The effect of genotype on anther culture response of cultivated and wild oats Elina Kiviharju, MattiPuolimatka Agricultural Research Centre ofFinland, Plant Production Research, Crops and Soil, FIN-31600 Jokioinen, Finland, e-mail: elina.kiviharju@mtt.fi Marketta Saastamoinen, Simo Hovinen Boreal Plant Breeding, FIN-31600 Jokioinen, Finland Eija Pehu Department ofPlant Production, PO Box 27, FIN-00014 University ofHelsinki, Finland Anther culture ability was tested for 44 oat (Arena sotiva L.), six naked oat (A. saliva L., naked type) and 15 wild oat (Avena sterilis L.) genotypes, in addition to progeny of five intraspecific crosses of A. saliva and two interspecific crosses ofA. saliva x A. sterilis. Anther culture response was affected considerably by genotype. Thirty one oat genotypes responded by callus growth on induction medi- um and seven of them produced embryo structures, two of the lines consistently. All naked oat geno- types produced embryo structures. Embryo production rates for the wild oat lines were comparable with those for the naked oat genotypes, and higher than for oat: 13 of the 15 genotypes tested pro- duced embryo structures. Plant regeneration was possible only from wild oat. The regeneration abil- ity was inherited in the progeny of the A. saliva x A. sterilis cross cv. Puhti x CAV 2648. The response of anthers of oat genotypes was inhibited by auxin on the induction medium, while naked oat, wild oat and A. saliva x A. sterilis crosses responded better on a medium containing 2,4-dichlorophenoxy- acetic acid. Key words: androgenesis, haploids, wild oat Introduction Production of doubled haploid (DH) lines using anther culture is useful in plant breeding for in- creasing the speed and efficiency of selection. It is also a tool for production of homozygous ma- terial for breeding and basic research purposes in many crop species, including cereals. New varieties have been developed via haploidy in wheat, Triticum aestivum L., (Hu et al. 1986,De Buyser et al. 1987, Pauk et al. 1995) barley, Hordeum vulgäre L., (Kuhlmann and Foroughi- Wehr 1989, Ho and Jones 1980) and rice, Oryza sotiva L., (Croughan et al. 1985). Cultivated oat (Avena sotiva L.) has been shown to be a very recalcitrant cereal species regarding production of doubled haploids via © Agricultural and Food Science in Finland Manuscript received March 1998 409 Voi 711998): 409-422. AGRICULTURAL AND FOOD SCIENCE IN FINLAND anther culture. To date the only report of suc- cessful anther culture of oat is by Rines (1983) whoreported production of three anther-derived plants. Sun et al. (1991) recovered twelve green plants and one albino plant from anther culture of naked (hulless) oat. Recently, anther-derived plantlets were regenerated from wildoat, Avena sterilis L., however, at a low frequency (Kivi- harju et al. 1997). Callus or embryoid induction from oat anther culture was reported also by Chung (1980) and Polsoni (1991), but no plants were regenerated. Growth responses of cultured plant tissues are presumed to be under strong genetic control (Foroughi-Wehr et al. 1982,Bullock et al. 1982, Jones and Petolino 1987,Lazar et al. 1990, Hoff- mann et al. 1991), and both callus initiation and haploid plant production were shown to be highly heritable traits in wheat and barley (Lazar et al. 1990). A strong genotypic effect on anther cul- ture capacity was also reported in spring and winter wheat (Lazar et al. 1984,Tuvesson et al. 1989,Luetal. 1991,Hoffman etal. 1991,Masojc et al. 1993, Orlov et al. 1993, Ghaemi and Sarrafi 1994), barley (Logue et al. 1993), maize, Zea mays L., (Petolino and Thompson 1987, Hongchang et al. 1991), rye, Secale cereale L. (Rakoczy-Trojanowska et al. 1997) and triticale, X Triticosecale Wittmack, (Balatero et al. 1995). It was also reported to be an important factor in anther culture of oat (Rines and McCoy 1980, Rines 1983, Rines et al. 1997), as well as in so- matic embryogenesis from immature embryo explants (Rines and McCoy 1981, Gana et al. 1995). The aim of this study was to investigate the genotype effect of cultivated oat on