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Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines
Agricultural Sciences in China
April 2011
2011, 10(4): 490-498
Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou
Waxy Maize Inbred Lines ZHONG De-yi1, 3, ZHU You-yin1, 2, LIU qian1, 3, ZHOU ti1, 3 and ZHAO De-gang1, 2 Guizhou Key Laboratory of Agro-Bioengineering, Guizhou University, Guiyang 550025, P.R.China Laboratory of Green Pesticide and Agricultural Biengineering, Ministry of Education/Guizhou University, Guiyang 550025, P.R.China 3 College of Life Sciences, Guizhou University, Guiyang 550025 P.R.China 1 2
Abstract Immature embryos from three elite Guizhou waxy maize inbred lines (W21019, B7, and QCL5036) were evaluated for their ability of forming callus and regeneration into plants. Immature embryos harvested at different physiological stages were used as explants to initiate callus on N6 basal medium with 0-3.5 mg L-1 of 2,4-dichlorophenoxy acetic acid (2,4-D). The concentration of 2,4-D, physiological age of immature embryos and genotype had a significant effect (P<0.05) on the percentage of embryogenic callus formed. The optimum 2,4-D concentration for the initiation of embryogenic callus was varied among the waxy maize genotypes from 2.0 mg L-1 (B7 and QCL5036) to 3.0 mg L-1 (W21019). The shoots were generated from embryogenic callus which were transferred into the regeneration medium supplemented with 0-2.5 mg L-1 of 6-benzylaminopurine (6-BA). 6-BA in the medium significantly promoted the regeneration of embryogenic callus. Embryogenic size was also an important factor that affected regeneration capacity. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest number. The ability of the plant regeneration was also affected by genotype. W21019 had the highest number of shoots formed per primary embryogenic callus. With the optimum condition, the highest mean number of shoots per primary callus was up to 12.13, 5.73, and 3.33 in line W21019, B7, and QCL5036, respectively. The successful regeneration of the two inbred lines provides a basis for development of genetic transformation to improve priority traits such as enhanced insects and drought tolerance. Key words: waxy maize, immature embryos, embryogenic callus, regeneration, Zea mays L.
INTRODUCTION The importance of maize (Zea mays L.) has long been critical to our understanding in the world. Maize expresses its own history not only in its genetic makeup but in its major agricultural and economic contributions. By the year 2020, maize will surpass both rice and wheat in global demand (Pingali and Pandey 2001). In recent Recceived 22 April, 2010
years, special maize is needed in China to meet market demand. One significant change is that waxy maize as a vegetable and a process crop has become more popular, especially in South China (Jeff and Sarah 2009). Nowadays, about 80% of waxy maize is distributed in southwestern areas, particularly in Yunnan, Guangxi and Guizhou which are referred to the genetic diversity centre for waxy maize (Tian et al. 2009). The track record of maize biotechnology dates back
Accepted 4 June, 2010
ZHONG De-yi, MSc candidate, Tel: +86-851-3865027, E-mail: [email protected]; Correspondence ZHAO De-gang, Professor, Tel: +86-851-8292056, E-mail: [email protected] © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60029-1
Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines
to the mid-1970s (Frank and Christian 2010), with the first documented regeneration of maize plants (Green and Phillips 1975). Since then, maize regeneration has been reported from immature embryos (Duncan et al. 1985; Bohorova et al. 1995; Aguado-Santacruz et al. 2007; Binott et al. 2008; Ombori et al. 2008), mature embryos (Huang and Wei 2004; Abebe et al. 2008; Zhao et al. 2008), split seed (Al-Abed et al. 2006), nodal regions (Vladimir et al. 2006), leaf tissues (Zhang et al. 1997; Du and Wang 2001; Ahmadabadi et al. 2007), anthers (Ting et al. 1981; Katalin et al. 2005; He et al. 2006), tassel and ear meristems (Pareddy and Petolino 1990; Zhong et al.1992), protoplast (Morocz et al. 1990), and shoot apical meristems (Zhang et al. 2002, Sairam et al. 2003; Li et al. 2004; Chen et al. 2006). However, immature embryos remain the most popular choice for producing embryogenic callus (Alan and Brian 2009) and genetic transformation. A variety of basal mediums including MS (Murashige and Skoog 1962), N6 (Chu et al. 1975), N6/B5 combination (Duncan et al. 1985), and LS (Linsmaier and Skoog 1965) medium have been used to initiate maize callus cultures and regenerate plants. Growth regulators such as 2,4-D (2,4-dichlorophenoxyacetic acid) or dicamba (3,6-dichloro-o-anisic acid) have been used to initiate maize callus cultures from various genotypes. Similarly, cytokinin (benzylaminopurine, kinetin or thidiazuron) might promote development and germination of somatic embryos (Zhao et al. 2008). Amino acids and silver nitrate (AgNO3) are also important components of maize tissue culture medium. With the progress of plant genomic studies, high throughput transformation systems are one of the critical technologies for maize breeding (Zhao et al. 2001). However, maize transformation is largely dependent on availability in vitro regeneration protocols that are amenable for use. Most of the genotypes fail to produce embryogenic callus from explants or regenerate since they are recalcitrant in vitro response. The regeneration efficiency of immature embryo-derived callus is relatively low and more recalcitrant to tissue culture than normal dent maize in waxy maize inbred lines, as it has been indicated in the limited number of reports (Luo and Wu 2003; Liang et al. 2007; He et al. 2009b). Therefore, for practical purpose, the present study was conducted to find the maize genotypes and optimize
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those factors that typically affect in vitro regeneration potential. Our results provide a helpful basis for expanding the success for regeneration and transformation of waxy maize inbred lines.
