University of Illinois
Embryolike structure formation and subsequent callus induction from anther cultures of several genotypes
--Vincent Beaumont, Pierre Dieu and Jack Widholm
The anther culture ability of 12 genotypes of different origins was evaluated. The first group (6 genotypes) had previously been evaluated by Dieu, P and Beckert (Maydica 31:246-259, 1986) (RC, progeny from RC1XRC20 and NS) and FR16, Pa91XFR16, H99XFR16, H99XPa91 which had been cultured by Petolino and Jones (Crop Sci. 26:1072-1074, 1986). The second group came from crosses between RC20, RC1 and genotypes which are presently being used for breeding purposes by Rustica Semences, France (Table 1).
The material was derived from field-grown plants during the summer of 1990 in Urbana, Illinois. Tassels with uninucleate microspores were harvested in the morning, wrapped in moist paper towels and aluminium foil and cold-treated at 8 C. After 14 days, tassels were sterilized with a 0.5% sodium hypochlorite solution and 30 anthers were plated in 20x60mm dishes containing 10ml of medium. The medium was YP salts and vitamins with the addition of 500mg/l enzymatic casein hydrolysate, 0.1mg/l TIBA (2,3,5-triiodobenzoic acid), 60g/l sucrose and 5g/l activated charcoal which was filtered after autoclaving. The pH was set at 5.8 before autoclaving.
Table 1. Origin of the genotypes tested.
|Code||Progenitors||Origin of the parents|
|S1||RC20 x 6402 Sib1 Self1||640: group Mo17 and Oh43|
|S4||RC20 x 90002 Sib1 Self1||9000: group lodent|
|S9||(RC20 x 3454)x M7||3454: group Oh43 and Mo17|
|S10||(RC20 x 9000)x 3546||3546: group B73|
|S11||[RC20 x(RC525 x Co158)] x 710||710: group B14 and Oh43|
|S13||(RCC1 x 3454)x 3461||3461: group Mo17|
The dishes were kept in the dark at 28 C. After 3 weeks, the anthers started to produce some embryos. Embryos 2mm and larger were transferred onto D medium (Duncan et al., Planta 165:322-332, 1985) in order to produce haploid regenerable calli lines.
Despite the high number of anthers plated, the variability of the results is very high (Table 2). Thus, embryo induction appears to be very dependent upon the conditions of the donor-plant. The yield in embryos is around 1.4 per 100 anthers, which matches with the results from other authors (Dieu and Beckert, 1986; Petolino and Jones, 1986).
Table 2. Embryos and regenerable calli production from 12 genotypes.
|Genotype||No. tassels tested||No. anthers plated||No. embryos produced||Embryos/100 anthers||Regenerable calli produced|
|S1||14||1170||21||1.00 ± 3.10||1|
|S9||32||2760||61||2.20 ± 2.00||4|
|S11||28||2430||30||2.23 ± 2.00||6|
|S13||34||2940||36||1.23 ± 1.07||2|
|RC||15||1290||41||3.17 ± 2.23||0|
|PF||10||900||17||1.90 ± 1.63||8|
|HF||24||1980||35||1.73 ± 1.17||0|
|Fr16||17||1470||18||1.23 ± 0.70||0|
Twenty-one callus lines which appeared to be regenerable were obtained from the 259 embryos produced. Three of the genotypes (RC, FR16 and H99xFR16) which produced a high number of embryos, did not produce any regenerable calli. Other results in this laboratory show that FR16 is very difficult to obtain regenerable callus from but H99xFR16 usually responds well.
Four of the genotypes in the second group gave some results, both in
embryo induction and regenerable calli production. These results suggest
that it should be possible to introduce anther culture ability into populations
used for breeding purposes.
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