Plant Cell Reports
Plant Cell Reports (1996) 16:l 6 7 - 1 7 3
© Springer-Verlag 1996
High regeneration rates in anther culture of interspecific sunflower hybrids Tengku Nurhidayah, Renate Horn, Thomas Riicher, and Wolfgang Friedt [nstitut fiir Pflanzenbau und Pflanzenzfichtung I, Justus-Liebig-Universit/it, Ludwigstr. 23, D-35390 Giessen, G e r m a n y Received 3 January 1996/Revised version received 6 April 1996 - C o m m u n i c a t e d by W. Barz
Abstract. Optimization of anther culture with regard to the induction of callus formation and direct embryogenesis was obtained for interspecific hybrids of H. annuus with H. tuberosus, H. laetiflorus, and H. resinosus by investigating six different induction media and four regeneration media. One media combination (MS-I3, MS-R3 and MS-R4) used under different culture conditions (30°C/35°C and different dark treatments) gave up to 92.7% embryogenic anthers with an average of 8.5 embryos per anther. However, direct embryogenesis as well as callus formation showed a strong genotypic and treatment specific reaction. From 5,600 anthers of the four investigated genotypes more than 2,000 plants could be regenerated. Regenerants were characterized by morphological traits and isozyme analyses to prove their androgenetic origin.
Abbreviations: BAP =
6-benzylaminopurine, MNR = menadione reductase, NAA = a-naphthalene acetic acid, PGM = phosphoglucomutase, PGI = phospoglucoisomerase, PVP = polyvinylpyrrolidone, CMS = cytoplasmic male sterile, MS = Murashige & Skoog
Introduction Anther and microspore culture allow the acceleration of breeding programmes by providing homozygous doubled haploids within a comparatively short time. Homozygous lines are required for commercial hybrid production. In addition, doubled haploid populations have proven useful in the development of molecular maps and in tagging important agronomic traits. Application of haploid techniques in breeding programmes affords high regeneration rates. In sunflower, haploids have been obtained via anther culture, but none of the reports so far allowed the regeneration of plants in a number interesting for breeding programmes, and the success of haploid techniques has been strongly dependent on genotype (reviewed in Friedt et al. 1996). Employment of wild species and interspecific hybrids Correspondence to: W. Friedt
allowed the first successful reports of anther culture in sunflower (Mix 1985, Bohorova et al. 1985). Shoot formation was observed for HI divaricatus and H. annuus x I-L decapetalus. For other wild species, e.g. H. tuberosus or H. resinosus, Jonard and Mezzarobba (1990) observed callus formation and embryogenesis which allowed regeneration of plants. From interspecific hybrids involving hr. resinosus, H. rigidus, and H. occidentalis plants were also regenerated from anther culture, although with very low rates in the case of the interspecific hybrids involving the last two species. Furthermore, interspecific hybrids of H. annuus to /42. smithii or H. eggertii allowed the regeneration of plants from anther culture by Nenova et al. (1992). Cultivated sunflower proved to be very recalcitrant in anther culture (Giirel et al. 1991, Mezzarobba and Jonard 1986). More recently, Jonard and Mezzarobba (1990) have reported the induction of callus formation from anthers of cultivated lines but no embryogenesis or organogenesis could be observed, although Mezzarobba (1988) was able to regenerate 91 plants from 2,278 plated anthers using other cultivated sunflower lines. Anther culture in sunflower still needs considerable improvement before it can be routinely included in breeding programmes. We here report on an improved anther culture protocol using interspecific hybrids which allowed the production of a large number of plants from anther culture without any multiplication by subculturing. Using anther culture, it still remains a problem to prove the androgenetic origin of regenerated plants. Jonard and Mezzarobba (1990) assessed the ploidy level of plants regenerated from anthers of different sunflower genotypes. However, as spontaneously doubled haploids appear, cytological investigations do not always offer evidence for androgenesis. In sunflower, isozyme analysis has been successfully used to characterize interspecific hybrids obtained by
168 embryo rescue (Dahlhoff et al. 1992) or somatic hybridization (Barth et al. 1993, Krasnyanski and Menczel 1994). The value of isozyme analysis for the identification of androgenic sunflower plants will be discussed and compared with results obtained by characterizing the plants on the basis of morphological traits.
Materials and Methods Plant material Five genotypes of three interspecific hybrid combinations (Tab. 1) which were obtained earlier by embryo rescue (Kr/iuter et al. 1991) were investigated for their performance in anther culture. Donor plants were grown in the field or in the greenhouse. Tab.l: Interspecific hI ,brids of line HA89(CMS) with three wild species GenoWpe Male parent 40/3 H. resinosus (RES-1545) 30/61 H. laetiflorus (LAET-Hung) 10/25, 10/28 H. tuberosus (TUB-5) 11/51 H. tuberosus (TUB- 1705) Flower buds were surface sterilized by immersion for 30 s in 96% ethanol, followed by a 15 rain incubation in 2% (v/v) commercial bleach. After three washings with sterile distilled water anthers were dissected from the first 3-4 rows of disc fitters. Only anthers with microspores at the early uninueleate stage were used for culture in vitro.
Anther culture Six induction media modified according to Murashige and Skoog (1962) which differed in the concentration of vitamins and hormones (Tab. 2) were investigated with regard to their ability to induce androgenesis. Twenty anthers per Petri dish ( ~ 5 cm) were cultured under permanent light (600 - 800 lux) or in the dark at 30°C for four weeks. Green calli formed during this time were transferred to different regeneration media to induce shoot formation. Calli on MS-I1, MS-I3 and MS-I5 were transferred to MS-R1, while calli from MS-I2, MS-I4 and MS-I6 were put on MS-R2 (Tab. 2). Culture temperature was reduced to 25°C at 16 h light (2,000 lux). Calli showing shoot induction were placed on MSR3 for 3-4 weeks for shoot formation. Single shoots carrying 4-6 leaves were transferred to tubes on MS-R4 medium for root induction. Successfully rooted plantlets were further grown on soil and adapted to greenhouse conditions. For further improvement of anther culture one media combination (MS-I3, MS-R3 and MS-R4) was used and the culture conditions were modified. Anthers were either cultured at 30°C or 35°C with 0, 6, 12 or 18 days of dark treatment on the induction medium MS-I3. Afterwards the Petri dishes were transferred to permanent light (2,000 lux) for the remaining period. In total, all anthers were incubated for four weeks on the induction medium MS-I3. Afterwards, developing embryos were transferred to the regeneration medium MS-R3 and cultured at 25°C and 16 h light (2,000 lux). Shoots carrying 4-6 leaves were transferred to tubes on MS-R4 medium for rooting. Successfully rooted plantlets were adapted in soil to greenhouse conditions.
