wide range of genetic variation was studied in a randomized block design with three replications. Each replication consisted of 2 pots with 3 plants. The day ...
Euphytica 65: 81-85, 1993. (~ 1993 Kluwer Academic Publishers. Printed in the Netherlands.
Influence of genotype and culture conditions on the production of embryos from anthers of tetraploid wheat (Triticum turgidum) M. Ghaemi, A. Sarrafi & G. Alibert
Laboratoire de Biotechnologie et Am~lioration des Plantes (BAP) 1NP-ENSAT145, Avenue de Muret 31076, Toulouse, France Received6 March 1992, accepted9 November1992
Key words: androgenesis, embryo formation, genetic effect, tetraploid wheat, Triticum turgidum Summary
Anther culture of 10 tetraploid wheat (Triticum turgidum) genotypes and two backcross lines representing a wide range of genetic variation was studied in a randomized block design with three replications. Each replication consisted of 2 pots with 3 plants. The day length was 16h and temperature 25°C/15°C for day/night in a controlled greenhouse where the anther donor plants were grown. Two different treatments were used for anther culture. The first one was potato 2 medium (Chuang et al., 1978) modified by adding 0.5 mg/1 glutamine and solidified by gelrite (4 g/l) (Henry & De Buyser, 1981). Cultures were incubated in light (15/x E m -2 S -1) at 26°C at 16 h day length. The second medium was described by Fadel & Wenzel (1990), differing from the first by the nature of the sugar (maltose) and consistency of the medium (semiliquid by ficoll). Anther cultures were incubated in the dark at 28 ° C. The study of about 1300 anthers per genotype and treatment showed that both genotype and treatment affected embryo formation of tetraploid wheat. The backcross lines exhibited significant differences for androgenic abilities when compared to their common parent. Most of the genotypes were medium dependent for androgenesis and revealed significant interactions with the two treatments. Five green plantlets were regeneratedand fertile doubled haploid plants were obtained from three out of the 12 studied genotypes.
Introduction
Haploids derived from microspores by anther culture have considerable potential as breeding material in crop improvement programs because of the time saved by the reduction of the classical selection cycle period and the genetic value of highly homozygous doubled haploids obtained. The practical application of doubled haploid breeding has been adequately demonstrated in tobacco (Deaton et al., 1982), potato (Mendiburu et al., 1974) and barley (Kasha & Reinbergs, 1980; Sarrafi et al., 1986). In other species, such as hexaploid wheat (Triticum aestivum) androgenesis is not yet widely
used in breeding because of its relatively low yield of haploid plants (Picard et al., 1978). Two lines of investigation have been pursued to improve the success of this method in hexaploid wheats.
Cultural conditions. Progress has been made by using cold pretreatment of anthers (Picard & De Buyser, 1975), ficoll medium (Kao, 1981; Zhou & Konzak, 1989), and a maltose medium (Hunter, 1987). The effect of temperature (Ouyang et al., 1983) and light (Bernard, 1980) was also determined. Genetic control. Considerable variation has been
82 SAT 508 - Opale' and 'Opale - ENSAT 508' resulted from 8 back crosses of 'ENSAT 508 x Opale' and 'Opale x ENSAT 508' with the two parents, Opale and ENSAT-508, in our department. Seeds of all genotypes were vernalized for six weeks at 2 ° C + 1 in a refrigerator, prior to planting in pots of 18 cm diameter in a controlled greenhouse. The experimental design was a randomized block with 3 replications. Each replication consisted of 2 pots with 3 plants. The day length was 16 h and temperature 25 ° C/15°C (day/night). Lighting was provided by H L R G 400 W lamps and natural light. The light intensity on the greenhouse bed at pot level ranged from 130 to 300/z Em-2S -1. The primary tiller and two secondary tillers from each plant were used for anther culture. The tillers were collected when most of the microspores were at the uninuclear stage, wrapped with aluminium foil to maintain high humidity and placed in flasks containing water at 4 ° C + i for one week in a refrigerator. Before anther culture, spikes were surface sterilized for 7 rain in 20% (v/v) sodium hypochlorite and washed three times with sterile distilled water. Two treatments were used. The first one was using potato 2 medium (Chuang et al., 1978) modified by adding 0.5 mg/l of glutamine (Henry & De Buyser, 1981) and solidified by gelrite (4 g/l). Cultures were then incubated in light (15/x E m -2 S -1) at 26°C at 16h day length. In the second treatment, the medium of Fadel & Wenzel (1990) was used differing from the first by the nature of the sugar (maltose) and the consistency of the medium
identified among hexaploid wheat genotypes for anther culture response (Bullock et al., 1982; Lazar et al., 1984; Liang et al., 1982; Sheaffer et al., 1979). In contrast to hexaploid wheat, relatively little research has been done using tetraploid wheat (T. turgidum) for androgenesis. As far as we know, the few investigations done with tetraploid wheats have shown that this species is recalcitrant because of the low frequency of embryo production and the low yield of regenerated plants, which are always albinos (Zhu et al., 1979; Hadwieger & HeberleBors, 1986; Foroughi-Wehr & Zeller, 1990). The present investigation was undertaken to determine the influence of genotype on anther culture response using different genotypes and backcross lines of tetraploid wheat (T. turgidum). The effect of two treatments was also studied.
