Plant Cell Reports (1985) 4:307-310 Plant Cell Reports © Springer-Verlag 1985

Effect of the 1B/1R translocation on anther culture ability in wheat ( Triticum aestivum L.)

Y. Henry and J. de Buyser

A.D.A.R., Laboratoire d'Am61ioration des plantes, Universit6 Paris-Sud, F-91405 Orsay, France

Received June 26, 1985 / Revised version received September 27, 1985 - Communicated by A. M. Boudet

ABSTRACT Using two varieties, their reciprocal hybrids,F 8 lines and doubled haploids, results confirmed that three genetic components are involved in wheat anther culture ability, viz embryo induction frequency, regeneration ability and the frequency of albinism. In these experiments, no significant maternal effects were noticed. For embryo yields, transgressive lines were obtained from hybrids between distant genotypes. Regeneration of green plants depended upon two inde- pendent traits: regeneration ability and the frequency of albinos. F 8 lines and two doubled haploids equaled the 50% regeneration rate of the hybrids, but they on- ly regenerated green plants. Based upon cytological em amination and gliadin patterns, it is suggested that genes favoring regeneration ability could be linked to the IBL-IRS translocated chromosome from Aurora.

INTRODUCTION Marked effects of genotype on anther culture response have been observed in a number of cereals, including rice (Oone 1975), barley (Foroughi-Wehr et al. 1976, 1982), rye (Wenzel et al. 1977), wheat (De Buyser and Henry 1979) and Triticale (Charmet and Bernard 1984). Furthermore, significant genotype effects and genotype x environment interactions have been reported (Lazar et al. 1984a - Charmet and Bernard 1984). A particular feature concerned the frequency of albinos : 20 to 60% in rice (Oono 1975), 20 to 40% in wheat (De Buyser and Henry 1979), 75% in rye (Wenzel et al. 1977), 70 to 80Z in Triticale (Charmer and Bernard 1984) and 30 to 98% in barley (Foroughi- Wehr et al. 1976, 1982). In the case of rice (Oone 1975) and wheat (De Buyser and Henry, unpublished results) the appearance of albinos gradually decreased over a series of selfing generations. These facts seem to indicate that albinism is at least partly under ge- netic control. In order to investigate further the genetic contribu- tion to anther culture response in wheat, we compared two parental strains, their two reciprocal hybrids, two F 8 breeding lines and four doubled haploids extra~ ted from four F 8 lines. Comparisons were made for formation of multicellular pollen grains, embryo yields, regeneration ability, IB chromosome structure and gliadin patterns. The variety Aurora possesses a IB/IR translocation (Mettin et al. 1976) in which the short arm of IB is replaced by the short arm of IR chromosome from rye. Genes controlling gliadin production are located on I BS and 1RS chromosome arms (Miller 1984).

Offprint requests to." Y. Henry

MATERIAL AND METHODS Plant material (Triticum aestivum L.) was developed as described in Figure i. The variety Talent (T) and the B3, B4, B5 and B6 (H 77022) F 8 pedigree lines were provided by the C.C Benoist Company, and the variety Aurora (A) by the INRA wheat laboratory at Versailles. Four doubled haploids (DH) were randomly chosen from 40 obtained from B3, 24 from B4, 75 from B5 and 90 from B6.

