Factors infl uencing somatic embryogenesis and regeneration ability in somatic tissue culture of spring and winter rye

Rye is an important crop in Northern and Eastern Europe. However, the application of various biotechnologies in rye breeding has been limited duo to its recalcitrant in tissue culture. In order to improve somatic tissue effi ciency, key factors affecting somatic embryogenesis and reproducible green plant regeneration of rye (Secale cereale L.) were evaluated and optimised. In this study, a total 27 rye genotypes including 10 spring and 17 winter genotypes were involved in the investigation. Genotype, culture medium, sugar, gel agent and auxin infl uenced somatic embryogenesis of immature embryo signifi cantly. One-two weeks cold pretreatment of young embryo enhanced somatic embryogenesis and green plant regeneration. In culture of immature embryos, infl orescences and leaf segments of the seedlings, explants signifi cantly infl uenced the culture effi ciency. Highest embryogenic callus yield resulted from rye immature embryo as explant compared to young infl orescence and leaf segment of seedling. Developmental stage of embryo played an important role in somatic embryogenesis. Late spherical coleoptile stage (embryo size 0.5–1mm in length) was optimal developmental stage of immature embryo for culture. Morphogenetic potential of embryogenic callus decreased with an increasing number of subcultures, and this ability could be maintained in vitro for a maximum of 8 months of culturing.


Introduction
Various biotechnological applications in plant breeding, such as gene transformation and in vitro selection, rely on the availability of effi cient plant tissue culture systems.Regeneration of plants from in vitro-cultured cells is an important advance in the genetic manipulation of plants (Lörz et al. 1988).Plant tissue and cell culture techniques Ma, R. & Pulli, S. Somatic embryogenesis and regeneration ability of rye have long been recognized as valuable tools in crop improvement programs, particularly in crop breeding.Effi cient plant tissue culture systems have been established and advances made in the genetic transformation of staple cereals, including wheat (Vasil et al. 1993, Zhang et al. 2000), rice (Dong et al. 2001, Pragya et al. 2002) and barley (Wan andLemaux 1994, Trifonova 2001).
Rye (Secale cereale L. 2n = 2x = 14) is an important cereal crop in Europe, and its adaptability to adverse conditions, such as low temperatures and diseases, among others, is also of major interest to plant breeders.However, rye has proved to be among the most recalcitrant graminaceous species in plant tissue culture and genetic transformation.So far, transgenic rye plants have been reported (Castillo et al. 1994, Popelka and Altpeter 2003a, b, Altpeter et al. 2004), and the target materials were limited in specifi c inbred line.The lack of an effi cient in vitro culture system is still a limitation for genetic transformation and manipulation of this species.Early investigations of rye somatic embryogenesis and plant regeneration from various explant sources of immature embryo (Rybczynski 1979, Zimny and Lörz 1989, Rakoczy-Trojanowska and Malepszy 1995, Popelka and Altpeter 2001), young infl orescence (Krumbiegel-Schroeren et al. 1984, Linacero and Vazquez 1990, Rakoczy-Trojanowska and Malepszy 1993), young leaf segment (Linacero and Vazquez 1986) and root organ cultures (Whitney 1996) have been described in several reports.However, the low embryogenesis and green plant regeneration are problematical.In particular, the effects of some important factors, such as different explants, cold pretreatment, genotype, medium, auxins and sugars, infl uencing somatic embryogenesis remain obscure.
In this study, the important physiological and physical factors infl uencing embryogenic callus induction and reproducible green plant regeneration were evaluated and optimised to improve effi ciency of plant tissue culture systems of rye in practical rye breeding work for genetic transformation, manipulation and in vitro selection.Embryogenic calli from somatic tissue culture were also suitable sources for cell suspension culture initiation and protoplasts culture.

Plant materials
A total of 27 rye cultivars including 10 spring and 17 winter genotypes served as donor plants in this experiment (Table 1).Plant materials, which were used for immature embryo culture and infl orescence culture, were grown in a greenhouse (18/16ºC, day/night temperature, 16-h photoperiod at about 200 µmol m -2 s -1 ).Seedlings of winter genotypes were vernalized in a cold room (4ºC, 12-h photoperiod at 70 µmol m -2 s -1 light intensity) for 10-12 weeks then transferred to the greenhouse.For leaf segment culture, sterilized seed (surface sterilized by 1.6% sodium hypochlorite with 0.5 Tween 20 for 15 min then rinsed several times with sterile water) of rye cultivars was germinated in Magenta boxes with sterilized fi lter paper at