Materials and methods
The experiment was carried out with 12 genetically diverse tetraploid wheat genotypes. ENSAT 508 is an Ethiopian line with a high grain protein content (Sarrafi et al., 1989). ENSAT 1, ENSAT 2, ENSAT 3 and Opale are pure high yielding lines selected in our Department. Primadur and Mondur are French commercial cultivars. Omrabi, Hourani and Cham are drought-resistant cultivars obtained through the International Center for Agricultural Research in Dry Areas (ICARDA). Finally 'EN-
Table 1. Values of mean squares in the analysis of variance of embryogenic ability in tetraploid wheats
Source of variation
ES
EA
E
E/EA
Genotype (G) Protocol(P) Genotype × Protocol Block Error
405.39*** 804.14"** 279.90*** 39.94 ns 62.59
471.08"** 3875.42*** 585.43*** 71.75 Ns 84.68
917.53"** 4365.90*** 611.11"** 76.44 Ns 63.14
1682.75"** 943.26*** 1915.24"** 119.01Ns 200
ES: Embryogenic spikes per 100 spikes. EA: Embryogenic anthers per 100 spikes. E: Embryos per 100 spikes. E/EA: Embryos per 100 embryogenic anthers. ***: Significant at p = 0.001 level. r~s: Not significant.
83 (semiliquid through ficoll). Cultures were incubated in the dark at 28 ° C. In both treatments approximately 48 anthers from each spike were aseptically plated on the medium. Approximately, 1300 anthers per genotype and per treatment were studied. The number of embryos was recorded 30 to 40 days after culture. Then they were transfered to 'R8' regeneration medium (Henry & De Buyser, 1980) and put in a culture room with a photoperiod of 14 h and a temperature of 26 ° C + 1. Five green plantiers were obtained and treated with a solution of 1 g/l colchicine containing 2% DMSO (dimetyl sulfoxide) for 5 h at 20 ° C + 1° C (Arabi et al., 1991). Fertile doubled haploid plants were grown in the greenhouse. Statistical analysis was carried out in order to determine the main effects of genotype, culture treatment and their interactions. The Newman-Keuls test was used for comparing means for the following traits: - Embryogenic spikes expressed as the number of spikes giving at least one embryo, per 100 spikes (ES). - Embryogenic anthers expressed as the number of anthers giving embryos per 100 spikes (EA).
Embryo production rate expressed as the number of embryos obtained per 100 spikes (E). - Embryos produced per embryogenic anther (anthers giving at least one embryo) expressed as the number of embryos, per 100 embryogenic anthers (E/EA). In order to normalize the distributions, all data were transformed by the Arc s i n V ~ function before statistical analyses (Rasch et al., 1978).
-
Results
Results of the analyses of variance for all traits studied are summarized in Table 1. There are highly significant differences between all of the genotypes, treatments, and their interactions for all characters studied. As the interaction between genotype and treatment is statistically significant, the comparison of treatment means across genotypes and genotype means across treatments may not be appropriate. The relative response frequencies of genotypes for the two treatments, as summarized in Table 2, show that for the first treatment: ENSAT 508 and Opale are significantly different in
Table 2. M e a n p erformance of genotype and t r e a t m e n t for embryogenic ability in tetraploid wheats Genotype
ES
EA
E/EA
E
CP1
CP2
CP1
CP2
CP1
CP2
CP1
CP2
E N S A T 508 Opale E N S A T 508 - Op ale
24.73 ab 15.86 a~ 19.47 abe
0 12.89 ~b~ 15.86 a~
41.75 ab 18.75 b~a 32.88 abe
0 12.98 ~a 15.86 b°a
39.49 bc 16.59 a~f 29.15 b~a
0 10.33 d~f 12.57 aer
46.06 ab~a 26.75 ~a~ 37.44 °a°
0 23.51 ~a~ 23.51 cae
Opale - E N S A T 508
22.35 abe
ENSAT 1 ENSAT 2
35.06 a 0
27.62 ab 6.49 b~
50.42 a 0
25.24 ~d 6.49 °a
ENSAT 3 Primadur
30.50 a 22.35 "b~
22.35 abe 0
30.50 ~bc 30.50 abe
22.35 b~a 0
Omrabi Hourani
27.62 ~b 15.86 "b~
22.35 abe 19.47 abe
32.88 ~ 21.13 b°a
22.35 b~a 19.47 b~a
Cham
15.86 ab~
Mondur
0
0
0 22.35 a~
41.55 ab
25.69 b~a 0
0
0 25.24 b~a
42.23 b "69.23 a 0
0
50.90 ab~a
0
32.19 b°a 5.17 ~f
59.73 abe 0
70.21 ab 11.75 ae
39.40 ~ 29.15 b~
17.74 a~f 0
62.38 abe 45.00 abca
35.26 b ~ 0
31.8 b~a 19.96 °~f
19.53 a~f 19.97 ~a~f
43.94 "~a 28.94 ~d~
38.51 b°a~ 48.28 abed
12.57 a~f 0
0 25.79 ~a
14.97 a~ 0
Means (Arc sin V ~ ) followed by different letters are significantly different at p = 0.05 level (Newman-Keuls test). ES: E m b r y o g e n i c spikes per 100 spikes. E A : E m b r y o g e n i c anthers per 100 spikes. E: E m b r y o s p er i00 spikes. E / E A : E m b r y o s p e r 100 embryogenie anthers. CP1 and CP2: T r e a t m e n t N ° 1 and 2 (see Materials and methods).