Fig.l : Origin of the Triticum aestivum L. genotypes

Varieties Talent (T) Aurora (A) I

F1 T ~ A ~ AxT I

F6 family B 3~56 / \

F7 family B 34 family B 156 I t I

F8 B3 B4 B5 B6 I I J (-anther culture

DHB3 DHB4 DHB5 DHB6

At mitosis in the variety Aurora, only one pair of satellited chromosomes (6B) was seen, instead of the two pairs in normal wheat. In rye the I R chromo- some appeared satellited, with a marked secondary constriction on its short arm, but in a wheat back- ground the appearence of the constriction is suppres- sed (Miller 1984). Plants were grown and anthers cultured as described previously (Henry et al. 1984). The anther culture medium contained 0.2 g/l glutamine, 70 ml/l potato extract and was gelled with 6 g/l agarose (Sigma type I, low EEO). The regeneration medium was MS medium developed by He and Ouyang (1984), gelled with 6 g/l agarose. Only a proportion of the embryos obtained were cultured to determine the regeneration rate. After 13 days culture, i00 anthers (i0 per spike) were randomly removed from i0 spikes each of T, TxA, AxT, A, DHB5, B6 and DHB6 genotypes and fixed in acetic alcohol (1:3). Pollen grains were stained with acetic carmine. 200 pollen grains from each anther were observed in order to determine the multicellular pollen grains (MPG) frequency. For chromosomal counts, root tips were treated by the Feulgen technique, and squashed in acetic carmine. Polyacrylamide gels were run to examine the gliadin composition (Branlard 1983).

RESULTS Large differences in response between batches of anthers obtained from a single genotype have been

308

previously observed (De Buyser and Henry 1979). An experiment was performed in order to illustrate this problem, thirty-two spikes from one genotype were divided randomly into two lots of 16, called X and Y. From these 32 spikes, a total of 2000 anthers were plated. After 45 days culture, the X spikes produced 5 embryos and the Y spikes 14. The propor- tions were significantly different at the 1% level. In order to reduce this "spike effect" (De Buyser and Henry 1979), we decided to plate, for each geno- type, except B5 line, the anthers from a minimum of 40 spikes. After 13 days culture, the induction rate was similar for all genotypes tested (Tablel). Several exceptional anthers were observed in all genotypes, corresponding to a high embryogenic initiation rate reaching 32Z of the pollen grains. So the potential of anther cultu- re response was high with an average of 9 MPG with more than i0 nuclei per anther. In these MPG, many mitoses were observed. The frequency of diploid MPG (2n=42), 26,5% was identical to the frequency of spontaneous diploid regenerated plants, showing that the diploid state did not confer any selective advan- tage. Abnormalities leading to micronuclei production were observed and these could be the related to some abor- tion.

Table 1 : Induction of multicellular pollen grains (MPO)

Genotype T TxA AxT A DHB5 B5 DHB6

Induction rate (a) 84 !04 75 81 102 !!i 68

(a) : induction rate is the number of MPG containing more than i0 nuclei. The mean is 89.3, from 20000 pollen grains observed for each genotype (confidence limits 95Z : 72-111, 99% : 68-118).

Embryo yields are shown in Table 2. Parental strains A and T were the least productive (2,3%) for embryos.

Reciprocal hybrids (5,5%) were identical and better than their parents. More interesting were the B5 and B6 breeding lines (17,5%), which consistently out-yielded the hybrids. There was no correlation bet- ween the number of MPG with more than i0 nuclei and the yield of embryos.

Data on regeneration of plants from the embryos are shown in Table 3. For total plant regeneration (green + albino), the DHB3, BS, DHB5 and B6 genotypes equal- led the two hybrids and were better than the parents. These lines had retained the good regeneration ability of the hybrids. DHB4 equalled the parental varieties, and DHB6 was inferior to B6. For green plant regeneration, the hybrids and DHB4 equalled the A parent and were significantly better than the T parent. DHB3, B5, DHB5 and B6 lines were significantly better than the hybrids. DHB6 was infe- rior to the B6 line. For albino regenerat~n ability, A, DHB3, DHB4, BS, DHB5 and B6 produced significantly less albinos than T, TxA and AxT. The hybrids resembled the T parent in producing the highest albino frequency, and the DHB3, DHB4, B5, DHB5 and B6 lines performed as well as A. The yield of green plants per I00 cultured anthers are summarized on Figure 2. The hybrids and DHB4 exceeded the parents, giving more than 1 green plant from i00 anthers. Highest yields were obtained with DHB5, B5 and B6 which yielded about 8 green plants from I00 anthers.

Cytological examination revealed those wheat lines which inherited the IB/IR translocation from Aurora.

The variety Talent and DHB4 had 4 satellited chromo- somes (IB and 6B pairs). Aurora, which possessed a IB/IR transloeation (Mettin et al. 1976) had two satellited chromosomes as did B3, B5, B6, DHB3, DHB5 and DHB6 (Table 4). Three satellited chromosomes were observed in mitosis of the two hybrids. The F 8 line B4 was heterogeneous for this trait and can have 2, 3 or 4 satellited chromosomes.