Embryogenic callus induction
Spikes were harvested 10-20 days after isolated pollination (spikes isolated with cellophane bags to prevent cross pollination from other genotypes), and were stored at 4ºC in darkness with stalks in water.Immature caryopses were surface-sterilized by 1.6% sodium hypochlorite with 0.5% Tween 20 for 15 min then rinsed several times with sterile water.Immature embryos were placed on an induction medium with scutellum up.Tillers containing young infl orescence 0.5-2 cm in length were collected, and surface-sterilized in 70% ethanol for 30 seconds.Infl orescences were cut into 2-mm-long segments and cultured on an induction medium.
For leaf segment culture, basal part of shoot (approx.30 mm, 2-3 weeks old) grown in Magenta boxes were cut into 3-mm-long sections and cultured on induction medium.Approximately 10 immature embryos, infl orescence segments or leaf sections were cultured in a Petri dish (diameter 95 mm) containing 30 ml solid induction medium.Media MS (Murashige and Skoog 1962), CC (Potrykus et al. 1979), AA (Müller and Grafe 1978, omitted kinetin and GA3), mMS (modifi ed MS medium consisting of MS basic salts and vitamins, 2 mg l -1 glycine, 146 mg l -1 glutamine, 200 mg l -1 casein hydrolysate, Li et al. 1992) and S 1 (Yin et al. 1993) consisting of macronutrients of AA medium, micronutrients and vitamins of B5 medium (Gamborg et al. 1968), 500 mg l -1 Proline, 877 mg l -1 glutamine, 266 mg l -1 aspartic acid, 288 mg l -1 arginine, 75 mg l -1 glycine, 20 mg l -1 coconut milk were used as induction media.pH of the media was adjusted to 5.8 before autoclaving.Coconut milk was added after autoclaving by fi lter sterilization.Cultures were incubated at 27ºC in darkness.The effects of different auxins (2 mg l -1 2,4-D, Sigma, 4 mg l -1 Dicamba, Sigma, 2 mg l -1 NAA, Sigma, the concentrations used were optimal concentrations in our pre-tests), sugars (3% of sucrose, maltose and glucose) and gel agents (0.3% Phytagel, Sigma and 0.7% Agar, Difco) in AA medium on embryogenesis of immature embryos were tested.Totally 9 050 embryos were used in these three tests.
In cold treatment test, spikes with immature embryos were cold treated at 4ºC in darkness with stalks in water for different durations (0-5 weeks).A total of 3 600 immature embryos were used in the cold treatment experiment.Totally 1 808 immature embryos were used for test of genotypic effects.The culture abilities of different explants (immature embryos, infl orescence segments or leaf sections of seedling) were tested and 850 immature embryos, 850 immature infl orescence segments and 850 leaf sections were cultured.AA medium was used as embryogenic induction medium in these experiments.
Effects of developmental stages of immature embryos on embryogenesis were studied.Immature embryos in different developmental stages (1-6 scale of developmental stages of Zimny and Lörz 1989, embryo size from < 0.5 mm, 0.5-1 mm, 1-2 mm, 2-3 mm) were cultured on AA medium in this experiment.Totally 3200 embryos were used in this experiment.

Data collection and statistical analysis
Frequencies of explants forming embryogenic calli (embryogenic calli / 100 explants) and frequencies of regenerated calli producing green plants (green Ma, R. & Pulli, S. Somatic embryogenesis and regeneration ability of rye plants / 100 calli) were recorded.In the experiments, the completely randomized designs (CRD) were used.Each Petri dish was considered an experimental unit and each treatment contained fi ve replicates.Response variables were callus induction and green plant regeneration.Data analyses with more than two treatment levels were carried out by the ANOVA procedure.Multi-range comparisons were performed by LSD test.

Medium effects on somatic embryogenesis
In the fi ve media tested, AA medium produced a signifi cantly (P < 0.05) higher embryogenic ef-fi ciency than CC and mMS media, and CC and mMS media, in turn, gave a signifi cantly (P < 0.05) higher embryogenic effi ciency than S 1 and MS media.No interactions of genotype × medium on somatic embryogenesis were detected (P > 0.05) (Table 2).
Culture medium is another important factor infl uencing somatic embryogenesis.In previous work on the somatic tissue culture of cereal crops, different basal culture media have been employed for embryogenic callus induction.MS or modifi ed MS medium are most commonly used as induction media for somatic embryogenesis of wheat, rice and barley.CC medium has been recommended for embryo culture of rye (Zimny and Lörz 1989) and barley (Lührs and Lörz 1987).AA is an amino acid based medium preferred by Immonen (1996) for embryo culture of triticale.In this study, AA  (Yin et al. 1993) Vol. 13 (2004): 363-377.medium produced the highest number of embryogenic calli, suggesting AA medium to be the induction medium of choice in practical plant tissue culture for somatic embryogenesis of rye.The rest experiments of this study were performed with AA medium (Tables 3, 4 5, 7, 8, 9, 10).

