A COMPARISON OF CALLUS INDUCTION AND PLANT REGENERATION FROM DIFFERENT EMBRYO EXPLANTS OF TRITICALE (x Triticosecale Wittmack)

CELLULAR & MOLECULAR BIOLOGY LETTERS Volume 9, (2004) pp 353 361 http://www.cmbl.org.pl Received 8 March 2004 Accepted 22 April 2004 Short Communication A COMPARISON OF CALLUS INDUCTION AND PLANT REGENERATION FROM DIFFERENT EMBRYO EXPLANTS OF TRITICALE (x Triticosecale Wittmack) MELAHAT A. BIRSIN and MURAT ÖZGEN* Department of Field Crops, Faculty of Agriculture, University of Ankara, 06110 Diskapi, Ankara, Turkey Abstract: Immature, mature and endospermsupported mature embryos of six triticale cultivars (BDMT988S, Melez2001, Mikham2002, Presto, Tacettin Bey and Tatlicak97) were cultured in vitro to compare the levels of callus induction and plant regeneration. Immature embryos, 1518 days after anthesis, were aseptically excised and placed with the scutellum upwards on a callus culture medium consisting of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l 1 2,4dichlorophenoxyacetic acid (2,4D). Mature embryos were aseptically excised from the imbibed seeds and placed scutellum up on MS medium supplement with 2 mg l 1 2,4D. Endospermsupported mature embryos were moved slightly in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l 1 2,4D for callus induction. The developed calli and regenerated plants were maintained on hormonefree MS medium. Variability among the genotypes was observed for all the types of embryo culture. Immature embryos from Presto and endospermsupported mature embryos from Mikham 2002 had excellent regeneration capacities (92.0% and 97.3%, respectively) and the highest number of plants regenerated growing in soil (9 and 13, respectively). A comparison of the responses of the three explants used indicated that the endospermsupported mature embryo was the most useful explant for plant regeneration in triticale. Key Words: Callus Induction, Embryo Culture, Plant Regeneration, Triticale, Tissue Culture * Corresponding author, email: mozgen@tr.net Abbreviations used: 2,4D 2,4dichlorophenoxyacetic acid; MS Murashige and Skoog

354 CELL. MOL. BIOL. LETT. Vol. 9. No. 2. 2004 INTRODUCTION Triticale (x Triticosecale Wittmack) is an amphiploid cereal crop obtained by crossing wheat (Triticum sp.) and rye (Secale sp.). It has been considerably improved through breeding and is currently grown on about 3 million hectares worldwide [1]. It is an important alternative crop due to its resistance to some diseases and tolerance to drought [2]. Triticales are an especially valuable alternative to feed barley and oats under dryland conditions. Wheat X rye crosses exhibit poor seed set and endosperm development. Hybrid embryos are often abnormal, and usually abort during the early stages of seed development [3]. Therefore, it is essential to establish efficient callus induction and plant regeneration techniques for rearing hybrid embryos and the production of multiple plants from one embryo. Intensive research on triticale was started in the early 1950s [4]. During the last decade, somatic tissue culture [58], molecular genetics [9] and transgenic studies [10] have been reported on for this plant. Triticale plants have been regenerated via organogenesis or somatic embryogenesis from explants such as young inflorescences [11], leaf bases [8], anthers [9, 1214], and immature [68, 15] and mature embryos [5, 8]. However, there are only a few reports on the somatic embryogenesis of triticale employing mature embryos [5, 8]. Immature embryos proved optimal as starting material for in vitro regeneration of triticale [8], but they cannot be obtained throughout the year, and their suitable stage for culture is also strictly limited. In cereals, mature embryos which are easily available without limit and at any time are the least frequently used explant sources, because of the low obtainable frequency of callus induction. However, some new techniques such as endospermsupported mature embryo culture were successfully used in callus induction and plant regeneration in wheat [16]. The purpose of this study was to establish an efficient method of callus induction and plant regeneration from mature embryo culture, to compare the responses of immature, mature and endospermsupported mature embryo cultures, and to determine genotypic influences within embryo culture in triticale. MATERIALS AND METHODS Six genotypes of hexaploid winter triticale cv. BDMT988S, Melez2001, Mikham2002, Presto, Tacettin Bey and Tatlicak97 were used as sources of immature and mature embryos. Plants were grown in the field during the winter of 20022003 and headed during May. Immature seeds were harvested from main spikes 1518 days after anthesis, surfacesterilized in 70% (v/v) ethanol for 1 min and in commercial bleach (5% sodium hypochlorite) for 30 min, and then washed several times in sterile distilled water. Immature embryos were aseptically excised from caryopsis and