Isozyme electrophoresis For isozyme analyses about 150 mg of leaf material were taken from each sunflower plant and 100 p.1 extraction buffer according to Soltis et al. (1983) were added. The leaf material was stored frozen at -80°C. For the extraction 200 ixl of modified extraction buffer (containing PVP, ascorbic acid and B-mercaptoethanol) and quartz sand were added. The samples were homogenized and centrifuged at 6,200g for 5 min at 4°C. The clear supematant was used for electrophoresis on 12% (w/v) starch gels. Buffer systems for electrophoresis were modified for PGM and PGI according to Stuber et al. (1977) and for MNR according to Soltis et al. (1983). Staining was performed for MNR (Rieseberg and Soltis 1989), PGI and PGM (Vallejos 1983).
Results Effect of different induction and regeneration media In total, 2,375 anthers were cultured in order to investigate the ability of different induction and
regeneration media (Tab.2) to support androgenesis. For the genotypes 11/51, 30/61 and 10/28 callus induction was obtained on each of the six investigated induction media under permanent light (Tab.3) and dark conditions (data not shown). For the dark treatment no embryogenesis or organogenesis could be induced on the regeneration media. However, under permanent light pronounced effects of medium and genotype were observed after the transfer to a regeneration medium. For genotype 11/51 representing an Fl-hybrid H. annuus x H. tuberosus only green calli transferred from MS-I3 and MS-I5 to MS-R1 showed shoot induction. Upon transfer to the hormone free MS-R3 medium, green vigorous calli gave rise to shoots. Seventy-five (MS-I3 medium) and 85 shoots (MS-I5 medium) could be isolated from this genotype and transferred to the rooting medium MS-R4, respectively. MS-R4 contains only 10% (w/v) sucrose and thiamine HC1 is reduced to 0.1 mg 1-1. About 6070% of the shoots could be rooted. Thirty-seven and 41 plantlets were finally transferred to the greenhouse, respectively. For genotype 30/61 representing an F1hybrid of//. annuus with H. laetiflorus only green calli from MS-I1 showed shoot induction on the regeneration medium MS-R1. A very high number of 187 shoots could be isolated but rooting proved to be very difficult for this genotype. Often multiple shoots developed on the rooting medium or no roots were formed. Therefore, only 96 plantlets could be planted into soil and only 44 plantlets could be finally transferred to the greenhouse. All plantlets that were regenerated in this experiment were developed from calli. The genotype 10/28 representing another F~hybrid (H. annuus x H. tuberosus) did not show any response besides callus formation on each of the six induction media. Direct embryogenesis and callus formation under different culture conditions For further improvement of anther culture the media combination MS-I3, MS-R3 and MS-R4 was applied. The cultivation step on MS-R1 was omitted and the culture conditions were modified. In total, 5,600 anthers were cultured at different dark treatments (0, 6, 12 and 18 days) either at 30°C or 35°C (Tab. 4). In addition to the three genotypes 11/51, 30/61 and 10/25 an interspecific hybrid of H. annuus with tt. resinosus (genotype 40/3) was included in these studies. Direct embryogenesis was observed on MS-I3 medium within 10 to 28 days for genotype 40/3 (Fig. 1A). Up to 92.7% embryogenic anthers were obtained and a mean of 8.5 embryos per anther was observed for this genotype (Tab. 4). Anthers from the interspecific hybrids involving H. tuberosus and [t. laetiflorus also showed direct embryogenesis, although at a much lower rate (Tab. 4). Embryoids were transferred to MS-R3 medium for further development. However, many of the primary embryoids ceased growth at the
169 Table 2: Composition of the different induction and regeneration media Compounds
MS-I1
MS-I2
MS-I3
MS-I4
MS-I5
MS-I6
MS-R1
MS-R2
MS-R3
MS-R4
2.0 100.0 0.5 0.5 0.5 0.01 0.5 0.5 30.0 3.0 5.7
2.0 100.0 0.5 0.5 0.5 0.01 1.0 1,0 30.0 3,0 5,7
2.0 100.0 1.0 1.0 1.0 0.01 0.5 0.5 30.0 3.0 5.7
2.0 100.0 1.0 1.0 1.0 0.01 1.0 1,0 30.0 3.0 5.7
2,0 100.0 1.5 1.5 1.5 0.01 0.5 0.5 30.0 3.0 5.7
2.0 100.0 1.5 1.5 1.5 0.01 1.0 1.0 30.0 3.0 5.7
2.0 100.0 1.0 1.0 1.0
2.0 100.0 1.0 1.0 1.0
2.0 100.0 0.5 0.5 0.5
2.0 100,0 0.5 0.1 0.5
0.5 0.5 30.0 3.5 5.8
1.0 1.0 30.0 3.5 5.8
30.0 4.0 5.8
I0.0 4,0 5.8
(mg 1") glyeine myo-inositol nicotinic acid thiamine-HCl pyridoxine-HC1 biotin BAP NAA sucrose (g 1"1) Gelrite (g 1-1) pH
Table 3: Plant regeneration on different induction media under permanent light Genotype
Media
11/51
MS-I1 MS-I2 MS-I3 MS-I4 MS-I5 MS-I6 Mean MS-I1 MS-I2 MS-I3 MS-I4 MS-I5 MS-I6 Mean MS-I1 MS-I2 MS-I3 MS-I4 MS-I5 MS-I6 Mean
30/61
10/28
NO. of anthers plated 100 100 100 100 100 100 100 60 60 60 60 60 60 60 50 50 50 50 50 50 50
Initiated calli No. 90 75 71 71 75 80 77.0 58 57 59 52 56 59 56.8 36 42 36 48 27 44 38.8
% 90.0 75.0 71.0 71.0 75.0 80.0 77.0 96 95 98 86 93 98 94.3 72 84 72 96 54 88 77.7
cotyledonary stage. After a period of two to four weeks of dormancy, primary embryoids developed into multiple secondary embryos before regeneration of shoots occurred (Fig.lB). For a successful rooting shoots had to be transferred to MS-R4 medium. Culture temperature had a considerable influence on the percentage of embryogenic anthers which could be obtained for the four investigated genotypes (Tab. 4). In general, anthers cultured under 35°C show a better response in vitro regarding direct embryogenesis. For genotype 10/25 embryogenic anthers could only be observed when culW.red at 35°C whereas no embryogenesis occurred at 30°C. The genotypes 11/51 and 30/61 also had a higher percentage of direct embryogenesis at 35°C. However, there was one exception. The genotype 40/3 showing the best overall performance with regard to embryogenesis has a mean value of 67.2% for all treatments at 30°C compared to 12.9% at 35°C (Tab. 4). For this genotype the mean percentage of callus formation is comparably low at
Morphogenic caUi No. % 0 0.0 0 0.0 5 5.0 0 0.0 7 7.0 0 0.0 2.0 2.0 8 13.3 0 0 0 0 0 0 0 0 0 0 1.3 3.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0
No. of isolated shoots 0 0 75 0 85 0 26.7 187 0 0 0 0 0 31.2
Plants transferred to soil 0 0 57 0 59 0 19.3 96 0 0 0 0 0 16.0
No. of vital plants 0 0 37 0 41 0 13.0 44 0 0 0 0 0 7.3
0
0
0
30°C with a value of 25.1%. All other investigated genotypes showed callus formation of about 70% with an average of about 3% of anthers showing direct embryogenesis under these conditions. In general, the rate of callus formation is much higher at 30°C than at 35°C for all investigated genotypes. Dark treatments of 6, 12 and 18 days were applied in order to try to improve response in this way (Tab. 4). For all four genotypes the elongation of the dark period from 0 to 18 days resulted in a considerable increase in anthers forming callus. With regard to direct embryogenesis the genotypes differed in their response to various dark treatments. For genotype 40/3 any dark period led to a remarkable reduction in direct embryogenesis at 30°C as well as at 35°C. Genotype 30/61 also showed this reduction in embryogenesis at 30°C but not at 35°C. However, for the genotypes 11/51 and 10/25 involving H. tuberosus, dark treatments of six days at 30°C or 12 days at 35°C increased the rate of direct embryogenesis to up to 9%. A dark treatment of 18 days had a
170
Fig. 1:
Anther culture of an interspecific hybrid between H. a n n u u s and H. r e s i n o s u s (genotype 40/3). (A) Multiple embryos induced on an anther after 15 days, 30°C, and 03) Shoot development from embryos on MS-R3 after six weeks, 30°C.
