Xylem is an essential conductive tissue in vascular plants, and secondary cell wall polymers found in xylem vessel elements, such as cellulose, hemicellulose, and lignin, are promising sustainable bioresources. Thus, understanding the molecular mechanisms underlying xylem vessel element differentiation is an important step towards increasing woody biomass and crop yields. Establishing in vitro induction systems, in which vessel element differentiation is induced by phytohormonal stimuli or by overexpression of specific transcription factors, has been vital to this research. In this review, we present an overview of these in vitro induction systems, and describe two recently developed in vitro induction systems, VISUAL (Vascular cell Induction culture System Using Arabidopsis Leaves) and the KDB system. Furthermore, we discuss the potentials and limitations of each of these new in vitro induction systems for advancing our understanding of the molecular mechanisms driving xylem vessel element differentiation.
Existing methods to quantify fluorescent signals are primarily limited to non-moving objects or tracking a limited number of cells. These techniques, however, are unsuitable for measuring fluorescent signals in time-lapse experiments using plant specimens that move naturally during a course of imaging. We developed an automated method to measure fluorescent signal intensities in transgenic Arabidopsis plants using a stereomicroscope with standard microscopy software. The features of our technique include: 1) recognizing the shape of plant specimens using autofluorescent signals; 2) merging targeted fluorescent signals to specimen outlines; 3) extracting signals within the shape of specimens from their background signals. Our method facilitates the measurement of fluorescent signals on freely moving plant leaves that are physically unrestrained. The method we developed addresses the challenge of recognizing plant shapes without relying on: a) manual definition which is prone to subjectivity and human error; b) introducing stable fluorescent markers to define plant shapes; c) recognizing plant shapes from bright field images which include a wide range of colors and background noise; d) unnecessarily stressing plants by immobilizing them; e) the use of multiple software packages or software development expertise.
Neolamarckia cadamba is a miracle tree species with considerable economic potential uses as a timber wood, woody forage and traditional medicine resource. The present study aimed to establish a highly efficient and robust protocol of plant regeneration for N. cadamba. Greenish callus was induced from very young leaf explants of sterile in vitro plantlets cultured on Murashige and Skoog’s (MS) medium supplemented with 3 mg l−1 thidiazuron (TDZ), 0.1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg l−1 α-naphthaleneacetic acid (NAA). The callus could differentiate into nodular embryogenic structures or adventitious shoots, and these two regeneration pathways often occurred in the same callus clumps. The micro-shoots developed roots in MS supplemented with 0.05 mg l−1 NAA and 0.05 mg l−1 indole-3-butyric acid (IBA), while the nodular embryogenic structures germinated directly and developed into plantlets on induction medium contained with 0.5 mg l−1 (or 1 mg l−1) 6-benzyladenine (6-BA) and 0.05 mg l−1 NAA. The rooted plantlets could be successfully acclimatized to a greenhouse with more than 92.0% survival. This regeneration protocol can be used in large scale cultivation needs and may be useful for future genetic modifications of N. cadamba.
Transgenic ever-bearing strawberry (Fragaria×ananassa Duch. ‘HS 138’) was cultivated in a closed plant production system to produce functional proteins that can enhance human immune functions. We investigated the effects of air temperature before harvest on fruit growth and the concentration of human adiponectin (hAdi) at harvest in transgenic strawberry. During the different stages of maturity (mature white and immature green stages), hAdi-expressing plants were exposed to four air temperature treatments (15, 20, 25, and 30°C) under 24-h illumination provided by fluorescent lamps. Fruits were harvested at the mature red stage. The number of days to the mature red stage decreased with increasing air temperature, being the least at 30°C. Fruit total soluble protein (TSP) concentration increased with decreasing air temperature, particularly at 15°C, whereas fruit hAdi concentration tended to be higher under the 30°C treatment than under any other of the temperature treatments. There was no significant relationship between fruit fresh weight at harvest time and hAdi concentration within treatments, nor between the number of days to harvest and hAdi or TSP concentration. Although there were no significant differences in fruit hAdi content among treatments, hAdi production rate was the highest at 30°C because of the shortest duration to harvest. These results indicate that a higher air temperature promoted fruit maturation and accelerated the production of functional hAdi proteins in the fruit. For hAdi-expressing strawberry plants, exposure to 30°C may reduce energy consumption (lighting and air conditioning) for functional protein production under controlled environments.
In Solanum lycoperisicum (tomato), a transcription factor of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family, JASMONATE-RESPONSIVE ERF 3 (JRE3), is a closest homolog of JRE4, a master transcriptional regulator of steroidal glycoalkaloid (SGA) biosynthesis. In tomato genome, JRE3 resides in a gene cluster with JRE4 and related JRE1, JRE2, and JRE5, while JRE6 exists as a singleton on a different chromosome. All of the JREs are induced by jasmonates (JAs), whereas sodium chloride (NaCl) treatment drastically increases the expression of the JREs except for JRE4 and JRE6. In this study, to get insights into the regulatory function of the JA- and NaCl-inducible JRE3, a series of genes upregulated by β-estradiol-induced overexpression of JRE3 are identified with microarray analysis in transgenic tomato hairy roots. No gene involved in the SGA pathway has been identified through the screening, confirming the functional distinction between JRE3 and JRE4. Among the JRE3-regulated genes, we characterize the stress-induced expression of genes encoding malate synthase and tonoplast dicarboxylate transporter both involved in malate accumulation. In transient transactivation assay, we reveal that both terminal regions of JRE4, but not a central DNA-binding domain, are indispensable for the induction of a gene involved in the JRE4 regulon. Functional differentiation of the JREs is discussed.
