Optimizing anther culture for barley breeding Outi Manninen Agricultural Research Centre ofFinland, Institute ofCrop and Soil Science, Plant Breeding Section, FIN-31600 Jokioinen, Finland, e-mail: outi.manninen@mtt.fi Bailey anther culture methods were optimized for the production of doubled haploid lines from Finn- ish spring barley (Hordeum vulgare) breeding material. 22 F ( -progenies of two-rowed barley culti- vars (‘Bonus’, ‘lnari’, JolölO, ‘Kustaa’, ‘Kymppi’, ‘Prisma’) and six-rowed barley cultivars (‘Arve’, ‘Botnia’, ‘Larker Mutant’, 08264, ‘Rolfi’, WW7860) were used for the experiments. The effect of basic induction media, pretreatment on mannitol medium, density of anthers, incubation temperature and light regime were tested. Pretreatment of anthers for 4 days on medium containing 0.175 M mannitol was beneficial for all 8 genotypes tested and increased production of green plants per 100 anthers from 26% to 74% for the best genotype (‘lnari’ x ‘Kymppi’ F ( ). A lower anther density (1.6 anthers per cm 2 ) was better than a more dense one. A modified MS-medium with ammonium nitrate partly replaced with glutamine (MMS-MG) was slightly better than a medium based on N 6 salts (N6 - MG), and addition of 100 pM silver nitrate reduced both plant and green plant production. No signif- icant differences were observed between the effects of incubation temperatures (20°C vs. 25°C) or the light regime (darkness vs. weak light) during incubation of anthers. In each experiment the gen- otypic effect was prominent and the recalcitrance of some genotypes was apparent. Green plants were produced however from all genotypes. Key words: doubled haploid, Hordeum vulgare, lighting, mannitol, pretreatment, temperature Introduction Breeding new barley varieties is based on creat- ing new gene combinations by controlled cross- es and subsequent testing and selection during the selfing generations. This takes 12-15 years using conventional methods. The early genera- tions following crossing are highly heterozygous. making reliable selection difficult until an ac- ceptable level of homozygosity is reached. In heterozygous plants recessive genes are 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 new barley varieties single head selections must be made during later generations when adequate homozygosity has been reached, © Agricultural and Food Science in Finland Manuscript received August 1997 389 Vol. 6 (1997): 389-398. AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=5sBvqno3Hpay-gSf.2xQ1y9zqHJ_87XJlsLIdPQ.iQnKt4VVuPSSl9I8knAdVW9n3StQEDlx9UKrcTO-5AJCuRIMtrGN1-caifEFayWc-murkxzYT7zt_DLP9ienV47aIuqszoZhhY__Un05m6wtETs_Qg-dTP9xsmIrcLdx1NhTKQ1SkboA8cn5RUUVWwNZMHDR5Wlxcc5_XMaLHuntemxRsPrHsip7MllCZkVMcUYL-fYxKo8H7Z2kYjIbM_Tv9glk7byqCSlK0RxgtP8q8MZAJQ0V677ctNWH27oel0iyNQ Manninen, O. Optimizinganther culture ofbarley and subsequent multiplication of seed on a com- mercial scale requires several years. A short cut to homozygosity can be achieved by producing doubled haploid lines from the microspores of the early, segregating generations (F-F 3 ). Success in production of microspore- derived barley plants through anther culture was first reported 25 years ago by Clapham (1973). Since then barley anther culture methods have been developed into a practical breeding meth- od by modifying the donor plant growth condi- tions (Foroughi-Wehr and Mix 1979), by using a cold pretreatment of tillers or spikes (Powell 1988b, Huang and Sunderland 1982), or manni- tol pretreatment ofanthers (Roberts-Oehlschlag- er and Dun well 1990), and by replacing ammo- nium nitrate with glutamine in the induction medium (Olsen 1987). A major breakthrough was achieved whenalternative sugars, including maltose, were used in induction media inplace of sucrose (Hunter 1987, Sorvari and Schieder 1987,Finnic et al. 1989). Much of the work with barley anther culture was 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 easy to use, efficient and applicable to all breed- ing lines. In addition, the anther culture meth- ods used should not invoke any unconscious se- lection of the variation existing within the bar- ley material. The aim of this study was to optimize the anther culture method to be able to produce 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 data were compared. Material and methods 22 F 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- gramme were used for the experiments. Differ- ent genotypes were used in each of the experi- ments. Plants were grown in the greenhouse in pots with peat soil mix and fertilized regularly (11% N, 4% P, 25% K). Natural light was 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 plants was planted in August and the second in December. Plants from both lots were used in each experiment. Donor spikes were collected when the dis- tance between the flag leaf ligule and the penul- timate leaf was 3-7 cm and the microspores were at the mid- or late uninucleate stage. If not oth- erwise stated, freshly collected spikes were used for anther isolation. Usually MMS-MG medium was used for induction. This was modified MS- medium (Murashige and Skoog 1962) with am- monium nitrate partly replaced with glutamine as proposed by Olsen (1987). All induction me- dia contained 175 mM maltose as their carbohy- drate source, 4,4 pM 6-benzylaminopurine and 5,7 pM indolate-3-asetic acid, and were solidi- fied with 2 g/1 Gelrite. All media were sterilizer by autoclaving 20 min. at 120°C. If otherwise stated, the dishes were first kept in darkness for four weeks and then in light, the temperature be- ing +25°C. Induction media Anthers from theF, generation oftwo six-rowed and four two-rowed barley crosses were used. Two differentbasic media were used: MMS-MG and N.