Genotypic differences in callus induction and plant regeneration from mature embryos of barley (Hordeum vulgare L.)

Genotypic differences in callus induction and plant regeneration from mature embryos of barley ( Hordeum vulgare L.) first-author corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:jinxl@zju.edu.cnzhanggp@zju.edu.cn †E-mail:jinxl@zju.edu.cnzhanggp@zju.edu.cn jinxl@zju.edu.cn corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:jinxl@zju.edu.cnzhanggp@zju.edu.cn †E-mail:jinxl@zju.edu.cnzhanggp@zju.edu.cn jinxl@zju.edu.cn Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China An efficient induction system and regeneration protocol based on mature barley embryos were developed. Embryos isolated from mature seeds, dehusked by hand and inoculated with longitudinally bisected sections, showed low contamination and high primary callus-forming capability. The influences of nine culture media on primary callus induction and germination from the mature embryos of barley cultivars Golden Promise and Zaoshu 3 were analyzed. The results showed that the two cultivars had much higher values of primary callus induction in the B16M6D medium as compared to the other eight medium formulations, with a frequency of 74.3% and 78.4% for Golden Promise and Zaoshu 3, respectively. Furthermore, Zaoshu 3 demonstrated particularly high stability in callus induction over the different media, indicating its potential utilization in callus induction and regeneration for its good agronomic traits and wide adaption. There were significant differences amongst 11 barley genotypes in terms of primary callus induction in the optimum medium, with percentages of callus induction and germination response ranging from 17.9% to 78.4% and 2.8% to 47.4%, respectively. Green plantlets of Dong 17, Golden Promise, and Zaoshu 3 were successfully developed from primary calli through embryogenesis, with green plant differentiation frequencies ranging from 9.7% to 21.0% across genotypes. Barley Mature embryo Genotypic difference Primary callus Plant regeneration Introduction 1 The improvement of abiotic stress tolerance in barley depends on the understanding of genetic variation within this crop and the availability of precise methods for desirable genetic transformation (Ellis et al., 2000 2000 ). Once the genotypes for desirable characters are identified by preliminary evaluation, the transferral of desired genes to an elite barley germplasm can be attempted (Özgen et al., 2005 2005 ). However, molecular enhancement in barley remains hampered due to the lack of efficient and long-term regenerable culture systems. Despite the fact that barley presents as a promising model plant for the development methods of genetic engineering, its transformation is marked by some complications. For barley, the protocols for callus induction and plant regeneration are not efficiently and widely used because of high genotype-specific dependency (Taniguchi et al., 1991 1991 ; Akula et al., 1999 1999 ; Ganeshan et al., 2003 2003 ) and low regeneration efficiency (Rengel, 1987 1987 ; Bregitzer et al., 1998 1998 ; Chen et al., 2006 2006 ). Therefore, the application of genetic engineering has been restricted to only a few barley germplasms that possess high tissue culture capability; the so-called model cultivars mostly contain a low agronomic value. Most culture systems for callus formation and plant regeneration were developed for model barley varieties such as Golden Promise (Wan and Lemaux, 1994 1994 ; Tingay et al., 1997 1997 ; Holme et al., 2006 2006 ; Kumlehn et al., 2006 2006 ). It is imperative, therefore, to explore and establish regeneration systems for more elite barley genotypes that contain not only high tissue culture potential but also reasonable agronomic traits. In cereals, green plantlets can be successfully developed from a variety of explants, including embryos (Chauhan et al., 2007 2007 ), mature embryos (Cho et al., 2004 2004 ), leaves (Chugh and Khurana, 2003 2003 ) or seedling segments (Becher et al., 1992 1992 ), apical meristems (Goldman et al., 2003 2003 ), coleoptiles (Sahrawat and Chand, 2004 2004 ), and microspores (Jähne et al., 1994 1994 ). For barley, morphogenic calli can be induced more frequently from immature embryos (Caligari et al., 1987 1987 ; Bregitzer et al., 1998 1998 ). Moreover, highly efficient and reproducible plant regeneration protocols were primarily established through the use of immature embryotic materials (Repellin et al., 2001 2001 ). However, immature embryotic materials require the continuous growth of donor barley plants in environmentally-controlled