Plant Cell Rep (2005) 23:716–720 DOI 10.1007/s00299-004-0871-2
GENETIC TRANSFORMATION AND HYBRIDIZATION
S. J. Li · X. M. Deng · H. Z. Mao · Y. Hong
Enhanced anthocyanin synthesis in foliage plant Caladium bicolor
Received: 19 February 2004 / Revised: 4 August 2004 / Accepted: 5 August 2004 / Published online: 15 September 2004 Springer-Verlag 2004
Abstract A protocol was developed for Agrobacteriummediated genetic transformation of monocotyledon foliage plant Caladium bicolor cv. Jackie Suthers using leaf disc and petiole as the explants. The explants were inoculated with Agrobacterium strain LBA4404 harboring a binary vector with the maize anthocyanin regulatory gene Lc under the control of the cauliflower mosaic virus promoter. Callus formation was induced in MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (6BA), 0.1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g/l sucrose and kanamycin 50 mg/l for selection. Resistant calli were induced for shoot generation in MS medium with 2 mg/l 6-BA and 0.2 mg/l a-naphthaleneacetic acid. As much as 10% of the explants gave rise to kanamycin-resistant shoots with our procedure. Transformed plants had enhanced anthocyanin accumulation in the roots, leaves and stems (epidermis and vascular bundles). Integration of the transgene into the host genome was confirmed by genomic Southern blot hybridization, and RNA blot hybridization analysis indicated that the expression of the transgene correlated with anthocyanin accumulation. This investigation illustrates the utility of anthocyanin regulatory genes in the genetic manipulation of the color of foliage plants. It also supports the premise that the Lc gene can be used as a powerful non-destructive cell autonomous visual marker in a wide variety of plants, as exemplified by the perfect symmetrical half-green/halfred plant presumably derived from the symmetrical division of one transgenic and one non-transgenic precursor meristematic cell. Keywords Caladium · Anthocyanin · Anthocyanin regulatory gene · Genetic transformation · Foliage plant Communicated by I.S. Chung S. J. Li · X. M. Deng · H. Z. Mao · Y. Hong ()) Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore, Singapore, 117604 e-mail:
[email protected] Tel.: +65-687-27095 Fax: +65-687-27007
Abbreviations 6-BA: 6-Benzylaminopurine · 2,4-D: 2,4-Dichlorophenoxyacetic acid · KT: Kinetin · NAA: a-Naphthaleneacetic acid
Introduction Extensive studies on the synthesis of anthocyanin pigments in maize plants revealed that two classes of upstream transcription factors—Myc and Myb homologs— coordinately regulate structural genes for anthocyanin synthesis (review by Dooner et al. 1991). The same anthocyanin regulatory system has also been found in other plants, such as antirrhinum (Goodrich et al. 1992) and Petunia (Avila et al. 1993). The Lc (for leaf color) gene is a member of the maize R-gene family. It encodes a protein with the basic-helixloop-helix (b-motif) found in Myc proteins (Ludwig and Wessler 1990). Ectopic expression of Lc-mediated protein enhanced the expression of anthocyanin in Arabidopsis, tobacco (Lloyd et al. 1992) and petunia (Quattrocchio et al. 1993). Goldsbrough et al. (1996) observed that the Lc gene conferred augmented anthocyanin accumulation when introduced into tomato under the control of the cauliflower mosaic virus (CaMV) promoter. Lc was found to mediate enhanced pigmentation in all vegetative tissues. The development of transformation methods for economically important ornamental crops would enable molecular genetic methods to be used for modifying characteristics such as flower color, shape, longevity, plant morphology and resistance to environmental stresses, insects and diseases. During the past few years, tremendous progress has been made in transforming ornamental crops, mostly the cut-flower crops. Useful genes have been introduced and expressed in some major flower crops resulting in, for example, a color-modified chrysanthemum (Courtney-Gutterson et al. 1994) and a carnation with an extended vase life (Savin et al. 1995). Caladium bicolor is a plant species with erect, fleshy stems and large, colorful and waxy leaves. Its fancy-
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leaved and strap-leaved types of cultivars are grown both as landscape bedding plants and as potted plants (Cantwell 1994). There has been one report on genetic transformation and the expression of human growth hormone gene in this species (Li et al. 1994), but it lacked technical details and a comparative study on the genetic transformation protocol. In the investigation reported here, we set up and optimized a highly efficient genetic transformation system for an albino cultivar of this plant species and introduced the maize anthocyanin regulatory gene Lc under the control of the CaMV promoter. Expression of Lc gene promoted enhanced pigmentation in all vegetative tissues, including the roots, stems and leaves. One rare plant with perfect symmetrical green/red leaves was also obtained.