Table 4: Reactions of the genotypes 40/3, 11/51, 10/25 and 30/61 to different dark treatment at 30°C and 35°C Genotype
Dark period (days)
40/3
0 6 12 18 Mean 0 6 12 18 Mean 0 6 12 18 Mean 0 6 12 18 Mean
11/51
10/25
30/61
No. of anthers plated
30°C 220 220 220 220 220 360 360 360 360 360 160 160 160 160 160 120 120 120 120 120
35°C 120 120 120 120 120 200 200 200 200 200 120 120 120 120 120 100 100 100 100 100
% of anthers with % of anthers with i % of anthers with direct callus formation root formation embryogenesis 30°C 92.7 69.1 60.9 45.9 67,2 3.9 6.1 5.6 1.4 4.2 0 0 0 0 0 5.8 3.3 1.7 0.0 2.7
35°C 21.7 13.3 10.0 6.7 12.9 4.5 6.0 9.0 5.0 6.1 2.5 2.5 3.3 1.7 2.5 7.0 8.0 5.0 2.0 5.5
negative effect on direct embryogenesis for all genotypes. The hybrid 11/51 differs from the other investigated genotypes by the fact that it showed pronounced root formation from the anthers. At 35°C the formation of roots is reduced in hybrid 11/51 but
30°C 5.9 18.6 34.5 41.2 25.1 49.7 77.2 81.7 89.2 74.4 56.6 63.1 73.1 80.6 68.1 67.5 72.5 81.7 93.3 78.8
35°C 0 0 0 0 0 5.5 9.0 195 28.0 15.5 26.7 29.2 30.8 37.5 30.0 11.0 33.0 35.0 38.0 29.3
30°C 0 0 0 0 0 0.6 2.8 6.7 8.9 4.7 0 0 0 0 0 0.0 0.0 0.8 0.8 1.7
35°C 0 0 0 0 0 0.0 0.0 1.5 1.5 0.8 0 0 0 0 0 0 0 0 0 0
Average number of direct embryos per anther 30°C 35°C 8.5 1.5 2.4 1.0 2.0 1.0 2.0 1.0 3.7 1.0 1.0 1.0 1.5 1.0 1.5 1.0 1.0 1.0 1.3 1.0 0 1.0 0 1.0 0 1.0 0 1.0 0 1.0 1.8 1.0 1.0 1.0 1.0 1.0 0.0 1.0 1,0 1.0
still observed in treatments with 12 and 18 days of darkness. The percentage of root formation observed for genotype 11/51 increased with longer period in the dark.
171 Characterization of regenerants from anther culture by _isozvme electrophoresis The three isozyme systems menadione reductase (MNR), phosphoglucoisomerase (PGI) and phosphoglucomutase (PGM) were analyzed to investigate the origin of the regenerated plants. For the interspecific hybrid of H. a n n u u s and H, r e s i n o s u s (genotype 40/3) 1,211 plants regenerated from anther culture were characterized. The isozyme system menadione reductase proved to be the most informative (Fig. 2A). The parental lines as well as the F:-hybrid showed a clearly different pattern. For the regenerated plants four groups can be distinguished: (1) plantlets that have the same pattern (five bands) as the Fl-hybrid, (2) plantlets that show a single upper band, (3) plantlets that have a single lower band, and (4) a single plant with a pattern of three upper bands. From the investigated 1,211 regenerants eight plantlets showed the upper band and seven plantlets the lower band. The segregation of the pattern indicates that these plants are of androgenetic
origin. For the plants that showed the pattern of the respective F:-hybrid no definitive answer can be given. For PGI, HA89(CMS), the F:-hybrid and all regenerants showed a single band (Fig. 2B). However, RES-1545 has three bands. Also for PGM, only RES1545 showed a divergent pattern (three bands) whereas the female parent, the F:-hybrid and regenerated plantlets have the same pattern (four bands, Fig. 2B). Characterization of the regenerants on the basis of morphological traits Morphological characters such as~ e.g., plant height, anthocyanin colouring or leaf shape (Tab. 5) were evaluated for 1,000 regenerated plants. Fifteen groups distinguishable by morphological parameters could be observed (data not shown). Special attention was paid to the plants that segregated with regard to the pattern of the isozyme MNR as isozyme analysis of these plantlets proved that they represent regenerants of androgenetic origin. The morphological parameters
A:
B"
Fig. 2: Isozyme pattern of the plants regenerated from anther culture compared to the parental lines of H. a n n u u s (HA89) and H. r e s i n o s u s (RES-1545) as well as the Fl-hybrid 40/3. Starch gels were stained for activity of (A) MNR (for each sample duplicates were applied) and 03) PGI and PGM (each lane represents a single plant).
172 Table 5: Morphological traits of the regenerated plants from genotype 40/3 which have been characterized by isozyme electrophoresis Trait
Donor plant
Plant height (cm) Anthocyanin colouring Branching Leaf arrangement Petiole (cm) Leaf length (cm) Leaf width (cm) Angel at leaf top (°) Leaf margin Hairiness of leaves Capitulum (cm) Anthers (mm) Fertility Pollen viability (%) Pollen size (~tm)
100-135 medium fully branched opposite/alternate 3-5 18-25 4-8 30-32 serrated slightly hairy 3-4 4-5 fertile 30-60 30-45
taken (Tab. 5) support this hypothesis as the regenerated plants showing the upper band of MNR have a very similiar habitus and can be clearly distinguished from the group of plants showing the lower band of MNR. Pollen viability did not differ significantly between the two groups.