MATERIALS AND METHODS Plant materials Three elite Guizhou waxy maize inbred lines W21019, B7 and QCL5036 were used in the study. Immature ears of the self pollinated waxy maize were harvested 10 to 13 d after pollination. Ears were stored up to 3 d at 4°C before dissection when necessary (Frame et al. 2002).
Embryo dissection The 4-6 ears were placed into a sterilized container in laminar flow beach. 75% ethanol was then added to cover ears for 30 s. Then, the ears were sterilized for 8 min with 0.1% (w/v) mercuric chloride (HgCl2). During this disinfection, the containers were occasionally shaken and forcep was used to dislodge air bubbles for surface sterilization of ear effectively. Subsequently, the ears were rinsed five times with sterilized distilled water. Aseptic immature embryos were dissected from ear and placed with scutellar side up and flat surface down on the callus induction medium for callus induction. Plates were wrapped with Parafilm and incubated in the dark at 28°C.
Culture medium The components of basal medium are described in Table 1. The pH of the medium was adjusted to 5.8 with NaOH and/or HCl, then 8.5 g L -1 agar was added, and the medium was sterilized by autoclaving for 20 min. AgNO 3 and N6 vitamins were added to the medium after autoclaving.
Callus induction and embryogenic callus formation Various levels of 2,4-D (0, 1.0, 1.5, 2.0, 3.0, and 3.5 mg L-1)
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Table 1 Composition of medium used Medium Induction medium Proliferation medium Regeneration medium Rooting medium 1)
Composition1) N6 salts and vitamins, 2,4-D 0.0-3.5 mg L , inositol 100 mg L-1, CH 100 mg L-1, AgNO3 5 mg L-1, L-proline 0.69 g L-1, sucrose 30 g L-1 N6 salts and vitamins, 2,4-D 0.0-3.5 mg L-1, inositol 100 mg L-1, CH 100 mg L-1, AgNO3 10 mg L-1, L-proline 2.88 g L -1, sucrose 30 g L-1 N6 salts and vitamins, 6-BA 0.0-2.5 mg L-1, inositol 100 mg L-1, CH 100 mg L-1, L-proline 2.88 g L-1, sucrose 30 g L-1 N6 salts and vitamins, IBA 0.5 mg L-1, inositol 100 mg L-1, CH 100 mg L-1, L-proline 0.69 g L-1, sucrose 30 g L-1 -1
Vitamins and AgNO3 stocks were filter-sterilized and stored at 4°C. CH, casein hydrolysate.
were added to determine the effect of this auxin on induction efficiently of embryogenic callus. Similarly, ears harvested at different physiological stages were used. Due to the special environment in Guiyang, various size of immature embryos (1.0-3.0 mm in length) instead of days after pollination (DAP) were cultured. The experiment was replicated 3 times. Embryogenic calli were visible as early as 2 wk after induction, and number of explants (in percentage) producing primary callus was recorded in each genotype. Then the calli were sub-cultured onto proliferation medium for every 2 wk. After 4 wk incubation on proliferation medium, the number of the callus that converted to embryogenic callus was recorded in each genotype.
Regeneration Embryogenic calli were transferred onto regeneration medium supplemented with 0.0, 0.5, 1.0, 1.5, and 2.5 mg L-1 6-benzylaminopurine (6-BA). In addition, embryogenic calli were divided into small pieces (3.0, 3.05.0, 5.0-7.0, and 7.0-10.0 mm in length), and were subsequently placed onto regeneration medium. Regenerating cultures were incubated at 16 h photoperiod and 28°C. Plantlet shoots were arose from somatic embryos after 2-3 wk, the number of shoots formed per culture was recorded after 3 wk of culture. Regenerated shoots were transferred to rooting medium for 2 wk. Well-rooted plantlets (Fig. 1-F) from Magenta box were rinsed with water to remove the medium and then transferred into pots containing peat moss.
Statistical analysis Statistical assays were carried out by the SPSS software. An analysis of variance (ANOVA) was performed for each experiment. The Duncan’s multiple range test (P=0.05) was used.
RESULTS Embryogenic callus formation Effects of 2,4-D on embryogenic callus inductions The results have shown that 2,4-D is an important factor in the initiation of primary callus and maintenance of embryogenic callus from immature embryos of maize. In our research, different levels of 2,4-D was evaluated to study the effect on embryogenic callus induction of waxy maize inbred lines. Embryos were readily germinated to form shoots and roots, and no primary callus was formed on induction medium devoid of 2,4-D (Fig. 1-A). The concentration of 2,4-D had a significant effect on the percentage of the embryogenic callus (P<0.05) (Table 2). The optimum 2,4-D concentration for the initiation of embryogenic callus varied among the waxy maize genotypes 2.0 mg L-1 (B7 and QCL5036) and 3.0 mg L-1 (W21019) (Table 2). Non-embryogenic callus which was soft, watery and yellow or white in color was also formed, and turned brown in subsequent subculture. With further increasing of 2,4-D concentration, the callus become brownish. After 4-6 d of culture, the scutellum was swelled and primary callus was formed on the surface of scutellum (Fig. 1-B, C). Within 7 d of incubation, the swollen scutellum was developed into irregular callus. Small globular somatic embryos were seen at the surface of the soft friable callus (Fig. 1-D). During the first 14 d of culture on induction medium, the zygotic embryos of line W21019 responded similar to lines B7 and QCL5036. Embryogenic callus maintenance required bi-weekly subculture on proliferation medium. Among the three waxy maize genotypes investigated in this study, inbred line QCL5036 yielded the highest frequency (94.05%) of embryogenic callus induction, but the size of embryogenic calli was only 0.4-0.5 cm in comparison with the size of W21019 (0.9-1.5 cm) and B7 (0.8-1.3 cm) after 6 wk culture.