Infl uence of auxins on somatic embryogenesis
Different auxins 2,4-D, Dicamba and NAA were tested in this experiment.Signifi cantly highest embryogenesis was from Dicamba comparing 2,4-D and NAA.No statistically signifi cant interactions between genotype and auxin on embryogenesis were found.The highest embryogenesis frequency was from 'Florida dwarf' by Dicamba, the lowest was from 'Auvinen' with NAA (Table 3).Effi ciency of auxins on somatic embryogenesis in cereals has been assessed in previous works (Lührs andLörz 1987, Popelka andAltpeter 2001).The type and concentration of auxins in the induction medium is important for obtaining a high efficiency of somatic embryogenesis (Zimny and Lörz 1989).Dicamba has proved to be more effective on somatic embryogenesis than 2,4-D in embryo culture of barley (Lührs andLörz 1987, triticale (Immonen 1996) and rye (Zimny and Lörz 1989).In our study, Dicamba gave the best somatic embryogenesis compared to 2,4-D, while NAA was in accordance with the observations of Zimny and Lörz (1989).

Sugar and gelling agent in culture medium
In this study (Table 4) somatic embryogenesis was signifi cantly (P < 0.001) infl uenced by sugars used in the induction medium.Signifi cantly highest embryogenesis was produced by sucrose than by  maltose and glucose.No signifi cant genotype and sucrose interactions were detected.Somatic embroygenesis was signifi cantly improved by using the gel agent Phytagel (0.3%) (P < 0.001) compared to agar (0.7%) (Table 4).
Sugar is an essential requirement for all culture media, providing carbon, energy and osmotic regulation.Sugar infl uences callus induction and green plant regeneration.In cereal crop somatic tissue culture, sucrose is most frequently used in culture media for somatic embryogenesis.Maltose has been shown to be the most effective sugar for increasing androgenesis and green plant regeneration in rye (Flehinghaus et al. 1991), wheat (Moieni 1997) and barley (Kuhlmann and Foroughi-Wehr 1989, However, our results revealed maltose (3%) to be an inferior carbohydrate source in compari-son with sucrose (3%) for somatic embryogenesis of rye.
Infl uences of gelling agent in plant tissue culture have been reported in previous studies (Kohlenbach and Wernicke 1978, Lührs and Lörz 1987, Flehinghaus et al. 1991).Agar was widely used for culture medium solidifi cation.However, since Kohlenbach and Wernicke (1978) found the inhibitory effects of agar, alternative gelling agents, such as agarose and gelrite, have been employed to replace agar.Agarose was superior to agar for increasing the effi ciencies of somatic embryogenesis in rye (Zimny and Lörz 1989) and barley (Lührs and Lörz 1987) embryo culture.Gelrite was better than agrose for embryogenic induction and regeneration rates in rye anther culture (Flehinghaus et al. 1991) and embryo culture (Popelka and Table 5.Effects of ecotypes, genotypes and cold pre-treatment on somatic embryogenesis (embryogenic calli per 100 embryos) in immature embryo culture of rye.AA medium with 3% sucrose, 4 mg l -1 Dicamba and 0.3% Phytagel was used for induction medium.Each treatment contained fi ve replicates.Cold pre-treatment on somatic embryogenesis and plant regeneration

Eco-type
Cold pre-treatment of immature embryo at 4ºC in darkness for 1-2 weeks signifi cantly improved somatic embryogenesis (Table 5) and plant regeneration (Table 6).Duration of cold pre-treatment longer than three weeks reduced the culture efficiencies.No interactions of genotype × cold treatment on embryogenesis and plant regeneration were found.
In cereals cold pre-treatment of anthers is among the key factors infl uencing androgenesis and commonly employed to enhance the frequency of embryogenesis and green plant regeneration (Thomas et al. 1975, Guo andPulli 2000).Cold pre-treatment of immature embryo was found to be favourable to somatic embryogenesis and plant regeneration in wheat (Maes et al. 1996) and triticale (Immonen 1996).A three-week cold pre-treatment of immature embryo at 4ºC before culture for wheat somatic embryogenesis and plant regeneration, and a two-week cold pre-treatment of immature embryos at 8ºC in culture for triticale plant regeneration were optimal.According to the results of our studies, one to two weeks of cold pre-treatment was benefi cial for both the embryogenesis and plant regeneration of rye.