anther cul- ture response in auxin-containing and hormone- free culture media. Different A. sterilis geno- types were also tested to establish whether this wild oat species has better responsiveness in anther culture (see Kiviharju et al. 1997). Fur- thermore, inheritance of anther culture ability and possible positive effect of heterozygosity were studied by culturing anthers of progenies of A. saliva x A. saliva and A. saliva x A. steri- lis crosses. Material and methods Oat Forty four oat (A. saliva) genotypes (Table 1) were selected for testing. The genotypes were selected primarily from theFinnish oat gene pool and boreal vegetation zone. However, a few gen- otypes originating from other areas were also included. Donor plants were grown in the green- house under controlled conditions: 16°C/12°C day/night temperatures, 16h photoperiod. Light intensity supplementedby fluorescent lamps was 350 mol m Vl . Seeds were sown in a peat soil mix (0 11 cm pots), two seeds per pot and ferti- lized with 1% solution of 5-Superex™Kekkilä, Finland; 11%N, 4%P, 25%K). Culms were collected when the tip of the panicle inside the leaf sheath reached the level of the second leaf base. For cold pretreatment, excised culms were placed in conical flasks con- taining tap water at +4°C in the dark for seven days. After the pretreatment the culms with pani- cles still enclosed in the leaf sheath were sur- face sterilized with 70% ethanol. Anthers con- taining late uninucleatestage microspores (Dun- well 1985) were isolated aseptically. The devel- opmental stage of the microspores was deter- mined using light-microscopy after squashing anthers in aceto-carmine. Because of the wide diversity of developmental stages of the anthers in one panicle of oat, the length and color of anthers was used as a morphological marker for selecting anthers containing microspores at the uninucleate stage. These characters were deter- mined independently for each genotype. Two induction media were used for anther culture: A-medium contained no growth regula- tors; B-medium was supplemented with 1.0 mg L 1 2,4-dichlorophenoxyacetic acid (2,4-D). Both media contained MS-salts and vitamins (Mu- rashige and Skoog 1962), 10% sucrose, and were adjusted to pH 5.8. Media were solidified with 0.3% Phytagel™ (Sigma), and sterilized by auto- claving for 20 min. at a temperature of 120°C and pressure of 1 kg cm 2 . Isolated anthers (60/ 410 Kiviharju, E. et al. The ejfect ofgenotype on anther culture ofoats AGRICULTURAL AND FOOD SCIENCE IN FINLAND AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 7(1998): 409^22. Table 1. Antherculture response (callus and embryos) of different oat (Avena saliva) genotypes after eight week incubation on induction media without 2,4-D (A) and with 1 mg I' 1 2,4-D (B). 1 number of anthers is equal to isolated anthers minus contaminated anthers. genotype no. ofanthers 1 %of anthers embryos/anthers %of anthers embryo responding producing emb- producing structures ryos, no./no. embryos /100 anthers AB AB AB AB AB Stout 600 300 10.5 5.0 8/8 3/3 1.3 1.0 1.3 1.0 Puhti 600 300 9.5 0.7 000 00 0 Sisko 600 300 0.3 0.3 000 00 0 Virma 600 540 0 0 0 0 0 0 0 0 Ryhti 600 300 2.20.7 000 0 0 0 Nasta 600 300 0 0 0 0 0 0 0 0 Sv 86432 600 300 0 0 000 00 0 Kolhu 600 300 1.2 0 0 0 0 0 0 0 WW 18019 600 300 2.21.0 14/7 3/3 1.21.0 2.31.0 Cascade 600 300 1.50.7 1/1 2/2 0.20.7 0.20.7 Heikki 600 300 7.73.0 000 00 0 Hankkijan Vouti 600 300 0.2 0 000 00 0 Yty 600 300 1.2 0 1/1 0 0.2 0 0.2 0 Roope 600 300 0.5 0 0 0 0 0 0 0 Veli 600 300 0 0 0 0 0 0 0 0 Aarre 600 300 8.00.3 000 0 0 0 Freja 600 300 0 0 0 0 0 0 0 0 Pol 600 300 13.23.7 000 0 0 0 Park 600 300 0 0 0 0 0 0 0 0 Titus 600 300 0 0 0 0 0 0 0 0 Katri 600 300 0.5 0 0 0 0 0 0 0 ME 7539 600 300 6.5 5.0 000 00 0 Aio 600 300 2.2 1.0 000 00 0 Sisu 600 300 4.22.0 0 1/1 0 0.3 0 0.3 Pegaz 600 300 2.80.3 000 0 0 0 STH 180 480 300 1,0 0.7 000 00 0 Wiesel 600 300 0 0.3 000 00 0 Maldwyn 540 300 0.4 0.3 000 00 0 Myriane 600 300 3.8 0 0 0 0 0 0 0 Avesta 600 300 0 0.3 000 00 0 Ogle 600 300 0 0 0 0 0 0 0 0 Ceal 360 300 10.8 2.7 000 00 0 0T257 480 300 0.2 