Several selection markers for the screening of transformants have been developed; however, simple and reliable methods are generally preferred. We have developed a novel visible selection system for the identification of transformants in Arabidopsis thaliana that does not require any special reagent and/or equipment except using the albino-cotyledon mutant cyo1. In this system, the pCYO vector carrying the CYO1 genomic fragment as a selection marker is introduced into the cyo1 mutant. Transformation is performed by the Agrobacterium-mediated floral dip method and resultant T1 seeds are sown in soil. Seedlings with green cotyledons, not albino, are expected to be ‘complemented’ transformants with the transgene of interest. This system provides a very simple selection method that can be performed without any special equipment, reagent, sterile conditions, or UV illumination. We have constructed three vectors, (1) pCYO1, an empty vector; (2) pCYO2, an overexpression vector carrying CaMV35S promoter; and (3) pCYO3, a vector for genome editing, carrying the CRISPR/Cas9 cassette. Example transformation experiments using these vectors, including genome editing, are shown.
Sorghum (Sorghum bicolor L.) ranks as the fifth most widely planted cereal in the world and is used for food as well as a biomass plant for ethanol production. Use of the TX430 non-tannin sorghum variety has enhanced Agrobacterium-mediated sorghum transformation. These protocols could not be applied, however, to other tannin producing sorghum varieties such as the BTx623 model cultivar for sorghum with full genome information of sorghum. Here we report an improved protocol for Agrobacterium-mediated genetic transformation of tannin-producing sorghum variety BTx623. We successfully developed modification of root regeneration condition for generation of transgenic plant of BTx623. We inoculated immature embryos with Agrobacterium tumefaciens strain EHA105 harboring pMDC32-35S-GFP to generate transgenic plants. In the root regeneration step, we found that regeneration from transformed calli was affected by tannin. For root regeneration, shoots that appeared were not transferred to agar plate, but instead transferred to vermiculite in a plastic pod. Direct planting of regenerated shoots into vermiculite prevented the toxic effect of tannin. Root regeneration efficiency from calli emerged shoots in vermiculite was 78.57%. Presence of sGFP transgene in the genome of transgenic plants was confirmed by PCR and sGFP expression was confirmed in transgenic plants. This improved protocol of Agrobacterium-mediated transformation for tannin-producing sorghum BTx623 could be a useful tool for functional genomics using this plant.
A peptide-mediated DNA delivery system for several plant species has been recently developed. This system uses ionic complexes of DNA and fusion peptides containing several domains, such as DNA-binding and cell-penetrating peptides. Although the peptide-DNA complexes are capable of penetrating into plant cells through the cell wall by mechanical pressure using a syringe, sample throughput is limited. Here, we describe a Centrifugation-Assisted Peptide-mediated gene Transfer (CAPT) method for improving sample throughput with reproducible gene transfer efficiency. We optimized the parameters of CAPT for transient gene transfer efficiency by using Nicotiana tabacum cotyledons as a model plant material. The optimal parameters for centrifugation were 10,000×g for 60 s. Furthermore, we successfully transferred the peptide–DNA complex into rice cotyledons using the optimized CAPT method. Thus, the CAPT method is superior to the previous syringe-mediated infiltration method in terms of sample throughput in multiple plant species.
Somatic embryogenesis in flowering dogwood (Cornus florida) has been achieved, but not the regeneration of plants with active shoot growth. To improve plant regeneration, eight media treatments were tested for induction of somatic embryogenesis from immature zygotic embryos. Somatic embryogenesis was obtained on three media containing the plant growth regulators (PGRs) 0.1 mg l−1 picloram, 2.0 mg l−1 2,4-dichlorophenoxyacetic acid, or 0.1 mg l−1 indole-3-butyric acid (IBA). Somatic embryogenesis was also induced on Woody Plant Medium without PGRs. Overall, 92% of the somatic embryos examined converted into plants with active root and shoot growth. This is the first report of somatic embryo-derived plants of C. florida that have active shoot growth and that could be transferred to soil. Embryogenic suspensions were established from IBA-treated cultures that could serve as a target for C. florida bioengineering.
The majority of tissue culture and transformation studies in cassava (Manihot esculenta Crantz) focus on the modification of conditions in order to establish a better protocol. Although this is a standard approach for making progress in genetic transformation technology for a target plant variety, serious difficulty still remains due to the limited applicability and adaptability of the given protocol. In the present study, we aim to develop a new concept that focuses on the development of simple but adaptable genetic transformation technology in cassava. In order to establish an efficient transformation protocol, two local edible cassava varieties, R-type, with a broad leaf with reddish petiole, and S-type, with a thin leaf with shiny greenish petiole, were obtained from Okinawa, Japan. Three detection methods, i.e., fluorescence microscopy, thin-layer chromatography (TLC) with detection under an ultraviolet (UV) illumination (254 nm) and light emitting diode (LED) illuminations (365 nm and 500 nm) without any staining, and a spectrum scanning (250–700 nm) by a microplate reader system were employed to identify a series of unique features of the petioles and leaves. Antimicrobial activity of methanol extracts from the tissues was also assayed. We succeeded in the transient expression of the GUS gene using cassava leaves and also established stable introduction of the GUS gene into three organogenic cassava calli by adapting Agrobacterium-mediated transformation. With all the findings, we have identified a flexible tool to create a better match between explants and Agrobacterium strains.