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Fig. 1 Embryogenesis and regeneration of plantlet from immature embryos of waxy maize inbred line, B7. A, embryos germinated to form shoots and roots on induction medium devoid of 2,4-D. B, embryogenic callus was induced on callus induction medium supplemented with 1.0 mg L-1 2,4-D. C, callus was induced on callus induction medium supplemented with 2.0 mg L-1 2,4-D. D, globular embryos induced on proliferation medium. E, germination of somatic embryo and formation of green shoots of embryogenic callus on regeneration medium. F, plantlet with healthy roots on rooting medium in Magenta box. Table 2 Effects of 2,4-D on embryogenic callus inductions from immature embryos (IEs) of three waxy maize elite inbred lines W21019 Conc. of 2,4-D (mg L-1 ) 0.0 1.0 1.5 2.0 3.0 3.5
QCL5036
B7
No. of IEs cultured
No. of embryogenic callus
No. of IEs cultured
No. of embryogenic callus
No. of IEs cultured
No. of embryogenic callus
70 69 77 76 79 67
0 (0.00%) 51 (73.91%) 61 (79.22%) 65 (85.53%) 70 (88.61%) 55 (82.09%)
65 73 75 84 75 72
0 (0.00%) 66 (90.41%) 56 (74.67%) 79 (94.05%) 68 (90.67%) 61 (84.72%)
68 75 63 76 80 93
0 (0.00%) 65 (86.67%) 58 (92.06%) 70 (92.11%) 65 (81.25%) 83 (89.25%)
Embryogenic callus induction frequency was defined as the number of IEs that produces embryogenic callus at the end of 4 wk of induction on medium relative to the total number of IEs cultured.
Effect of immature embryo physiological age on callus induction Calli were induced from embryos excised from ears at different physiological ages. Embryo size didn’t contribute significantly to differences in callus induction among inbred lines (P<0.05) (Fig. 2). However, both quality and size of callus induced from immature embryos (0.7-1.0 mm and 2.0-3.0 mm long) were lower than that of immature embryos which were 1.0-2.0 mm long. When the embryos were 0.71.0 mm long, callus initiation was slow or even no re-
sponse and the embryos gradually died. When the embryos were 2.0-3.0 mm long, the embryos gradually glowed and induced callus often turned soft, loose and brown.
Regeneration of embryogenic callus Effect of 6-BA on regeneration capacity from embryogenic callus After embryogenic calli were transferred to regeneration medium, plantlet regeneration oc-
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Fig. 2 Effect of embryo size on frequencies of callus induction from immature embryos (IEs) of three inbred lines after 4 wk culture on medium supplemented with 2.0 mg L-1 2,4-D. Experiments were performed three times. Columns denoted by different letters are significantly different at P<0.05 according to Duncan’s multiple range test. Vertical bars represent standard errors for three sets of the results. The same as below.
curred within 15-20 d (Fig. 1-E). 6-BA at 0.5 mg L-1 appears to be the optimal level, showing the highest regeneration frequency, 92.31% in line W21019, while in line B7 and QCL5036, the highest frequencies were 89.29 and 84.21% using 6-BA at 1.0 and 1.5 mg L-1, respectively. Using the embryogenic callus of three waxy maize inbred lines, plantlets were regenerated and the frequencies were ranged from 72.41 to 92.31% (Fig. 3). Moreover, the number of shoots formed by W21019 was significantly (P<0.05) higher compared
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to those formed by line B7 and QCL5036 (Fig. 4). Analysis of variance suggested highly significant effect of genotype on both shoot percentage and shoot number per callus. This genotype dependence is likely to be due to the genetic diversity caused by helitron transposable elements (Buckler et al. 2006). In this study, in terms of establishment of complete plants from embryogenic callus, W21019 was the best genotype followed by B7. Though embryogenic callus induction percentage in QCL5036 was higher than in B7, both shoot percentage and shoot number per callus were lower than B7, allowing more established plantlets. Effect of callus size on regeneration capacity from embryogenic callus Embryogenic calli were divided into small pieces (3.0, 3.0-5.0, 5.0-7.0, and 7.0-10.0 mm in length), and were subsequently placed onto regeneration medium supplemented with 0.5 mg L-1 6-BA. Significant differences (P<0.05) were detected on the effect of callus size on the number of shoots formed per primary embryogenic callus in three inbred lines. Though increase in callus size caused an increase in the mean number shoots per secondary callus (Fig. 5-A), the situation in the mean number shoots per primary embryogenic callus was different. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest
Fig. 3 Effects of 6-BA in regeneration medium on shoot regeneration from the embryogenic callus of three inbred lines after 6 wk of culture.