greenhouses, thereby placing high demands on labor, time, and space. Furthermore, it is difficult to control the developmental stage of tissue culture within immature embryos. The use of mature embryos has remarkable advantages, such as a simplified cultivation of tissues excised from mature dry seeds and the availability of large amounts of initial materials without growth season restrictions. The establishment of embryogenesis and plant regeneration system based on mature embryos has been reported in recent studies (Sharma et al., 2004 2004 ; He and Jia, 2008 2008 ), but optimal systems for the isolation and culture mode of explants either are underdeveloped or lack adequate recognition. Described in this paper is an optimized primary callus induction medium, as well as an accessible and reproducible isolation and induction system for mature barley embryos. The differences in primary callus induction and plant regeneration among barley genotypes are evaluated. Also reported is a preliminary screening of several local Chinese barley cultivars with high primary callus induction and regenerability. The Chinese cultivars selected for the present study were planted locally, in wide distribution, and are deemed of specific value in agronomy. Finally, from the somatic embryogenesis of mature embryos, an efficient and reproducible regeneration protocol was established for Zaoshu 3. Materials and methods 1 Plant materials 2 Mature seeds of 12 barley ( Hordeum vulgare L.) genotypes were used, consisting of 11 hulled barley genotypes (Dong 17, Fuxuan 48, Golden Promise, Humai 1, Igri, TL43, Triumph, Weisuobuzhi, Zaoshu 3, ZAU 3, and Zhepi 1) and one hulless landrace (Yuyaohuanghumimai). Due to their high yield potential, wide adaptability, and high tolerance to waterlogging, the representative cultivars Fuxuan 48, Humai 1, Zaoshu 3, ZAU 3, and Zhepi 1 were widely planted in the Yangtze River Delta region during the last two decades. Effects of isolation and sterilization methods of mature embryos on primary callus induction 2 Fuxuan 48 was inoculated in four induction treatments to determine the callus induction response. Seeds were soaked in water for 2 h, and then dehusked by hand or by 50% (v/v) concentrated sulfuric acid. The sections containing whole mature embryos (3–5 mm in length) were, then, dissected from the dehusked seeds, surface-sterilized in 75% (v/v) ethanol for 30 s, and then immersed in 15% (v/v) sodium hypochlorite for 15 min. Meanwhile, the intact, dehusked seeds of Fuxuan 48 were surface-sterilized in 75% ethanol for 30 s, and then immersed in 50% sodium hypochlorite for 30 min. The isolated embryos or caryopses were rinsed three times with sterile distilled water and longitudinally bisected into two halves. Approximately 20–25 explants were placed, bisected section down, in Petri dishes containing medium B13S2D. Effects of induction medium and genotype on primary callus induction 2 The basal media used in this study were MS (Murashige and Skoog, 1962 1962 ), B5 (Gamborg et al., 1968 1968 ), and N6 (Chu, 1978 1978 ), and were supplemented with different concentrations of 2,4-dichlorophenoxy-acetic acid (2,4-D) and carbon sources (Table 1 1 ). Added to all media were 0.5 g/L of proline and 0.5 g/L of casein hydrolysate. Unless otherwise indicated, all medium components, except plant hormone, were stirred, adjusted to pH 5.8, solidified with 0.55% agar, and autoclaved at 115 kPa and 120 °C for 20 min. All growth factors were filter-sterilized before added to the autoclaved medium. To optimize the primary callus inductions of cvs. Zaoshu 3 and Golden Promise, explants were induced on each of the nine induction media. To detect the genotypic difference in primary callus induction, mature embryos of 11 genotypes were dehusked by hand, extracted of endosperm, and inoculated on B16M6D medium. Culture medium Macronutrient Micronutrient Vitamin Carbon source 2,4-D (mg/L)  B13S2D N6 MS B5 30 g/L sucrose 2  B13S6D N6 MS B5 30 g/L sucrose 6  B13M6D N6 MS B5 30 g/L maltose 6  MS3M2D MS MS MS 30 g/L maltose 2  MS3M6D MS MS MS 30 g/L maltose 6  B16M6D N6 MS B5 60 g/L maltose 6  B5MS6M6D MS MS B5 60 g/L maltose 6  MS6M6D MS MS MS 60 g/L maltose 6  N6MS6M6D N6 MS MS 60 g/L maltose 6 After two weeks incubation in darkness at 23–25 °C, the frequencies of primary callus induction/germination response were calculated by counting the explants that formed callus/germinated out of the total number of mature embryos. Since most explants exposed to this germination intensity would likely have ceased callusing, the germination response value represents the frequency of explants