Seed plant zygotes differentiate into two components, the embryo proper and the suspensor. Previous studies have led to the generally accepted view that development of the embryo proper is regulated by the suspensor connecting the embryo proper to donor tissue. However, biochemical, biological, and molecular analyses of embryogenesis are difficult, since zygotic embryos in higher plants are deeply embedded in mother tissues. To find a way out of the difficulties, some embryo-defective mutants of Arabidopsis have been used to discuss embryogenesis and suspensor function. On the other hand, somatic embryogenesis is widely used as a model system for studying the process of zygotic embryo formation. Because somatic embryo of gymnosperms has a well-developed suspensor, it has been successfully used to observe the suspensor directly and to identify factors modulating the interaction between the embryo proper and the suspensor. Various stimulatory and inhibitory factors are correlated with the interaction. Here, we review the results of studies employing Arabidopsis mutants and some gymnosperm tissue culture, and we discuss the possibility of using somatic embryogenesis as a new model for studies of suspensor biology.
Cytokinin is an essential plant developmental regulator that requires precise temporal and spatial control for synergistic action on morphogenesis. To identify the cellular target for proper cytokinin function, the bacterial gene IPT, encoding an isopentenyl transferase for de novo cytokinin biosynthesis, was expressed in transgenic plants using promoters with different specificities. Analysis of the transgenic plants revealed that ectopic IPT expression was detrimental to plant development, whereas exclusive expression of IPT in cycling cells led to normal plant development with increased growth and final organ size. The enlarged organ size was a result of increase in both cell number and cell size, which was accompanied by increased expression of CycD3 and CycB1, indicating that cytokinin controls organ size by regulating cyclin expression. The cell cycle-specific cyclin promoters were active in multiple organs, including root, leaf and flower, suggesting the biological significance of the locally produced cytokinin on morphogenesis, and the amplified cytokinin biosynthesis in the pre-existing dividing cells of these organs is necessary and sufficient for coordinated cell division, cell growth, pattern formation and organ development. To our knowledge, this is the first case that cytokinin was genetically manipulated in transgenic plants to produce dramatically enhanced phenotypes without noticeable negative effect, providing a promising opportunity for crop improvement.
We have previously developed an antibiotic-free system for the selection of plant transformants that is based on a gene (OASA1D) for a mutant α subunit of rice anthranilate synthase. The product of this gene shows a reduced sensitivity to negative feedback regulation by tryptophan. Whereas 5-methyltryptophan (5MT) is lethal for normal plant cells because it causes tryptophan deficiency, expression of OASA1D confers resistance to this tryptophan analog. We used this OASA1D-5MT system for the transformation of Arabidopsis thaliana. An expression vector containing OASA1D under the control of the 35S promoter of cauliflower mosaic virus was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants that harbored OASA1D exhibited resistance to 5MT but did not manifest any other differences in growth, morphology, or fertility. The OASA1D-5MT selection system performed as well as the HPT-hygromycin system for the transformation of Arabidopsis. Given the limited number of conventional marker genes currently available, OASA1D should prove to be a useful tool in Arabidopsis transformation, especially for the generation of plants carrying multiple transgenes.
In developing a technique for plantlet regeneration in vitro by somatic embryogenesis and adventitious bud formation, callus formation must be avoided to prevent somaclonal variation. However the somaclonal variation caused by callus formation is considered to be important because it potentially promotes diversity in the breeding material. In the present study, we developed an efficient method for somatic embryogenesis from callus derived from shoot apex of tea Camellia sinensis (L.) O. Kuntze. The addition of mannitol at 0.31 M improved somatic embryogenesis relative to that on medium without mannitol (25% and 7%, respectively). When the callus was cultured on medium with small amounts of hygromycin (5–10 mgl−1), the differentiation rate increased up to 43% that of control. Results suggested that culturing with osmotica or antibiotics was quite effective to induce somatic embryogenesis from vegetative-derived callus tissue of tea plants.
We have developed a selection system for transformation of Arabidopsis thaliana via Agrobacterium tumefaciens. This selection system uses human cytochrome P450 monooxygenases that metabolize herbicides. Herbicide-tolerant seedlings transformed with CYP1A1, CYP2B6, CYP2C9, or CYP2C19 were selected with the herbicides acetochlor, amiprophos-methyl, chlorpropham, chlorsulfuron, norflurazon, and pendimethalin. The herbicide-tolerant plants transformed with CYP1A1, CYP2B6, and CYP2C19 expressed the corresponding P450 cDNAs. Inheritance and segregation of the P450 genes were analyzed in T2 progeny of herbicide-tolerant T1 seedlings. Metabolism of [14C]norflurazon by transgenic T2 seedlings expressing CYP1A1 produced non-toxic N-demethylated norflurazon. This result suggests that the P450 species expressed in transgenic Arabidopsis plants coordinately functioned as selectable markers because of active metabolism of the herbicides.
