Plant Biotechnology Reports

A protocol for in vitro propagation from pseudobulb sections of Lycaste armomatica (Graham ex Hook) Lindl., an ornamental and fragrant orchid, was developed. The effect of four cytokinins: kinetin (K), meta-topolin (mT), N 6-benzyladenine (BA), and thidiazuron (TDZ), in equimolar concentrations, was investigated. Shoot formation from apical and basal pseudobulb sections was obtained in all treatments. A few medial sections cultured in media supplemented with BA formed protocorm-like bodies. Shoot formation was greater from the basal section than the apical, and mainly occurred in explants cultured in media containing TDZ. The highest average numbers of shoots per explant were achieved from basal sections cultured in media supplemented with TDZ at 4.4, 8.87 and 2.2 µM, forming on average 9.9, 8.6 and 7.3 shoots per explant, respectively. Since the medial pseudobulb section was the worst explant for propagation of L. aromatica, we recommend that pseudobulbs be divided into two sections; the basal half should be cultured in MS medium supplemented with TDZ at 4.4 µM and the apical half with TDZ at 2.2 µM. Subculturing individual shoots in MS medium without plant growth regulators allows further development and rooting. A survival rate of more than 90% under greenhouse conditions was achieved. This research represents a direct contribution to the conservation and sustainable use of this valuable natural resource.

Some edible Leguminous are toxic when raw, and the Chinese are particularly fond of beans, so Leguminous poisoning is very common in China. Rapid and accurate identification of poisoned species and determination of their toxic components would better assist physicians in treating patients. However, traditional morphology-based identification methods possess many limitations. DNA barcoding technique is a new species identification technique developed in recent years, which is expected to make up for the shortcomings of traditional morphological identification. In this study, a comprehensive evaluation system based on DNA barcoding and ELISA kits was attempted. A total of 30 Leguminous toxic plants were collected, involving 9 genera and 10 species. We used simulated gastric fluid (SGF) to simulate the human gastric environment. Three markers (rbcL, trnH-psbA, and ITS) were amplified and sequenced for all untreated and 15 mock-digested samples. The validity of DNA barcoding for species identification was assessed using the Basic Local Alignment Search Tool (BLAST) method and the tree construction method. The levels of three toxic components (saponin, phytoagglutin and trasylol) were determined in all samples using ELISA kits. The amplification success rate of all three regions was high (rbcL 96.67%, trnH-psbA 100%, and ITS 100%), but the sequencing of the trnH-psbA region was less satisfactory (66.67%), and SGF had a significant impact on the sequencing of the ITS region (After 40 min of SGF treatment, the sequencing success rate decreased by 46.67%). The samples from different species and origins contained different levels of toxic components, and the levels of all three substances decreased significantly after undergoing SGF digestion. After 1 h of SGF treatment, the saponin content decreased to 0–8.60% in untreated content (PHA decreased to 8.62–36.88%, trasylol decreased to 4.70–47.06%). The current results suggest that DNA barcoding has great potential for rapid identification of Leguminous poisoning in clinical settings. Toxins are probably not detectable in the patient for longer periods of poisoning. We recommend DNA barcoding technology as a first step for rapid screening and combined with toxin analysis for clinical diagnosis.

Floral architecture plays a pivotal role in developmental processes under genetic regulation and is also influenced by environmental cues. This affects the plant silique phenotype in Arabidopsis and grain yield in crops. Despite the relatively small number of family members of zinc finger homeodomain (ZF-HD) transcription factors (TFs) in plants, their biological role needs to be investigated to understand the molecular mechanisms associated with plant developmental processes. Therefore, we generated HB31SRDX and HB21SRDX repressor mutant lines to understand the functional role of ZF-HD TFs. The mutant lines showed severe defects in plant architecture, including increased branching number, reduced plant height, distorted floral phenotype, and short silique. We found that HB31 and HB21 are paralogs in Arabidopsis, and both positively regulate cell size-related genes, cell wall modification factor-related genes, and M-type MADS-box TF families. In addition, HB31 and HB21 are negatively associated with abiotic stress-related genes, vegetative-to-reproductive phase transition of meristem-related genes, and TCP and RAV TFs. microRNA164 (miR164), miR822, miR396, miR2934, and miR172 were downregulated, whereas miR169, miR398, miR399, and miR157 were upregulated in the two repressor lines. Phenotypic and molecular analyses demonstrated that the miR396/GRF modules regulated by HB31 and HB21 are involved in the plan floral architecture of Arabidopsis. The findings of this study will help elucidate the role of ZF-HD TFs in maintaining the floral architecture.