Materials and methods Gene construct A construct containing the maize Lc gene was kindly provided by Dr. Lim Saw Hoon, Department of Biological Science, National University of Singapore. Full-length Lc cDNA was excised with BamHI/Hind III and ligated to BamHI/SacI-cut pBI121 (BD Sciences Clonetech, Palo Alto, Calif., www.bdbiosciences.com) in the presence of a Hind III-SacI adaptor AGCTTGAGCT. The junction was sequenced to confirm that the Lc gene was cloned downstream of the 35S promoter. The resultant pBI-Lc was introduced into Agrobacterium tumefaciens strain LBA4404 through electroporation.
duction medium ( basal medium supplemented with 0.5 mg/l 6-BA, 0.1 mg/l 2,4-D, 500 mg/l cefotaxime and 50 mg/l kanamycin). Explants were subcultured every 2 months. Regeneration of transgenic shoots After 6 months in the induction medium, the red-colored calli were transferred to regeneration medium for shoot generation (basal medium supplemented with 2 mg/l 6-BA, 0.2 mg/l NAA, 500 mg/l cefotaxime, 50 mg/l kanamycin). Following two to three monthly subcultures, small shoots (approx. 1.5 cm high) were transferred to a hormone-free medium for rooting (basal medium supplemented with 500 mg/l cefotaxime, 50 mg/l kanamycin). Rooted plantlets 3– 4 cm high were transferred to soil for further growth in a greenhouse. Genomic Southern blot hybridization DNAs were isolated from C. bicolor leaves according to a modified CTAB method (Xie and Hong 2002). Twenty-microgram aliquots of genomic DNA were digested with BamHI, separated on an 0.8% agarose gel and transferred to a nylon membrane (Hybond N+, Amersham, UK, www.amershambiosciences.com) according to a standard protocol (Sambrook et al. 1989). The 1.8-kb fulllength cDNA fragment of the Lc gene was labeled with the PCR DIG Probe Synthesis Kit (Roche, Indianapolis, Ind., www. roche-applied-science.com) as the hybridization probe. Hybridization, washing and detection were performed according to manufacturer’s instructions. Briefly, the membrane was washed at 68C in 0.1 washing solution after hybridization, incubated with alkaline phosphate conjugated anti-Digoxigenin followed by the chemiluminescent substrate CDP-Star and finally exposed to X-ray film. Northern blot hybridization
Media and culture conditions The basal medium consisted of MS medium (Murashige and Skoog 1962) supplemented with 30 g/l sucrose, 0.75 g/l MgCl2·6H2O and 2 g/l phytagel. All plant growth regulators and antibiotics were filter-sterilized and added to autoclaved (121C for 25 min) media. All media were adjusted to pH 6.2 with 1N KOH after autoclaving. All cultures were maintained at 28C under a 16/8-h photoperiod with light supplied by warm-white fluorescent lamps at an irradiance of 26 mmol/m2 per second. Optimization of the regeneration system Young leaves and petioles of Caladium bicolor cv. Jackie Suthers were sterilized with 70% (v/v) ethanol for 1 min, then immersed in 10% Clorox (containing 5.23% NaClO, w/v) for 10–15 min and washed three times with sterile ddH2O. Young leaves were cut into discs (5–67–8 mm), and petioles were cut into 0.3- to 0.5-cm-long segments. The explants were put onto media supplemented with different combinations of phytohormones for callus induction and shoot induction. Inoculation and callus induction Agrobacterium strain LBA4404 harboring the pBI-Lc construct was grown overnight in the dark at 28C in LB medium containing 50 mg/l streptomycin and 50 mg/l kanamycin. The overnight culture was pelleted and resuspended in MS medium supplemented with 2 mg/l acetosyringone to an O.D.600=0.3. Sterile segments of young leaves and petiole were precultured on MS medium for 48 h before being immersed in the activated Agrobacterium suspension for 5 min, washed with sterile H2O, blotted dry on sterile filter paper and cocultured for 2 days before being transferred to in-
Total RNAs from C. bicolor leaves were isolated according to Chang et al. (1993) with modifications. Briefly, leaves were ground in the presence of liquid nitrogen and mixed with 10 ml of the extraction solution (2% CTAB, 2% PVP, 2 M NaCl, 25 mM EDTA, 0.1 M Tris, pH 8.0, 2% b-ME) for each gram of powder. The mixture was incubated at 65C for 30 min and spinned down. The supernatant was extracted four times with chloroform before the RNA was precipited with 2 M LiCl. RNAs were redissolved in H2O and then extracted with chloroform once before precipitation with sodium acetate and ethanol. Total RNAs (20 mg) were separated on a 1.2% agarose gel containing formaldehyde. Denatured RNA markers (Promega, Madison, Wis., www.promega.com) and 1-kb DNA markers (New England Biolabs, Beverly, Mass., www.neb. com) were run together with samples as size standards. RNAs were transferred to Hybond N+ (Amersham) membranes through capillary transfer in 20 SSC buffer and fixed on the membrane by a UV cross-linker. Before hybridization, the membrane was stained in 0.04% methylene blue to check 18S and 28S ribosomal RNA bands for loading uniformity and integrity of the RNAs. Hybridization, washing and detection were carried out as described for genomic Southern blot hybridization.