We developed a new chimeric promoter to express a selectable marker gene specifically at the selection stage during transformation of rice (Oryza sativa L.). The promoter consists of a 135-bp upstream sequence of the rice RezA gene and 4 or 8 copies of an enhancer fragment of the CaMV35S promoter. The β-glucuronidase (GUS) reporter gene under the control of this promoter was strongly expressed in transgenic rice calli but not in endosperm, the edible tissue in rice grains. When the hygromycin B phosphotransferase gene was fused with this chimeric promoter and introduced into rice, we were able to select transgenic plants. We demonstrated that the chimeric promoter did not influence expression of the endogenous RezA gene in the transgenic calli. These results indicate that this chimeric promoter could be useful for the selection of transgenic rice free of marker gene products in the edible tissue.
Pharbitis nil Choisy has a large number of mutants and is well known in classical genetics. The mutants exhibit particularly rich and varied floral colors and patterns compared to other plants. P. nil is a typical short-day plant and has therefore been used as a model plant for the genetic analysis of floral colors and patterns and for the photoperiodic induction of flowering. In this paper, we describe an efficient transformation protocol mediated by Agrobacterium tumefaciens in P. nil. The binary vector pBI121, containing the neomycin phosphotransferase II (NPT II) gene, was used as a selectable marker, and the luciferase (Luc+) gene was used as a reporter instead of β-glucuronidase (GUS). Agrobacterium tumefaciens strain GV3101 is carried in the binary vector, while that of LBA4404 is contained the binary and ternary vector, which expresses a constitutive virG mutant gene (virGN54D). We infected 393 somatic embryos with the Agrobacterium strain LBA4404/virGN54D/pBI121-Luc+. Fifty-seven kanamycin-resistant shoots were obtained after 2–3 months of culturing in a selection medium, and over 20% of the regenerated shoots were transgenic. Transformation was confirmed with PCR analysis, Southern hybridization, and Luc assay of the transgenic plants. All transgenic plants were morphologically normal and were fertile. The transformation efficiency in this study reached 3.1% of treated explants, which is high enough to produce transgenic P. nil with genes of interest.
We have developed a sensitive system to investigate the possible roles of protein phosphorylation in ABA signaling in Arabidopsis. We used staurosporine, a protein kinase inhibitor, to suppress the inhibitory effects of ABA on seed germination and the opening/greening of cotyledons. Using staurosporine, we performed a large genetic screen for asa (altered sensitivity to staurosporine and ABA) mutants and identified novel weak alleles of abi4 and abi5 mutants as asa1 and asa2 mutants. This method will be useful in isolating weak alleles of mutants and in selecting mutants with altered sensitivity to chemical compounds.
Two myb-related proteins, LHY and CCA1 are essential for circadian clock function in Arabidopsis. Double loss-of-function of lhy cca1 shows a photoperiod-insensitive phenotype and a shortened generation time under short-days (SDs). To understand the molecular mechanisms underlying early flowering of lhy cca1 mutants, we screened for mutations that enhanced the phenotype of lhy cca1 under SDs. Here we show that one of the enhancer mutations is a novel allele of a shoot-identity gene, terminal flower 1 (tfl1). Triple loss-of-function of lhy cca1 tfl1 causes precocious and ectopic expression of LFY and AP1 and dramatically reduces the generation time of Arabidopsis. The additive phenotype in lhy cca1 tfl1 may be due to convergence of the autonomous and photoperiod pathways. Reduction of generation time of crops is an important issue for molecular breeding. Our results highlight a possibility that combining loss-of-function of the circadian clock and one of the shoot-identity genes can be applied for the marker-assisted breeding to manipulate flowering time of crops.
Two myb-related proteins, LHY and CCA1, play key roles in circadian clock function and flowering in Arabidopsis. GI mediates between LHY/CCA1 and floral activators (CO and FT) to promote flowering. The effect of GI on flowering probably involves FT-independent pathways, because ft only partially suppresses the early flowering caused by lhy cca1 or overexpression of GI (GI-ox). LFY, FT, and SOC1 integrate four flowering pathways: the photoperiod, gibberellic acid (GA), vernalization, and autonomous pathways. Roles of SOC1 and LFY in mediating between the clock and control of flowering time have not been elucidated. Here, we demonstrate that SOC1 functioned redundantly with FT to promote flowering via the LHY/CCA1/GI pathway. GI-ox and lhy cca1 increased mRNA levels of the SOC1 and gi partially suppressed the up-regulation in lhy cca1 under SD. The overexpression of LHY (lhy-1) shifted the phase of SOC1 expression, and the gi mutation did not affect the phase shift, suggesting that LHY regulates SOC1 expression both in GI-dependent and independent manners.