0.3 0 1/1 0 0.3 0 0.3 Talgai 600 300 0.2 0 1/1 0 0.2 0 0.2 0 Semu 4.004 600 300 0 0 000 00 0 Fuchs 420 300 0 0 0 0 0 0 0 0 Hja 86008 600 300 0.7 1.0 000 00 0 Hja 85013 600 300 0 0.3 000 00 0 Mostyn 480 240 0 0 0 0 0 0 0 0 Salo 420 300 0 0 0 0 0 0 0 0 Amby 240 180 0 0 0 0 0 0 0 0 STH 7518 240 180 0 0 000 00 0 Ebene 300 300 0.3 0 0 0 0 0 0 0 Rollo 300 240 0.3 0 0 0 0 0 0 0 all x 2.09 0.67 s.d. 3.50 1.27 responding x 3.41 1.41 0.84 0.66 sal 3.95 1.55 0.94 0.35 411 dish) were placed in Petri dishes (0 6 cm, Grein- er) containing 10 ml of culture medium. In to- tal, 600 anthers of each genotype were cultured on A-medium and 300 anthers on B-medium. Dishes were incubated at +25°C in the dark and anther response was assessed after eight weeks. The experiment was repeated with eight geno- types which produced embryo structures. As the carbohydrate source, 10% sucrose or 10% mal- tose was used in filter-sterilized A-medium (4 ml medium in 3.5 cm 0 Petri dishes.Falcon). Three hundred anthers were cultured per geno- type (30 anthers/dish). In total, 13 MS or N - based differentiation media were used contain- ing various concentrations of auxins (2,4-D, 1-naphthaleneacetic acid (NAA), indoleacetic acid (IAA)) and/or cytokinins (kinetin, 6-ben- zylaminopurine (BAP), zeatin), carbohydrates (maltose or sucrose, 2-10%), glutamine, myo- inositol and in some cases activated charcoal. Naked oat, wild oat and crosses Six naked oat (A. sutiva L., naked type) geno- types and 15 wild oat (A. sterilis) genotypes were included in this study. Seeds of F-2 progeny of five different oat (A. sotiva x A. sotiva) cross- ings and two different oat x wild oat (A. sotiva x A. sterilis) crossings were sown to produce the anther-donor plants. Genotypes are given in Ta- ble 2. Donor plant growing conditions were simi- lar to those for oat, except that 14 cm diameter pots were used and three seeds sown in each. Two induction media were used: A-medium contained no growth regulators; B-medium was supple- mented with 5.0 mg T 1 2,4-D. Both media con- tained MS-salts and vitamins, 10% maltose, and were adjusted to pH 5.8. The media were lay- ered in solid and liquid phases: the lower layer was solidified with 0.3% Phytagel™ (Sigma), the upper liquid layer contained 10% (w/v) Ficoll 400. The solid layer was filter-sterilized (Phytagel™ was autoclaved in a small amount of water) and the liquid part autoclaved (20 min at 120°C and pressure of Ikg cm'2). After cold pretreatment at +4°C for seven days, naked oat and wild oat culms were sterilized as above, whereas panicles of crosses were removed from the leaf sheaths and sterilized in 2.0% NaOCl solution for 20 min and rinsed five times with sterilized water. Isolated anthers (30/dish) were placed in Petri dishes (0 35 mm, Falcon) con- taining 2.5 ml of solid culture medium and 1.0 mg 1 1 liquid medium. The uninucleate state of microspores was determined as in the A. saliva experiments. In total, 150-450 anthers were cul- turedper genotype per medium depending on the number of seeds available. Plated anthers were pretreated at +32°C for five days before incuba- tion at +25 °C in the dark.Anther culture response was assessed after eight weeks. Embryo struc- tures were transferred onto solidified, filter-ster- ilized differentiation medium E-31 containing MS salts and vitamins, 1.0 mg T 1 kinetin, 4% maltose and 0.3% Phytagel™ at pH 5.8. For na- ked oat embryos, E-65 differentiation medium (E-31 supplemented with 0.2 mg T 1 NAA) was also used. Regenerated plantlets were transferred onto filter-sterilized, or autoclaved, rooting me- dium containing MS salts and vitamins, 3% su- crose, 0.3% Phytagel™ and no growth regula- tors at pH 5.8. Ploidy level of regenerants was determined by flow cytometry analysis (FACSort, Becton Dickinson) of propidium iodide stained nuclei isolated from leaf tissue. The barley cultivar ‘Sultan’ was used as the control (4C = 22.24 pg DNA; Michaelson et al. 1991, Bennett et al. 1982). Results Oat Response of anthers of different genotypes fell into two categories: callus production and direct embryogenesis (Kiviharju et al. 1997). Thirty of 412 Kiviharju, E. et al. The effect ofgenotype on anther culture ofoats AGRICULTURAL AND FOOD SCIENCE IN FINLAND AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 7 (1998): 409^22. Table 2. Anther culture response of wild oat (Arena sterilis), naked oat (A. saliva, naked type), A. saliva xA, saliva and A. saliva xA. sterilis crosses after eight weeks on hormone-free (A) and smg I 1 2,4-D (B) containing induction media, gh = greenhouse, transferred = transferred onto differentiation media. 