Results and discussion Optimized conditions for calli formation and shoot generation Efforts were made to optimize media for calli formation (Table 1). Basal MS medium supplemented with 0.5 mg/l 6-BA and 0.1 mg/l 2,4-D was the most effective for callus induction: up to 100% of the petiole explants formed calli
718 Table 1 Caladium bicolor callus induction by different phytohormones
Phytohormones
Explant
Callus induction efficiency calli/explanta (%)
0.5 mg/l 6-BA + 0.1 mg/l 2,4-D
Petiole Leaf Petiole Leaf Petiole Leaf Petiole Leaf
46/46 66/73 5/41 10/47 49/68 28/58 0/42 9/52
0.1 mg/l KT + 0.1 mg/l 2,4-D 0.5 mg/l 6-BA + 0.5 mg/l NAA 0.1 mg/l KT + 0.5 mg/l NAA a
(100) a (90.4) a (12.2) e (21.3) e (72.0) c (48.3) d (0) f (17.3) e
Data followed by different letters are significantly different from each other at P=0.05
Table 2 C. bicolor shoot regeneration Phytohormones
Shoot regeneration shoot/callia (%)
1 mg/l 6-BA + 0.2 mg/l NAA 2 mg/l 6-BA + 0.2 mg/l NAA 3 mg/l 6-BA + 0.2 mg/l NAA
32/70 (45.7) b 48/70 (68.6) a 34/70 (48.6) b
a Data followed by different letters are significantly different from each other at P=0.05
in this media. Calli formation from leaf explants was equally efficient—approx. 90%. Callus induction efficiency decreased with the substitution of NAA for 2,4-D—72% for petiole explants and 48% for leaf explants (Table 1). In both phytohormone combinations, petiole explants formed callus more efficiently than leaf explants. KT was found to be less efficient than 6-BA for the induction of callus. When KT was used in combination with 0.1 mg/l 2,4-D, only 12% of the petiole explants and 21% of the leaf explants formed calli; when it was combined with 0.5 mg/l NAA, petiole explants could not form calli at all and only 17% of the leaf explants formed calli. In summary, the combination of 0.5 mg/l 6-BA and 0.1 mg/l 2,4-D effectively induced callus formation in both petiole and leaf explants. Consequently, this combination was used for subsequent genetic transformation experiments. The explants formed large and green calli after 6 months in the induction medium. 6-BA and NAA were efficient in shoot induction (data not shown). Consequently, different combinations of 6-BA and NAA were tested to optimize shoot regeneration (Table 2). The ratio of 6-BA to NAA was found to be important to efficient shoot regeneration, with the most optimal ration being 10:1 (2 mg/l 6-BA and 0.2 mg/l NAA). Genetic transformation of C. bicolor C. bicolor is a foliage species with more than 1,000 registered cultivars. Many of these cultivars have different patterns of anthocyanin distribution. We chose a white cultivar—Jackie Suthers—as the target of our genetic transformation experiments (Fig. 1a). Both the petiole and leaf were used as the explants, and both were successfully regenerated and genetically transformed. Up to
10% of the explants generated kanamycin-resistant shoots (Fig. 1b). Following rooting, transgenic plantlets were visually distinguishable from a non-transgenic plantlet (Fig. 1c, right and left) by the accumulation of anthocyanin in the root (Fig. 1d) and leaf of the former. When these plantlets elongated and grew, the tuber and stem also had anthocyanin accumulation (Fig. 1g,i). Goldsbrough et al. reported that Lc-mediated pigmentation accumulation was limited to the outmost cell layers of transgenic tomato plants (Goldsbrough et al. 1996). However, we found that anthocyanin accumulated in other parts of the stem as well as the epidermis, most noticeably in vascular bundles (Fig. 1f). No other phenotypic differences were observed between transformed and control primary regenerants, and none of the control regenerants (no Agrobacterium infection) showed anthocyanin accumulation. The absolute levels and pattern of anthocyanin accumulation varied (visually) among the transformants (Fig. 1). Two mosaic phenotypes were identified: one with a single green plant derived from the same tuber as several other anthocyanin-expressing plants (Fig. 1h); the other plant had a perfect symmetrical half-green/half-red leaves and stem (Fig. 1e). To verify that the enhanced anthocyanin level was due to the Lc transgene, we used genomic Southern blot hybridization and RNA Northern blot hybridization to verify the integration