sharing a similar germination intensity and having an average shoot length of 3–5 cm and root length of 1–2 cm. Embryogenic callus formation and plant regeneration 2 After three weeks of induction, primary callus was separated from explants, then transferred to a B13M medium supplemented with 3.0 mg/L 2,4-D and 0.1 mg/L 6-benzylaminopurine (BA) to commence embryogenesis at 23–25 °C in darkness for three weeks. The proliferated callus was then cultured in a regeneration medium B13M containing 0.4 mg/L BA under low light conditions [20–30 μE/(m 2 ∙s)] at 24 °C and during a 14-h/10-h (light/dark) photoperiod. Embryogenic callus with green structures and leaf primordial could be obtained after five to six weeks of incubation. Eleven to twelve weeks after culture initiation, green shoots and plantlets were successfully developed from embryogenic callus through a biweekly subculture. To induce a strong root system, well-developed shoots or plantlets (2–3 cm in length) were separated from the regenerating embryoids and transferred to a rooting medium consisting of 1/2 MS medium, 20 g/L sucrose, 2.0 mg/L α-naphtalene acetic acid (NAA), and 0.4% agar. Conditions selected for plant regeneration and rooting of plantlets were based on those described by Sharma et al. ( 2005 2005 ). The well-rooted plants were transferred to soil and placed in a growth camber (16-h light period, 15 °C). Statistical analysis 2 Identical and independent experiments were performed for each cultivar. The data were analyzed using the statistical package statistical analysis system (SAS) Version 8 (SAS Institute Inc., Cary, NC, USA). A two-way factorial analysis was conducted to study the effects of medium component and genotype on induction response. Differences in primary callus induction, germination response, and plant regeneration amongst the 12 studied genotypes were evaluated by analysis of variance (ANOVA) testing. The significance of group differences was determined by a least significant difference (LSD) formulation. Results 1 In vitro responses of mature embryos in four induction treatments 2 Explants of Fuxuan 48 had more stable induction responses but were more vulnerable to contamination as compared to Golden Promise; therefore, Fuxuan 48 was selected for this experiment. During initial experiments, the mature embryos showed differential induction responses in medium B13S2D (Fig. 1 1 ). Explants dehusked by hand, with endosperm removed, showed relatively great primary callus-forming capability. Moreover, isolating embryos from mature seeds could effectively eliminate contamination, even if sodium hypochlorite strength was reduced from 50% (for 30 min) to 15% (for 15 min) during surface-sterilization. Removing the hull with concentrated sulfuric acid is more convenient than when performed by hand, regardless of a statistically higher frequency of germinating embryos. Seven to nine days following culture initiation, the caryopsis dehusked by sulfuric acid germinated intensively and main shoots elongated rapidly, with callus forming 5–7 d later. The callusing response took 3–4 d later for the explants dehusked by sulfuric acid as compared to those dehusked by hand, probably due to the injury of sulfuric acid to the embryos. Induction treatment: 1, dehusked by sulfuric acid/endosperm contained; 2, dehusked by sulfuric acid/endosperm removed; 3, dehusked by hand/endosperm removed; 4, dehusked by hand/endosperm contained. * indicates that values are significantly different at P =0.05 according to the ANOVA test. A total of 40 to 50 explants were used as starting materials Optimization of culture medium for induction of primary callus 2 The purpose of this experiment was to determine the influences of nine different media on primary callus induction of two barley cultivars, Zaoshu 3 and Golden Promise (Fig. 2 2 ). The statistical analysis showed that primary callus induction of the mature embryos differed among medium types between the two cultivars, thereby indicating an interaction between cultivar and medium. On the whole, the value of primary callus induction in medium B16M6D was significantly higher than those within the other media. Regardless of genotype, explants induced in B13S2D resulted in a slightly higher (about 10%) frequency of germinating embryos as compared to the other media, indicating that germination may be reasonable at low concentrations of sucrose and 2,4-D. Moreover, the results suggest that a higher maltose level (60 g/L) combined with a higher 2,4-D concentration (6 mg/L) could efficiently promote callusing response. Frequencies of primary callus and germinating embryos