Fi@. 2 : Green plant production

i0

green plants per i00 cultivated anthers

T TxA AxT

<

V] A DHB3 DHB4 B5 DHB5 B6 DHB6

The patterns of gliadins, especially the omega glia- dins, were also used to determine those lines having the IB/IR translocation. The IRS chromosome arm of rye carries genes for secalins (Miller 1984). Talent and Aurora were different in their electrophoretic patterns (Figure 3) and reciprocal hybrids were similar.

Fi@. 3 : Electrophoretic patterns of gliadins

From left to right : Talent (T), Aurora (A), TxA, AxT, B3, DHB3, B4, DHB4, B5, DHB5, B6, DHB6. + : seeds possessing a IBL-IRS translocated chromosome.

The most important point concerned the omega gliadins. A, TxA, AxT, BS, DHB3, B5, DHB5, B6 and DHB6 genotypes possessed the IB/IR translocation : their omega glia- din patterns were similar. B4 and DHB4 resembled Talent.

Table 2 : Embryos yields

309

Genotype T TxA AxT A DHB3 DHB4 B5 DHB5 B6 DHB6

Cultivated spikes 50 44 42 40 50 40 12 42 49 52

Embrogenic spikes 28 38 32 19 45 36 12 38 42 34

% 56.0 86.4 76.2 43.2 90.0 90.0 i00 90.5 85.7 65.4

Cultivated anthers 2690 2450 2240 2090 3310 2380 780 2410 2880 2880

Embryogenic anthers 42 91 63 36 171 134 47 154 169 75

% 1.6 3.7 2.8 1.7 5.2 5.6 6.0 6.4 5.9 2.6

Embryos 65 141 114 48 371 263 138 384 497 127

% (i) 2.4a 5.8c 5.1be 2.3a ll.2d ll. Od 17.7e 15.6e 17.3e 4.4b

( 1 ) : number of embryos per 100 anthers 2

Entries followed by the same letter are not significantly different (X).

Table 3 : Regeneration ability

Genotype T TxA AxT A DHB3 DHB4 B5 DHB5

Cultured embryos 64 115 82 48 278 206 47 298

Total regenerated plants 16 60 43 12 153 54 23 167

% (i) 25.0a 52.2c 52.4c 25.0a 55.0c 26.2a 48.9bc 56.0c

Regenerated green plants 2 37 21 Ii 144 41 21 144

% 3.1a 32.2c 25.6bc 22.9bc 51.8d 19.9b 44.7d 48.3d

Regenerated albinos plants 14 23 22 1 9 13 2 23

20.Ob 26.8b 2.1a 3.2a 6.3a 4.3a 7.7a % 21.9b

B6

415

215

51.8e

183

44.1d

32

7.7a

DHB6

85

3O

35.3ab

16

18.8b

14

16.5b

(i) % of embryos giving plants Entries within lines followed by the same letter are not significantly different

Table 4 : Presence of IB chromosomes

Genotype T TxA A B3 DHB3 B4 DHB4 B5 DHB5

Number of satellited chromosomes 4 3 2 2 2 2-3-4 4 2 2

Number of IB satellited chromosomes 2 1 0 0 0 0-1-2 2 0 0

B6 DHB6

2 2

0 0

Study of the gliadin patterns showed that B3, DHB3, B5, B6 and DHB6 were identical, but each of T, TxA (or AxT), B4, DHB4 and DHB5 patterns was distinct. Doubled haploid patterns were identical to the star- ting F 8 lines, in the case of DHB3 and DHB6, or diffe- rent in the case of DHB4 and DHB5. Although B6 and DHB6 had the same gliadin patterns, they showed different behaviour for anther culture ability. The doubled haploid line was less productive than the parental line. In the case of B5 and DHB5, which had slightly different gliadin patterns,anther culture yields were similar. F 8 parental lines B5 and B6 were used in our work since they were unstable in the field, and DHB6 had kept this unstability.