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Fig. 4 Effect of 6-BA on mean number of shoots per embryogenic callus.
number. In line W21019, B7 and QCL5036, the highest mean number of shoots per primary callus were up to 12.13, 5.73 and 3.33, respectively (Fig. 5-B). In comparison with embryogenic calli which were not divided, the mean number of shoots per callus were 5. 08, 2.48 and 1.37, respectively (Fig. 4).
generation from different genotype inbred lines are in progress. In the present study, it was established that the opti-
DISCUSSION Tissue culture systems are vital to many areas of maize research and improvement, particularly in mutant selection (Guo and Zhang 1992; Gao et al. 1994; Wang et al. 2005; Liu et al. 2006; He et al. 2009a) and plant transformation (Liang et al. 2007). The ability to regenerate shoots from callus is essential for establishing a successful plant culture system. The success of regeneration procedure is affected predominantly by genotype, explant materials and medium composition such as plant regulators, amino acids, silver nitrate, and other antioxidants. It has been found that embryogenic callus induction and plantlet regeneration were significantly associated with the genotypes (Zhao et al. 2008), particularly in the inbred lines which showed a lower percentage of green shoots and regenerated plantlets. The present studies have confirmed that callus induction, embryogenic callus formation and plant regeneration were genotype independent. Further studies to optimize re-
Fig. 5 Effect of callus size on frequencies of regeneration capacity from the embryogenic callus of three inbred lines after 6 wk of culture. A, mean no. of shoots per secondary callus. B, mean no. of shoots per primary embryogenic callus.
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mal explant material for callus induction and regeneration was immature embryo in the range of 8-13 DAP. This is true for waxy maize tested in this study. The results showed that the embryos which were 1.0-2.0 mm long (11-13 DAP) were critical for callus induction and embryogenic callus formation. However, this result is contrary to Binott et al. (2008). According to Bohorova et al. (1995), immature embryos of maize less than 0.5 mm in length did not respond in culture. Plant regulators play the most important role in callus culture. Generally, medium for induction and proliferation of maize callus requires a strong auxin such as 2,4-D. Che et al. (2006) found that gene expression patterns change extensively during somatic embryo maturation and germination. There is a progressive decline in the expression of genes involved in cell proliferation and growth, such as genes encoding histones and ribosomal proteins throughout embryo maturation when the callus was transferred to medium lacking 2,4-D. Bronsema et al. (2001) reported that at least 0.2 mg L-1 2,4-D is needed to cross the threshold level for the transition from germination to callus induction. Other reports have confirmed the use of 1-3 mg L-1 2,4D to induce callus from maize immature embryos is a critical factor (Armstrong and Green 1985). The results of this research also showed that the presence of 2.0-3.0 mg L-1 2,4-D in culture medium was critical for embryogenic callus formation. The addition of cytokinins into regeneration medium may promote development and germination of somatic embryos. BA is more effective than kinetin for shoot induction. In agreement with that, the present study suggests that the addition of BA to regeneration medium significantly increased the frequency of embryogenic callus formation. In the present study, we found that adding 0.5-1.5 mg L-1 6-BA into regeneration medium was more efficient than regeneration medium without 6-BA. However, in comparison with other studies on maize immature embryo culture where multiple shoots per callus were recovered (Sujay et al. 2010), the shoots per callus in this study were rather high except in QCL5036. Amino acids and silver nitrate (AgNO3) are important components of maize tissue culture medium. The beneficial effects of L-proline were first reported by Armstrong and Green (1985). The later experiments
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indicated that proline had a stimulating effect on formation of embryogenic callus but was not essential for callus growth during the time of the experiment (Zhao et al. 2008). AgNO3 has been shown to affect ethylene action by competing for its binding site but without affecting its biosynthesis. Songstad et al. (1991) evaluated AgNO3 in promoting plant regeneration using immature embryos of elite inbred B73. Songstad et al. (1992) also reported beneficial effects of AgNO3 on the type II callus response from cultured immature tassel explants, indicating that explants other than immature embryos responded favorably to blocking ethylene action (Huang and Wei 2004). In the present study, 5 mg L-1 AgNO3 and 0.69 g L-1 L-proline were used in induction medium and 10 mg L-1 AgNO3 and 2.88 g L-1 L-proline were used in proliferation medium. The regeneration method reported here is quick, efficient and highly reproducible and perhaps could be used in transformation studies. Thus, the established regeneration protocol for inbred lines W21019 and B7 might make it possible to use the protocol in transformation studies towards development of genetically modified waxy maize readily adapted to Guizhou’s condition.
Acknowledgements This study was supported by the Genetically Modified Organisms Special Projects in Guizhou Province of China (2004 NZ004), the National Key Technology R&D Program (2007BAD59B) and the Genetically Modified Organisms Breeding Major Projects (2008ZX08010-003). We thank the Institute of Upland Food Crops, Guizhou Academy of Agricultural Sciences and Guizhou Center of Maize Engineering Techniques, China, for providing the waxy maize seeds.
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Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines
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Sairam R V, Paran M, Franklin G, Lifeng Z, Smith B, MacDougall J, Wilber C, Sheikhi H, Kashikar N, Meeker K, et al. 2003.
