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2Department of Plant Physiology, Eötvös Loránd University, H-1518 Budapest Pf. 32, Hungary (. ∗ request for offprints: Fax: +36-22-460-213; E-mail: ...
Plant Cell, Tissue and Organ Culture 79: 39–44, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

39

Effect of combined changes in culture medium and incubation conditions on the regeneration from immature embryos of elite varieties of winter wheat Cec´ılia Tam´as1,∗ , P´eter Sz˝ucs1 , Mariann Rakszegi1 , L´aszl´o Tam´as2 & Zolt´an Bed˝o1 1 Agricultural

Research Institute of the Hungarian Academy of Sciences, H-2462 Martonv´as´ar, POB 19, Hungary; of Plant Physiology, Eötvös Lor´and University, H-1518 Budapest Pf. 32, Hungary (∗ request for offprints: Fax: +36-22-460-213; E-mail: [email protected])

2 Department

Received 12 August 2003; accepted in revised form 29 February 2004

Key words: immature embryo, in vitro culture, light, shoot regeneration, temperature, Triticum aestivum L.

Abstract In this study, tissue culture method for plant regeneration from immature embryos of elite Hungarian winter wheat varieties was established. The influence of the growth regulators and the concentration of macroelements in the regeneration medium and of the incubation temperature and light intensity on regeneration frequency were investigated. The most noticeable effect on regeneration frequency was achieved by simultaneously reducing both the incubation temperature to 23 ◦ C and the concentration of macroelements in the regeneration medium to halfstrength. This modification increased the average regeneration frequency from about 10–78%. Changes in the light intensity and temperature gave an average plant regeneration frequency of 83%.

Introduction The achievement of wheat transformation (Vasil et al., 1992; Altpeter et al., 1996; Pastori et al., 2001; Pellegrineschi et al., 2002) has opened the way for the genetic engineering of new transgenic wheat plants with improved agronomic traits, grain quality and composition. The application of genetic modification to elite wheat varieties, however, is not always possible due to their low regeneration capacity (Iser et al., 1999; Rakszegi et al., 2001). In most cereal species, plant regeneration still depends strongly on the genotype (Fennell et al., 1996; Zhang et al., 2000; Varshney and Altpeter, 2002), the explant source (Ozgen et al., 1998; Barro et al., 1999; Folling and Olesen, 2001) and the medium composition (He et al., 1989; Barro et al., 1999). This is important as the transformation frequency is largely determined by the ability of the explant tissue to regenerate fertile plants. In addition, incubation conditions, such as temperature and light intensity, are major factors influencing the embryogenic response and plant regeneration (Thorpe, 1994). Although the temperature has a ma-

jor effect on plant growth and development both in vivo and in vitro, this factor has not usually been examined thoroughly. Generally, cultures are kept at a constant temperature between 20 and 30 ◦ C. However, the optimum temperature for growth and differentiation for a particular species should be determined, as different species have different optima (Hughes, 1981; Chalupa, 1987). Even within the same tissue, different optimum temperatures for shoot formation and rooting have been reported (Rumary and Thorpe, 1984). Light, another major factor in the culture environment, has also been shown to affect plant differentiation and development in vitro (Thorpe, 1994). The light requirements for differentiation involve a combination of several components, including intensity, spectral composition and photoperiodism. Though most researchers do not critically evaluate the light requirements for optimum growth and differentiation there are several reports that indicate clearly the importance of this factor (Thorpe, 1980; Hughes, 1981). For example, maximum callus growth often occurs in darkness, and very low light intensity

40 Table 1. Culture media used for the culture of immature embryos of wheat Medium

Composition

BEG 2

MS macroelements, MS microelements (Murashige and Skoog, 1962), 0.77 mg l−1 glycine, 0.13 mg l−1 nicotinic acid, 0.025 mg l−1 thiamine hydrochloride, 0.025 mg l−1 pyridoxine hydrochloride, 0.025 mg l−1 calcium pantothenate, 100 mg l−1 myo-inositol, 200 mg l−1 casamino acids, 200 mg l−1 L-asparagine, 146 mg l−1 L-glutamine, 30 g l−1 sucrose, 9 µM 2,4- D , 8 g l−1 agar, pH 5.8