LHY and CCA1 are clock components and TFL1 encodes a shoot-identity gene in Arabidopsis. When combined with tfl1, lhy cca1 results in precocious and ectopic expression of a floral integrator gene, LFY. We have shown that lhy cca1 and GI-ox cause early flowering and increase the expression of two floral integrator genes, FT and SOC1. FT and SOC1 are required for the early flowering of GI-ox under SD. Here we demonstrate that tfl1 dramatically reduced the generation time of GI-ox as well as lhy cca1 plants. tfl1 enhanced the precocious expression of the meristem-identity gene AP1 in GI-ox in a similar way to that in lhy cca1. However, tfl1 did not affect the mRNA levels of FT and SOC1 in lhy cca1 and GI-ox, suggesting that the additive phenotypes in lhy cca1 tfl1 (and GI-ox tfl1) are attributable to the concurrent up-regulation of three genes, FT/SOC1 and LFY. The terminal flower phenotype of tfl1 was enhanced by lhy cca1 and GI-ox under SD, suggesting that a proper balance between FT and TFL1 with antagonistic roles is important for the photoperiodic control of architecture in Arabidopsis. Our results indicate that GI mediates between the circadian clock and three floral integrator genes, FT, SOC1 and LFY, to control the photoperiodic flowering.
Signal transduction events which are involved in methyl jasmonate (MeJA)-enhanced production of the tetracyclic diterpene, scopadulcic acid B (SDB), were investigated in leaf organ cultures of Scoparia dulcis. Pretreatment of leaf organ cultures with Ca2+-channel blocker, verapamil, resulted in a dose-dependent inhibition of MeJA enhanced SDB production. Treatments with Ca2+ ionophore, A23187, stimulated SDB production in the absence of MeJA. In addition, preincubation of the leaf organ cultures with calmodulin (CaM) antagonist, trifluoperazine (TFP), diminished MeJA enhanced SDB production. MeJA-enhanced production of SDB was suppressed by addition of protein kinase inhibitors such as staurosporine and 2,6-diamino-N-([1-(1-oxotridecyl)-2-piperdinyl]methyl)hexanamide (NPC-15437). An activator of protein kinase C, 1-oleoyl-2-acetyl-sn-glycerol (OAG), stimulated this production in the absence of MeJA. These results indicate that Ca2+ and CaM mediates signaling events leading to enhancement of SDB production upon MeJA treatment. It was also suggested that phosphorylation of protein by protein kinases might be involved in this signal pathway.
Although the relationship between leaf senescence and programmed cell death (PCD) has been actively researched, no research had been carried out previously on juvenile and young leaves. We showed evidence of PCD (DNA laddering by agarose gel electrophoresis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays) for juvenile and young leaves of Nicotiana tabacum. Additionally, confocal laser scanning microscopy of nuclei in protoplasts from juvenile leaves clarified the beginning stages of nuclear fragmentation. We concluded that even juvenile leaves of tobacco exhibit PCD and that tobacco leaves grow while undergoing PCD from the initial stages of development until senescence.
Features of programmed cell death (PCD), including nuclear fragmentation and DNA ladders were detected in hybrid cells of Nicotiana suaveolens×N. tabacum expressing hybrid lethality at 28°C, but not in cells kept at 36°C. Heat treatment (HT, 50°C for 15 min) before transfer to 28°C from 36°C temporarily suppressed the increase in the percentage of dead cells. In hybrid cells without HT, the percentage of Sub G1 nuclei, corresponding to those with nuclear fragmentation increased at 6 h after transfer to 28°C and DNA ladders were detected at 9 h after transfer to 28°C. On the other hand, in hybrid cells with HT, the percentage of Sub G1 increased and DNA ladders were detected 15 h after transfer to 28°C. These results suggest that HT temporarily suppresses PCD during expression of hybrid lethality.
Eurycoma longifolia Jack is well known among the communities in Southeast Asia because of its aphrodisiac properties and its effectiveness as the cytotoxic, anti-malarial, anti-ulcer, anti-tumor promoting and anti-parasitic agent. Micropropagation through direct plant regeneration from in vivo shoot tip explants was carried out. The highest regeneration percentage (90%) and multiple shoots formation were obtained with the basal Murashige and Skoog (MS) medium supplemented with 5.0 mg l−1 kinetin. Roots were induced after 14 days of culture in the basal MS medium supplemented with 0.5 mg l−1 of indole-3-butyric acid. Plantlets regenerated from shoot tip explants survived well with no morphological differences from parent plants after two months of transplantation to soil.