Reports, 21, 263-270. Zhao C H, Zhang L J, Ge C, Hu K. 2008. Establishment and optimization of the regeneration system of mature embryos
Shoot meristem an ideal explant for Zea mays L. transformation. Genome, 46, 323-329. Songstad D D, Armstrong C L, Peterson W L. 1991. Silver nitrate increases type II callus production from immature embryos of maize inbred B73 and its derivatives. Plant Cell Reports, 9, 699-702. Songstad D D, Peterson W L, Armstrong C L. 1992. Establishment of friable embryogenic (type II) callus from immature tassels of Zea mays (Poaceae). American Journal of Botany, 79, 761764. Sujay R, Zerka R, Sekhar J C, Fatma T, Sain D. 2010. Callus
of maize (Zea mays L.). Agricultural Sciences in China, 7, 1046-1051. Zhao Z Y, Gu W N, Cai T S, Laura T, David H, Diane B, Sheryl S, Marjorie R, Dottie P. 2001. High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Molecular Breeding, 8, 323-333. Zhong H, Srinivasan C, Mariam B S. 1992. In-vitro morphogenesis of corn (Zea mays L.): II. Differentiation of ear and tassel clusters from cultured shoot apices and immature inflorescences. Planta, 187, 490-497. (Managing editor SUN Lu-juan)
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2011, 10(4): 490-498
Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou
Waxy Maize Inbred Lines ZHONG De-yi1, 3, ZHU You-yin1, 2, LIU qian1, 3, ZHOU ti1, 3 and ZHAO De-gang1, 2 Guizhou Key Laboratory of Agro-Bioengineering, Guizhou University, Guiyang 550025, P.R.China Laboratory of Green Pesticide and Agricultural Biengineering, Ministry of Education/Guizhou University, Guiyang 550025, P.R.China 3 College of Life Sciences, Guizhou University, Guiyang 550025 P.R.China 1 2
Abstract Immature embryos from three elite Guizhou waxy maize inbred lines (W21019, B7, and QCL5036) were evaluated for their ability of forming callus and regeneration into plants. Immature embryos harvested at different physiological stages were used as explants to initiate callus on N6 basal medium with 0-3.5 mg L-1 of 2,4-dichlorophenoxy acetic acid (2,4-D). The concentration of 2,4-D, physiological age of immature embryos and genotype had a significant effect (P<0.05) on the percentage of embryogenic callus formed. The optimum 2,4-D concentration for the initiation of embryogenic callus was varied among the waxy maize genotypes from 2.0 mg L-1 (B7 and QCL5036) to 3.0 mg L-1 (W21019). The shoots were generated from embryogenic callus which were transferred into the regeneration medium supplemented with 0-2.5 mg L-1 of 6-benzylaminopurine (6-BA). 6-BA in the medium significantly promoted the regeneration of embryogenic callus. Embryogenic size was also an important factor that affected regeneration capacity. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest number. The ability of the plant regeneration was also affected by genotype. W21019 had the highest number of shoots formed per primary embryogenic callus. With the optimum condition, the highest mean number of shoots per primary callus was up to 12.13, 5.73, and 3.33 in line W21019, B7, and QCL5036, respectively. The successful regeneration of the two inbred lines provides a basis for development of genetic transformation to improve priority traits such as enhanced insects and drought tolerance. Key words: waxy maize, immature embryos, embryogenic callus, regeneration, Zea mays L.
INTRODUCTION The importance of maize (Zea mays L.) has long been critical to our understanding in the world. Maize expresses its own history not only in its genetic makeup but in its major agricultural and economic contributions. By the year 2020, maize will surpass both rice and wheat in global demand (Pingali and Pandey 2001). In recent Recceived 22 April, 2010
years, special maize is needed in China to meet market demand. One significant change is that waxy maize as a vegetable and a process crop has become more popular, especially in South China (Jeff and Sarah 2009). Nowadays, about 80% of waxy maize is distributed in southwestern areas, particularly in Yunnan, Guangxi and Guizhou which are referred to the genetic diversity centre for waxy maize (Tian et al. 2009). The track record of maize biotechnology dates back
Accepted 4 June, 2010
ZHONG De-yi, MSc candidate, Tel: +86-851-3865027, E-mail: [email protected]; Correspondence ZHAO De-gang, Professor, Tel: +86-851-8292056, E-mail: [email protected] © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60029-1
Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines
to the mid-1970s (Frank and Christian 2010), with the first documented regeneration of maize plants (Green and Phillips 1975). Since then, maize regeneration has been reported from immature embryos (Duncan et al. 1985; Bohorova et al. 1995; Aguado-Santacruz et al. 2007; Binott et al. 2008; Ombori et al. 2008), mature embryos (Huang and Wei 2004; Abebe et al. 2008; Zhao et al. 2008), split seed (Al-Abed et al. 2006), nodal regions (Vladimir et al. 2006), leaf tissues (Zhang et al. 1997; Du and Wang 2001; Ahmadabadi et al. 2007), anthers (Ting et al. 1981; Katalin et al. 2005; He et al. 2006), tassel and ear meristems (Pareddy and Petolino 1990; Zhong et al.1992), protoplast (Morocz et al. 1990), and shoot apical meristems (Zhang et al. 2002, Sairam et al. 2003; Li et al. 2004; Chen et al. 2006). However, immature embryos remain the most popular choice for producing embryogenic callus (Alan and Brian 2009) and genetic transformation. A variety of basal mediums including MS (Murashige and Skoog 1962), N6 (Chu et al. 1975), N6/B5 combination (Duncan et al. 1985), and LS (Linsmaier and Skoog 1965) medium have been used to initiate maize callus cultures and regenerate plants. Growth regulators such as 2,4-D (2,4-dichlorophenoxyacetic acid) or dicamba (3,6-dichloro-o-anisic acid) have been used to initiate maize callus cultures from various genotypes. Similarly, cytokinin (benzylaminopurine, kinetin or thidiazuron) might promote development and germination of somatic embryos (Zhao et al. 2008). Amino acids and silver nitrate (AgNO3) are also important components of maize tissue culture medium. With the progress of plant genomic studies, high throughput transformation systems are one of the critical technologies for maize breeding (Zhao et al. 2001). However, maize transformation is largely dependent on availability in vitro regeneration protocols that are amenable for use. Most of the genotypes fail to produce embryogenic callus from explants or regenerate since they are recalcitrant in vitro response. The regeneration efficiency of immature embryo-derived callus is relatively low and more recalcitrant to tissue culture than normal dent maize in waxy maize inbred lines, as it has been indicated in the limited number of reports (Luo and Wu 2003; Liang et al. 2007; He et al. 2009b). Therefore, for practical purpose, the present study was conducted to find the maize genotypes and optimize
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those factors that typically affect in vitro regeneration potential. Our results provide a helpful basis for expanding the success for regeneration and transformation of waxy maize inbred lines.
