effects ofgenotype,treatmentandseasonplus all thetwo-

way interactions. The data from all experiments wereanalysedascompletely randomised designs.

Pairwise comparisons between significantly dif- ferent treatment means according to the results ofANOVA, weredone with Tukey’stest.Results of parametrictestswerecompared with those of the non-parametric Friedmans test in _one of the experiments. All statistical analyseswere run us- ingSPSS®, ^version6.1.

Results

Effect of induction media

The per plate values for plant production ranged from _{zero to} 172.0% and for green plant pro- duction from zero ^to 126.0%. All the main ef- fects for PPA and GPAwere statistically signif- icant(P<0.05), but the propotion of green plants was effected only bygenotype and season, not the treatment used. No statistically significant interactionswere detected. The MMS-MG me- diumwas the best induction medium for most genotypes(Fig. 1).The addition of silver nitrate had no positive effect onany of the variables measured, but hindered the formation of green plants. Large genotypic differenceswere seen in anther culture response and thetreatmentsused could not overcome the recalcitrance of some genotypes (Fig. I^.). The second sowing time (December) was significantly better for anther culture response than the first (August).

Effect of light and temperature condi-

tions during embryoid induction

The plants per 100 anthers values ranged from zeroto 106.0% and the green plants per 100an- thers values from zero to 46.0%. A significant interaction forgenotypex treatmentwasdetect- ed for GPA(P=0.032).The genotypic effectwas significant for_PPA,GPA and proportion of green plants when tested against genotype-treatment

Fig. 1. Effect of^{inductionmedia,}

seasonand genotype. Black bars represent theproduction of green plantsper 100 anthers,bars with different letters aresignificantly different (P<0.05) from others within thesamemain effect.

Fig. 2.Effect oflightand temper- ature conditionsduring embryoid

induction. Treatment 1) 20°C, weaklight, 2)20°C,darkness, 3) 25°C, weaklight,4) 25°C, dark- ness.Results frompairwise com- parisonsof treatmentsare^shown for only those genotypes,were one way ANOVA showedsignificant differences. Treatments with dif-

ferent letter^aresignificantlydif- ferent (P<0.05) for their green plant production.

Table 1.Mean squares and P-values for main effects and two-way interactionsinpretreatmentexperiment.

Plants per 100anthers Greenplantsper 100 Proportionof green

anthers plants

df MS P MS P MS P

Genotype^(G) 7 0.2600.001" 0.426 <0.001" 1.286 <0.001"

Treatment (T) 3 0.785 <0.001" ^0.387 <0.001" 0.0610.407"

Season (S) 1 0.1580.01 0.1400.003 0.0270.323

GxT 21 0.0480.006 0.0350.004 0.0600.004

GxS 7 0.0120.816 0.0300.075 0.0220.588

TxS 3 0.0380.187 0.0030.889 0.0130.693

Residual 139 0.0230.016 0.028

"

Tested against GxT interaction term.

interaction(P-values 0.007, <O.OOl and <O.OOl respectively), but the treatment effect _was not (P-values0.095,0.091 and0.057). Values of PPA and GPA for each genotypex treatmentcombi- nationare shown in Fig. 2. Treatment 1(induc- tion in dim light, ^20°C)hadapositive effecton greenplant production fora few crossingcom- binations. Several genotypes remained recalci- trant after thetreatmentsused. The secondsow- ing time (December) wassignificantly better for anther culture response than the first one(Au-

gust).The effect of three transformationstonor- malize the distribution of residuals for GPAwas compared graphically. The

arcsinV-transforma-

tion was the mosteffective. None of the trans- formations equalized variances satisfactorily.

When ANOVAwasperformedonall transformed data as wellas on untransformeddata, no sub- stantial differenceswereobserved. Even though the P-values varied, this did not alter thecon- clusions drawn from the analyses.