MS9

MS macroelements, MS microelements, MS vitamins (Murashige and Skoog, 1962), 2.0 mg l−1 glycine, 20 g l−1 sucrose, 2.7 µM IAA, 4.4 µM BAP, 8 g l−1 agar, pH 5.8

MS9N

MS9 medium minus IAA, plus 2.7 µM NAA

MS92

MS9 medium with half-strength MS macroelements

MS92N

MS9N medium with half-strength MS macroelements

(90 nmol m−2 s−1 ) may enhance organogenesis and embryogenesis (Villalobos et al., 1984). Genotype is the primary factor influencing the successful regeneration and transformation of cereals. Efforts have been made to extend the transformation technology to elite genotypes, which are either agronomically-important breeding lines or current commercial cultivars (Machii et al., 1998; Barro et al., 1999; Pastori et al., 2001; Varshney and Altpeter, 2002). Although these reports study tissue culture variables on model and elite winter wheats as well, there is still a clear need to improve the regeneration frequency of commercial cultivars. The objective of the present work was to screen for Hungarian elite winter wheat varieties with high callus induction and plant regeneration ability, suitable for breeding purposes. To identify responsive genotypes for tissue culture and to reduce the genotypic variability of agronomically important varieties, studies were made on the combined effect of incubation temperature, light intensity and medium composition on the regeneration frequency of calli derived from immature embryos of elite winter wheat (Triticum aestivum L.) varieties.

Materials and methods Plant materials Currently cultivated Hungarian winter wheat varieties from Martonvásár (Mv), ‘Mv Emese’, ‘Mv Magvas’, ‘Mv Martina’, ‘Mv Pálma’, and two genotypes of ‘Mv Emma’ (differing in their HMW glutenin subunit composition: Glu-D1x5 + Glu-D1y10 (5 + 10) or

Glu-D1x2 + Glu-D1y12 (2 + 12)) were used as donor plants. Three different lines (B2, B70, and B8) of ‘Bánkúti 1201’, an old Hungarian wheat variety, were also studied. The plants were grown under controlled conditions in a phytotron chamber with supplementary light providing a 16-h photoperiod, at a day/night temperature regime of 16/14 ◦ C. Prior to transfer to these conditions, the imbibed seeds were vernalised for 6 weeks at 6 ◦ C. To ensure continuous production of immature embryos, seeds of each variety were planted every week. Particular care was given to maintaining the growing conditions at the same level for all the experiments, and to avoid any stress to the donor plants. Every 15 days, routine pesticide and fertilizing treatments were applied to the donor plants. The experiments were done during the winter season in Hungary. In vitro culture of immature embryos Seeds from spikes 8–10 days after pollination, containing slightly translucent immature embryos, were harvested, peeled and surface-sterilised in 1.3% hypochlorite solution with a drop of surfactant for 20 min (Larkin, 1982). After five washes with sterile distilled water, embryos approximately 0.5–1.0 mm in length were dissected. Thirty embryos were placed in a Petri dish containing BEG 2 callus induction medium (Table 1), and incubated in darkness for 1 week, thereafter only explants producing callus or embryos with a bumpy scutellum, and no precocious germination, were subcultured to fresh callus induction medium. The cultures were spaced evenly so that the density was not more than 13–15 calli per plate. After another 4-week period, calli were randomly checked for embryogenesis and transferred to shoot regeneration me-