MATERIALS AND METHODS Plant materials Three elite Guizhou waxy maize inbred lines W21019, B7 and QCL5036 were used in the study. Immature ears of the self pollinated waxy maize were harvested 10 to 13 d after pollination. Ears were stored up to 3 d at 4°C before dissection when necessary (Frame et al. 2002).
Embryo dissection The 4-6 ears were placed into a sterilized container in laminar flow beach. 75% ethanol was then added to cover ears for 30 s. Then, the ears were sterilized for 8 min with 0.1% (w/v) mercuric chloride (HgCl2). During this disinfection, the containers were occasionally shaken and forcep was used to dislodge air bubbles for surface sterilization of ear effectively. Subsequently, the ears were rinsed five times with sterilized distilled water. Aseptic immature embryos were dissected from ear and placed with scutellar side up and flat surface down on the callus induction medium for callus induction. Plates were wrapped with Parafilm and incubated in the dark at 28°C.
Culture medium The components of basal medium are described in Table 1. The pH of the medium was adjusted to 5.8 with NaOH and/or HCl, then 8.5 g L -1 agar was added, and the medium was sterilized by autoclaving for 20 min. AgNO 3 and N6 vitamins were added to the medium after autoclaving.
Callus induction and embryogenic callus formation Various levels of 2,4-D (0, 1.0, 1.5, 2.0, 3.0, and 3.5 mg L-1)
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Table 1 Composition of medium used Medium Induction medium Proliferation medium Regeneration medium Rooting medium 1)
Composition1) N6 salts and vitamins, 2,4-D 0.0-3.5 mg L , inositol 100 mg L-1, CH 100 mg L-1, AgNO3 5 mg L-1, L-proline 0.69 g L-1, sucrose 30 g L-1 N6 salts and vitamins, 2,4-D 0.0-3.5 mg L-1, inositol 100 mg L-1, CH 100 mg L-1, AgNO3 10 mg L-1, L-proline 2.88 g L -1, sucrose 30 g L-1 N6 salts and vitamins, 6-BA 0.0-2.5 mg L-1, inositol 100 mg L-1, CH 100 mg L-1, L-proline 2.88 g L-1, sucrose 30 g L-1 N6 salts and vitamins, IBA 0.5 mg L-1, inositol 100 mg L-1, CH 100 mg L-1, L-proline 0.69 g L-1, sucrose 30 g L-1 -1
Vitamins and AgNO3 stocks were filter-sterilized and stored at 4°C. CH, casein hydrolysate.
were added to determine the effect of this auxin on induction efficiently of embryogenic callus. Similarly, ears harvested at different physiological stages were used. Due to the special environment in Guiyang, various size of immature embryos (1.0-3.0 mm in length) instead of days after pollination (DAP) were cultured. The experiment was replicated 3 times. Embryogenic calli were visible as early as 2 wk after induction, and number of explants (in percentage) producing primary callus was recorded in each genotype. Then the calli were sub-cultured onto proliferation medium for every 2 wk. After 4 wk incubation on proliferation medium, the number of the callus that converted to embryogenic callus was recorded in each genotype.
Regeneration Embryogenic calli were transferred onto regeneration medium supplemented with 0.0, 0.5, 1.0, 1.5, and 2.5 mg L-1 6-benzylaminopurine (6-BA). In addition, embryogenic calli were divided into small pieces (3.0, 3.05.0, 5.0-7.0, and 7.0-10.0 mm in length), and were subsequently placed onto regeneration medium. Regenerating cultures were incubated at 16 h photoperiod and 28°C. Plantlet shoots were arose from somatic embryos after 2-3 wk, the number of shoots formed per culture was recorded after 3 wk of culture. Regenerated shoots were transferred to rooting medium for 2 wk. Well-rooted plantlets (Fig. 1-F) from Magenta box were rinsed with water to remove the medium and then transferred into pots containing peat moss.
Statistical analysis Statistical assays were carried out by the SPSS software. An analysis of variance (ANOVA) was performed for each experiment. The Duncan’s multiple range test (P=0.05) was used.