1 number of anthers is equal to isolated anthers minus contaminated anthers. genotype no. of anthers' embryos transferred regenerants plants in gh /100 anthers embryos /100 anthers /100 anthers /100 anthers green albino ABABABABABAB wild oat: CAV 1126 420 360 0 13.9 0 0.8 0 0 0 0 0 0 16 300 420 5.6 54.3 2.7 25.0 0 2.6 0.3 0.2 0 2.6 3a 300 300 1.0 1.0 0 0.3 0 0 0 0 0 0 CW 537 150 - 0 - 0 - 0 0 0 0 0 0 Ciav 2321 450 450 0 0.7 0 0 0 0 0 0 0 0 55 360 270 1.7 33.3 1.7 9.2 0 0 0 0 0 0 CW 533 300 300 111.7 189.0 67.7 98.3 1.3 13.3 0 0.3 1.3 12.3 CW 453 300 300 0,3 4.3 0 0.7 0 0 0 0 0 0 CAV 1095 300 300 0000000000 CD 7983 300 270 1.0 5.5 0.3 4.1 0 0 0 0 0 0 CAV 1191 300 300 0.3 17.3 0 2.7 0 0.3 0 0.3 0 0.3 CAV 2941 300 300 4.7 116.7 4.7 70.0 0.3 2.0 0 0.3 0 2.0 CAV 3175 300 300 10.7 11.7 0 1.0 0 0 0 0 0 0 WAHL 6 300 300 1.0 2.7 0 0 0 0 0 0 0 0 CAV 2057 300 270 1.1 0 0 0 0 0 0 0 0 0 all x 9.27 32.17 5.14 15.15 s.d. 28.49 55.18 17.36 30.49 responding it 12.65 37.53 15.42 21.21 s.d. 33.00 58.14 29.27 34.64 naked oat: Lisbeth 450 450 0 0,4 0 0 0 0 0 0 0 0 Jo 1418 450 450 3.1 13.6 0.9 13.6 0 0 0 0 0 0 Jo 1419 450 450 0.4 2.7 0.2 2.7 0 0 0 0 0 0 Bor 1335 450 450 0 0.7 0 0.2 0 0 0 0 0 0 Bor 1267 450 450 0.4 2.7 0.2 0.9 0 0 0 0 0 0 Bor 1306 390 390 13.1 169.2 2.1 79.5 0 0 0 0 0 0 all x 2.83 31.55 0.57 16.15 s.d. 5.16 67.61 0.82 31.46 responding x 4.25 0.85 19.38 s.d. 6.04 0.90 34.04 A. saliva x A. saliva: Fuchs x PC 62 300 150 0 0.7 0 0 0 0 0 0 0 0 80r70623x80r70818 450 300 0 1.0 0 0 0 0 0 0 0 0 Hja 88612 x Bor 70584 300 210 0 2.5 0 1.3 0 0 0 0 0 0 NS 126-93x1186-4189 270 240 1.9 13.3 0 4.2 0 0 0 0 0 0 HjaB77l2xAPR 166 450 300 3.8 4.0 3.1 0.7 0 0 0 0 0 0 all x 1.14 4.30 0.62 1.24 s.d. 1.70 5.20 1.39 1.74 responding it 2.85 4.30 3.1 2.07 s.d. 1.34 5.20 0 1.87 A. saliva x A. sterilis: Puhti x CAV 2648 450 390 23.1 67.9 11.6 31.4 0.2 0.5 0 0.5 0.2 0.25 KP 9304 x CAV2648 300 150 15.7 138.7 0.3 50.0 0 0 0 0 0 0 all it 19.40 103.30 5.95 40.70 sal 5.23 50.06 7.99 13.15 413 the 44 oat genotypes included in the study pro- duced callus on either of the induction media (Table 1). Callus induction among different va- rieties ranged from 0 to 13.2% on hormone-free medium (x = 2.1, s.d. = 3.50) and 0 to 5.0% on 2,4-D containing medium (x = 0.7, s.d. = 1.27). Over 5% induction was achieved in ‘Puhti’ (13.5%), ‘Pol’ (13.2%), ‘Ceal’ (10.8%), ‘Stout’ (10%), ‘Aarre’ (8%), ‘Heikki’ (7.7%) and line ME 7539 (6.5%) on hormone-free medium. Fourteen genotypes did not respond on the in- duction medium. Seven of the genotypes pro- duced callus only on medium without 2,4-D, while three genotypes produced callus only on medium with 2,4-D. Mean callus induction per- centage over all genotypes cultured on 2,4-D containing medium was only 32% of the mean of the induction percentage on hormone-free medium. Seven genotypes ofoat produced microspore- derived embryo structures (WW 18019, Stout, ‘Cascade’, ‘Sisu’, ‘OT2S7’, ‘Yty’ and ‘Talgai’) (Table 1, Fig. la). WW 18019 and Stout showed markedly higher responses than the other five genotypes. In WW 18019 1.2% of the anthers produced embryos (2.3 embryos/100 anthers) and in Stout 1.3% of the anthers produced em- bryos (1.3 embryos/100 anthers). When the ex- periment was repeated with these seven geno- types, only WW 18019 and Stout produced em- bryo structures. Maltose was shown to be a bet- ter carbon source than sucrose as WW 18019 and Stout produced 4.3 and 2.7 embryos/100 anthers respectively on the maltose containing medium, while only WW 18019 produced embryos (0.7 embryos/100 anthers) on sucrose-containing medium. As