41 dium (Table 1), and cultured for 4–8 weeks. Cultures were illuminated with different light intensities at different temperatures as described below. The light was provided by white cool fluorescent tubes. The plantlets then were counted and the regeneration frequency was calculated. The plantlets were then placed on root regeneration medium (a hormone-free MS medium, Murashige and Skoog, 1962) and cultured at the same temperature and lighting conditions as for shoot regeneration were applied, until roots formed. In all experiments two plants per genotype were grown up in soil. Media and culture conditions Preliminary experiments were made using standard media (Larkin, 1982) of BEG 2, MS9 and MS media (Table 1) for callus induction, shoot- and root regeneration, respectively, to examine the tissue culture response of various cultivated Mv varieties and lines of ‘Bánkúti 1201’. For the three consecutive tissue culture phases, the same incubation temperature of 26 ± 1 ◦ C was applied because cultures are generally kept at a constant temperature, usually 26 ◦ C (Thorpe, 1994). After callus induction in the dark, the calli were illuminated with low intensity light (20 µmol m−2 s−1 , 16-h photoperiod) for 4–8 weeks to regenerate plantlets. In the first set of experiments the effect of the auxin type (IAA or NAA) and of the concentration of MS macroelements on shoot regeneration were studied. Embryos and calli were cultured under the same temperature and lighting conditions as in the preliminary experiments but were regenerated on three MS9-type media (MS9N, MS92, MS92N) differing in their auxin composition and in the concentration of macroelements (Table 1). To test the effect of reduced incubation temperature on regeneration, in a second set of experiments embryos and calli were cultured at 23 ± 1 ◦ C in the three consecutive tissue culture phases. After callus induction in the dark, the calli on all four MS9-type media were illuminated with low intensity light for 4–8 weeks to regenerate plantlets. In a third set of experiments the effect of a further reduction of temperature to 20 ± 1 ◦ C in the callus induction phase was also investigated by testing two Martonvásár varieties (‘Mv Emese’ and ‘Mv Martina’). The embryos and calli of these varieties were cultured at 20 ± 1 ◦ C in the dark, but the calli on MS92 medium (Table 1) were cultured at 23 ± 1 ◦ C and were illuminated with low intensity light for 4–8 weeks to regenerate plantlets.

In a fourth set of experiments the effect of constant higher intensity light, applied in the shoot regeneration phase, on plant regeneration was studied. Embryos and calli were cultured at 23 ± 1 ◦ C in the three consecutive tissue culture phases as in the second set of experiments. However, after callus induction in the dark, the calli on all four MS9-type media were illuminated with constant higher intensity light for 4–8 weeks to regenerate plantlets. In a fifth set of experiments the effect of changes in light intensity and alterations in the incubation temperature and shoot regeneration medium composition on plant regeneration were studied. Immature embryos and calli were cultured on BEG 2 medium at 23 ± 1 ◦ C in the dark for 5 weeks. For shoot regeneration only media with half-strength MS macroelements (MS92 and MS92N) were used. Calli were illuminated with low intensity light at 23 ± 1 ◦ C for 2 weeks, then calli and green structures were cultured under constant higher intensity light at 26 ± 1 ◦ C until they formed plantlets, after approximately another 2 weeks. Plantlets were placed in root regeneration medium, and incubated under the same temperature and lighting conditions as for the second part of shoot regeneration in this experiment was applied. Statistical analysis For each genotype, each treatment was replicated in five Petri dishes each containing 30 embryos, and each experiment was repeated two or three times. Regeneration frequency was assessed as the number of plantlets produced per number of explants × 100, where calli and embryos with bumpy scutellum transferred to fresh BEG 2 medium after 1 week of culture were considered as explants. In each experiment, 25–27 embryos per plate (85–90% of the dissected embryos) were subcultured to fresh BEG 2 medium and were the basis for calculating regeneration frequency. Within experiments, the statistical evaluation of the data was done by two-factor analysis of variance to determine significant differences in regeneration capacities among the wheat varieties tested.

Results and discussion Preliminary experiments The tissue culture response of various cultivated Mv varieties and lines of ‘Bánkúti 1201’ was tested initially using standard media and culture conditions. Amongst the nine genotypes tested, B1201/B8 line

42 Table 2. Effect of regeneration medium composition and temperature on regeneration from immature embryo-derived calli of elite varieties of winter wheat Variety/line

Regeneration frequency (%) 26 ± 1 ◦ C

23 ± 1 ◦ C

MS9

MS9N

MS92

MS92N

MS9

MS9N

MS92

MS92N

Mv Emese Mv Emma (5 + 10) Mv Emma (2 + 12) Mv Magvas Mv Martina Mv P´alma B1201/B2 B1201/B70 B1201/B8