RESULTS Embryogenic callus formation Effects of 2,4-D on embryogenic callus inductions The results have shown that 2,4-D is an important factor in the initiation of primary callus and maintenance of embryogenic callus from immature embryos of maize. In our research, different levels of 2,4-D was evaluated to study the effect on embryogenic callus induction of waxy maize inbred lines. Embryos were readily germinated to form shoots and roots, and no primary callus was formed on induction medium devoid of 2,4-D (Fig. 1-A). The concentration of 2,4-D had a significant effect on the percentage of the embryogenic callus (P<0.05) (Table 2). The optimum 2,4-D concentration for the initiation of embryogenic callus varied among the waxy maize genotypes 2.0 mg L-1 (B7 and QCL5036) and 3.0 mg L-1 (W21019) (Table 2). Non-embryogenic callus which was soft, watery and yellow or white in color was also formed, and turned brown in subsequent subculture. With further increasing of 2,4-D concentration, the callus become brownish. After 4-6 d of culture, the scutellum was swelled and primary callus was formed on the surface of scutellum (Fig. 1-B, C). Within 7 d of incubation, the swollen scutellum was developed into irregular callus. Small globular somatic embryos were seen at the surface of the soft friable callus (Fig. 1-D). During the first 14 d of culture on induction medium, the zygotic embryos of line W21019 responded similar to lines B7 and QCL5036. Embryogenic callus maintenance required bi-weekly subculture on proliferation medium. Among the three waxy maize genotypes investigated in this study, inbred line QCL5036 yielded the highest frequency (94.05%) of embryogenic callus induction, but the size of embryogenic calli was only 0.4-0.5 cm in comparison with the size of W21019 (0.9-1.5 cm) and B7 (0.8-1.3 cm) after 6 wk culture.
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Fig. 1 Embryogenesis and regeneration of plantlet from immature embryos of waxy maize inbred line, B7. A, embryos germinated to form shoots and roots on induction medium devoid of 2,4-D. B, embryogenic callus was induced on callus induction medium supplemented with 1.0 mg L-1 2,4-D. C, callus was induced on callus induction medium supplemented with 2.0 mg L-1 2,4-D. D, globular embryos induced on proliferation medium. E, germination of somatic embryo and formation of green shoots of embryogenic callus on regeneration medium. F, plantlet with healthy roots on rooting medium in Magenta box. Table 2 Effects of 2,4-D on embryogenic callus inductions from immature embryos (IEs) of three waxy maize elite inbred lines W21019 Conc. of 2,4-D (mg L-1 ) 0.0 1.0 1.5 2.0 3.0 3.5
QCL5036
B7
No. of IEs cultured
No. of embryogenic callus
No. of IEs cultured
No. of embryogenic callus
No. of IEs cultured
No. of embryogenic callus
70 69 77 76 79 67
0 (0.00%) 51 (73.91%) 61 (79.22%) 65 (85.53%) 70 (88.61%) 55 (82.09%)
65 73 75 84 75 72
0 (0.00%) 66 (90.41%) 56 (74.67%) 79 (94.05%) 68 (90.67%) 61 (84.72%)
68 75 63 76 80 93
0 (0.00%) 65 (86.67%) 58 (92.06%) 70 (92.11%) 65 (81.25%) 83 (89.25%)
Embryogenic callus induction frequency was defined as the number of IEs that produces embryogenic callus at the end of 4 wk of induction on medium relative to the total number of IEs cultured.
Effect of immature embryo physiological age on callus induction Calli were induced from embryos excised from ears at different physiological ages. Embryo size didn’t contribute significantly to differences in callus induction among inbred lines (P<0.05) (Fig. 2). However, both quality and size of callus induced from immature embryos (0.7-1.0 mm and 2.0-3.0 mm long) were lower than that of immature embryos which were 1.0-2.0 mm long. When the embryos were 0.71.0 mm long, callus initiation was slow or even no re-
sponse and the embryos gradually died. When the embryos were 2.0-3.0 mm long, the embryos gradually glowed and induced callus often turned soft, loose and brown.
Regeneration of embryogenic callus Effect of 6-BA on regeneration capacity from embryogenic callus After embryogenic calli were transferred to regeneration medium, plantlet regeneration oc-
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Fig. 2 Effect of embryo size on frequencies of callus induction from immature embryos (IEs) of three inbred lines after 4 wk culture on medium supplemented with 2.0 mg L-1 2,4-D. Experiments were performed three times. Columns denoted by different letters are significantly different at P<0.05 according to Duncan’s multiple range test. Vertical bars represent standard errors for three sets of the results. The same as below.
curred within 15-20 d (Fig. 1-E). 6-BA at 0.5 mg L-1 appears to be the optimal level, showing the highest regeneration frequency, 92.31% in line W21019, while in line B7 and QCL5036, the highest frequencies were 89.29 and 84.21% using 6-BA at 1.0 and 1.5 mg L-1, respectively. Using the embryogenic callus of three waxy maize inbred lines, plantlets were regenerated and the frequencies were ranged from 72.41 to 92.31% (Fig. 3). Moreover, the number of shoots formed by W21019 was significantly (P<0.05) higher compared
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to those formed by line B7 and QCL5036 (Fig. 4). Analysis of variance suggested highly significant effect of genotype on both shoot percentage and shoot number per callus. This genotype dependence is likely to be due to the genetic diversity caused by helitron transposable elements (Buckler et al. 2006). In this study, in terms of establishment of complete plants from embryogenic callus, W21019 was the best genotype followed by B7. Though embryogenic callus induction percentage in QCL5036 was higher than in B7, both shoot percentage and shoot number per callus were lower than B7, allowing more established plantlets. Effect of callus size on regeneration capacity from embryogenic callus Embryogenic calli were divided into small pieces (3.0, 3.0-5.0, 5.0-7.0, and 7.0-10.0 mm in length), and were subsequently placed onto regeneration medium supplemented with 0.5 mg L-1 6-BA. Significant differences (P<0.05) were detected on the effect of callus size on the number of shoots formed per primary embryogenic callus in three inbred lines. Though increase in callus size caused an increase in the mean number shoots per secondary callus (Fig. 5-A), the situation in the mean number shoots per primary embryogenic callus was different. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest
Fig. 3 Effects of 6-BA in regeneration medium on shoot regeneration from the embryogenic callus of three inbred lines after 6 wk of culture.