The efficient plant regeneration system from embryogenic cell suspension cultures of Gynura procumbens (Lour.) Merr. is described. Leaf, stem and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) in various concentrations (0, 0.1, 0.3, 1.0 and 3.0 mg l−1). Leaf, stem and petiole explants formed pale-yellow nodular callus and off-white calluses at a frequency of 100% when cultured on MS medium supplemented with more than 1 mg l−1 of 2,4-D after 4 weeks incubation. However, only 20% of pale-yellow nodular callus derived from petiole explants developed into white embryonic structures. Upon transfer to MS basal medium without growth regulators, these white embryonic structures differentiated into somatic embryos. Embryogenic cell suspension cultures were initiated from petiole-derived pale-yellow nodular calluses. More than 73.2% of regenerated plantlets via somatic embryogenesis produced roots on MS medium supplemented with 0.1 mg l−1 α-naphthaleneacetic acid and 1 mg l−1 indole-3-butyric acid (IBA), respectively. Rooted plantlets were successfully transplanted to soil mixture of sterile vermiculite and potting soil (1:1) and grown to maturity in a growth chamber, achieving a survival rate of > 95%. The plant regeneration system from embryogenic cell suspension cultures of G. procumbens established in this study could be applied as an alternative for mass proliferation as well as molecular breeding for quality improvement of G. procumbens.

Tocopherols belong to the plant-derived poly phenolic compounds known for antioxidant functions in plants and animals. Activation of mitogen-activated protein kinases (MAPK) is a common reaction of plant cells in defense-related signal transduction pathways. We report a novel non-antioxidant function of α-tocopherol in higher plants linking the physiological role of tocopherol with stress signalling pathways. Pre-incubation of a low concentration of 50 μM α-tocopherol negatively interferes with MAPK activation in elicitor-treated tobacco BY2 suspension culture cells and wounded tobacco leaves, whereas pre-incubated BY2 cells with α-tocopherol phosphate did not show the inhibitory effect on stimuli-induced MAPK activation. The decreased MAPK activity was neither due to a direct inhibitory effect of α-tocopherol nor due to the induction of an inhibitory or inactivating activity directly affecting MAPK activity. The data support that the target of α-tocopherol negatively regulates an upstream component of the signaling pathways that leads to stress dependent MAPK activation.

In this study, genetic engineering methods, ranging from gene transformation to gene editing, were efficiently conducted on cotyledon explants in Solanum nigrum. Organogenic calli were observed on the explants following 10 days of growth on medium supplemented with 2 mg/L zeatin and 0.2 mg/L indole acetic acid (IAA), which we suggest to be an efficient shoot initiation medium (SM1). In vitro shoot production was enhanced in explants grown on media supplemented with 1 mg/L zeatin and 0.1 mg/L IAA, which we used as a proper shoot differentiation medium (SM2) in S. nigrum shoot regeneration. Direct infection of explants with Agrobacterium harboring CRISPR/Cas9 constructs can simplify the method of Agrobacterium-mediated T-DNA transformation by skipping the preincubation step. The method was applied to deliver transgenes for genome editing, such as CRISPR/Cas9 DNA. SnLazy1 locus, considered to be orthologs of tomato Lazy1, was edited using the CRISPR/Cas9 system in S. nigrum. Two independent deletion snlazy1-cr alleles were successively inherited and showed stem growth in a relatively downward direction. Our method offers rapid and simple procedure for gene transformation and genome editing that could be applicable for the enhancement of beneficial traits for precision plant breeding and engineering quantitative trait loci in S. nigrum.

Arsenic is a major pollutant that contaminates land, water, and air. We previously reported that the expression of tobacco Ntcyc07 confers arsenite (As III) tolerance by decreasing the arsenic level by promoting the transcription of the arsenite export carrier ACR3 and inhibiting that of the As(III) uptake transporter FPS1 in S. cerevisiae (Mok et al. 2008). To examine if Ntcyc07 is also involved in As(III) tolerance and accumulation in plants, we generated transgenic tobacco and Arabidopsis that overexpress Ntcyc07. The Ntcyc07 tobacco exhibited increased tolerance to but decreased accumulation of arsenite, but the Ntcyc07 Arabidopsis showed enhanced As(III) tolerance and no change in the As level. In contrast, Atcyc07 knockout Arabidopsis (Atcyc07 is a homolog of Ntcyc07) displayed reduced As(III) tolerance and an increased As level. Taken together, cyc07 promotes As(III) tolerance by decreasing the cellular accumulation of arsenite in plants.