Application of an inducible transposon with anther culture in generation of di-haploid homologous mutants
© Yang and Charng; licensee Springer. 2014
Received: 16 December 2013
Accepted: 19 February 2014
Published: 24 February 2014
Insertional mutagenesis represents one of the most effective ways to acquire information about a plant gene’s function. However, it is hindered by the autosomal genome being diploid and therefore, most mutations being recessive. The problem is addressed by inducing the transposition during anther culture so that selected mutations can be transmitted and then regenerated to a homozygous state.
To this end, we treated transgenic rice floral tissues containing the inducible transposon with an inducer, salicylic acid. Excision events were detected in regenerated calli and subsequent plantlets. DNA blot and PCR assay were used to determine the homogeneity of knockout mutants. About 5% of the mutants containing transposition events were homozygous. Furthermore, the inducible transposon was active during calli regeneration.
This strategy could be applicable to improve transposition efficiency in microspore development stages to create stable di-haploid mutants in plants.
KeywordsAnther culture Rice Inducible transposon Knockout mutant Di-haploid mutagenesis
Transposable element (TE) tagging has become a powerful tool to create mutants for isolating new genes in animal and plant systems (Lin et al. 2006; Horie et al. 2011; Veilleux et al. 2012). Several experimental approaches have been used to develop transgenic plants with genes randomly tagged by insertion elements (Greco et al. 2001; Hirochika 2001; Hirochika et al. 2004; Izawa et al. 1997; Jeon and An 2001). One limitation of tagging experiments is the problem of the autosomal genome being diploid and thus, most mutations are recessive. Therefore, loss-of-function screens with mutant cells that lack expression of a particular gene are difficult in diploid cells, because in most cases both alleles of a gene must be knocked out to result in a phenotype. Homozygous lines are required for screening to obtain the desired mutant phenotype. Therefore, creation of biallelic TE-insertion mutants is time-consuming and requires more breeding processes.
For animal systems, insertional mutagenesis usually produces a heterozygous individual and then homozygous mutants by further intercrossing. An alternative technique has been used for phenotypic screening directly from somatic cells -- inducing loss of heterozygosity of the mutated gene, which is a less demanding technique with the application of high-concentration G418 selection in heterozygous cells (Huang et al. 2012). However, this technique has relatively low efficiency and sometimes creates a mutation with altered function in a non-targeted locus, with false results (George et al. 2007).
For plant systems, anther culture is used to produce haploids and di-haploids (DHs) via gametic embryogenesis for a single-step development of complete homozygous lines from heterozygous parents. Regeneration from male gametes has been reported in more than 200 species belonging to the Solanaceae, Cruciferae and Gramineae families (Dunwell 1986; Hu and Yang 1986). In rice, the regeneration rate is in general is more than 5 green plantlets/100 anthers. This technique has been further used with TE mutagenesis to obtain homozygous mutants (Kikuchi et al. 2003; Dong et al. 2012). TE allows for creating mutagenesis in abundant germline cells and the subsequent anther culture allows for producing DHs. Yet, these experiments have involved use of native TEs without attempting to promote transposition efficiency or controlling the transposition specifically in germinal cells. Thus, the transposition events may occur in regenerated somatic DH cells, which results in heterozygous mutant plantlets. Several successful transposon-tagging experiments in plants indicated that the Ac/Ds system is a valuable tool for rice functional genomic studies (Zhu et al. 2003,2004; Komatsu et al. 2003). Previously, we constructed a one-time inducible transposon, COKC, by fusing the transposase gene with a chemically inducible PR-1a promoter (Charng et al. 2008; Tai et al. 2011). COKC was introduced into rice plants and could be successfully induced by the inducer salicylic acid (SA) to trigger transposition events. Here, we assessed the use of TE mutagenesis with rice anther culture to produce biallelic mutants from COKC-containing transgenic rice.
Donor plants and pre-treatments of panicles
According to our previous experiments (Tai et al. 2011), we used five independent transgenic lines (K-17, K-19, K-20, K-21 and K-24) containing a single copy of COKC as anther donor plants. All transgenic rice plants were self-pollinated to obtain homozygous COKC. Transgenic rice seedlings were set in pods and grown in a greenhouse.
Anther culture and SA induction
For controlled anther culture, the collected tillers were placed in a bottle containing water, covered with a polyethylene bag, and pretreated at 10°C for 10 days (Ogawa et al. 1995). Then, spikes with the leaf sheaths removed were surface-sterilized with 70% ethanol for 30 sec. Spikeles were removed from the sheath leaf, soaked in 0.6% NaOCl for 3 min, then rinsed thoroughly with sterile distilled water three times. Anthers at mid- to late-uninucleate microspore stages were inoculated onto callus induction medium (CIM) consisting of 1/2 MS salts except with full-strength Fe-EDTA and vitamins, supplemented with 4 mg NAA, 2 mg kinetin, 6% sucrose and 0.8% agar (Sigma Chemical Co., St. Louis, MO). In total, 50 anthers were plated in each Petri dish (87 × 15 mm) containing 25 ml solid medium. Dishes were sealed with paraffin taps (Whatman Ltd.) and maintained at 24°C for 7 to 9 weeks. Calli were transferred to plant regeneration medium, which consisted of MS salt and vitamins, supplemented with 1 mg/L NAA, 4 mg/L kinetin and 3% sucrose, and gelled with 0.3% phytagel. Calli were cultured under a 24-h photoperiod, and temperatures were maintained at 28 ± 2°C. After 4 to 6 weeks, plants regenerated from calli were transferred into glass jars (Sigma) containing 100 ml MS basal semi-solid medium and 3% sucrose until they fully developed roots, then were transplanted to sterile soil in a greenhouse.