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Fig. 4 Effect of 6-BA on mean number of shoots per embryogenic callus.
number. In line W21019, B7 and QCL5036, the highest mean number of shoots per primary callus were up to 12.13, 5.73 and 3.33, respectively (Fig. 5-B). In comparison with embryogenic calli which were not divided, the mean number of shoots per callus were 5. 08, 2.48 and 1.37, respectively (Fig. 4).
generation from different genotype inbred lines are in progress. In the present study, it was established that the opti-
DISCUSSION Tissue culture systems are vital to many areas of maize research and improvement, particularly in mutant selection (Guo and Zhang 1992; Gao et al. 1994; Wang et al. 2005; Liu et al. 2006; He et al. 2009a) and plant transformation (Liang et al. 2007). The ability to regenerate shoots from callus is essential for establishing a successful plant culture system. The success of regeneration procedure is affected predominantly by genotype, explant materials and medium composition such as plant regulators, amino acids, silver nitrate, and other antioxidants. It has been found that embryogenic callus induction and plantlet regeneration were significantly associated with the genotypes (Zhao et al. 2008), particularly in the inbred lines which showed a lower percentage of green shoots and regenerated plantlets. The present studies have confirmed that callus induction, embryogenic callus formation and plant regeneration were genotype independent. Further studies to optimize re-
Fig. 5 Effect of callus size on frequencies of regeneration capacity from the embryogenic callus of three inbred lines after 6 wk of culture. A, mean no. of shoots per secondary callus. B, mean no. of shoots per primary embryogenic callus.
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mal explant material for callus induction and regeneration was immature embryo in the range of 8-13 DAP. This is true for waxy maize tested in this study. The results showed that the embryos which were 1.0-2.0 mm long (11-13 DAP) were critical for callus induction and embryogenic callus formation. However, this result is contrary to Binott et al. (2008). According to Bohorova et al. (1995), immature embryos of maize less than 0.5 mm in length did not respond in culture. Plant regulators play the most important role in callus culture. Generally, medium for induction and proliferation of maize callus requires a strong auxin such as 2,4-D. Che et al. (2006) found that gene expression patterns change extensively during somatic embryo maturation and germination. There is a progressive decline in the expression of genes involved in cell proliferation and growth, such as genes encoding histones and ribosomal proteins throughout embryo maturation when the callus was transferred to medium lacking 2,4-D. Bronsema et al. (2001) reported that at least 0.2 mg L-1 2,4-D is needed to cross the threshold level for the transition from germination to callus induction. Other reports have confirmed the use of 1-3 mg L-1 2,4D to induce callus from maize immature embryos is a critical factor (Armstrong and Green 1985). The results of this research also showed that the presence of 2.0-3.0 mg L-1 2,4-D in culture medium was critical for embryogenic callus formation. The addition of cytokinins into regeneration medium may promote development and germination of somatic embryos. BA is more effective than kinetin for shoot induction. In agreement with that, the present study suggests that the addition of BA to regeneration medium significantly increased the frequency of embryogenic callus formation. In the present study, we found that adding 0.5-1.5 mg L-1 6-BA into regeneration medium was more efficient than regeneration medium without 6-BA. However, in comparison with other studies on maize immature embryo culture where multiple shoots per callus were recovered (Sujay et al. 2010), the shoots per callus in this study were rather high except in QCL5036. Amino acids and silver nitrate (AgNO3) are important components of maize tissue culture medium. The beneficial effects of L-proline were first reported by Armstrong and Green (1985). The later experiments
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indicated that proline had a stimulating effect on formation of embryogenic callus but was not essential for callus growth during the time of the experiment (Zhao et al. 2008). AgNO3 has been shown to affect ethylene action by competing for its binding site but without affecting its biosynthesis. Songstad et al. (1991) evaluated AgNO3 in promoting plant regeneration using immature embryos of elite inbred B73. Songstad et al. (1992) also reported beneficial effects of AgNO3 on the type II callus response from cultured immature tassel explants, indicating that explants other than immature embryos responded favorably to blocking ethylene action (Huang and Wei 2004). In the present study, 5 mg L-1 AgNO3 and 0.69 g L-1 L-proline were used in induction medium and 10 mg L-1 AgNO3 and 2.88 g L-1 L-proline were used in proliferation medium. The regeneration method reported here is quick, efficient and highly reproducible and perhaps could be used in transformation studies. Thus, the established regeneration protocol for inbred lines W21019 and B7 might make it possible to use the protocol in transformation studies towards development of genetically modified waxy maize readily adapted to Guizhou’s condition.
Acknowledgements This study was supported by the Genetically Modified Organisms Special Projects in Guizhou Province of China (2004 NZ004), the National Key Technology R&D Program (2007BAD59B) and the Genetically Modified Organisms Breeding Major Projects (2008ZX08010-003). We thank the Institute of Upland Food Crops, Guizhou Academy of Agricultural Sciences and Guizhou Center of Maize Engineering Techniques, China, for providing the waxy maize seeds.
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Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines
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