Differences of somatic embryogenesis between plant organs of rye
Somatic embryogenesis of different plant organs (embryo, infl orescence and leaf segment) of rye was tested (Table 7).Culture effi ciencies (embryogenic calli per explant) for both spring and winter genotypes were considerably higher by embryo culture than by infl orescence culture, which were signifi cantly higher than by leaf tissue culture (P < 0.01).Callus induction frequencies of winter genotypes exceeded those of spring genotypes, but not signifi cantly.Frequencies for embryogenesis were 55.3% in embryo culture, 37.3% in infl orescence culture and 5.0% in leaf tissue culture.Signifi cant genotype × explant interaction in embryogenesis was found (P < 0.01).

Ma, R. & Pulli, S. Somatic embryogenesis and regeneration ability of rye
In cereal crops immature embryo, immature infl orescence and leaf tissue have been commonly used as primary explant in the tissue culture of wheat (Ozias-Atkins andVasil 1982, Gonzalez et al. 2001), rice (Jyoti and William 1996) and barley (Thomas and Scott 1985, Lührs and Lörz 1987, Timothy et al. 1993, Chang et al. 2003).In rye species, the somatic embryogenesis and plant regeneration from different explant sources of immature embryo (Lu et al. 1984, Zimny and Lörz 1989, Popelka and Altpeter 2001), immature infl orescence (Linacero andVazquez 1990, Rakoczy-Trojanowska andMalepszy 1993) and leaf tissue culture have been investigated.Immature embryo culture gave the best result, although other factors may have infl uenced the results.In our study, the highest embryogenesis frequency was from immature embryo culture, being in agreement with previous studies.The lowest frequency was from leaf tissue culture, which was much lower than that reported by Linacero and Vazquez (1986).This result is likely explained by the different genotypes used in the experiments.In this study, interaction of genotype × explant played an important role in the somatic embryogenesis of rye.A similar interaction has been found in barley (Ruiz et al. 1992).
Genotype is an important infl uencing factor in the plant tissue culture of cereal crops.In most species of cereals, genetic transformation is strongly dependent on genotype and effi ciency is largely determined by culture ability of the tissue (Maddock et al. 1983, Fennel et al. 1996).In the anther and microspore culture of rice (Gosal et al. 1997), wheat (Hu 1997) and rye (Guo and Pulli 2000), culture abilities are genetically controlled and culture effi ciencies are genotype dependent.In the immature embryo and infl orescence culture of wheat (Felföldi and Purnhauser 1992, Machii et al. 1998, Gonzalez et al. 2001), rice (Jyoti and William 1996) and barley (Lührs and Lörz 1987).The genotypic effects on somatic embryogenesis and plant regeneration have also been noticed.For barley, somatic embryogenesis and plant regeneration are two independent processes, and are controlled by independent genetic systems (Komatsuda et al. 1989, Stirn et al. 1995).In hexaploid wheat, the day length-sensitive allele ppd1 play major role in somatic embryogenesis and plant regeneration (Ben et al. 1992).Rye is a crossing-pollinating species, and has a great variability due to its allogamous character.Even plants from the same genotype were not exactly genetically identical, but plants from same genotype are genetically more homogeneous than plants from different genotypes.Different genotypes could show different responses in tissue culture (Linacero and Vazquez 1990).Rakoczy-Trojanowska and Malepszy (1995)   gested that in vitro response of rye seems to be a complex trait controlled by many genes co-operating in different ways, and the regeneration ability were determined by recessive genes.
In the present study on the embryo culture of rye, both embryogenesis and regeneration differed signifi cantly among genotypes, thus proving the importance of genotype.Genotypic effects also have been observed in infl orescence culture of rye (Linacero andVazquez 1990, Rakoczy-Trojanowska andMalepszy 1993).A correlation between embryogenesis and regeneration for all genotypes tested in the present study was observed.The genotypes with high embryogenesis also showed comparatively high regeneration.There was no difference in embryogenesis and regeneration between winter and spring genotype groups.Similar results were also demonstrated in embryo, Vol. 13 (2004): 363-377.infl orescence and scutellum culture of wheat and barely (Felföldi andPurnhauser 1992, Fransisco et al. 1999).