of the transgene into host genome and its expression. Genomic DNAs from 11 putative transformed plants together with a non-transformed control (CK) were cut with BamHI (with no restriction site on Lc gene) prior to hybridization to the Lc probe. We discovered that these transformed plants belonged to few independent transgenic lines (Fig. 2a). The control plant and the green sibling (1G) had no Lc gene integrated while all of the red plants had single or multiple copies of Lc genes integrated into the genome. RNA blot hybridization (Fig. 2b) verified that the 2.0-kb transcript was present in all of the plants expressing anthocyanin (red leaves) but absent in the control plant, the green sibling and the green half of the mosaic plant (3G). These results confirmed that the Lc transgene was responsible for the enhanced anthocyanin phenotype. Anthocyanin expression in maize is known to be dependent on the presence of both the Myb and Myc types of regulatory factors. Lloyd et al. (1992) suggested that functional copies of both the R-gene family members
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Fig. 1 Genetic transformation of Caladium bicolor cv. Jackie Suthers with the maize Lc gene. a The explant, b calli and shoots in shoot induction medium, c a transformed plantlet (right) and a nontransformed plantlet (left), d anthocyanin accumulation in roots, e the half-transgenic, half-non-transgenic plant, f stem cross-sections
of control plant (lower left) and transformed plants (bar: 2 mm); g one single transgenic plant, h–I transgenic plants with various level and patterns of anthocyanin accumulation (bar: 2 cm for all except f)
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advantage of Lc over many other reporters is that its use results in a visual phenotype without the requirements for cofactors or substrates. The perfect symmetrical halftransgenic and half-non-transgenic plant suggests that only one of the two precursor meristematic cells be transformed. These two cells divided symmetrically, and each gave rise to half of the plant. It is possible that, with more batches of transformation at different stages of regeneration, mosaic phenotypes from cells infected at the four-cell or eight-cell stage or during subsequent differentiation stages could be generated. Such visual mosaic plants would help build up cell lineages and, eventually, the development blueprint.
References
Fig. 2 Integration and expression of the transgene. a Genomic Southern blot hybridization. Lanes: CK Control plant, 1G, 1R green and red sibling plant, respectively, from the same tuber (Fig. 1h), 2– 12 red plants. b RNA blot hybridization. Lanes: CK Control plant, 1G, 1R green and red plant, respectively, from one tuber, 3G, 3R green half and red half of the mosaic plant (Fig. 1e), 2–12 red plants
(Myc type) and a C1-like gene (Myb type) were both required for anthocyanin expression. In our study, the expression of Lc alone under the control of the CaMV 35S promoter was sufficient to enhance anthocyanin accumulation in all green vegetative parts of C. bicolor cv. Jackie Suthers. One possible explanation for this observation is that this cultivar may express a Myb-type transcription regulator but somehow lacks the expression of the Myc-type transcription factor. The exogenous Lc gene complemented endogenous Myb-type transcription factor(s) to enhance the anthocyanin synthesis. This is in line with the known fact that C. bicolor has more than 1,000 registered cultivars, many of which have various levels and patterns of anthocyanin accumulation on leaves, stem and roots. We suggest that the presence of such a Myb factor in vascular bundles led to anthocyanin accumulation in both the vascular bundles and epidermis (Fig. 1f), a result that differs from that of Goldsbrough et al. (1996). We observed no abnormal phenotype in the transgenic C. bicolor plants, suggesting that integration and expression of Lc did not alter other morphological features. This conclusion is strongly supported by the perfect symmetrical half-transgenic and half-non-transgenic plant. This finding also supports the proposal of Goldsbrough et al. (1996) that Lc is a non-destructive visual reporter. The
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