for callus induction, the best em- bryo production rate was obtained on medium without 2,4-D (WW 18019 2.3 embryo struc- tures/100 anthers); four genotypes produced more embryos on hormone-free medium, three on 2,4-D containing medium, but in general embryo induction rates were very low. No plants were regenerated. Naked oat In contrast to the results for oat, naked oat, wild oat and progeny of the interspecific crosses dif- ferentiated directly into embryo structures and no callus initiation was observed. The six geno- types testedproduced embryo structures, two of them only on 2,4-D containing medium (Table 2). Higher induction rates were achieved on medium containing 2,4-D compared with hor- mone-free medium, but the variation between the genotypes was high (in medium A, x = 2.8, s.d. = 5.16; in medium B, x = 31.55, s.d. = 67.61). Line Bor 1306 produced up to 169.2 embryos/ 100anthers, of which 79.5 embryos/100 anthers reached adequate size to be transferred onto dif- ferentiation media. The second best line, Jo 1418, produced only 13.6 embryos/100 anthers, all of which were transferred onto differentiation me- dia. Wild oat Thirteen of the 15 lines tested produced embryo structures (Table 2, Fig. lb). There was great variation in anther culture ability between the lines. Embryo induction varied between 0 and 111.7embryos/100 cultured anthers on hormone- free medium (x = 9.3 embryos/100 anthers, s.d. = 28.5), and 0 to 189.0on 2,4-D containing me- dium (x = 32.2 embryos/100 anthers, s.d. = 55.2). As for naked oat genotypes, embryo induction was generally higher on medium containing 2,4- D, although one line produced embryos only on hormone-free medium (CAV 2057). Ten geno- types produced embryos, which reached the size when they could be transferred onto the differ- entiation medium. Three genotypes produced 25 or more embryos/100 cultured anthers of trans- ferable size during eight weeks of culture: CW 533 (67.7 on A-medium; 98.3 on B-medium), CAV 2941 (70.0 on B-medium) and 16 (25.0 on B-medium). 414 Kiviharju, E. et al. The effect ofgenotype on anther culture of oats AGRICULTURAL AND FOOD SCIENCE IN FINLAND Fig. I. a) An embryo structure developed from an anther of oat Avena sotiva cv. Sisu on an induction medium containing auxin, b) Embryo structures initiated from anthers of wild oat Avena sterilis line CAV 2941 on an induction medium containing auxin, c) Embryo structure developing from an anther of A. saliva x A. sterilis cross Puhti x CAV 2648 on an induction medium containing auxin, d) Regenerated plantlet of A. saliva x A. sterilis cross Puhti x CAV 2648 on rooting medium, e) Panicle of non-fertile, haploid, mature plant from A. saliva x A. sterilis cross Puhti x CAV 2648. 415 Vol. 7 (1998): 409-422. AGRICULTURAL AND FOOD SCIENCE IN FINLAND AGRICULTURAL AND FOOD SCIENCE IN FINLAND Kiviharju, E. et al. The effect ofgenotype on anther culture ofoats Four lines, 16,CW 533,CAV 1191 andCAV 2941, produced green plants. Most of the plants were regenerated from embryos produced on 2,4- D containing medium. In total, 41 green plants of the most responsive line, CW 533, (600 an- thers cultured) survived in the greenhouse. Four of the plants (1.3 plants/100 anthers) were in- duced on hormone-free medium and 37 (12.3 plants/100 anthers) on 2,4-D containing medi- um. Few albinos were produced, most originat- ing from 2,4-D containing medium. Approxi- mately two thirds of the plants of the two best responsing lines (CW 533 and 16) regenerated from embryos induced on B-medium were hap- loids (n=3x=2l) (Table 3, Fig. 2c). In addition to haploids and diploids (2n=6x=42) (Fig. 2b), 8% of the plants regenerated from the line CW 533 had chromosome numbers of higher ploidy levels (9x, 12x) (Fig. 2d). Of the plants regener- ated on hormone-free medium one plant was haploid, two were diploids and one of a higher ploidy level (Fig. 2). Lines CAV 2941 and CAV 1191 regenerated only haploid plants. Crosses The best embryo induction from the A. saliva x A. sotiva crosses (13.3 embryos/100 anthers) was achieved for the cross NS 126-93 x II 86—4189 on 2,4-D containing medium (Table 2). 