with 3–5 cm long shoot were recorded in each combination two weeks after culture initiation. Average data were from two independent experiments each with 60 to 70 mature embryos as materials Golden Promise showed a frequency of callus induction ranging from 23% to 74% in the nine media, indicating that the cultivar has specificity in its response to culture media during callus induction. By contrast, the local cultivar Zaoshu 3 had a smaller variation in the frequency of callus induction over the different media, ranging from 50% to 60%, suggesting that the cultivar is relatively insensitive to the media. For Golden Promise, however, increasing the 2,4-D level from 2 mg/L to 6 mg/L resulted in a significant increase of callus induction frequency and decrease of germination. Interestingly, Zaoshu 3 showed higher primary callus induction when macronutrients in the MS medium were substituted by those in the N6 medium. Moreover, the same pattern took place for Zaoshu 3 when vitamins in the MS medium were replaced by those in the B5 medium. This provides conclusive evidence that the medium B16M6D was optimal for the primary callus induction of the mature embryos. It was, therefore, used as the initial culture medium for callus induction in the subsequent experiments. Genotypic difference in primary callus induction in B16M6D medium 2 Due to the low concentration of sodium hypochlorite in surface-sterilization, as well as the direct contact of longitudinally bisected embryos with the induction media, the developments of plumule and radicle were initiated within 24 h, and became visible after 3 d of incubation. Meanwhile, the callusing response was observed as showing the non-embryogenic callus, termed “primary callus,” maintaining proliferation coupled with elongation of the main shoot (Fig. 3a 3a ). The primary callus could be obtained after two weeks of induction culture (Fig. 3b 3b ). At this time, the plumule and radicle lengths of most germinated explants were 3–5 cm and 0.5–1.0 cm, respectively. A few explants germinated significantly to a plumule length of 3–5 cm after 7-d induction and moreover, the plumule would continue growing for one week further, reaching a length of up to 15 cm and lifting the explants out of the medium. Callus proliferation was, therefore, inhibited. Consequently, in order to maintain the embryogenic potential, it was deemed reasonable to discard the primary calli from the callusing explants after two to three weeks. (a) Developing primary callus along with plumule and radicle from mature embryos after 3 d of incubation; (b) Induction response of isolated embryos two weeks after culture initiation; (c) The soft, translucent, and watery primary callus discarded from mature embryos; (d) Embryogenic callus with nodular and compact structures in the Petri dish; (e) Regenerating embryos with small shoots, plantlets, and clusters of embryoids; (f) Green plantlets developed from embryoids 13 weeks after culture initiation Frequencies of primary callus induction and germination responses of mature embryos in 11 barley genotypes are shown in Table 2 2 . The nutrient-modified medium B1, containing 60 g/L maltose and 6 mg/L 2,4-D, bore a high frequency of primary callus induction higher than 50% for Zaoshu 3, Golden Promise, Dong 17, and Zhepi 1. These four genotypes, in addition to ZAU 3, were, therefore, selected for the plant regeneration experiment. The results indicated that Zaoshu 3 had the highest percentage of callus induction, followed by Golden Promise and Dong 17; whereas other genotypes had significantly lower percentages. Humai 1, Triumph, Yuyaohuanghumimai and Igri showed particularly low percentages of primary callus induction, with Igri being the lowest (17.9%), indicating a remarkable genotypic difference in the frequency of primary callus induction. The germination response of Humai 1 was 47.4%; the highest amongst the 11 genotypes. This high germination response could be attributed to low callus induction. The primary callus derived from embryos of hulless barley was generally loose, petal-like, and low in embryogenesis potential. The soft, translucent, and watery calli, dissected from the explants of the five barley cultivars with high callus induction (Zaoshu 3, Golden Promise, Dong 17, Zhepi 1, and ZAU 3), were transferred to the subculture medium for proliferation and embryogenesis (Fig. 3c 3c ). n ei : number of explants inoculated Three independent experiments with three replicates each were carried out for each cultivar; for Zhepi 1 and ZAU 3, six and eight replicates were done, respectively Values (mean±SE) followed by