CELLULAR & MOLECULAR BIOLOGY LETTERS 355 placed with the scutellum upwards on solid agar medium in sterile Petri dishes for 14 days at 26±1ºC in continuous darkness. The agar medium contained the mineral salts of Murashige and Skoog (MS) [17] and 20 mg l 1 sucrose, 2 mg l 1 2,4D, and 7 mg l 1 agar. For shoot and root initiation, calli were transferred to the same medium without 2,4D and maintained for 5 weeks at 26±1ºC in a 16 h light (2000 lux), 8 h dark photoperiod. Mature seeds were surfacesterilized in 70% (v/v) ethanol for 5 min, rinsed twice with sterile distilled water, incubated further in commercial bleach for 30 min, and rinsed several times in sterile distilled water. The surfacesterilized seeds were incubated at 33ºC for 2 h in sterile distilled water for imbibitions. The mature embryos were easily separated from the endosperm in imbibed seeds and placed scutellum up on MS medium supplemented with 20 mg l 1 sucrose, 2 mg l 1 2,4D, and 7 mg l 1 agar. Incubation was at 26±1ºC for 14 days in darkness. After this incubation, the calli were transferred to hormonefree MS medium for root and shoot initiation, and maintained for 5 weeks at 26±1ºC in a 16 h light, 8 h dark photoperiod. Endospermsupported mature embryo culture was carried out according to Özgen et al. [16] with minor modifications as follows. Mature seeds were surfacesterilized with 70% (v/v) ethanol for 5 min, washed twice with sterile distilled water, treated for 25 min with commercial bleach, and rinsed several times in sterile distilled water. The seeds were then imbibed in sterile distilled water for 2 h at 33ºC. For callus induction, mature embryos were aseptically moved (not set free) with a scalpel from the imbibed seeds. The seeds with moved embryos were placed furrow downwards in sterile 10cm Petri dishes containing 7 ml of 2, 4D solution (8 mg l 1 ). The dishes were kept at 26±1ºC in total darkness for 14 days. The developed calli were removed from the seed and transferred onto a hormonefree shoot initiation medium containing MS mineral salts, glycine (2 mg l 1 ), sucrose (20 mg l 1 ) and agar (7 mg l 1 ) in Petri dishes. The transferred callus cultures were kept at 26±1ºC in total darkness for 3 weeks, and then were placed in a growth cabinet in the light with 16 h light, 8 h dark photoperiod at 26±1ºC. When the shoots and roots were established, plantlets of 12 cm height were transferred to a babyjar containing a hormonefree MS medium for 1 month. When the roots of these plantlets reached 1012 cm in height, they were transferred to soil in pots. Each pot was covered with a plastic bag for 1 week to maintain high humidity under a 16 h light, 8 h dark photoperiod at 26±1ºC. After 3 weeks, the plants were vernalized for 2 weeks at 4ºC and subsequently transferred to soil and grown in the greenhouse to maturity to ensure fertility and seed set. The media were adjusted to ph 5.8 and autoclaved for 20 min at 121ºC and 1.1 kg/cm 2 pressure. Callus fresh weight data were aseptically obtained before the calli were transferred to the shoot initiation medium in all the embryo cultures. Each treatment consisted of 20 embryos or seeds per Petri dish with four replicates. The effect of embryo type on culture responses was determined by the