4.2 em- bryos/100 anthers were transferred onto differ- entiation media. For all of the progenies, 2,4-D containing medium gave better results (x = 4.3 embryos/100 anthers, s.d. = 5.2) than hormone- free medium (x = 1.1, s.d. = 1.7). Three of the five crosses showed no response on medium without 2,4-D. No plants regenerated from em- bryos transferred onto differentiation media (in total 29 embryos were transferredfrom the three crosses). Embryo induction rates for the two A. saliva xA. sterilis crosses tested were markedly high- er than embryo inductionrates for A. sotiva x A. sotiva crosses (Table 2, Fig. 1c). Embryo induc- tion was 3 to 8 times higher on 2,4-D containing medium (x = 103.3,s.d. = 50.1) than on hormone- free medium (x = 19.4, s.d. = 5.2). KP 9304 x CAV 2648 produced 138.7 embryos/100 anthers on 2,4-D containing medium, of which 50 em- bryos/100 anthers were transferred onto differ- entiation medium. No plant regeneration was achieved for this cross. Although Puhti x CAV 2648 gave lower embryo induction results (67.9 embryos/100 anthers, of which 31.4 transferred embryos/100 anthers on B-medium), three green plants and two albinos were regenerated (Fig. Id, le). One diploid (2n=6x=42) green plant re- generated on the hormone-free medium, and the other two green plants (one haploid (n=3x=2l), the other one died before transfer into green- house) and the two albino plants on auxin-con- taining medium. Table 3. Ploidy level of green plants of Avena sterilis and Avena sativa x A. sterilis crosses produced by anther culture. A = hormone-free, B=s mg I' 1 2,4-D, gh = greenhouse, a no seed set Genotype plants induction haploid diploid higher ploidy dead not in gh medium (n=3x=2l) (2n=6x=42) (2n=9x,l2x) ingh analyzed no. no. (%) no. (%) no. (%) no. (%) no. no. (%) 16 11 B 7(64) 4(36) 0 0 CW 533 4 A I (25) 2 (50) 1 (25) 0 CW 533 37 B 24 (65) 9 (24) 3 (8) 1 1 (3) • CAV 2941 6 B 6(100) 0 0 0 CAVII9I 1 B 1(100) 0 0 1 Puhti x CAV 2648 1 A 0 1(100) 0 0 Puhti x CAV 2648 1 B 1(100) 0 0 0 416 AGRICULTURAL AND FOOD SCIENCE IN FINLAND 417 Fig, 2, Flow cytometer analysis ofDNA contentof regenerated plants, 2C indicating normal diploid (eg. hexaploid in Avena sterilis) ploidy level and 4C doubled genome justprior to cell division, a) ControlHordeum vulgare, cv. Sultan, b) Diploid (2n=6x=42) A. sterilis regenerant line CW 533, c) Flaploid (n=3x=2l) A. sterilis regenerant line CW 533, d) A. sterilis regenerant line CW 533 with DNA content over 6x ploidy level. Vol. 7 (1998): 409^122. Discussion In line with the report of Rines (1983) on A. sali- va, a strong genotype effect was demonstrated for anther culture response of oat, as well as for naked oat and A. sterilis. Although cultivated oat is recalcitrant in anther culture, callus induction rates obtained in our experiments [16% (7/44) of genotypes demonstrated over 5% induction] were at the same level as Han-Min et al. (1990) reported for winter wheat [17% (37/215) ofgen- otypes exceeded 5% induction], but lower than that reported by Orlov et al. (1993) for common wheat [43% (13/30) of tested genotypes exceed- ed 4% response level]. Of 100 oat genotypes, two cultivars. Stout and ‘Clintford’ were report- ed to exceed 6% callus induction and produce callus consistently (Rines and McCoy 1980, Rines 1983, Rines et al. 1992). Under optimal conditions, 30% of anthers of these cultivars were reported to produce calli. In our experi- ments, cv. Stout had a high induction rate (10.5% on hormone-free medium; 5.0% on 2,4-D-medi- um). In addition to Stout, six other genotypes of the 44 tested, exceeded 6% callus initiation fre- quency. Thirty two percent (14/44) of genotypes showed no response in our experiments, which is considerable, but still less than for maize, where Hongchang et al. (1991) reported that nearly half of the genotypes failed to develop calli or embryos (13/25; 13/30). Sixteen percent (7/44) of the oat genotypes produced embryo structures, of which only 4,5% (2/44) produced themconsistently. Another gen- otype capable of consistent, and even better embryo