Introduction
Wheat progenitors are often used as sources of desirable agronomic characters that could be introduced into cultivated new varieties. It has long been known that Aegilops are resistant to various wheat diseases of which most important are rusts (Puccina spp.) (Özgen, Reference Özgen1987) and to salinity (Colmer et al., Reference Colmer, Flowers and Munns2006). Especially, Aegilops umbellulata (Özgen, Reference Özgen1983), Aegilops triaristata (Özgen, Reference Özgen1985) and Aegilops biuncialis (Özgen, Reference Özgen1984; Damania and Pecceti, Reference Damania and Pecetti1990) are resistant to yellow rust. Ae. Biuncialis is also resistant to brown rust (Dimov, Reference Dimov, Zaharieva, Mihova and Damania1993). On the other hand, Triticum monococcum (Datta et al., Reference Datta, Kumar, Varma and Angrish1995), Ae. biuncialis (Gorham, Reference Gorham1990), Aegilops cylindrica (Farooq et al., Reference Farooq, Niazi, Iqbal and Shah1989; Gorham, Reference Gorham1990), Aegilops triuncialis (Farooq et al., Reference Farooq, Niazi, Iqbal and Shah1989), Ae. umbellulata (Gorham, Reference Gorham1990) contain genes for salt tolerance. Therefore, plant genetic resources are necessary to food security for the future. Genetic resources are usually considered as a public good and shared internationally. Wild relatives of crop species represent the store of genetic diversity that will help to provide the food requests for billions of people by 2050. New technologies from genomics support classical plant breeding for enhancing traits to meet these food requests. Genetic diversity is the lifeblood of all kinds of plant breeding. The increase in the number of biotech crops reveals the value of global food security (Philip, Reference Philip2014).
Successful plant regeneration from cultured tissues is required for the application of biotechnology in crop improvement. The achievement of tissue culture depends upon appropriate culture techniques and genotypes. The tissue culture parameters such as callus induction and plant regeneration of wheat are generally affected by the genotype (Sears and Deckard, Reference Sears and Deckard1982; Chowdhury et al., Reference Chowdhury, Kato, Yamamato and Hayashi1991; Fennel et al., Reference Fennel, Bohorova, Ginkel, Crossa and Hoisington1996; Özgen et al., Reference Özgen, Türet, Altınok and Sancak1998; Bi et al., Reference Bi, Kou, Chen, Mao and Wang2007), explant source (Ozias-Akins and Vasil, Reference Ozias-Akins and Vasil1982; Zhang and Seilleur, Reference Zhang and Seilleur1987; Redway et al., Reference Redway, Vasil, Lu and Vasil1990), and culture medium (Mathias and Simpson, Reference Mathias and Simpson1986; Fennel et al., Reference Fennel, Bohorova, Ginkel, Crossa and Hoisington1996; Barro et al., Reference Barro, Martin, Lazzeri and Barceló1999; Mendoza and Kaeppler, Reference Mendoza and Kaeppler2002; Satyavathi, Reference Satyavathi, Jauhar, Elias and Rao2004).
Immature embryos were shown to be the most suitable explant for callus induction and plant regeneration via in vitro cultivation of wheat (Maddock, Reference Maddock, Bright and Jones1985; Tuberosa et al., Reference Tuberosa, Ravaglia and Lucchese1988; Redway et al., Reference Redway, Vasil, Lu and Vasil1990; Ke et al., Reference Ke, Chen, Yang, Li and Chen1995; Tang et al., Reference Tang, Zhang, Zhang, Yan and Ren2004). Therefore, immature embryos have been used mostly as an explant in wheat tissue culture. Recently, it has also been used in molecular studies in wheat and its relatives (Zhao et al., Reference Zhao, Jiang, Ma, Zhang, Zhao, Wang, Wang, Cao, Lu, Zhen and Wei2014). However, it is usually difficult to obtain immature embryos throughout the year, and their appropriate stage for culture is also limited. Whereas, the use of mature embryos reduces greenhouse costs, and saves space and time (Zale et al., Reference Zale, Borchardt-Wier, Kidwell and Steber2004). Mature embryos are also available in a large amount all year round.
Researches on the embryo culture of wheat progenitors are very inadequate. The purpose of this study was to screen a diverse set of wild wheat species (Aegilops sp. and Triticum sp.), which are very important progenitors of wheat cultivars, for their ability to induce callus and regenerate plants from mature embryos, for ultimate use in genetic transformation of wheat genotypes.
Materials and methods
Plant materials
A total of 12 accessions of Aegilops aucheri (2n= 2x= 14, BB), Ae. Biuncialis (2n= 2x= 28, CUCUMbMb), Ae. cylindrica (2n= 2x= 28, CCDD), Ae. triaristata (2n= 2x= 28, CUCUMtMt), Ae. triuncialis CUCUCC), Ae. umbellulata (2n= 2x= 14, CUCU), Aegilops speltoides (2n= 2x= 14, BB), Aegilops ligustica (2n = 2x = 14, BB), Triticum monococcum (2n= 2x= 14, AmAm), Triticum dicoccum (2n= 2x= 28, AABB), Triticum spelta (2n= 2x= 42, AABBDD), Triticum vavilovii (2n= 2x= 42, AABBDD) were used as sources of mature embryos. Dry seeds of these wheat progenitors were supplied from Prof. Dr M. Özgen, Ankara University, Faculty of Agriculture, Ankara, Turkey. For naming of the Aegilops and Triticum species, the classifications of van Slageren (Reference van Slageren1994) and Hammer et al. (Reference Hammer, Filatenko and Pistrick2011) were taken as a base, respectively.