Discussion Interspecific hybrids have been successfully used in anther culture of sunflower (Alissa et al. 1985, Bohorova et al. 1985, Mix 1985, Jonard and Mezzarobba 1990). However, this is the first report of successful anther culture involving interspecific hybrids of H. annuus with H. tuberosus or H. laetiflorus, respectively. Especially, genotypes 11/51 and 10/28 involving H. tuberosus are of special interest for sunflower breeding programmes, as H. tuberosus is a potential source of resistance, e.g., to Phomopsis helianthi or Sclerotinia sclerotiorum (cited in Atalagic et al. 1993). Interspecific hybrids of H. annuus with H. resinosus have already been reported to have a high regeneration potential in anther culture (Jonard and Mezzaroba 1990). With our best protocol for anther culture 92.7% of the anthers from genotype 40/3 showed direct embryogenesis with an average number of 8.5 embryos per anther. In total, more than 2,000 plants could be regenerated starting with 5,600 anthers. Application of anther culture allowed to create a considerable diversity in the breeding material with regard to morphological traits evaluated. As in other reports (Jonard and Mezzarobba 1990) induction media on MS basis with NAA (0.5 mg 1-1) and BAP (0.5 mg 1-I) gave the best results in our experiments. A higher concentration of these two hormones (1 mg 1"1) gave high percentages of callus induction but after transfer to the regeneration media no organogenesis could be observed. One media
Regenerants with upper band 92-146 light fully branched opposite/alternate 2-5 16-25 4-8 28-32 serrated slight hairy 2-4 4-5 fertile 23-37 30-50
Regenerants with lower band 125-231 intensive fully branched opposite/alternate 3-6 18-32 7-11 30-40 partly serrated slightly hairy 2-5 4-5 semi-sterile 15-35 30-50
combination (MS-I3, MS-R3 and MS-R4) resulted in high rates of embryogenic anthers and callus formation under different culture conditions although it is still dependent on genotype. Cultivation at 35°C had a stimulating effect on direct embryogenesis in the genotypes which showed low rates at 30°C. However, the positive effect of a higher culture temperature is genotype specific and advantage of this culture condition has to be investigated for each new genotype. In the study of Mezzarobba and Jonard (1986) the percentage of embryogenic anthers could be increased for all investigated genotypes by raising the temperature from 25°C to 35°C and an additional pretreatment of 12 days darkness. In our experiments a dark treatment of 12 days was only advantageous to embryogenesis for the two genotypes 11/51 and 10/25 of the interspecific hybrid H. annuus x H. tuberosus. In general, dark treatment seems to increase the number of anthers forming callus but to reduce the percentage of embryogenic anthers. Cold pretreatment was demonstrated to have a very strong effect on embryo induction in the study of Thengane et al. (1994). A combination of our method of plant regeneration from anthers with this cold pretreatment might further improve the percentage of embryogenic anthers, especially from the interspecific hybrids of H. annuus with H. tuberosus or H. laetiflorus.
Microspore culture has the potential advantage that the regenerated plants can be assumed to be of androgenetic origin (cf. Coumans and Zhong 1995), whereas in anther culture plants might have also been regenerated from the diploid cells of the anther wall. In fact, the regeneration of diploid plants from the somatic anther tissue remains a major problem. For example, Zhong et al. (1995) have recently reported the recovery of embryo-derived plants from sunflower anthers, where all
173 plants proved to be diploid (2n =34). Consequently, it is necessary to unequivocally determine the origin of the regenerants. For this purpose, the value of different isozyme systems was investigated. The two isozyme systems PGI and PGM allowed a differentiation of the two parental lines of the investigated FI-hybrid. However, the hybrid and the regenerants had the same pattern as HA89(CMS) which did not allow the detection of an androgenetic origin. Since no divergence from the pattern was observed, somaclonal variation detectable by these isozymes did not occur. Menadione reductase was comparably informative as [t. a n n u u s and H. r e s i n o s u s were distinguishable from each other as well as from the corresponding Fl-hybrid. However, the hybrid did not have an additive pattern of the parental lines alone, instead new bands appeared. For the regenerants having the same pattern as the F1hybrid no information on their origin has been obtained so far. However, the fifteen regenerated plants of genotype 40/3 which segregated into two groups, one with the upper band and one with the lower band, are likely to be of androgenetic origin. The pattern obtained for these regenerants is very simple compared to the pattern of the Fl-hybrid which can be regarded as another hint for their homozygosity. The single regenerant showing a totally divergent pattern might be a case of somaclonal variation or a different segregation pattern. For the investigated regenerants of genotype 40/3 a prediction of the results is complicated because this genotype represents an F~-hybrid between the diploid H. a n n u u s (2n=2x=34) and the hexaploid wild species H. resinosus (2n=6x=102). Alissa et al. (1985) cytologically investigated plants regenerated from anthers of the interspecific hybrid H. a n n u u s x H. r e s i n o s u s . Of the investigated 64 plants 47 were aneuploid, only 7 were haploid, and 10 diploid. Therefore, only the observed segregation of the isozyme pattern observed in our regenerants proves their androgenetic origin. An aneuploid plant analyzed by isozymes can show the same pattern as the Fl-hybrid but can nevertheless be of androgenetic origin. However, isozyme analyses only allowed the identification of little over 1% of the plants regenerated from anther culture of the interspecific hybrid (genotype 40/3) to be definitely developed from microspores. Segregation of the morphological traits of the investigated 1,000 plants which allowed a grouping into 15 distinct types indicates that the major number of the plants are of androgenetic origin. One major factor limiting isozyme analysis is the low number of loci that can be investigated. So far, there are only few reports about molecular investigations verifying the haploid or doubled haploid nature of regenerants. For example, in asparagus, microsporeoriginated plants could be identified by isozyme electrophoresis (Colby and Peirce 1988). Corres-
pondingly, oligo-fingerprinfing was successfully used in sugar beet (Schmidt et al. 1993). However, using interspecific hybrids between H. a n n u u s and /-/2. r e s i n o s u s as donor plants for anther culture the molecular characterization of the regenerated plants with regard to their androgenetic origin will be considerably hampered by the unpredictable genome rearrangements in the meiosis of the Fl-hybrid plants (2n=4x=68).
Acknowledgements We wish to thank Alexandra Runkel for excellent assistance in the isozymeanalysis. In addition we are grateful to Ulrike Ott and Silke Knollmarmfor technical help in cultivation and multiplication of regenerated plants, respectively. This research was supported by grants from the DAAD (German Academic Exchange Service) and the GFP (Association for Promotionof Private GermanPlant Breeding), Bonn.