production efficiency than Stout, was WW 18019, despite its low overall response. In contrast to oat, all of the six naked oat genotypes testedproduced embryo structures, one of them (Bor 1306) in very high numbers (up to 169.2 embryos/100 anthers). However, no plant regen- eration was achieved from embryo structures of oat, naked oat or A. sativa x A. sativa crosses. Contrary to wheat, where hybrid vigour has been strongly expressed in anther culture competence (Hu Han 1986, Ekiz and Konzak 1991, Pauk et al. 1991), heterozygosity did not lead to better anther culture performance in our study on cul- tivated oat. Correlationbetween the level of embryo pro- duction and plant regeneration was reported to be very low (Foroughi-Wehr et al. 1982, Deaton et al. 1987), and these two phenomena seem to be genetically unrelated (Knudsen et al. 1989, Larsen et al. 1991). Examples can be found from triticale (Balatero et al. 1995), barley (Larsen et al. 1991, Logue et al. 1993), wheat (Orlov et al. 1993) and rice (Chaleff and Stolarz 1981, Man- dal and Gupta 1995). In our study on cultivated oat, plants were not regenerated, although em- bryo production was quite high is some geno- types. Immature embryo cultures ofA. sterilis gen- otypes were reported to have lower regeneration frequencies compared with oat and naked oat (Rines and McCoy 1981). However, according to the results of our experiments, anther culture characteristics of wild oat are better than those of cultivated oat. Eighty seven percent (13/15) of the A. sterilis lines tested produced embryo structures, of which three genotypes produced 25 or more transferrable embryos/100 anthers. Embryo induction rates were also markedly high- er in tested wild oat genotypes than in A. sativa x A. sterilis crosses. Naked oat performed better than oat and the means of embryo induction rates of naked oat and wild oat were about the same in the most responsive induction medium con- taining 2,4-D. Plant regeneration was achieved from four wild oat lines and this regeneration capacity was also inherited in the A. sativa x A. sterilis cross Puhti x CAV 2648: three green and two albino plants were produced in anther culture. Wild species have been reported to respond better than cultivated species in other crops including pota- to, Solanum tuberosum L., (Rokka et al. 1996, Veilleux 1996). Crosses between cultivated and wild rye (Secale cereale x Secale vavilovii L.) were also shown to respond better than cultivat- ed rye genotypes (Rakoczy-Trojanowska et al. 1997). In contrast, wild wheat species (Orlov et al. 1993) were reported to be weaker in response 418 Kiviharju, E. et al. The effect ofgenotype on anther culture ofoats AGRICULTURAL AND FOOD SCIENCE IN FINLAND in anther culture than common wheats. Success- ful production of plants from the Avena sativa x Avena sterilis cross opens up also the possibili- ty for transferring anther culture ability from wild oat to breeding material of cultivated oat through a crossing program. At least within spe- cies, Balatero et al. (1995) suggested, that high embryo induction and green plant regeneration can easily be transferred and fixed in non-respon- sive or low-responsive lines through breeding and selection. Hou et al. (1994) also found se- lection of recombinant genotypes for higher green plant regeneration ability effective and considered improvement of anther culture re- sponse possible by crossing and selection. In two wild oat genotypes (CW 533, 16), pro- ducing the highest numbers of green plants, ap- proximately two thirds of the plants initiated on medium containing 2,4-D were haploid (64- 65%) and one third diploid (24-36%). CW 533 produced three (8%) plants with DNA content close to higher ploidy levels (2n = 9x or 12x) according to flow cytometry analysis. Seed set of these plants was disturbed, but some seeds did develop in each panicle. These results are in agreement with those of Masojc et al. (1993), who in wheat anther culture obtained plants of which 45% were haploids, 29% spontaneous dip- loids and 26% had abnormal chromosome num- bers. In our