the same superscript letters are not significantly different at P =0.05 according to the ANOVA test Genotype n ei Primary callus induction (%) 2 2 Germination after two weeks (%) 2 2 Dong 17 161 64.8±4.4 b 32.7±2.6 ab Golden Promise 154 74.3±3.7 ab 15.2±2.7 efg Igri 148 17.9±5.7 f 7.0±3.2 fg Zhepi 1 362 53.7±3.3 c 20.2±3.5 ce Weisuobuzhi 145 42.8±7.8 cd 42.3±9.7 a ZAU 3 511 49.3±1.9 cd 30.2±3.1 bd Zaoshu 3 171 78.4±2.6 a 17.3±2.0 ef Humai 1 126 24.5±5.0 ef 47.4±5.1 a Yuyaohuanghumimai 166 27.1±3.0 ef 2.8±1.4 g TL43 175 38.8±6.4 de 18.2±3.2 def Triumph 149 27.3±6.6 ef 19.9±6.3 def Embryogenesis and plant regeneration from five barley cultivars 2 The basic medium B1 containing 30 g/L maltose and 0.4 mg/L BA was found to be efficient for the induction of embryogenic callus with biweekly subculture when incubated for 3 d in the dark, followed by two to four weeks under light. The proliferated calli with shiny, nodular, and compact structure were of great embryogenic potential (Fig. 3d 3d ). Embryogenic callus with excellent regenerability was obtained after nine to ten weeks of culture. The successive subcultures promoted the formation of numerous clusters of embryoids at different developmental stages with leaf primordial and/or small plumule (Fig. 3e 3e ). During embryogenesis, the cultivars Zhepi 1 and ZAU 3 presented great callus proliferative ability and the embryogenic calli were also capable of turning green under light conditions. Nevertheless, the calli were not effective for differentiation due to genotype dependence and they eventually turned brown. Plumules and plantlets spontaneously derived from embryoids were observed in the next three weeks for Zaoshu 3 (Fig. 3f 3f ). In addition, the media free of plant hormone or supplemented with 3 mg/L 2,4-D and 0.1 mg/L BA were also efficient for plant regeneration, whereas embryogenesis took place on the hormone-free medium two to three weeks later than on the hormone-containing medium (data not shown). Regenerated plantlets of 3 cm in length were transferred into the rooting medium, and a strong root system developed within two to three weeks. The well-rooted plantlets were transferred to soil and placed in a growth cabinet. Generally, no phenotypic difference was found between the regenerants and seed-initiated seedlings of Zaoshu 3. Table 3 3 shows percentages of embryogenic callus induction, plant regeneration efficiency, and average number of plants per regenerated callus from the five barley cultivars. The percentage of green plants was 21.0%, 14.4%, and 9.7% for Zaoshu 3, Golden Promise, and Dong 17, respectively, being much higher than those for Zhepi 1 and ZAU 3. The cultivar Golden Promise had strong regenerability in terms of average number of plants per regenerated callus. Interestingly, Dong 17, which had the highest frequency of embryogenic callus, showed a relatively weak regenerability when compared with Zaoshu 3 and Golden Promise. Values (mean±SE) are calculated after three subcultures on regeneration medium Frequency was estimated as the ratio of the number of embryogenic calli or greenish calli to the total number of calli used Regeneration capacity of calli was calculated as the ratio of the number of calli with green or albino shoots to the number of calli plated on regeneration medium Values followed by the same superscript letters are not significantly different at P =0.05 according to the ANOVA test Cultivar Number of callus incubated Embryogenic callus (%) 2 2 , 4 4 Green plantlets (%) 3 3 , 4 4 Albinos (%) 3 3 Average number of plants per regenerated callus 4 4 Zaoshu 3 319 67.7±3.5 b 21.0±3.7 a 2.2±1.1 3.1±0.3 ab Golden Promise 143 63.2±7.2 b 14.4±6.9 ab 1.2±1.1 4.2±0.9 a Dong 17 71 93.7±3.6 a 9.7±3.2 ab 9.7±4.4 1.7±0.2 b Zhepi 1 81 71.0±5.2 b 0 b 0 0 b ZAU 3 48 51.6±8.9 b 0 b 0 0 b Discussion 1 Recently, mature embryos of barley were evaluated as starting materials in different culture modes, including endosperm-supported culture (He and Jia, 2008 2008 ) and meristematic shoot segment culture, in which explants are excised from germinated mature embryos (Sharma et al., 2004 2004 ). However, the isolation and obtaining of explants were not at that time readily available; therefore, the reliability needed further confirmation. We have optimized the conditions of callus induction and plant regeneration. The frequency of somatic embryogenesis was improved with segmentation from mature embryos into pieces in wheat (Delporte et al., 2001 2001 ). In the present study, when the 2/3 endosperm of soaked seeds was removed, the isolated embryos were easily dehusked by hand, and the embryos were cut