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Plant Cell Reports (1996) 16:l 6 7 - 1 7 3
© Springer-Verlag 1996
High regeneration rates in anther culture of interspecific sunflower hybrids Tengku Nurhidayah, Renate Horn, Thomas Riicher, and Wolfgang Friedt [nstitut fiir Pflanzenbau und Pflanzenzfichtung I, Justus-Liebig-Universit/it, Ludwigstr. 23, D-35390 Giessen, G e r m a n y Received 3 January 1996/Revised version received 6 April 1996 - C o m m u n i c a t e d by W. Barz
Abstract. Optimization of anther culture with regard to the induction of callus formation and direct embryogenesis was obtained for interspecific hybrids of H. annuus with H. tuberosus, H. laetiflorus, and H. resinosus by investigating six different induction media and four regeneration media. One media combination (MS-I3, MS-R3 and MS-R4) used under different culture conditions (30°C/35°C and different dark treatments) gave up to 92.7% embryogenic anthers with an average of 8.5 embryos per anther. However, direct embryogenesis as well as callus formation showed a strong genotypic and treatment specific reaction. From 5,600 anthers of the four investigated genotypes more than 2,000 plants could be regenerated. Regenerants were characterized by morphological traits and isozyme analyses to prove their androgenetic origin.
Abbreviations: BAP =
6-benzylaminopurine, MNR = menadione reductase, NAA = a-naphthalene acetic acid, PGM = phosphoglucomutase, PGI = phospoglucoisomerase, PVP = polyvinylpyrrolidone, CMS = cytoplasmic male sterile, MS = Murashige & Skoog
Introduction Anther and microspore culture allow the acceleration of breeding programmes by providing homozygous doubled haploids within a comparatively short time. Homozygous lines are required for commercial hybrid production. In addition, doubled haploid populations have proven useful in the development of molecular maps and in tagging important agronomic traits. Application of haploid techniques in breeding programmes affords high regeneration rates. In sunflower, haploids have been obtained via anther culture, but none of the reports so far allowed the regeneration of plants in a number interesting for breeding programmes, and the success of haploid techniques has been strongly dependent on genotype (reviewed in Friedt et al. 1996). Employment of wild species and interspecific hybrids Correspondence to: W. Friedt
allowed the first successful reports of anther culture in sunflower (Mix 1985, Bohorova et al. 1985). Shoot formation was observed for HI divaricatus and H. annuus x I-L decapetalus. For other wild species, e.g. H. tuberosus or H. resinosus, Jonard and Mezzarobba (1990) observed callus formation and embryogenesis which allowed regeneration of plants. From interspecific hybrids involving hr. resinosus, H. rigidus, and H. occidentalis plants were also regenerated from anther culture, although with very low rates in the case of the interspecific hybrids involving the last two species. Furthermore, interspecific hybrids of H. annuus to /42. smithii or H. eggertii allowed the regeneration of plants from anther culture by Nenova et al. (1992). Cultivated sunflower proved to be very recalcitrant in anther culture (Giirel et al. 1991, Mezzarobba and Jonard 1986). More recently, Jonard and Mezzarobba (1990) have reported the induction of callus formation from anthers of cultivated lines but no embryogenesis or organogenesis could be observed, although Mezzarobba (1988) was able to regenerate 91 plants from 2,278 plated anthers using other cultivated sunflower lines. Anther culture in sunflower still needs considerable improvement before it can be routinely included in breeding programmes. We here report on an improved anther culture protocol using interspecific hybrids which allowed the production of a large number of plants from anther culture without any multiplication by subculturing. Using anther culture, it still remains a problem to prove the androgenetic origin of regenerated plants. Jonard and Mezzarobba (1990) assessed the ploidy level of plants regenerated from anthers of different sunflower genotypes. However, as spontaneously doubled haploids appear, cytological investigations do not always offer evidence for androgenesis. In sunflower, isozyme analysis has been successfully used to characterize interspecific hybrids obtained by
168 embryo rescue (Dahlhoff et al. 1992) or somatic hybridization (Barth et al. 1993, Krasnyanski and Menczel 1994). The value of isozyme analysis for the identification of androgenic sunflower plants will be discussed and compared with results obtained by characterizing the plants on the basis of morphological traits.
Materials and Methods Plant material Five genotypes of three interspecific hybrid combinations (Tab. 1) which were obtained earlier by embryo rescue (Kr/iuter et al. 1991) were investigated for their performance in anther culture. Donor plants were grown in the field or in the greenhouse. Tab.l: Interspecific hI ,brids of line HA89(CMS) with three wild species GenoWpe Male parent 40/3 H. resinosus (RES-1545) 30/61 H. laetiflorus (LAET-Hung) 10/25, 10/28 H. tuberosus (TUB-5) 11/51 H. tuberosus (TUB- 1705) Flower buds were surface sterilized by immersion for 30 s in 96% ethanol, followed by a 15 rain incubation in 2% (v/v) commercial bleach. After three washings with sterile distilled water anthers were dissected from the first 3-4 rows of disc fitters. Only anthers with microspores at the early uninueleate stage were used for culture in vitro.
Anther culture Six induction media modified according to Murashige and Skoog (1962) which differed in the concentration of vitamins and hormones (Tab. 2) were investigated with regard to their ability to induce androgenesis. Twenty anthers per Petri dish ( ~ 5 cm) were cultured under permanent light (600 - 800 lux) or in the dark at 30°C for four weeks. Green calli formed during this time were transferred to different regeneration media to induce shoot formation. Calli on MS-I1, MS-I3 and MS-I5 were transferred to MS-R1, while calli from MS-I2, MS-I4 and MS-I6 were put on MS-R2 (Tab. 2). Culture temperature was reduced to 25°C at 16 h light (2,000 lux). Calli showing shoot induction were placed on MSR3 for 3-4 weeks for shoot formation. Single shoots carrying 4-6 leaves were transferred to tubes on MS-R4 medium for root induction. Successfully rooted plantlets were further grown on soil and adapted to greenhouse conditions. For further improvement of anther culture one media combination (MS-I3, MS-R3 and MS-R4) was used and the culture conditions were modified. Anthers were either cultured at 30°C or 35°C with 0, 6, 12 or 18 days of dark treatment on the induction medium MS-I3. Afterwards the Petri dishes were transferred to permanent light (2,000 lux) for the remaining period. In total, all anthers were incubated for four weeks on the induction medium MS-I3. Afterwards, developing embryos were transferred to the regeneration medium MS-R3 and cultured at 25°C and 16 h light (2,000 lux). Shoots carrying 4-6 leaves were transferred to tubes on MS-R4 medium for rooting. Successfully rooted plantlets were adapted in soil to greenhouse conditions.