0 78.24 a
84 their in vitro androgenic capacity. ENSAT 508 has a much better androgenic ability when compared with Opale. The comparison of backcross line 'ENSAT 508 - Opale' and Opale showed that 'ENSAT 508 - Opale', which is supposed to have the cytoplasm of ENSAT 508, is more efficient for rate of embryogenic anthers (EA) and embryo production (E) (32.88 and 29.15) than Opale (18.75 and 16.59), probably because of the favorable action of the ENSAT 508 cytoplasm. Not only the cytoplasm of ENSAT 508, but also its nucleus exerts great influence when it is combined with Opale cytoplasm. All traits studied are superior in 'Opale - ENSAT 508', which is supposed to have Opale cytoplasm and the ENSAT 508 nucleus, when compared with Opale. The ENSAT 508 nuclear effect is very important for the rate of embryogenic anther and embryos production (EA), where values are 41.75 for ENSAT 508 and 41.55 for 'Opale - ENSAT 508'. Anyhow, it must be mentioned that the effect of any residual nuclear differences cannot be separated from possible cytoplasmic effects in the backcross lines. Treatment means can also be compared for the same genotype. Treatment n ° 1 is usually more efficient than treatment n ° 2 for the backcross lines and all genotypes, except for ENSAT 2, and for the cultivar Mondur. These genotypes produce embryos only with the second treatment. The first treatment usually gives better resuits than the second one for all genotypes and for all traits studied.
and Sagi & Barnabas (1989). The backcross lines used in our study are good examples of favorable combinations of nucleus and cytoplasm in tetraploid wheats. The residual nuclear effect which cannot be separated from cytoplasmic effect could also be the possible origin of these differences. Some genotypes, like ENSAT 508, 'Opale - ENSAT 508', Primadur and Cham, did not produce any embryos using the second treatment. It seems that they are highly medium dependent and reveal significant interactions between genotype and anther donor environment, as has been shown by Lazar et al. (1990) in hexaploid wheats. However, the second treatment was more efficient for the production of embryos from ENSAT 2 and Mondur (Table 2). In contrast to previous work in hexaploid wheat (Fadel, 1990), the second treatment was less efficient for its androgenic abilities in the tested genotypes. The best genotype of the 12 tested was ENSAT 1 which gives the highest number of embryos by both culture treatments. Finally, we may conclude that the production of haploid embryos in tetraploid wheats depends on the genotype as well as on treatment. Five green plants were regenerated and fertile doubled haploids were obtained in the two backcross lines ENSAT 508, 'Opale - ENSAT 508' and in ENSAT 3 but low frequencies of embryo development and the production of albino plants remain problems to be solved.
Acknowledgements Discussion In our experiment, both genotype and treatment affect embryo formation in anther culture of tetraploid wheat (T. turgidurn) (Table 2). Such effects have already been demonstrated in hexaploid wheats (Bullock et al., 1982; Lazar et al., 1984; Picard & De Buyser, 1977). Both backcross lines, 'ENSAT 508 - Opale' and 'Opale - ENSAT 508', show significant differences for androgenic ability when compared with Opale, their common parent, and represent favorable nuclear and cytoplasmic effects. These two effects were also demonstrated in hexaploid wheats by Becraft & Taylor (1989)
The authors thank the Caussade Semences Company for supporting this work, and Dr. John Bull for correcting the English.
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