In Vitro Cell.Dev.Biol.—Plant (2014) 50:1–8 DOI 10.1007/s11627-013-9544-6
BIOTECHNOLOGY
Development of an efficient regeneration and Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants Hongyan Dai & Wenran Li & Wenjuan Mao & Lei Zhang & Guofen Han & Kai Zhao & Yuexue Liu & Zhihong Zhang Received: 20 March 2013 / Accepted: 2 July 2013 / Published online: 23 July 2013 / Editor: J. Forster # The Society for In Vitro Biology 2013
Abstract Apple has become a model species for Rosaceae genetic and genomic research, but it is difficult to obtain transgenic apple plants by Agrobacterium-mediated transformation using in vitro leaves as explants. In this study, we developed an efficient regeneration and Agrobacterium-mediated transformation system for crab apple (Malus micromalus) using cotyledons as explants. The proximal cotyledons of M. micromalus, excised from seedlings that emerged from mature embryos cultured for 10–14 d in vitro, were suitable as explants for regeneration and Agrobacterium-mediated transformation. Cotyledon explants were cocultivated for 3 d with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA2301 on regeneration medium. Kanamycin-resistant buds were produced on cotyledon explants cultured on selective regeneration medium containing 20 mg/L kanamycin. Acetosyringone supplemented in the Agrobacterium suspension or in the cocultivation medium slightly enhanced the regeneration of kanamycin-resistant buds. The maximum percentage of explants with kanamycin-resistant buds was 11.7%. The putative transformed plants were confirmed by histochemical analysis of β-glucuronidase activity and the polymerase chain reaction amplification of the neomycin phosphotransferase II gene. This transformation system also enables recovery of nontransformed isogenic controls developed from embryo buds and is therefore suitable for functional genomics studies in apple. Hongyan Dai and Wenran Li contributed equally to this work. H. Dai : W. Li : W. Mao : G. Han : K. Zhao : Y. Liu : Z. Zhang (*) College of Horticulture, Shenyang Agricultural University, Dongling Road 120, Shenyang, Liaoning 110866, China e-mail:
[email protected] L. Zhang College of Bioscience and Biotechnology, Shenyang Agricultural University, Dongling Road 120, Shenyang, Liaoning 110866, China
Keywords Apple . Regeneration . Agrobacterium-mediated transformation . Cotyledon . Cocultivation time
Introduction Malus, the apple genus, includes over 30 primary species belonging to the Rosaceae family (Korban 1986). Apples were certainly one of the earliest fruits to be gathered. Ancient civilizations were growing apples at least 2,500 yr ago, and superior seedlings were actively selected for, as was propagation through budding and grafting around 2,000 yr ago (Roach 1985). The domestic apple (Malus × domestica Borkh.) is widely grown throughout the temperate zones of both the northern and southern hemispheres, and recently, it has been expanding into subtropical and tropical zones. Apple is an extremely versatile crop. Fruits can be eaten directly from the tree or stored for up to a year in a controlled atmosphere. They can be processed into juice, sauce, and slices and are a favorite ingredient in cakes, pies, and pastries, whereas crab apples are primarily known for their floral display and attractive foliage (Tian et al. 2011). Apple has become a model for understanding important traits in fruiting tree crops. Desirable apple cultivars are clonally propagated by grafting vegetative material onto rootstocks to produce a genetically uniform crop (Jesen et al. 2010). There are many interesting and important traits in apple, including dwarfing and some insect resistance, flavor, taste, health benefit compounds found in the skin and flesh of the fruit, and an autocatalytic burst of ethylene production late in fruit development (Newcomb et al. 2006). A high-quality draft genome sequence of the domesticated apple has been completed (Velasco et al. 2010), and there are more than 336,000 expressed sequence tags in GenBank. Gene silencing and overexpression by transformation are important approaches to elucidate gene function. Apple plants are susceptible to infection by both Agrobacterium
