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Plant Biotechnology (Tokyo, Japan) Jun 2020The family Liliaceae (Cronquist system) contains various important ornamental plants. We have been examining for about 20 years the establishment of plant regeneration...
The family Liliaceae (Cronquist system) contains various important ornamental plants. We have been examining for about 20 years the establishment of plant regeneration and genetic transformation systems in liliaceous ornamental plants for their biotechnological breeding and elucidation of the molecular mechanisms determining ornamental traits. In this review, studies on plant regeneration in 7 genera and on -mediated production of transgenic plants in 4 genera are described. Plant regeneration was achieved via callus cultures in , , , , and . Auxins (2,4-dichrolophenoxyacetic acid, α-naphthaleneacetic acid and/or picloram) were effective for inducing regenerable calli. species and cultivars were very recalcitrant to callus induction and plant regeneration. -mediated transformation was examined in , , and , and transgenic plants were obtained in all genera by using regenerable calli as a target material for inoculation, inoculation and co-cultivation with in the presence of acetosyringone, and selection of transgenic tissues and plantlets on hygromycin-containing media. Among 4 genera, has several advantages for transformation studies: higher transformation efficiency, relatively small plant size, ease of cultivation, and taking only 1 year from regeneration to flowering. We are now investigating the molecular mechanisms for determining plant form, flower color and flower form by using spp. as liliaceous model plants.
PubMed: 32821219
DOI: 10.5511/plantbiotechnology.20.0114a -
Frontiers in Plant Science 2021is a specialized plant pathogen that causes crown gall disease and is commonly used for -mediated transformation. As a pathogen, triggers plant immunity, which affects...
is a specialized plant pathogen that causes crown gall disease and is commonly used for -mediated transformation. As a pathogen, triggers plant immunity, which affects transformation. However, the signaling components and pathways in plant immunity to remain elusive. We demonstrate that two mitogen-activated protein kinase kinases (MAPKKs) MKK4/MKK5 and their downstream mitogen-activated protein kinases (MAPKs) MPK3/MPK6 play major roles in both -triggered immunity and -mediated transformation. Agrobacteria induce MPK3/MPK6 activity and the expression of plant defense response genes at a very early stage. This process is dependent on the MKK4/MKK5 function. The loss of the function of and or their downstream and abolishes plant immunity to agrobacteria and increases transformation frequency, whereas the activation of MKK4 and MKK5 enhances plant immunity and represses transformation. Global transcriptome analysis indicates that agrobacteria induce various plant defense pathways, including reactive oxygen species (ROS) production, ethylene (ET), and salicylic acid- (SA-) mediated defense responses, and that MKK4/MKK5 is essential for the induction of these pathways. The activation of MKK4 and MKK5 promotes ROS production and cell death during agrobacteria infection. Based on these results, we propose that the MKK4/5-MPK3/6 cascade is an essential signaling pathway regulating -mediated transformation through the modulation of triggered plant immunity.
PubMed: 34659297
DOI: 10.3389/fpls.2021.731690 -
The New Phytologist Jan 2022Agrobacterium tumefaciens colonizes the galls (plant tumors) it causes, and the roots of host and nonhost plants. Transposon-sequencing (Tn-Seq) was used to discover...
Agrobacterium tumefaciens colonizes the galls (plant tumors) it causes, and the roots of host and nonhost plants. Transposon-sequencing (Tn-Seq) was used to discover A.tumefaciens genes involved in reproductive success (fitness genes) on Solanum lycopersicum and Populus trichocarpa tumors and S.lycopersicum and Zea mays roots. The identified fitness genes represent 3-8% of A. tumefaciens genes and contribute to carbon and nitrogen metabolism, synthesis and repair of DNA, RNA and proteins and envelope-associated functions. Competition assays between 12 knockout mutants and wild-type confirmed the involvement of 10 genes (trpB, hisH, metH, cobN, ntrB, trxA, nrdJ, kamA, exoQ, wbbL) in A.tumefaciens fitness under both tumor and root conditions. The remaining two genes (fecA, noxA) were important in tumors only. None of these mutants was nonpathogenic, but four (hisH, trpB, exoQ, ntrB) exhibited impaired virulence. Finally, we used this knowledge to search for chemical and biocontrol treatments that target some of the identified fitness pathways and report reduced tumorigenesis and impaired establishment of A.tumefaciens on tomato roots using tannic acid or Pseudomonas protegens, which affect iron assimilation. This work revealed A.tumefaciens pathways that contribute to its competitive survival in plants and highlights a strategy to identify plant protection approaches against this pathogen.