Callus induction and plant regeneration
Dry seeds were surface sterilized with 70% (v/v) ethanol for 5 min, washed several times with sterile distilled water, treated for 20 min with commercial bleach, and rinsed several times with sterile distilled water. The seeds were then imbibed in sterile water for 2 h at 33°C. Thereafter, mature embryos were aseptically dissected slightly with a scalpel from the endosperm of imbibed seeds for callus induction. The embryos were cultured, with the scutellum upwards, in sterile 10-cm Petri dishes containing the mineral salts of MS (Murashige and Skoog, Reference Murashige and Skoog1962), 20 g/l sucrose, 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 7 g/l agar. The dishes were kept at 25 ± 1°C in total darkness for 14 d (method modified from Özgen et al., Reference Özgen, Türet, Özcan and Sancak1996, Reference Özgen, Türet, Altınok and Sancak1998).
Embryogenic calli were then removed from the embryos and transferred onto a hormone-free shoot initiation medium containing MS mineral salts (Murashige and Skoog, Reference Murashige and Skoog1962), glycine (2 mg/l), sucrose (20 g/l) and agar (7 g/l) in the plates. Callus fresh weight data were obtained before calli were transferred to shoot initiation medium in embryo cultures. The transferred callus cultures were incubated at 25 ± 1°C for 3 weeks in the dark, and then grown under 16 h/8 h (light/dark) photoperiod at 25 ± 1°C. At the end of 6–8 weeks, the frequency of differentiation for each species was calculated, based on the number of calli with green spots. Culture efficiency was based on regenerable calli out of the number of mature embryos cultured. When the regenerated shoots elongated to 10–15 mm, were transferred to Magenta boxes containing the same medium. Cultures for rooting were exposed to 50 W cool white fluorescent light (50 μmol/m2/s) at 25 ± 1°C under 16 h/8h (light/dark) photoperiod for 3 weeks. The all media were adjusted to pH 5.8 and autoclaved for 20 min at 121°C and 1.1 kg/cm2 pressure. Callus fresh weight data were obtained before calli were transferred to shoot initiation medium in embryo cultures (Özgen et al., Reference Özgen, Türet, Altınok and Sancak1998).
Statistical analysis
The tissue culture parameters included the callus induction frequency, callus weight, regeneration capacity of callus (number of nodular calli with green spots/number of calli induced × 100), and culture efficiency (number of nodular calli with green spots/number of embryos cultured × 100). A completely randomized design with three replications per species was used. Petri dishes containing 15 seeds were considered the units of replication. The effects of species on culture responses were determined by analysis of variance and least significant difference tests. Correlation coefficients between the different characters were calculated for each procedure (Steele and Torrie, Reference Steele and Torrie1960).
Results
In the experiment, mature embryos from a diverse set of 12 wheat progenitors were tested for tissue culture parameters. The variables for this experiment included callus induction frequency, weight of callus, regeneration capacity of callus, and culture efficiency.
Callus formation from embryos started after 2–3 d of culture. On the 6th day, the average diameter of the calli was 5–6 mm. Maximum callus induction occurred 14 d after placement on the mature embryo callus media. Calli with green spots rapidly developed shoots and leaves in shoot initiation medium. These plantlets did not establish an adequate root system when 2,4-D was present in the medium. Following transfer to 2,4-D-free MS medium, plants regenerated and produced multiple shoots from mature embryo calli (Fig. 1(a)–(c)). After about 3 months from callus initiation, regenerated plants (with shoot and root) were obtained in a jar (Fig. 1(b)–(d))
Fig. 1 Callus maintenance and plant regeneration from mature embryos of Aegilops biuncialis (a and b) and Aegilops umbellulata (c and d).
Culture responses were greatly influenced by the species for embryo culture (Table 1). On the other hand, a significant correlation was also observed between callus fresh weight and callus induction (r= 0.820, P< 0.01) or culture efficiency (0.740, P< 0.01) (Table 2). Therefore, frequencies of callus induction and regeneration capacity of callus of 12 wheat progenitors were significantly different. Callus induction frequency was highest in Ae. biuncialis (97.8%), T. dicoccum (97.8%), T. spelta (93.3%) and lowest in Ae. cylindrica (15.5%), Ae. aucheri (17.7%) and Ae. ligustica (26.6%) (Table 1). On the other hand, Ae. umbellulata (95.8%), Ae. biuncialis (93.1%), T. vavilovii (92.5%) and Ae. ligustica (91.7%) had an excellent regeneration capacity of callus, while Ae. aucheri (55.5%) and Ae. cylindrica (55.6%) had lowest regenerable callus frequencies (Table 1).