356 CELL. MOL. BIOL. LETT. Vol. 9. No. 2. 2004 analysis of variance and least significant difference tests. Percentage data were transformed to arcsine before analysis. Differences within three explant culture responses were evaluated with the chisquare test for independence. Correlation coefficients between the different characters were calculated for each procedure [18]. RESULTS AND DISCUSSION Callus induction Callus formation from all the explants started after 34 days of culture. The optimum immature embryo size (23 mm) for maximum callus induction was obtained from 1518 dayold embryos. Sirkka and Immonen [6] also reported that the optimal time for rescuing immature embryos was 1517 days after pollination in triticale. After 1314 days, maximum callus formation appeared from all types of embryo culture (Fig. 1a). The formed calli were nodular and white to cream in color. The culture responses were greatly influenced by the genotype in all types of embryo cultures (Tab. 1). Genotype effects on callusing ability from triticale mature embryo cultures were reported on previously [5, 13]. Immature embryos from BDMT988S, Mikham2002 and Tatlicak97 had an excellent callus induction frequency (98.8%) and a high regeneration capacity (96.3%, 94.8% and 93.8%, respectively). The significant correlation between callus induction frequency and regeneration capacity (r = 0.786 * ) in their immature embryo culture indicated that these characteristics are genetically dependent on each other (Tab. 2). Such genotypes which have high callus induction and regenerable callus frequencies are very desirable in tissue culture programs. However, it is known that callus induction and regeneration capacity may be controlled independently of each other [19]. The absence of significant relationships between callus induction frequency and the regeneration capacity of callus confirms that these characteristics are genetically independent [16]. Thus, BDMT988S had a high callus induction frequency (98.8%) but a low callus regeneration capacity (86.0%), while Tatlicak97 had a low callus induction frequency (87.5%) but most of its calli were regenerable (98.8%) in the mature embryo culture. On the other hand, in the endospermsupported mature embryo culture, while Melez2001 had an excellent callus induction frequency (100.0%) and a high regeneration capacity (97.5%), Tatlicak97 had a low callus induction frequency (92.5%) but most of its calli were regenerable (97.3%). The absence of a significant correlation between callus induction frequency and the regeneration capacity of callus clearly confirms that these characteristics are genetically independent in both mature and endospermsupported mature embryo culture (Tab. 2). These results were concurrent with the reports of Charmed et al. [20] and Marciniak et al. [13] which demonstrate that embryo production and green plant regeneration are independently controlled.

CELLULAR & MOLECULAR BIOLOGY LETTERS 357 Tab. 1. The embryo culture responses of six triticale genotypes. Genotypes Immature embryo Callus induction (%) Weight of callus (g) Regeneration capacity of callus (%) 2 Culture efficiency (%) 3 No. of plants growing in soil (per 80 embryos) BDMT988S Melez2001 Mikham2002 Presto Tacettin Bey Tatlicak97 1 92.5b 95.0ab 91.3b 1.7bc 2.1b 1.7bc 1.6c 2.6a 1.5c 96.3a 53.5c 94.8a 92.0a 76.5b 93.8a 95.0a 50.0d 93.8ab 87.5b 70.0c 92.5ab 8ab 5c 7b 9a 7b 7b Total Mean 96.0±3.43 11.2 1.9±0.17 84.4±16.8 81.4.0±17.9 43 7.1±0.61 Mature embryo BDMT988S Melez2001 Mikham2002 Presto Tacettin Bey Tatlicak97 95.0ab 81.3c 92.5ab 87.5bc 1.4ab 1.5a 1.4ab 1.2bc 1.4ab 0.9c 86.0b 91.3ab 97.3a 83.5b 83.5b 85.0ab 90.0a 92.5a 67.5c 77.5b 86.3ab 4ab 4ab 5a 3ab 2b 4ab Total Mean 92.3±2.80 7.8 1.3±0.09 90.1±2.79 83.1±3.76 22 3.4±0.35 Endospermsuppored mature embryo BDMT988S Melez2001 Mikham2002 Presto Tacettin Bey Tatlicak97 92.5b 100.0a 96.3ab 95.0ab 92.5b 1.3cd 2.1a 1.5bc 1.5bc 1.8ab 1.0d 87.3b 97.5a 97.3a 93.8ab 97.3a 81.3b 97.5a 93.8a 93.8a 92.5a 90.0ab 3d 9bc 13a 9bc 7c 11ab Total 9.2 52 Mean 95.8±3.14 1,5±0.16 95.4±4.27 91.5±5.55 8.6±1.42 1 Means followed by the same letter are not significantly different at 0.05 probability level, 2 No. of regenerable cali / No. of cali induced x 100, 3 No. of regenerable cali / No. of embryos cultured x 10.