-MG. The latter contained the macro- ando micro-salts and vitamins of the N 6 medium (Chu et al. 1975) inplace of those of the MS- medi- um. The third medium tested was MMS-MG sup- plemented with 100 mM silver nitrate. Anthers from each spike were distributed to all three media, 50 anthers per Petri dish (5 cm 0). A total of 700 anthers per genotype x medium combi- nation were used. 390 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Light and temperature conditions during embryoid induction Anthers from theF, generation offour six-rowed and four two-rowed barley crosses were used. Anthers from several spikes were randomly dis- tributed to four 5 cm diameter Petri dishes, 50 anthers per dish, 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 light at +2O°C, and the fourth in dim light at +25°C. A total of 600 an- thers per genotype x induction treatment combi- nation were used. Pretreatment Anthers from the F, generation offour six-rowed and four two-rowed barley crosses were used. Anthers from several spikes were randomly dis- tributed to four treatments: 1) MMS-MG medi- um, 5 cm dishes, 2) MMS-MG medium, 9 cm dishes 3) MMS-MAN medium, 5 cm dishes, 4) MMS-MAN medium, 9 cm dishes. 100 anthers were isolated per plate. MMS-MAN was the same medium as MMS-MG except that maltose was replaced by 175 mM mannitol.Anthers from the mannitol dishes were transferred to normal MMS-MG medium after four days pretreatment. A total of 600 anthers per genotype x pretreat- ment combination were 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 was tested with one of the data sets and 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), green plants per 100 cultured an- thers (GPA) and proportion ofgreen plants (PGP). When needed, normalized indices were produced by dividing all data by the maximum value in the experiment. The model included the main effects of genotype, treatment and season plus all the two- way interactions. The data from all experiments were analysed as completely randomised designs. Pairwise comparisons between significantly dif- ferent treatment means according to the results of ANOVA, were done with Tukey’s test. Results of parametric tests were compared with those of the non-parametric Friedmans test in one of the experiments. All statistical analyses were run us- ing SPSS®, version 6.1. 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 GPA were statistically signif- icant (P<0.05), but the propotion of green plants was effected only by genotype and season, not the treatment used. No statistically significant interactions were detected. The MMS-MG me- dium was the best induction medium for most genotypes (Fig. 1). The addition of silver nitrate had no positive effect on any of the variables measured, but hindered the formation of green plants. Large genotypic differences were seen in anther culture response and the treatments used 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 zero to 106.0% and the green plants per 100 an- thers values from zero to 46.0%. A significant interaction for genotype x treatment was detect- ed for GPA (P=0.032). The genotypic effect was significant for PPA, GPA and proportion ofgreen plants when tested against genotype-treatment 391 Vol. 6 (1997): 389-398. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Manninen, O. Optimizing anther culture ofbarley Fig. 1. Effect of induction media, season and genotype. Black bars represent the production of green plants per 100 anthers, bars with different letters are significantly different (P<0.05) from others within the same main effect. Fig. 2. Effect of light and temper- ature conditions during embryoid induction. Treatment 1) 20°C, weak light, 2) 20°C, darkness, 3) 25°C, weak light, 4) 25°C, dark- ness. Results from pairwise com- parisons of treatments are shown foronly those genotypes, were one way ANOVA showed significant differences.Treatments with dif- ferent letter are significantly dif- ferent (P<0.05) for their green plant production. 392 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 1. Mean squares and P-values formain effects and two-way interactions in pretreatment experiment. Plants per 100 anthers Green plants per 100 Proportion of 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,