Isozyme electrophoresis For isozyme analyses about 150 mg of leaf material were taken from each sunflower plant and 100 p.1 extraction buffer according to Soltis et al. (1983) were added. The leaf material was stored frozen at -80°C. For the extraction 200 ixl of modified extraction buffer (containing PVP, ascorbic acid and B-mercaptoethanol) and quartz sand were added. The samples were homogenized and centrifuged at 6,200g for 5 min at 4°C. The clear supematant was used for electrophoresis on 12% (w/v) starch gels. Buffer systems for electrophoresis were modified for PGM and PGI according to Stuber et al. (1977) and for MNR according to Soltis et al. (1983). Staining was performed for MNR (Rieseberg and Soltis 1989), PGI and PGM (Vallejos 1983).
Results Effect of different induction and regeneration media In total, 2,375 anthers were cultured in order to investigate the ability of different induction and
regeneration media (Tab.2) to support androgenesis. For the genotypes 11/51, 30/61 and 10/28 callus induction was obtained on each of the six investigated induction media under permanent light (Tab.3) and dark conditions (data not shown). For the dark treatment no embryogenesis or organogenesis could be induced on the regeneration media. However, under permanent light pronounced effects of medium and genotype were observed after the transfer to a regeneration medium. For genotype 11/51 representing an Fl-hybrid H. annuus x H. tuberosus only green calli transferred from MS-I3 and MS-I5 to MS-R1 showed shoot induction. Upon transfer to the hormone free MS-R3 medium, green vigorous calli gave rise to shoots. Seventy-five (MS-I3 medium) and 85 shoots (MS-I5 medium) could be isolated from this genotype and transferred to the rooting medium MS-R4, respectively. MS-R4 contains only 10% (w/v) sucrose and thiamine HC1 is reduced to 0.1 mg 1-1. About 6070% of the shoots could be rooted. Thirty-seven and 41 plantlets were finally transferred to the greenhouse, respectively. For genotype 30/61 representing an F1hybrid of//. annuus with H. laetiflorus only green calli from MS-I1 showed shoot induction on the regeneration medium MS-R1. A very high number of 187 shoots could be isolated but rooting proved to be very difficult for this genotype. Often multiple shoots developed on the rooting medium or no roots were formed. Therefore, only 96 plantlets could be planted into soil and only 44 plantlets could be finally transferred to the greenhouse. All plantlets that were regenerated in this experiment were developed from calli. The genotype 10/28 representing another F~hybrid (H. annuus x H. tuberosus) did not show any response besides callus formation on each of the six induction media. Direct embryogenesis and callus formation under different culture conditions For further improvement of anther culture the media combination MS-I3, MS-R3 and MS-R4 was applied. The cultivation step on MS-R1 was omitted and the culture conditions were modified. In total, 5,600 anthers were cultured at different dark treatments (0, 6, 12 and 18 days) either at 30°C or 35°C (Tab. 4). In addition to the three genotypes 11/51, 30/61 and 10/25 an interspecific hybrid of H. annuus with tt. resinosus (genotype 40/3) was included in these studies. Direct embryogenesis was observed on MS-I3 medium within 10 to 28 days for genotype 40/3 (Fig. 1A). Up to 92.7% embryogenic anthers were obtained and a mean of 8.5 embryos per anther was observed for this genotype (Tab. 4). Anthers from the interspecific hybrids involving H. tuberosus and [t. laetiflorus also showed direct embryogenesis, although at a much lower rate (Tab. 4). Embryoids were transferred to MS-R3 medium for further development. However, many of the primary embryoids ceased growth at the
169 Table 2: Composition of the different induction and regeneration media Compounds
MS-I1
MS-I2
MS-I3
MS-I4
MS-I5
MS-I6
MS-R1
MS-R2
MS-R3
MS-R4
2.0 100.0 0.5 0.5 0.5 0.01 0.5 0.5 30.0 3.0 5.7
2.0 100.0 0.5 0.5 0.5 0.01 1.0 1,0 30.0 3,0 5,7
2.0 100.0 1.0 1.0 1.0 0.01 0.5 0.5 30.0 3.0 5.7
2.0 100.0 1.0 1.0 1.0 0.01 1.0 1,0 30.0 3.0 5.7
2,0 100.0 1.5 1.5 1.5 0.01 0.5 0.5 30.0 3.0 5.7
2.0 100.0 1.5 1.5 1.5 0.01 1.0 1.0 30.0 3.0 5.7
2.0 100.0 1.0 1.0 1.0
2.0 100.0 1.0 1.0 1.0
2.0 100.0 0.5 0.5 0.5
2.0 100,0 0.5 0.1 0.5
0.5 0.5 30.0 3.5 5.8
1.0 1.0 30.0 3.5 5.8
30.0 4.0 5.8
I0.0 4,0 5.8
(mg 1") glyeine myo-inositol nicotinic acid thiamine-HCl pyridoxine-HC1 biotin BAP NAA sucrose (g 1"1) Gelrite (g 1-1) pH
Table 3: Plant regeneration on different induction media under permanent light Genotype
Media
11/51
MS-I1 MS-I2 MS-I3 MS-I4 MS-I5 MS-I6 Mean MS-I1 MS-I2 MS-I3 MS-I4 MS-I5 MS-I6 Mean MS-I1 MS-I2 MS-I3 MS-I4 MS-I5 MS-I6 Mean
30/61
10/28
NO. of anthers plated 100 100 100 100 100 100 100 60 60 60 60 60 60 60 50 50 50 50 50 50 50
Initiated calli No. 90 75 71 71 75 80 77.0 58 57 59 52 56 59 56.8 36 42 36 48 27 44 38.8
% 90.0 75.0 71.0 71.0 75.0 80.0 77.0 96 95 98 86 93 98 94.3 72 84 72 96 54 88 77.7
cotyledonary stage. After a period of two to four weeks of dormancy, primary embryoids developed into multiple secondary embryos before regeneration of shoots occurred (Fig.lB). For a successful rooting shoots had to be transferred to MS-R4 medium. Culture temperature had a considerable influence on the percentage of embryogenic anthers which could be obtained for the four investigated genotypes (Tab. 4). In general, anthers cultured under 35°C show a better response in vitro regarding direct embryogenesis. For genotype 10/25 embryogenic anthers could only be observed when culW.red at 35°C whereas no embryogenesis occurred at 30°C. The genotypes 11/51 and 30/61 also had a higher percentage of direct embryogenesis at 35°C. However, there was one exception. The genotype 40/3 showing the best overall performance with regard to embryogenesis has a mean value of 67.2% for all treatments at 30°C compared to 12.9% at 35°C (Tab. 4). For this genotype the mean percentage of callus formation is comparably low at
Morphogenic caUi No. % 0 0.0 0 0.0 5 5.0 0 0.0 7 7.0 0 0.0 2.0 2.0 8 13.3 0 0 0 0 0 0 0 0 0 0 1.3 3.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0
No. of isolated shoots 0 0 75 0 85 0 26.7 187 0 0 0 0 0 31.2
Plants transferred to soil 0 0 57 0 59 0 19.3 96 0 0 0 0 0 16.0
No. of vital plants 0 0 37 0 41 0 13.0 44 0 0 0 0 0 7.3
0
0
0
30°C with a value of 25.1%. All other investigated genotypes showed callus formation of about 70% with an average of about 3% of anthers showing direct embryogenesis under these conditions. In general, the rate of callus formation is much higher at 30°C than at 35°C for all investigated genotypes. Dark treatments of 6, 12 and 18 days were applied in order to try to improve response in this way (Tab. 4). For all four genotypes the elongation of the dark period from 0 to 18 days resulted in a considerable increase in anthers forming callus. With regard to direct embryogenesis the genotypes differed in their response to various dark treatments. For genotype 40/3 any dark period led to a remarkable reduction in direct embryogenesis at 30°C as well as at 35°C. Genotype 30/61 also showed this reduction in embryogenesis at 30°C but not at 35°C. However, for the genotypes 11/51 and 10/25 involving H. tuberosus, dark treatments of six days at 30°C or 12 days at 35°C increased the rate of direct embryogenesis to up to 9%. A dark treatment of 18 days had a
170
Fig. 1:
Anther culture of an interspecific hybrid between H. a n n u u s and H. r e s i n o s u s (genotype 40/3). (A) Multiple embryos induced on an anther after 15 days, 30°C, and 03) Shoot development from embryos on MS-R3 after six weeks, 30°C.