358 CELL. MOL. BIOL. LETT. Vol. 9. No. 2. 2004 Tab. 2. The correlation between different characteristics in callus cultures from immature, mature and endospermsupported mature embryos of six triticale genotypes (upper line: immature, middle line: mature, lower line: endospermsupported mature embryos). Characteristics 1. Callus induction (%) Correlation coefficients between characteristics 1 2 3 4 5 0.841 0.786* 0.848* 0.348 0.699 0.128 0.767 0.371 0.951** 0.339 0.759 0.242 2. Weight of callus (g) 0.639 0.392 0.179 0.693 0.140 0.630 0.425 0.109 0.031 3. Regeneration capacity of callus (%) 0.994** 0.599 0.868* 0.795* 0.690 0.858* 4. Culture efficiency (%) 0.741 0.654 0.723 5. Number of plants growing in soil (per 80 embryos) *, ** Significantly different from zero at 0.05 and 0.01 probability, respectively. On average, because the regeneration capacity of callus from endospermsupported mature embryos was high (95.4%), culture efficiency was also high (91.5%). The fresh weights of calli from all types of embryos were similar to each other (P>0.01). No significant correlation was observed between the fresh weight of callus, the regeneration capacity of callus and the culture efficiency (Tab. 2). Therefore, in tissue culture programs of triticale, genotypes should be chosen according to a high callus regeneration capacity. A comparative study of different explants in this study revealed that endospermsupported mature embryos (95.4%) were more responsive (χ 2 = 6.84, P<0.05) in producing shoots than those from immature (84.4%) and mature (90.1%) embryos. Plant regeneration Shoots and leaves formed from different embryos regenerated into young plantlets on 2,4Dfree MS medium (Fig. 1b, c). Plantlets were finally transferred to the soil (Fig. 1d) through a gradual acclimatization process. About 3 months after callus initiation, significant genotypic variation was observed in the number of regenerated plants growing in the soil, ranging from 5 to 8 in the immature embryo culture, from 2 to 4 in the mature embryo culture, and the from 3 to 13 in the endospermsupported mature embryo culture. In

CELLULAR & MOLECULAR BIOLOGY LETTERS 359 total, 43 plants were regenerated and grown from calli of the immature embryos, 22 from calli of the mature embryos and 52 from calli of the endospermsupported mature embryos (Tab. 1). Fig. 1. The production of plants regenerated from a endospermsupported mature embryo culture of winter triticale. (a) Callus induction from embryos after 1314 days of culture. (b) Plant regeneration on 2,4D free MS medium from 2monthold callus. (c) Regenerated plantlets at the optimum size for transplanting into a babyjar. (d) Regenerated plants in pots. Significant differences were observed in immature, mature and endospermsupported mature embryo culture for the number of plants regenerated from the total embryo cultured (χ 2 = 13.2, P<0.01). The genotype effect on plant regeneration from triticale embryo cultures was also reported on previously [15].

360 CELL. MOL. BIOL. LETT. Vol. 9. No. 2. 2004 Immature embryos from Presto and endospermsupported mature embryos from Mikham 2002 had an excellent regeneration capacity (92.0% and 97.3%, respectively) and the highest number of plants regenerated growing in soil (9 and 13, respectively). A significant correlation between the regeneration capacity of callus and number of regenerated plants in immature and endospermsupported mature embryos (r = 0.795 * and r = 0.858 * ) indicated that these characteristics are genetically dependent on each other (Tab. 2). However, no significant correlation was observed between the number of plants regenerated growing in soil and the other culture responses for all the types of embryo culture (Tab. 2). These results suggested that the number of plants regenerated was independent of the other characteristics except for the regeneration capacity of the callus. Therefore, in order to increase the number of regenerated plants in the embryo cultures suitable genotypes with a high regeneration capacity should be used in breeding programs. This study also showed that the regeneration capacity of callus and the number of plants regenerated from endospermsupported mature embryos may be higher than those from other embryo cultures. In conclusion, the transfer of foreign genes into triticale by genetic engineering techniques requires the development of efficient in vitro regeneration systems, such as the endospermsupported mature embryo culture, which may provide enough material for direct gene transfer studies. Therefore, endospermsupported mature embryos, which are readily available throughout the year, can be used as an effective explant source in triticale tissue culture as well as for wheat and oats [16, 21]. REFERENCES 1. FAO, FAOSTAT2002 Food and Agricuture Organization, UN, Italy (http://apps.fao.org.), 2002. 2. Gustafson, J.P., Bushuck, W. and Dera, A.R. Triticale: productional utilization. in: Handbook of Cereal Science and Technology (Lorenz, K.J. and Kulp, K. Eds). Marcel Dekker Incorporated, New York, 1991, 373399. 3. Hesemann, C.U. Application of tissue and cell culture techniques in developing productive Triticale. in: Proceeding of the Second International Triticale Symposium. Passo Fundo, Brazil, Mexico, D.F. CIMMYT, 1990, 295297 4. Pauk, J., Puolimatka, M., Toth, K.L. and Monostori, T. In vitro androgenesis of triticale in isolated microspore culture. Plant Cell. Tiss. Org. Cul. 61 (2000) 221229. 5. Padmaja,G., Reddy, V.D. and Redy, G.M. Somatic embryogenesis and plant regeneration from mature embryo callus cultures of triticale. Indian J. Exp. Biol. 30 (1992) 181184. 6. Sirkka, A. and Immonen, T. Comparison of callus culture with embryo culture at different times of embryo rescue for primary triticale production. Euphytica 70 (1993) 185190.

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