DISCUSSION - CONCLUSION Haploid plant production from anther culture seems to be controlled by at least three different and independently inherited traits. Embryo induction rate and regeneration ability have been previously emphasized (Raquin 1982 - Foroughi-Wehr et al 1982). The third factor for cereals is the ratio of green to albino plants. In our study, whp~t anther culture ability depended on nuclear genes and no significant maternal effects were acting during the androgenetic process, in agreement with results with barley (Foroughi-Wehr et al. 1982).

The embryo induction rate reflected the ability of genotypes to produce multicellular pollen grains

(MPG) and embryos from microspores. Genotypic diffe- rences obtained during.wheat anther cultures are not related to MPG induction but depend upon the different abortion rates of the MPGa (Henry et al. 1984). From parental strains giving low embryo yields we have obtained hybrids with improved embryogenic potential.

Embryo yield is known to increase with the heterozy- gosity of the anther donor plant (Oono 1975 - Raquin 1982), but our results show that transgressive lines can be extracted from hybrids between distant genotypes.

The regeneration ability also depended on the genotyp~ This trait is linked to embryo q~ality (De Buyser and Henry 1979 - Raquin 1982). In our experiment, the ability of embryos to regenerate plants was inhe- rited from both parents. The reciprocal hybrids were the sum of the two parents Talent and Aurora and no line or DH was superior to these hybrids.

Yet in the best lines, the frequency of grin or albino plants was not the same as from the hybrids. These latter gave half green plants and half albino plants but B5, B6, DHB3 and DHB5 gave mainly green plants. So we can conclude that there are two independent traits : one for the ability to regenerate plants and the other that determines the proportion of green and albino plants. It is possible to have lines which present a good ability for plant regeneration and which produce mostly green plants. Talent and Aurora

310

possess two different genetic systems for regenera- tion ability. So hybrids contain the two systems as do the best lines DHB3, B5, DHB5 and B6. These genes could act on the quality of the embryos at the sporophytic level.

Another point which can be discussed conc@rns the IB chromosome. In our experiments with wheat breeding material we have noticed that varieties such as Clement, which we have involved in some crosses and which carry a IBL-IRS translocated chromosome, give a good androgenetic response, in particular a high regeneration rate. Aurora also possesses the IB/IR translocation. We have observed that our best lines DHB3, B5, DHB5 and B6 possess the 1B/IR trans- located chromosome and a high regeneration rate. DHB4 possessing the Talent satellited IB chromosome gave a reduced regeneration ability compared to DHB3, but DHB3 and DHB4 were extracted from the sister families B3 and B4 separated in F7. So we propose that one of the genetic systems involved in regene- ration ability is located on the IRS chromosome arm. It does not affect the frequency of green plants because DHB4, which p,~ssessed the IB chromosome from Talent, also regenerated predominantly green plants. Results from the electrophoretic patterns of gliadins support this conclusion, because of the lack of rye bands in the omega gliadins in DHB4.

The short arm of IB and 1R chromosomes is known to carry a nucleolar organizer region (Snape et al. 1985). The 1B/1R translocation can also induce spon- taneous haploid plants in a particular cytoplasm, this technique being known as the Salmon method (Kob~ yashi and Tsunewaki 1978). Perhaps it is the same IRS genes which were acting on the regulation of the mitotic processes involved in normal and micro - spore embryogenesis.

So androgenetic ability clearly depends upon nuclear genes but environmental factors are known to modify their regulation. Recent studies have concentrated on genetic rather than on physiological strategy. Estimates of quantitative inheritance parameters have been established : most of the genotypic varian- ce is due to general combining ability, heritability is important (Lazar et al. 1984b Charmet and Bernard 1984). These genetical conclusions contribute to a better understanding of the androgenetic process but are not very useful for plant breeders. Crosses to improve androgenetic ability would limit the gene

pool and reduce the time saving effect of doubled haploids. Although our results indicate that androge- netic ability depends upon nuclear genes, the most effective method of improving anther culture response lies in the control of physiological parameters. Only when we are able to control such parameters more precisely will we be able to understand why the best DHB3 anther was able to give 16 embryos which regenerated 16 green plants.

ACKNOWLEDGEMENTS We thank Dr R.L. LYNE for helpful comments and impro- vements of the English text.

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ABBREVIATIONS

DH, doubled haploids; MS, Murashige and Skoog; MPG, multicellular pollen grains.