Table 4: Reactions of the genotypes 40/3, 11/51, 10/25 and 30/61 to different dark treatment at 30°C and 35°C Genotype
Dark period (days)
40/3
0 6 12 18 Mean 0 6 12 18 Mean 0 6 12 18 Mean 0 6 12 18 Mean
11/51
10/25
30/61
No. of anthers plated
30°C 220 220 220 220 220 360 360 360 360 360 160 160 160 160 160 120 120 120 120 120
35°C 120 120 120 120 120 200 200 200 200 200 120 120 120 120 120 100 100 100 100 100
% of anthers with % of anthers with i % of anthers with direct callus formation root formation embryogenesis 30°C 92.7 69.1 60.9 45.9 67,2 3.9 6.1 5.6 1.4 4.2 0 0 0 0 0 5.8 3.3 1.7 0.0 2.7
35°C 21.7 13.3 10.0 6.7 12.9 4.5 6.0 9.0 5.0 6.1 2.5 2.5 3.3 1.7 2.5 7.0 8.0 5.0 2.0 5.5
negative effect on direct embryogenesis for all genotypes. The hybrid 11/51 differs from the other investigated genotypes by the fact that it showed pronounced root formation from the anthers. At 35°C the formation of roots is reduced in hybrid 11/51 but
30°C 5.9 18.6 34.5 41.2 25.1 49.7 77.2 81.7 89.2 74.4 56.6 63.1 73.1 80.6 68.1 67.5 72.5 81.7 93.3 78.8
35°C 0 0 0 0 0 5.5 9.0 195 28.0 15.5 26.7 29.2 30.8 37.5 30.0 11.0 33.0 35.0 38.0 29.3
30°C 0 0 0 0 0 0.6 2.8 6.7 8.9 4.7 0 0 0 0 0 0.0 0.0 0.8 0.8 1.7
35°C 0 0 0 0 0 0.0 0.0 1.5 1.5 0.8 0 0 0 0 0 0 0 0 0 0
Average number of direct embryos per anther 30°C 35°C 8.5 1.5 2.4 1.0 2.0 1.0 2.0 1.0 3.7 1.0 1.0 1.0 1.5 1.0 1.5 1.0 1.0 1.0 1.3 1.0 0 1.0 0 1.0 0 1.0 0 1.0 0 1.0 1.8 1.0 1.0 1.0 1.0 1.0 0.0 1.0 1,0 1.0
still observed in treatments with 12 and 18 days of darkness. The percentage of root formation observed for genotype 11/51 increased with longer period in the dark.
171 Characterization of regenerants from anther culture by _isozvme electrophoresis The three isozyme systems menadione reductase (MNR), phosphoglucoisomerase (PGI) and phosphoglucomutase (PGM) were analyzed to investigate the origin of the regenerated plants. For the interspecific hybrid of H. a n n u u s and H, r e s i n o s u s (genotype 40/3) 1,211 plants regenerated from anther culture were characterized. The isozyme system menadione reductase proved to be the most informative (Fig. 2A). The parental lines as well as the F:-hybrid showed a clearly different pattern. For the regenerated plants four groups can be distinguished: (1) plantlets that have the same pattern (five bands) as the Fl-hybrid, (2) plantlets that show a single upper band, (3) plantlets that have a single lower band, and (4) a single plant with a pattern of three upper bands. From the investigated 1,211 regenerants eight plantlets showed the upper band and seven plantlets the lower band. The segregation of the pattern indicates that these plants are of androgenetic
origin. For the plants that showed the pattern of the respective F:-hybrid no definitive answer can be given. For PGI, HA89(CMS), the F:-hybrid and all regenerants showed a single band (Fig. 2B). However, RES-1545 has three bands. Also for PGM, only RES1545 showed a divergent pattern (three bands) whereas the female parent, the F:-hybrid and regenerated plantlets have the same pattern (four bands, Fig. 2B). Characterization of the regenerants on the basis of morphological traits Morphological characters such as~ e.g., plant height, anthocyanin colouring or leaf shape (Tab. 5) were evaluated for 1,000 regenerated plants. Fifteen groups distinguishable by morphological parameters could be observed (data not shown). Special attention was paid to the plants that segregated with regard to the pattern of the isozyme MNR as isozyme analysis of these plantlets proved that they represent regenerants of androgenetic origin. The morphological parameters
A:
B"
Fig. 2: Isozyme pattern of the plants regenerated from anther culture compared to the parental lines of H. a n n u u s (HA89) and H. r e s i n o s u s (RES-1545) as well as the Fl-hybrid 40/3. Starch gels were stained for activity of (A) MNR (for each sample duplicates were applied) and 03) PGI and PGM (each lane represents a single plant).
172 Table 5: Morphological traits of the regenerated plants from genotype 40/3 which have been characterized by isozyme electrophoresis Trait
Donor plant
Plant height (cm) Anthocyanin colouring Branching Leaf arrangement Petiole (cm) Leaf length (cm) Leaf width (cm) Angel at leaf top (°) Leaf margin Hairiness of leaves Capitulum (cm) Anthers (mm) Fertility Pollen viability (%) Pollen size (~tm)
100-135 medium fully branched opposite/alternate 3-5 18-25 4-8 30-32 serrated slightly hairy 3-4 4-5 fertile 30-60 30-45
taken (Tab. 5) support this hypothesis as the regenerated plants showing the upper band of MNR have a very similiar habitus and can be clearly distinguished from the group of plants showing the lower band of MNR. Pollen viability did not differ significantly between the two groups.