Effects of developmental stage of immature embryos on somatic embryogenesis
Infl uence of embryo developmental stages (stage 1-6 of Zimny and Lörz 1989, size ranging from under 0.5 mm to 3 mm) were tested in this study.Embryo developmental stages signifi cantly (P < 0.001) infl uenced the embryogenic induction (Table 9).Signifi cant highest embryogenesis (64.29%) was obtained from embryo sizes between 0.5-1 mm (about stage 3 of Zimny and Lörz 1989, Table 9).

Maintenance of morphological competence
Embryogenic calli were subcultured and green plant regeneration abilities were tested at each subculture.Regeneration ability signifi cantly (P < 0.001) decreased with increasing number of subcultures.With the exception of 'Zulpan' (19.4%) and 'Vågones' (11.2%), regeneration abilities of all genotypes were under 10% after 8 months of subculture (Table 10).
In cereal crops, embryogenic calli are suitable target tissues for genetic transformation, also being suitable starting materials for cell suspension and protoplast cultures.Immature embryo derived calli have been frequently used for establishment of embryogenic cell suspension used as a source of totipotent protoplasts (Maddock 1987, Ahmed andSagi 1993) and target tissue for genetic transformation (Becker et al. 1994, Dong et al. 2001).Maintenance of embryogenic capability and regeneration potential has been a critical problem in an effi cient in vitro culture system (Lührs andLörz 1987, Chang et al. 2003).Prolonged duration of subculture, has caused the gradual loss of embryogenic competence and regeneration potential (Bregitzer 1991, Jimenez andBangerth 2001).Somatic embryogenesis and plant regeneration are genetically controlled, and the frequency of embryogenesis and regeneration is genotype dependent.Medium composition and physiological state of the donor plant affects the reaction of the explant under in vitro conditions (Lührs andLörz 1987, Castillo 1998).Embryogenicity and regeneration ability has been maintained for 17 months in barley (Kachhwaha 1997), 36 months in wheat (Varshney 1996) and 34 months in rice (Utomo 1995).In our study, regeneration ability of embryogenic callus of rye was maintained for a maximum of 8 months for subculture.After 9 months subculture, regeneration ability was nearly lost.All green plants regenerated were normal in morphology and seed set.

Table 1 .
Source of rye materials in experiment.

Table 2 .
Effects of ecotypes, genotypes and media on somatic embryogenesis (embryogenic calli per 100 embryos) in immature embryo culture of rye (3% sucrose and 0.3% Phytagel were used).Each treatment contained fi ve replicates.

Table 3
. Effects of ecotypes, genotypes and different auxins on somatic embryogenesis (embryogenic calli per 100 embryos) in rye embryo culture.AA medium with 3% sucrose and 0.3% Phytagel was used for induction medium.Each treatment contained fi ve replicates.Ma, R. & Pulli, S. Somatic embryogenesis and regeneration ability of rye

Table 4
. Effects of ecotypes, genotypes, sugars and gel agents on somatic embryogenesis (embryogenic calli per 100 embryos) in rye embryo culture.AA medium with 4 mg l -1 Dicamba and 0.3% Phytagel was used for induction medium.Each treatment contained fi ve replicates.

Table 6
. Effects of ecotypes, genotypes and cold pre-treatment on green plant regeneration (green plants per 100 calli) in rye embryo culture.190-2 medium with 3% (w/v) sucrose, 3 mg l-1 BA and 0.45% Phytagel was used for regeneration medium.Each treatment contained fi ve replicates.

Table 7 .
Differences between plant organs in rye somatic embryogenesis.AA medium with 3% sucrose, 4 mg l-1 Dicamba and 0.3% Phytagel was used for induction medium.Each treatment contained fi ve replicates.

Table 8 .
Response of genotypes to embryogenesis and plant regeneration in immature embryo culture of rye.AA medium with 3% sucrose, 4 mg l-1 Dicamba and 0.3% Phytagel was used for induction medium.190-2 medium with 3% (w/v) sucrose, 3 mg l-1 BA and 0.45% Phytagel was used for regeneration medium.Each treatment contained fi ve replicates.

Table 9 .
Effects of ecotypes, genotypes and embryo size on somatic embryogenesis (embryogenic calli per 100 embryos) in rye embryo culture.AA medium with 3% sucrose, 4 mg l-1 Dicamba and 0.3% Phytagel was used for induction medium.Each treatment contained fi ve replicates.

Table 10 .
Infl uence of subculture age on green plant regeneration (green plants per 100 calli) in rye embryo culture.AA medium with 3% sucrose, 2 mg l-1 2,4-D and 0.3% Phytagel was used as culture medium for embryogenic callus maintenance.Each treatment contained fi ve replicates.and a higher percentage.These results were in agreement with of Krumbiegel-Schroeren et al. rate