Fig. 3.Effect of pretreatment and plating density.Treatment I) ^no pretreatment,5cmdish,2)_nopre- treatment,9cmdish,3) mannitol pretreatment, 5cmdish,4)man- nitol pretreatment,9cmdish. Re- sults frompairwise comparisons of treatmentsare^{shown foronly} those genotypes, where oneway anova^/Friedmans test showedsig- nificant differences. Treatments with different letteraresignificant- ly different (P<0.05) for their green plant production. Results from the parametric tests are shown above the black bars and those ofnon-parametricones ^be- low the treatment numbers.

Effect of pretreatment

Plants per 100 anthers values ranged from zero to279.0% and green plants per 100anthers from zeroto 151.0%. All main effects and the geno- type x treatmentinteraction had a statistically significant effect on ^PPA and GPA (Table 1).

accounted for43% and 22% respectively. In this experiment onlygenotype and G x T interaction hada statistically significant effect on the pro- portion of green plants. Themeanvalues of PPA and GPA for each genotype^x treatment combi- nationare shown in Fig. 3. The mannitol pre- treatmentpromoted PPA and GPA in several gen- otypes,especially when the lower anther densi-

ty(1.6 anthers percm²⁾ was used. The second sowing time(December) wassignificantly bet- terfor anther culture response than the firstone (August).

The results from one-way ANOVA andnon- parametric Friedmanstestfor GPAareshown in Fig. 3. Friedmans testappearedmore conserva- tive and did noteshtablish significant differenc- es between treatments in the cross ‘lnari’ x

‘Kymppi’ when ANOVA did. Some differences werealso found in pairwise comparisons.

Discussion

Genotypic ^effects

In all experiments thegenotypeeffectwashigh- ly significant for all characters measured and accounted for 8-21% of the variation in plant production per 100 anthers, 17-40% in green plant production per 100 anthers and 8-62% in proportion of green plants. Thegenotype effect on anther culture response is widely reported for various plant species. In barley, Knudsen etal.

(1989) tested the anther culture response of 17 varieties and reported that thecomponentofvar- iance fromgenotypes accounted for60% of the total variation in embryo formation.Intheir bar- ley material PPA ranged from 1 ^to47% and the

GPA fromzeroto40%. Inourexperiments green plants could be produced from each of the22 crossing combinations tested and the best geno- types onthe besttreatmentsgaveplants per 100 anthers valuesashighas 176% and green plants per 100 anthers values of 74%, although for manycrossing combinations the green plant pro- duction remained low(5-10%). The capacity for greenplant production did not depend on row type in any ofour experiments and it also ap- pears to be independent of the spring/winter growth habit(Larsen ^etal. 1991).The genetic background of anther culture response is com- plex and environment often affects gene action.

Both additive and dominance effects for embry- oid formation, total plant production and green plant production have been observed(Hou etal.

1994).Powell(1988a)reported_asignificantre- ciprocal effect in anther culture response in a diallel study, which indicated that the direction of the cross may be important for the develop- mentof microspore-derived plants. Reciprocal effects may indicate either cytoplasmic or ma- ternal effects on anther culture response. Large genotypic differences hinder effectiveuse of anther culture for breeding purposes. Houetal.

(1994)reported thatmostof the

F,

^{’s studiedwere}

intermediate in response between the parents even though transgressive segregation was also observed for some combinations. In practical breeding it is worthwhile testing the anther cul-

ture response of theparents of thecrosses used in doubled haploid production ^to allocate re- sourcesmosteffectively. Improvement in anther culture response by crossing and selection is possible, hut from the barley breeders’ point of view this may be of marginal interest.

Treatment effects

The ^mostcommonly used pretreatment in bar- ley anther culture is placing tillers orspikes at +4°C for 14to 28 days (Huang and Sunderland 1982,Powell 1988^b). This has been shown ^to been beneficial for several barleygenotypes,but notinvariably (Szarejko and Kasha 1991).In_our earlier studiesweconsistently obtaineda lower

response in anther culture ofcold pretreated till- ers orspikes compared with anthers isolated from fresh tillers (data unpublished). In thepresent

study all anthers were isolated from fresh till- ers.The mannitolpretreatmentfor four days was beneficial formostgenotypes studied. The gen- otype xtreatmentinteractions detected_werenot crossover interactions, which means thatman-

nitol didnotsuppress anther culture response in anygenotype. In practical useof anther culture it is impossible ^to optimize methods for each genotype, and the methods used should be ap- plicable to awide range ofgenotypes.The ben- eficial effect of mannitolpretreatment in barley anther culture was first detected on cv. Tgri’