Discussion Interspecific hybrids have been successfully used in anther culture of sunflower (Alissa et al. 1985, Bohorova et al. 1985, Mix 1985, Jonard and Mezzarobba 1990). However, this is the first report of successful anther culture involving interspecific hybrids of H. annuus with H. tuberosus or H. laetiflorus, respectively. Especially, genotypes 11/51 and 10/28 involving H. tuberosus are of special interest for sunflower breeding programmes, as H. tuberosus is a potential source of resistance, e.g., to Phomopsis helianthi or Sclerotinia sclerotiorum (cited in Atalagic et al. 1993). Interspecific hybrids of H. annuus with H. resinosus have already been reported to have a high regeneration potential in anther culture (Jonard and Mezzaroba 1990). With our best protocol for anther culture 92.7% of the anthers from genotype 40/3 showed direct embryogenesis with an average number of 8.5 embryos per anther. In total, more than 2,000 plants could be regenerated starting with 5,600 anthers. Application of anther culture allowed to create a considerable diversity in the breeding material with regard to morphological traits evaluated. As in other reports (Jonard and Mezzarobba 1990) induction media on MS basis with NAA (0.5 mg 1-1) and BAP (0.5 mg 1-I) gave the best results in our experiments. A higher concentration of these two hormones (1 mg 1"1) gave high percentages of callus induction but after transfer to the regeneration media no organogenesis could be observed. One media
Regenerants with upper band 92-146 light fully branched opposite/alternate 2-5 16-25 4-8 28-32 serrated slight hairy 2-4 4-5 fertile 23-37 30-50
Regenerants with lower band 125-231 intensive fully branched opposite/alternate 3-6 18-32 7-11 30-40 partly serrated slightly hairy 2-5 4-5 semi-sterile 15-35 30-50
combination (MS-I3, MS-R3 and MS-R4) resulted in high rates of embryogenic anthers and callus formation under different culture conditions although it is still dependent on genotype. Cultivation at 35°C had a stimulating effect on direct embryogenesis in the genotypes which showed low rates at 30°C. However, the positive effect of a higher culture temperature is genotype specific and advantage of this culture condition has to be investigated for each new genotype. In the study of Mezzarobba and Jonard (1986) the percentage of embryogenic anthers could be increased for all investigated genotypes by raising the temperature from 25°C to 35°C and an additional pretreatment of 12 days darkness. In our experiments a dark treatment of 12 days was only advantageous to embryogenesis for the two genotypes 11/51 and 10/25 of the interspecific hybrid H. annuus x H. tuberosus. In general, dark treatment seems to increase the number of anthers forming callus but to reduce the percentage of embryogenic anthers. Cold pretreatment was demonstrated to have a very strong effect on embryo induction in the study of Thengane et al. (1994). A combination of our method of plant regeneration from anthers with this cold pretreatment might further improve the percentage of embryogenic anthers, especially from the interspecific hybrids of H. annuus with H. tuberosus or H. laetiflorus.
Microspore culture has the potential advantage that the regenerated plants can be assumed to be of androgenetic origin (cf. Coumans and Zhong 1995), whereas in anther culture plants might have also been regenerated from the diploid cells of the anther wall. In fact, the regeneration of diploid plants from the somatic anther tissue remains a major problem. For example, Zhong et al. (1995) have recently reported the recovery of embryo-derived plants from sunflower anthers, where all
173 plants proved to be diploid (2n =34). Consequently, it is necessary to unequivocally determine the origin of the regenerants. For this purpose, the value of different isozyme systems was investigated. The two isozyme systems PGI and PGM allowed a differentiation of the two parental lines of the investigated FI-hybrid. However, the hybrid and the regenerants had the same pattern as HA89(CMS) which did not allow the detection of an androgenetic origin. Since no divergence from the pattern was observed, somaclonal variation detectable by these isozymes did not occur. Menadione reductase was comparably informative as [t. a n n u u s and H. r e s i n o s u s were distinguishable from each other as well as from the corresponding Fl-hybrid. However, the hybrid did not have an additive pattern of the parental lines alone, instead new bands appeared. For the regenerants having the same pattern as the F1hybrid no information on their origin has been obtained so far. However, the fifteen regenerated plants of genotype 40/3 which segregated into two groups, one with the upper band and one with the lower band, are likely to be of androgenetic origin. The pattern obtained for these regenerants is very simple compared to the pattern of the Fl-hybrid which can be regarded as another hint for their homozygosity. The single regenerant showing a totally divergent pattern might be a case of somaclonal variation or a different segregation pattern. For the investigated regenerants of genotype 40/3 a prediction of the results is complicated because this genotype represents an F~-hybrid between the diploid H. a n n u u s (2n=2x=34) and the hexaploid wild species H. resinosus (2n=6x=102). Alissa et al. (1985) cytologically investigated plants regenerated from anthers of the interspecific hybrid H. a n n u u s x H. r e s i n o s u s . Of the investigated 64 plants 47 were aneuploid, only 7 were haploid, and 10 diploid. Therefore, only the observed segregation of the isozyme pattern observed in our regenerants proves their androgenetic origin. An aneuploid plant analyzed by isozymes can show the same pattern as the Fl-hybrid but can nevertheless be of androgenetic origin. However, isozyme analyses only allowed the identification of little over 1% of the plants regenerated from anther culture of the interspecific hybrid (genotype 40/3) to be definitely developed from microspores. Segregation of the morphological traits of the investigated 1,000 plants which allowed a grouping into 15 distinct types indicates that the major number of the plants are of androgenetic origin. One major factor limiting isozyme analysis is the low number of loci that can be investigated. So far, there are only few reports about molecular investigations verifying the haploid or doubled haploid nature of regenerants. For example, in asparagus, microsporeoriginated plants could be identified by isozyme electrophoresis (Colby and Peirce 1988). Corres-
pondingly, oligo-fingerprinfing was successfully used in sugar beet (Schmidt et al. 1993). However, using interspecific hybrids between H. a n n u u s and /-/2. r e s i n o s u s as donor plants for anther culture the molecular characterization of the regenerated plants with regard to their androgenetic origin will be considerably hampered by the unpredictable genome rearrangements in the meiosis of the Fl-hybrid plants (2n=4x=68).
Acknowledgements We wish to thank Alexandra Runkel for excellent assistance in the isozymeanalysis. In addition we are grateful to Ulrike Ott and Silke Knollmarmfor technical help in cultivation and multiplication of regenerated plants, respectively. This research was supported by grants from the DAAD (German Academic Exchange Service) and the GFP (Association for Promotionof Private GermanPlant Breeding), Bonn.
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