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tumefaciens and Agrobacterium rhizogenes (James et al. 1989; Lambert and Tepfer 1992). To date, Agrobacterium-mediated transformation has been the preferred system for transforming apples. Transgenic lines have been obtained from a number of genotypes, including ‘Greensleeves’ (James et al. 1989), ‘M26’ (Lambert and Tepfer 1992), ‘Delicious’ (Sriskandarajah et al. 1994), ‘Royal Gala’ (Yao et al. 1995), ‘Golden Delicious’ (Puite and Schaart 1996), ‘New Jonagold’ (Zhang et al. 1997), ‘Marshall McIntosh’ (Bolar et al. 1999), ‘Elstar’ (Szankowski et al. 2003), ‘Fuji’ (Seong and Song 2008), ‘Makino’ (Zhang et al. 2006), ‘Pinova’ (Flachowsky et al. 2007), and ‘Hanfu’ (Yang et al. 2010). For Agrobacterium-mediated transformation of apples, in vitro-derived leaves were usually selected as the explants, but the efficiency of transformation is not high (James et al. 1989; Zhang et al. 1997; Maximova et al. 1998; Seong et al. 2005). Additionally, in some cases, the repeatability of apple transformation experiments is low, and it is still not easy to produce a large number of transgenic apple plants for most laboratories. Therefore, it is necessary to develop an efficient Agrobacterium-mediated transformation system for apples to facilitate analysis of gene function. Agrobacterium-mediated transformation using cotyledons as explants was widely used for annual plants (Schmidt and Willmitzer 1988; Santarem et al. 1998; Ellul et al. 2003; Sujatha et al. 2012) because, in most cases, cotyledons gave higher regeneration frequencies than leaf tissue (Guo et al. 2005; Dai et al. 2007). Cotyledons were seldom used in transgenic breeding of perennial fruit trees because agronomic traits are highly variable in seedling populations due to the high level of heterozygosity. However, for analysis of gene function by transformation, the cotyledon is a suitable explant because the primary interest to researchers relates to whether the transgenic and the nontransformed control plants have homogeneous genetic background, not whether the transgenic plants have good economic traits. For perennial fruit trees, transgenic lines derived from the cotyledons are suitable for comparison with nontransformed plants obtained from the apical bud belonging to the same seed. In this study, our aim was to develop an efficient regeneration and Agrobacteriummediated transformation system in crab apple (Malus micromalus) using cotyledons as explants. This system can be applied to apple functional genomic research.
inoculated onto embryo culture media. Two kinds of media were designed for in vitro culture of the mature embryos of M. micromalus; one was sweet cherry (SC) medium (Dai et al. 2004) supplemented with 2.22 μM 6-benzyladenine (BA) and 1.45 μM gibberellic acid (GA3), the other was SC medium supplemented with 2.22 μM BA and 2.89 μM GA3. The SC medium, designed for micropropagation of sweet cherry plants, differs from Murashige and Skoog (MS) medium (Murashige and Skoog 1962) in several respects: having a lower inorganic nitrogen content and proportion of NH4-N (the inorganic nitrogen content in SC and MS medium was 30 and 60 mM, respectively, while the ratio of NH4-N:NO3-N in SC and MS medium was 1:8 and 1:2, respectively) and replacing CaCl2 with Ca(NO3)2 without changing the final Ca2+ concentration (Dai et al. 2004). After 10 to 14 d of culture, cotyledons or cotyledonary nodes were excised from the seedling and used as the explants for adventitious bud regeneration and genetic transformation, while the apical bud was excised from the same seedling and cultured on subculture medium (MS medium [PhytoTechnology Laboratories, Overland Park, KS] supplemented with 1.33 μM BA, 1.14 μM indoleacetic acid, and 0.29 μM GA3) to obtain an isogenic nontransformed control plant. Bacterial strain and binary vector. A. tumefaciens strain EHA 105 harboring the binary vector pCAMBIA2301 was used for transformation (Fig. 1). The plasmid pCAMBIA2301 contains the neomycin phosphotransferase II (nptII) gene under control of the double CaMV 35S promoter, for selection on kanamycincontaining medium, and the encoding β-glucuronidase (uidA or gus) marker gene under control of CaMV 35S promoter (Luth and Moore 1999). The strains were maintained on solid yeast extract peptone (YEP) medium supplemented with 50 mg/L kanamycin and 200 mg/L rifampicin at 4°C. Plant transformation. A single colony from the bacterial strain was inoculated into liquid YEP medium with 50 mg/L kanamycin and 200 mg/L rifampicin and grown for 16–20 h at 28°C on an orbital shaker at 165 rpm, and then, the bacterial solution was diluted to OD600 =0.1–0.2 in 50 mL YEP with 100 μM acetosyringone and incubated at 25°C with shaking for 5 to 6 h until OD600 =0.5. The cotyledons were shaken gently in the bacterial suspension for 10 min and blotted on
Materials and Methods Plant material. Open-pollinated mature seeds of M. micromalus were collected from one tree in Hebei Province of China 150 d after blooming. The seeds were surface sterilized by immersion in 70% (v/v) ethanol for 1 min, followed by soaking in a solution of HgCl2 (0.1%, w/v) for 10 min, and then rinsed three times with sterile distilled water. Surface-sterilized seeds were peeled and the external coat removed; then, seeds were
Figure 1. Schematic representation of the T-DNA region of pCAMBIA2301. LB left border, nptII the coding region of neomycin phosphotransferase gene, P1 and P2 the pair of primers for nptII gene amplification, CaMV35S2 the double CaMV 35S promoter, uidA the coding region of β-glucuronidase gene, RB right border.