into two halves. The endosperm-excised explants resulted in a higher callus induction frequency than complete seeds. A similar observation was reported by Zapata et al. ( 2004 2004 ), who found that the percentage of callus induction from isolated embryos was generally higher than those from roots, shoots, and complete seeds. The success of in vitro regeneration via embryogenesis is probably attributed to excised explants containing embryonic axis, as the suitability of the embryonic axis explants was demonstrated in previous studies (Krishnamurthy et al., 2000 2000 ; Sharma et al., 2005 2005 ). Furthermore, barley regeneration systems via embryogenesis, based on different combinations of growth regulators, have been reported. Regenerated barley plantlets were successfully obtained on the medium containing 1.0 mg/L indole-3-butyric acid (IBA) and 0.1 mg/L kinetin (Zapata et al., 2004 2004 ), 4 mg/L 4-amino-3,5,6-trichloropicolinic acid (picloram) (Akula et al., 1999 1999 ), or 2 mg/L BA (He and Jia, 2008 2008 ). In contrast, our results showed that the plant regeneration is successful on the medium B13M containing 0.4 mg/L BA. Meanwhile, the current results prove that embryogenic callus formation and plant regenerability are also genotype-dependent. Similar results were found by Bi et al. ( 2007 2007 ). The hormone composition suitable for embryogenic callus formation and regeneration varies with genotypes, suggesting that the abilities of embryogenesis and regeneration for the callus derived from mature barley embryo may be dependent on not only hormone composition in the medium, but also on the hormone level in the callus. Specific modifications of the culture medium strongly influence the frequencies of primary callus induction and the germinating embryo. It has been reported that the formation of embryogenic callus in various barley cultivars was depended on 2,4-D concentration, and 2–3 mg/L was adequate in most cases (Bregitzer et al., 1998 1998 ; Zapata et al., 2004 2004 ). However, it has been further demonstrated that for cv. Golden Promise, the germination and callusing response of mature embryos are affected by 2,4-D concentration, with the optimum level being 6 mg/L, combined with an increase in maltose level (Sharma et al., 2005 2005 ). The inconsistence in the optimum 2,4-D concentration among the available reports may be attributed to the properties of embryos, which differ in developmental stages. In the current study, a marked increase in callus induction and decrease in germinating embryos were found when the media B1 and MS were supplemented with 6 mg/L 2,4-D and 60 g/L maltose for cv. Golden Promise, which was consistent with the results found by Sharma et al. ( 2005 2005 ). However, Golden Promise had a relatively high percentage of callus formation in this study, as compared to Sharma et al. ( 2005 2005 )’s protocol. Interestingly, the findings were not observed in cv. Zaoshu 3. Moreover, the macronutrients in the N6 medium, as well as vitamins in B5 medium, appeared favorable for callusing response for the cultivar, indicating that there are remarkable interactions between genotypes and medium components. In other words, the levels of auxins and nutrients for callus induction are cultivar specific. To date, Golden Promise has been well-known for its high tissue culture ability, and hence this genotype has been widely used in barley tissue transformation (Wan and Lemaux, 1994 1994 ; Tingay et al., 1997 1997 ; Holme et al., 2006 2006 ; Kumlehn et al., 2006 2006 ). In the current study, several genotypes were explored to establish callus induction and plantlet regeneration system. The results showed that genotype-specific dependency is universal and inevitable, and callus induction and plantlet regeneration vary with genotype. Therefore, the medium formulations have to be optimized according to the given genotype. However, Zaoshu 3 showed particularly high stability in callus induction over the different media and higher regenerability compared to Golden Promise, indicating its potential utilization in transgenic systems because of its reasonable agronomic traits and wide adaptation. 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Callus induction and in vitro regeneration from barley mature embryos 10.1631/jzus.B1000219 Project supported by the Ministry of Science and Technology of China (No. 2006BAD02B04-3), the Ministry of Agriculture of China (Nos. N20070331-01 and 2008zx08010-003), and the National Natural Science Foundation of China (No. 30630047) 2010-06-09 2011-03-31 2010-10-20 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 12