12.3 0.0 0.0 5.0 6.1 0.0 5.4 0.0 61.0

6.2 0.0 0.0 2.1 1.3 0.0 0.0 0.0 18.3

34.5 18.1 18.2 21.5 26.4 19.4 20.2 10.2 30.4

29.4 13.2 11.0 17.3 21.2 14.1 23.4 21.1 28.4

37.2 20.3 26.3 47.1 45.2 28.4 36.0 33.2 39.2

35.2 27.2 32.2 34.3 37.1 27.2 41.4 39.3 58.2

96.0 81.2 88.2 78.2 88.1 62.3 80.2 48.2 86.0

90.1 78.2 81.1 73.2 85.2 56.1 88.1 54.3 93.2

Mean

10.0

3.1

22.1

19.9

34.8

36.9

78.7

77.7

Embryos/calli were cultured at 26 ± 1 and 23 ± 1 ◦ C, respectively, illuminated with low intensity light (20 µmol m−2 s−1 , 16-h photoperiod) for differentiation. LSD5% = 7.2 (26 ± 1 ◦ C) and 7.8 (23 ± 1 ◦ C).

gave the highest frequency (61.0%) of regeneration (Table 2). The frequency in the other varieties varied greatly, ranging from zero (‘Mv Emma’, ‘Mv Pálma’ and ‘Bánkúti 1201’ line B70) to 5–12%, resulting in an overall mean frequency of 10.0%. Effect of medium composition and incubation temperature The optimisation of growth and plant regeneration from cultured tissues may require modifications rather than novel nutrient medium formulations. Thus, in the first set of experiments, embryos and calli were cultured under the same temperature and lighting conditions as in the preliminary experiments. To study the influence of the auxin type and of the concentration of MS macroelements on regeneration three more MS9type media were used for shoot regeneration. As a result of these experiments (Table 2), media containing half the concentration of the original MS macroelements (MS92 and MS92N) helped shoot regeneration and plant regeneration was achieved from all tested genotypes with a frequency between 10.2 and 34.5%. The mean frequencies on MS92 and MS92N were 22.1 and 19.9%, respectively. However, many calli exhibited hyperhydricity and necrosis, and microscopic observation revealed that a few of them produced somatic embryos in the callus induction phase. This observation suggested that the incubation temperature should be reduced.

In the second set of experiments, to study the effect of reduced temperature on regeneration embryos and calli were cultured at 23 ± 1 ◦ C instead of 26 ± 1 ◦ C. This modification reduced the size of the callus body and only a few calli appeared to be hydrated and necrotic. In addition, a higher number of embryogenic calli was observed with the stereomicroscope than when incubating at higher temperature. In this series of experiments, the regeneration frequency ranged from 20.3–96.0% (Table 2). The positive effect on regeneration of lowering the incubation temperature was also demonstrated by the increased overall regeneration frequency of 57.0%, which compares favourably with the 13.8% obtained after culturing at 26 ± 1 ◦ C. The regeneration frequency not only varied widely among the varieties tested, but was dependent on the type of MS9 media, too. The regeneration frequency from cultures on media with IAA was not significantly different from that on media with NAA. Again, those media which contained half the original concentration of MS macroelements (MS92 and MS92N) significantly increased plant regeneration, and a frequency of 48.2 and 96.0% was reached with a mean of circ. 78%, compared to 35–37% in full-strength (MS9 and MS9N) media. This finding is consistent with the suggestion of Constabel and Shyluk (1994) that plant shoot and root development may gain from a lower concentration of mineral salts, that is, from the use of half-strength MS or B5 media for plant regeneration. Indeed, He et al. (1989) obtained similar results

43 when testing the embryogenic capacity of immature embryos and protoplasts of wheat. The effect of a further reduction of temperature to 20 ± 1 ◦ C in the callus induction phase (third set of experiments) was also investigated by testing two Martonvásár varieties. This alteration had positive qualitative effect on callus induction, that is, there was a higher number of embryogenic calli with visible nodular outgrowth. However, regeneration from these cultures of ‘Mv Emese’ and ‘Mv Martina’ was 10 and 15% less, respectively, than from cultures induced at 23 ± 1 ◦ C. This finding suggests that plant regeneration from immature embryos of these varieties occurs mainly through organogenesis not somatic embryogenesis, at least in these media and environmental conditions applied.

modification had no positive effect on the regeneration frequency. This observation is consistent with the findings of Thorpe (1980) and Fischer-Iglesias et al. (2001), that it is mainly the endogenous rather than the exogenous cytokinin/auxin balance that influences shoot regeneration in wheat. Thorpe (1994) also indicated that in all cases of organised development in vitro, there was interplay between the genotype, the explant, the culture medium composition and the culture conditions. To achieve optimum responses, the interactions of these factors must be determined empirically.