EFFICIENT REGENERATION AND AGROBACTERIUM-MEDIATED TRANSFORMATION
sterile filter paper. They were then cocultivated on a solid regeneration medium (SC supplemented with 4.55 μM thidiazuron [TDZ], 1.08 μM α-naphthaleneacetic acid [NAA], and 1.45 μM GA3) in darkness for 3 d. Immediately after cocultivation, explants were washed three to four times with sterile distilled water to eliminate excess bacteria, blot dried, and transferred to the regeneration medium containing 20 mg/L kanamycin for selection. After 3 wk of culture in darkness, the cotyledon explants were transferred under cool, white fluorescent light (60 μmol m−2 s−1) with a 16-h light photoperiod. Subculturing was carried out every 2 wk to maintain selection pressure. The kanamycin-resistant buds were cultured on shoot elongation medium (MS medium supplemented with 8.88 μM BA, 1.08 μM NAA, and 0.58 μM GA3) containing 40 mg/L kanamycin. GUS histochemical assay. Histochemical assay of β-glucuronidase (GUS) activity was performed according to the method described by Jefferson et al. (1987). The M. micromalus kanamycin-resistant plantlets grown on shoot elongation medium supplemented with 40 mg/L kanamycin were used for GUS assay. The plantlets were incubated overnight at 37°C in 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) in phosphate buffer (pH 7.0) containing 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% (v/v) Triton X-100. The chlorophyll was removed by washing in 70% ethanol for 24 h after XGluc staining.
Figure 2. (a) Seedling of M. micromalus after 14 d of embryo culture (bar=1 cm). (b) Four types of explants: distal cotyledon, central cotyledon, proximal cotyledon (left, top to bottom), and cotyledonary node (right; bar=2 mm). (c) Adventitious buds occurred on the cotyledon explants (bar=1 cm). (d) Adventitious buds regenerated from the adaxial side of the cotyledon (bar=2 mm).
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Polymerase chain reaction amplification. The polymerase chain reaction (PCR) analysis was performed with isolated genomic DNA (gDNA) to check for the presence of the transgene in the putative transformed plants using primers for nptII gene. PCR analysis was also applied to check the absence of Agrobacterium contamination in the putative transformed tissues using primers for Agrobacterium chromosomal gene, chvA. Total DNA was isolated from the leaves of kanamycin-resistant plants with the modified CTAB method (Chang et al. 2007). The pair of primers for nptII amplification was P1: 5′-GTTCTTTTTGTCAAGACCGACC-3′ and P2: 5′-CAAGCTCTTCAGCAATATCACG-3′, which amplified a 562-bp fragment (Fig. 1), and the pair of primers for chvA amplification was 5′-TCCATCAGCAACGTGTCGGTGCT3′ and 5′-GTGGAAAGGCGGTGAGCGATGAT-3′, which amplified a 994-bp fragment. Plasmid and nontransformed plant DNA were used as the positive and negative controls, respectively. The thermal cycler was programmed with an initial denaturation of DNA at 94°C for 5 min, followed by 30 cycles of 94°C for 40 s, 51°C for 40 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The amplified products were analyzed by electrophoresis in 1% (w/v) agarose gels. Experimental design and data analysis. Experiments were set up in a completely randomized design and repeated two times, with 20 and 90 explants per treatment for the regeneration experiment and the transformation experiment, respectively. The
4 Table 1. Comparison of the regeneration abilities of four types of explants of M. micromalus x
Mean ± SD; means within a column followed by the same letter are not significantly different as indicated by Duncan’s multiple-range test (P