experiments, the ratio of haploid to diploid regenerants varied with regard to geno- type and medium used. Two low responding gen- otypes (CAV 1191, CAV 2941) produced only haploids. The A. sativa x A. sterilis cross pro- duced one haploid and one diploid plant (one died). In barley as many as 80% of the anther culture regenerants were reported to be doubled haploids (Gudu et al. 1993). In general, callus induction and embryo pro- duction of oat was better on hormone-free me- dium than in the presence of auxin, confirming the reported auxin inhibition of oat anther cul- ture (Rines 1983). In contrast, naked oat and wild oat genotypes were not sensitive to 2,4-D. In fact, the best embryo induction and plant regenera- tion results were obtained using induction me- dium containing 2,4-D (only one wild oat geno- type produced mature plants originating from hormone-free medium). Moreover, anthers of progeny from both A. sativa x A. sterilis crosses produced more embryos on medium containing 2,4-D, although plants did regenerate from em- bryos from both induction media. Two albino plants from the intraspecific cross, and almost all of the albinos regenerated from wild oat ma- terial, were initiated on induction medium con- taining auxin, indicating a possible albino pro- duction enhacing effect of 2,4-D. Nevertheless, three oat genotypes produced a few calli only on medium with 2,4-D, indicating the complex hormonal regulation of oat anther culture. Fur- ther investigations are needed, since although auxin inhibition seems clear, Rines (1983) re- ported regeneration of three anther-derived A. sativa plants from anther cultures initiated on medium containing 2,4-D. Maltose was a better carbohydrate source than sucrose, as embryo production in WW 18019 was about six times higher on maltose than on sucrose medium. Cultivar Stout produced embryos in this experiment only on maltose medium, although embryo induction also oc- curred on a medium containing sucrose. Acknowledgements. The authors wish to thank Ms. Sirpa Moisander for excellent technical assistance, M.Sc. Veli- Matti Rokka and M.Sc. Airi Tauriainen for expertise in flow cytometry analysis and Dr. Jonathan Robinson for his edi- torial assistance. Most of the genotypes of cultivated oats were kindly provided by Boreal Plant Breeding, Finland, cv. Stout by Prof. Rines from University of Minnesota, USA and line WW 18019by SvalöfWeibull. Sweden. 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AGRICULTURAL AND FOOD SCIENCE IN FINLAND SELOSTUS Genotyypin vaikutus kauran, kuorettoman kauran ja villikauran ponsiviljelyvasteeseen Elina Kiviharju, Matti Puolimatka, Marketta Saastamoinen, Simo Hovinen jaEija Pehu Maatalouden tutkimuskeskus, Boreal Suomen Kasvinjalostus ja Helsingin yliopisto Tutkimuksessa selvitettiin genotyypin vaikutusta kuo- rellisen ja kuorettoman kauran (Avena sotiva L.) ja villikauran {A. sterilis L.) ponsiviljelyvasteeseen auk- siinia sisältävällä ja hormonittomalla kasvatusalustal- la. Kauran 44:stä testatusta genotyypistä 70 % tuotti induktioalustalla kadusta tai alkiorakenteita. Kuoret- tomalla kauralla kaikki testatut kuusi genotyyppiä tuottivat alkiorakenteita. Alkionkehitys kuitenkin py- sähtyi eikä taimia erilaistunut kauran tai kuorettoman kauran ponsiviljelmistä. Villikauralla kolme geno- tyyppiä viidestätoista testatusta regeneroi vihreitä kasveja ponsiviljelyssä. Näistä paras linja, CW 533, tuotti 41 vihreää tainta 600 viljeltyä heteen pontta kohti. Villikauran taimien erilaistumiskyky periytyi villikaura x kaura -risteytysjälkeläisiin. Kaurageno- tyyppien ponsiviljelyvaste oli parempi hormonitto- malla kuin auksiinia sisältävällä alustalla. Sen sijaan kuoreton kaura, villikaura ja risteytykset tuottivat paremman ponsiviljelytuloksen auksiinia sisältäväl- lä kasvatusalustalla. Tulokset vahvistavat genotyypin vaikutuksen kauran ponsiviljelyssä ja identifioivat toimivia A. sterilis genotyyppejä sekä lupaavia vil- jellyn kauran genotyyppejä jatkotutkimuksiin. 422 Kiviharju, E. et al. The effect ofgenotype on anther culture ofoats AGRICULTURAL AND FOOD SCIENCE IN FINLAND