(Roberts-Oehlschlager and Dunwell 1990), which is awidely used modelgenotypefor bar- ley anther culture. Mannitol pretreatment has been considered_{as a}starvationtreatment(Hoek- stra et al. 1992), but in barley mannitoltreat- mentinduces enrichment of glucose intoanthers, which may promotethe earlystagesof develop- ment(Roberts-Oehlschlageretal. 1990). Man- nitolpretreatment has also been found_tobe ben- eficial in barley microspore culture^{(Kasha et}al.

1992,Hoekstraetal. 1996). However,Houetal.

(1993)reported that 28 days coldpretreatment

ofspikeswas moreefficient than3 dayspretreat- menton 0.3 M mannitol.

Anther densitycanaffect greenplant produc- tion, especially when numerous embryoids are formed. In ourexperiment, 1.6 anthers percm² was better5.1 anthers percm^{2 .} Xu and Sunder- land (1982) found that the optimal anther densi- ty for callus formation was as high as 8.5-17 anthers percm^{2 .}Subsequentlybarley anther cul-

ture methods have undergone several develop- mentsand in accordance withourresults 1-1.5 anthers percm²wasfoundtobe the optimal den- sity for mannitol pretreated anthers (Roberts- Oehlschlager and Dunwell 1990).

N-MG and MMS-MG differ in their_o macro- and microsaltcontents. In MMS ammonium ni- trate is partly replaced by an organic nitrogen source, glutamine. No statistically significant differences were detected between these two media, although MMS-MG was slightly better for most genotypes. The addition of silver ni-

trate to MMS-medium hindered the formation of green plantlets in anther culture. Silver nitrate is aninhibitor of ethylene production and Purn- hauser etal. (1987) were able to enhance shoot formation in wheat callus culture using silver nitrate. Cho and Kasha (1989)studied ethylene production and the effect of ethylene inhibitors andpromotors in barley anther culture and con- cluded that an optimum level of ethylene may be required for embryogenesis. Genotypes may differ in their production of ethylene in anther culture. Evans and Batty (1994) found that sil- ver nitrate hindered green plant production in anther culture ofbarley but positive effectswere achieved with another ethyleneinhibitor, silver thiosulphate. It may be that silver nitrate is not appropriate for barley anther cultureorthat the genotypes used in our experiment were of the low ethylene production type.

Incubation of anthers in weak light, together with the lower incubationtemperature (+2O°C), was beneficial for _afew genotypes. It has been found that green plant production increases in wheat anther culture by incubating isolated anthers in_weak,diffuse light (Bjprnstadetal. 1989).

Season

Season had a small, but significant effect on green plant production, December being better for sowing the donor plants in all experiments.

Even though a controlled greenhouse environ- ment wasused for growing the donor plant ma- terial, the amount of natural light differred be- tween seasons. Kuhlman and Foroughi-Wehr (1989) detected large seasonal variation in an- ther culture response of Tgri’, and speculated that temperatures occasionally exeeding 25°C duringsummer would make the donor plants less suitable for anther culture. In the practical use of anther culture for breeding, the yearly cycle of crossing and field testing will determine the suitable seasonfor anther culture. Inourcondi- tions anthers are mainly isolated during Octo- ber which leaves enough time for the doubled

haploid plants to setseed before sowing in the spring.

Statistical analysis of anther culture ^data

Statistical analysis of anther culture data is of-

tenproblematic since data may^notbe normally distributedand the variancesareoften heteroge- neous.Data often contain many zeros and due to occasional contamination may be unbalanced.

One way ^toavoid problems with non-normality and heterogeneous variances is touse transfor- mations. From the transformations tested with our data,the

arcsiiW

wasthe mostefficientone.