Effect of light intensity

To overcome the qualitative problems described above such as prolonged tissue culture period and excessive root formation, changes in the light intensity were combined with an alteration in the incubation temperature during the three consecutive tissue culture phases (fifth set of experiments). This modification improved regeneration further (Table 3). The highest regeneration was obtained from ‘Mv Emese’ (99.9%), but other varieties, such as ‘Mv Emma’, ‘Mv Martina’, and two lines of ‘Bánkúti 1201’ (B1201/B2 and B1201/B8), also regenerated well (78.2–96.2%) and no variety

The effect of light intensity on plant regeneration was studied in the shoot regeneration phase. After callus induction in the dark, calli were illuminated with low intensity or constant higher intensity light in separate experiments (second and fourth sets of experiments). Low intensity light stimulated the differentiation of the cells and very much improved the regeneration frequency as discussed previously (Table 2) but shoot regeneration was delayed. Under these culture conditions plant formation took 6–8 weeks to occur. This prolonged period of tissue culture may lead to genetic instability and somaclonal variation (Harvey et al., 1999). Attempts made to shorten the shoot regeneration period by incubating the calli under constant higher intensity light gave ambiguous results. The higher intensity light had a positive effect on differentiation and each callus became green or produced green spots and leafy structures. However, many of these green structures were unable to develop into shoots and the regeneration frequency was not improved. Data are not shown as the number of plantlets could not be counted precisely. Furthermore, many calli formed only short curled leaves instead of shoots, especially when cultured on MS9N. The effective exogenous cytokinin/auxin ratio was lower in MS9N medium than in media with IAA, as IAA is a less bioactive auxin (on the basis of equal molarity) than NAA. Although calli, which formed short curled leaves did not develop into plantlets, roots frequently formed. Using double the amount of cytokinin (benzylaminopurine) in this medium reduced root formation, however, this

Combined effect of changes in light intensity and alterations in the incubation temperature and shoot regeneration medium composition

Table 3. Effect of regeneration medium composition, and alterations in the temperature and light conditions during regeneration, on regeneration from immature embryo-derived calli of elite varieties of winter wheat Variety/line

Regeneration frequency (%) MS92

MS92N

Mv Emese Mv Emma (5 + 10) Mv Emma (2 + 12) Mv Magvas Mv Martina Mv P´alma B1201/B2 B1201/B70 B1201/B8

99.9 88.3 88.5 79.2 90.3 71.1 89.4 57.2 85.5

91.1 80.0 78.2 73.3 87.1 66.2 93.1 68.0 96.2

Mean

83.3

81.5

For shoot regeneration, calli and green structures were illuminated with low intensity light (20 µmol m−2 s−1 , 16-h photoperiod) at 23 ± 1 ◦ C for 2 weeks, then cultured under constant higher intensity light (50 µmol m−2 s−1 ) at 26 ± 1 ◦ C for another 2 weeks. LSD5% = 6.7.

44 showed less than 57.2% regeneration frequency, while the average frequency was increased to between 81.5 and 83.3%. Although significant genotypic variability was detected in regeneration frequencies the culture response of the genotypes was similar in all conditions. Close positive correlations (0.83∗∗–0.99∗∗∗) were found between regeneration frequencies of the various genotypes based on the 10 sets of culture conditions applied. The differences between the regeneration frequencies of three ‘Bánkúti 1201’ lines were similar to those observed in ‘Bobwhite’ (Pellegrineschi et al., 2002), indicating that it is possible to select or screen for regeneration capacity within a landrace type variety. In summary, the tissue culture conditions established in the present work were suitable for most of the elite Hungarian winter wheat varieties tested and appeared to elevate the plant regeneration frequency in all varieties more or less proportionally, so that the genotypic differences did not disappear completely. Therefore, these conditions appear to exert a general effect at least for winter type wheat.

Acknowledgements The authors thank László Sági, PhD for his critical reading of the manuscript. This research project was supported by grants received from the Research Funds of the Hungarian Academy of Sciences (AKP grant 98-36 3,1) and of the Hungarian Ministry of Education (OMFB grant 39/2000).

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