Transgenic rice plants were induced with SA at pot or culture stage. In the pot treatment, rice plants were subjected to flooding in 5 mM SA for 1 day when the distance of the flag leaf auricle (of the primary tiller) to that of the next leaf was about 5 cm. Then anthers were inoculated in CIM medium without SA. Culture treatment involved adding 0.1 mM SA to the CIM medium. At least 100 anthers of each transgenic line were collected for experiments and all treatments were tested in a completely randomized design.
PCR assay and DNA blot analysis
Genomic DNA was extracted from regenerated calli or plantlets of transgenic plants using a kit (Genemark, Tainan, Taiwan). Excision of COKC in transgenic plants was analyzed by PCR with three oligonucleotide primers: CF (5′-CGTTCAGTGCTGGTGGTCGT-3′), JR (5′-CTACAGCTCTTTTTGCAACTTTATC -3′) and DR (5′-CTTCTGCAGACTCCGGCGTG-3′) as described (Tai et al. 2011). The amplification protocol comprised 30 cycles of 1 min at 94°C, 2 min at 55°C, and 2 min at 72°C, and was performed in a T-gradient Thermocycler (Biometra, Göttingen, Germany).
The flanking sequences of the T-DNA or COKC integration sites in transgenic plants were determined by use of arbitrary degenerate primers and thermal assymeric interlaced PCR (TAIL-PCR) as described (Liu et al. 1995; Sha et al. 2004), with modification: primary TAIL-PCR involved approximately 150 ng of rice genomic DNA. The flanking sequences were amplified with the following oligonucleotide primers: TLnew4 (5′-GGTCAAGACCAATGTGGAGC-3′), TLnew3 (5′-GATTGTGTACGCCCGACAG-3′) and TLnew2 (5′-GGATTTTAGTACTGGATTTTGG-3′) for T-DNA and 3–1 (5′-GTGTGCTCCAGATTTATATGG-3′), 3–2 (5′-GATTTCGACTTTAACCCGACCGGA-3′) and 3–3 (5′-CGTTTTCGTTACCGGTATATCCCG-3′) for the 3′ end of COKC.
Genomic sequences flanking COKC insertion in transgenic rice plants
Insertion position (bp)
GenBank accession no.
Inducible transposon construct and generation of starter lines
Induction of COKC
Determination of transposition events
Determination of di-haploid (DH) mutants produced by COKC
Anther culture is used specifically in plant systems to produce haploids and DHs through gametic embryogenesis and allows for a single-step development of complete homozygous lines from heterozygous parents. In a few wheat species, the efficiency to regenerate DH plantlets from anther culture can be up to 30% (Lantos et al. 2013). In general, in rice, the regeneration rate is more than 5 green plantlets/100 anthers. Thus, this technique has been further used with native transposon mutagenesis (Kikuchi et al. 2003; Dong et al. 2012). To promote transposition efficiency and control transposition specifically in germinal cells, we assessed the use of an inducible transposon, COKC, to create DH mutant rice plants. To rule out spontaneous transposition events without induction (Tai et al. 2011), we preliminarily identifed 5 starter lines. We used two induction methods and, with pot SA treatment, the germinal transposition efficiency was up to 5% from regeneration calli. All regenerated plantlets were identified as homozygous mutants (Table 1 and Figure 5), so the germinal transposition events were successfully induced. With culture treatment, the transposition efficiency was up to 20%. Yet, many regenerated plantlets yielded untransposed COKC signals (Figure 3c), so the transposition events occurred during calli regeneration, which resulted in heterogeneity of the transposed COKC. Considering that rice anther culture yields a 5% regeneration rate, our results indicate that incubating 2,000 anthers from starter lines containing COKC can yield approximately 100 independent regenerated rice plantlets and 6 homozygous mutants. Since a high regeneration rate (up to 25%) has been reported in a commercial rice line (Islam et al. 2004), our technique can reduce the number of starter anthers needed by 5-fold.
Since the rice genome has been fully sequenced, the flanking sequences of the transposed COKC were aligned to monitor whether any desired gene was tagged. In the transposed line K24-C2, COKC inserted in the hypothetical gene, whose translated product is similar to flavonoid 4′–O-methyltransferase. Yet we observed no phenotypic alteration in this or the other 5 adult rice mutants (data not shown).
Although we have demonstrated that COKC can create DH rice mutants, the use of inducible transposon mutagenesis for other plant species needs further investigation. Plant species with high anther-culture efficiencies, such as Solanaceae and Cruciferae, are a prerequisite for application of germinal transposition mutagenesis. For plant species whose genomes have been completely sequenced and most genes annotated (or are in progress; e.g., papaya), a mutant with the tagged loci, which identified by flanking sequence alignment and matched the annotated gene, can be used to observe the mutant phenotype directly. For plants without complete genome sequencing, the flanking-sequence information of the tagged loci is needed to confirm the homozygous state, which is helpful for phenotype-driven genetic screening.
COKC has been designed for one-time induction, so the possibility of subsequent transposition of the transposed COKC by endogeneous stimuli can be ruled out. Yet, the transposition efficiency, specifically in germinal cells, needs to be improved. Additional developments for plant-gene tagging systems with anther culture could replace the promoter with a mid- to late-uninucleate microspore specific one so that the transposition is stable after integration. All of these promoters, combined with the one-time inducible transposon concept, will allow us to develop more efficient transposon systems for plant functional genomic studies.
This project was supported by the National Science Council of Taiwan (No. NSC101-2313-B-002-001-MY3).
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