Despite the fact that untransformed data didnot fulfil the expectations needed when using para- metrictests,results from ANOVA didnot differ markedly from those of transformeddata, which may beasign of the robustness of variance anal- ysis. Instead of usingtransformations, non-par- ametrictests,which do_notrequire _anormal dis- tribution _canbe used. Unfortunately, non-para- metric equivalentstovariance analysis withmore complicated models seldom exist in commercial computer packages. We compared the results from Friedmans test with those from the one- way analysis of variance. Some differencesex- isted among the pairwise comparisons and the Friedmanstestseemedtobe littlemore conserv- ative than ANOVA. When analysing results from a tissue culture experiment, one should keep in mind that it is oftenmore importanttoestablish the existence of differences large enoughto be of practical importance rather than ^to reveal small statistically significant, and in practice meaningless, differences.

Conclusions

The best method indicated by the results ofour experiments is isolation of fresh anthers on the mannitolpretreatment medium. Transfer ofan- thers after 4 days pretreatment to MMS-MG mediumcanbe aided with_apolyesternet, which has been placed onthe MMS-MAN medium be- fore isolation of anthers. Induction ofembryoids and germination of embryoids into plantletsoc- cur onthesame medium,and there isnorequire- ment to transfer the embryoids to a new medi- um. Thepresent method is efficient enough for most barley breeding lines and an adequate number of doubled haploid linescanbe produced for the Finnish barley breeding programs.

A practical method for production of doubled haploids provides the breeder withapossibility toproduce homozygous lines from crossingcom- binations withina year. Selection is more effi- cient when done on homozygous and homoge- neous lines. While using anther culture, one should be aware of the possibility of distorted segregation in the doubled haploid lines since segregation distortion has been detected insev- eral crosses with molecular markers (Graneret

al. 1991, Heunetal. 1991).

Acknowledgements. Ms.Maija Penttilä is thanked forex- cellent technical assistance andDr.Jonathan Robinson for correctingthelanguage.The financial support of theFinn- ishMinistry ofAgriculture and Forestry isgratefullyac- knowledged.

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SELOSTUS

Ponsiviljelymenetelmän optimointi ohranjalostuksen käyttöön

Outi Manninen

Maatalouden tutkimuskeskus

Ohran ponsiviljelymenetelmää optimoitiinkaksois- haploidien ohralinjojen tuottamiseksi suomalaisesta

ohran jalostusmateriaalista. Emokasveina käytettiin ohralinjojen ja -lajikkeiden (‘Arve’, ‘Bonus’, ‘Bot-

nia’, ‘lnari’, JOI6IO, ‘Kustaa’, ‘Kymppi’, ‘Larker Mutant', 08264, ‘Prisma’, ‘Rolfi’, WW7860) välis- tenristeytysten Tutkimuksessaselvi-

tettiin induktioalustan, mannitoli-esikäsittelyn, pon- sien maljaustiheyden sekä inkubaatiolämpötilan ja -valaistuksen ^vaikutustaponsiviljelyvasteeseen. Pon-

sienesikäsittely 0,175 M mannitolialustalla osoittau- tui hyväksiuseillagenotyypeillä, parhaalla genotyy-

pillä ^{(‘lnari’ x} ‘Kymppi’) vihreiden taimien tuotto 100pontta kohden parani 26 %:sta 74%:iin. Pienem- pi ponsitiheys maljalla (1,6 pontta/cm²⁾tuottienem- mänvihreitä taimia kuin suurempi (5,1 pontta/cm^2).

MMS-MG induktioalustaoli jonkin verran N6-MG alustaaparempi,kun taas 100 pM hopeanitraatin li- säys heikensi taimien tuottoa.Inkubaatiolämpötila ja -valaistus eivät vaikuttaneet ponsiviljelyvasteeseen.

Sensijaankaikissa kokeissa genotyyppivaikutti voi- makkaastiponsiviljelyvasteeseen. Kaikista tutkituis- tagenotyypeistä kyettiin tuottamaanvihreitäkasve- ja.