Table 1 Mature embryo culture responses of Aegilops and Triticum species
a The origin of the species is Turkey.
b No. of regenerable calli (nodular calli with green spots)/no. of calli induced × 100.
c No. of regenerable calli/no. of embryos cultured × 100.
d Means followed by the different letters are significantly different at the 0.05 probability level.
Table 2 Correlations between different characters in callus cultures from mature embryos of Aegilops and Triticum species
** Significantly different from zero at P= 0.01 probability.
Discussion
The effect of genotype on the regeneration of callus from mature embryos in wheat progenitors was similar to other wheat studies (Özgen et al., Reference Özgen, Türet, Özcan and Sancak1996; Özgen et al., Reference Özgen, Türet, Altınok and Sancak1998; Tan and He, Reference Tan and He2001; Ren et al., Reference Ren, Yin, Shi, Jiang and Wang2003; Yu et al., Reference Yu, Yin, Guo and Niu2003). On the other hand, it showed that culture efficiency was highest in Ae. biuncialis, T. vavilovii and T. dicoccum which all showed a better tissue culture response than the other genotypes (Table 1). These results showed that the genotype effect and genotype dependency were also high in the tissue culture of mature embryos from wheat genitors. Ae. biuncialis had both high callus induction (97.8%) and regenerable calli frequencies (93.1). Therefore, Ae. biuncialis had the most frequency of culture efficiency (91.1%) in the studied wheat progenitors. Such species are very desirable in tissue culture and plant breeding programmes.
Frequencies of callus induction were generally high but there was no significant relationship with the regeneration capacity of callus in the studied wheat progenitors (Table 2). Although some genotypes such as Ae. biuncialis had both high callus induction and plant regeneration capacity, the correlation coefficients between callus induction frequency and regeneration capacity or between regeneration capacity and culture efficiency were statistically insignificant (Table 2). Generally, it is known that callus induction and regeneration capacity may be controlled independently of each other (Sears and Deckard, Reference Sears and Deckard1982; Chowdhury et al., Reference Chowdhury, Kato, Yamamato and Hayashi1991; Özgen et al., Reference Özgen, Türet, Özcan and Sancak1996). The absence of significant relationships between callus induction frequency and regeneration capacity of callus clearly confirms that these characters are genetically independent. Thus, T. dicoccum (97.8%) and T. spelta (93.3%) had a high callus induction frequency but a low regeneration capacity of callus (82.0 and 76.7%, respectively), while Ae. ligustica (26.6%) and Ae. umbellulata (46.6%) had a low callus induction frequency but most calli were regenerable (91.7 and 95.8%, respectively) in the embryo culture (Table 1).
Genotype effects on plant regeneration from wheat embryo cultures have been reported previously (Sears and Deckard, Reference Sears and Deckard1982; Tuberosa et al., Reference Tuberosa, Ravaglia and Lucchese1988). These results suggested that the plant regeneration was independent on the other characters. It is known that, no significant correlations existed among the studied tissue culture parameters suggesting that different genes or gene combinations may control such responses (Özgen et al., Reference Özgen, Türet, Altınok and Sancak1998). However, a significant correlation between callus induction frequency and culture efficiency (r= 0.955, P< 0.01) in mature embryo culture indicated that culture efficiency tended to increase with increasing callus induction rate (Table 2). This result was consistent with the reports of Özgen et al. (Reference Özgen, Türet, Özcan and Sancak1996), Özgen et al. (Reference Özgen, Türet, Altınok and Sancak1998) and Zale et al. (Reference Zale, Borchardt-Wier, Kidwell and Steber2004).
This research has demonstrated that some species of wheat progenitors such as Ae. umbellulata and Ae. biuncialis can induce callus and regenerate plants from mature embryos at high frequencies. While immature embryos and immature inflorescences are an excellent source of explant for regenerating large numbers of plants, mature embryos are also an important source and can thus be used to save the time and growth space required to grow plants to post anthesis for embryo collection in wheat progenitors. To increase the plant regeneration capacity in mature embryo cultures of wheat progenitors, therefore, suitable species should be used. Mature embryos may also reduce some of the difficulties associated with obtaining explants of immature embryos and immature inflorescences for tissue culture of wheat progenitors.
In conclusion, there were strong genotypic influences on callus induction, callus weight, regeneration capacity and plant regeneration in mature embryo culture of wheat progenitors. There was no significant correlation between callus induction frequency and regeneration capacity of callus. These results suggest that regeneration capacity of callus was a key step in the tissue culture of wheat progenitors. The present study also showed that culture responses of mature embryos from wheat progenitors might be more useful than the other explants. The transfer of foreign genes into wheat by genetic engineering techniques requires the development of efficient in vitro regeneration systems. An efficient regeneration system such as the mature embryo culture may provide enough material for gene isolations and direct gene transfer studies in wheat progenitors. Therefore, mature embryos, which are readily available throughout the year, can be used as an effective explant source in the tissue culture of wild wheat species.