Topics: Agrobacterium tumefaciens; Carbon; Solanum lycopersicum; Plant Roots; Plant Tumors; Virulence
PubMed: 34655498
DOI: 10.1111/nph.17810 -
BMC Plant Biology Jun 2023With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a...
With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO and NHNO and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.
Topics: Melatonin; Dianthus; Agrobacterium tumefaciens; Glucuronidase; Nitrogen
PubMed: 37316783
DOI: 10.1186/s12870-023-04325-5 -
Plants (Basel, Switzerland) Jun 2020Banana and plantain are among the foremost staple food crops providing food and livelihood to over 500 million people in tropical countries. Despite the importance,...
Banana and plantain are among the foremost staple food crops providing food and livelihood to over 500 million people in tropical countries. Despite the importance, their production is hampered due to several biotic and abiotic stresses. Plant tissue culture techniques such as somatic embryogenesis and genetic transformation offer a valuable tool for genetic improvement. Identification and quantification of phytochemicals found in banana and plantain are essential in optimizing in vitro activities for crop improvement. Total antioxidants, phenolics, flavonoids, and tannins were quantified in various explants obtained from the field, as well as in vitro plants of banana and plantain cultivars. The result showed genotypic variation in the phytochemicals of selected cultivars. The embryogenic cell suspensions were developed for three farmer-preferred plantain cultivars, Agbagba, Obino l'Ewai, and Orishele, using different MS and B5-based culture media. Both culture media supported the development of friable embryogenic calli (FEC), while MS culture media supported the proliferation of fine cell suspension in liquid culture media. The percentage of FEC generated for Agbagba, Obino l'Ewai, and Orishele were 22 ± 24%, 13 ± 28%, and 9 ± 16%, respectively. Cell suspensions produced from FECs were successfully transformed by -mediated transformation with reporter gene constructs and regenerated into whole plants.
PubMed: 32599771
DOI: 10.3390/plants9060789 -
PloS One 2024Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle,...
Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.
Topics: Helianthus; Transformation, Genetic; Agrobacterium tumefaciens; Plants, Genetically Modified; Tissue Culture Techniques; Plant Roots; Plant Breeding; Crops, Agricultural
PubMed: 38722945
DOI: 10.1371/journal.pone.0298299 -
Frontiers in Plant Science 2023
PubMed: 36818863
DOI: 10.3389/fpls.2023.1149426 -
Plant Biotechnology Journal Jan 2020Development of marker-free and transgene insertion site-defined (MFTID) transgenic plants is essential for safe application of transgenic crops. However, MFTID plants...
Development of marker-free and transgene insertion site-defined (MFTID) transgenic plants is essential for safe application of transgenic crops. However, MFTID plants have not been reported for wheat (Triticum aestivum). Here, we prepared a RNAi cassette for suppressing lipoxygenase (LOX) gene expression in wheat grains using a double right border T-DNA vector. The resultant construct was introduced into wheat genome via Agrobacterium-mediated transformation, with four homozygous marker-free transgenic lines (namely GLRW-1, -3, -5 and -8) developed. Aided by the newly published wheat genome sequence, the T-DNA insertion sites in GLRW-3 and GLRW-8 were elucidated at base-pair resolution. While the T-DNA in GLRW-3 inserted in an intergenic region, that of GLRW-8 inactivated an endogenous gene, which was thus excluded from further analysis. Compared to wild -type (WT) control, GLRW-1, -3 and -5 showed decreased LOX gene expression, lower LOX activity and less lipid peroxidation in the grains; they also exhibited significantly higher germination rates and better seedling growth after artificial ageing treatment. Interestingly, the three GLRW lines also had substantially increased contents of several fatty acids (e.g., linoleic acid and linolenic acid) in their grain and flour samples than WT control. Collectively, our data suggest that suppression of grain LOX activity can be employed to improve the storability and fatty acid content of wheat seeds and that the MFTID line GLRW-3 is likely of commercial value. Our approach may also be useful for developing the MFTID transgenic lines of other crops with enhanced grain storability and fatty acid content.
Topics: Agrobacterium; DNA, Bacterial; Edible Grain; Fatty Acids; Mutagenesis, Insertional; Plants, Genetically Modified; Transgenes; Triticum
PubMed: 31141279
DOI: 10.1111/pbi.13178 -
International Journal of Molecular... Oct 2019Oil palm (, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands... (Review)
Review
Oil palm (, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands of palm oil consumption. However, tissue culture and biotechnological approaches can resolve these concerns. Over the past three decades, significant research has been carried out to develop tissue culture and genetic transformation protocols for oil palm. Somatic embryogenesis is an efficient platform for the micropropagation of oil palm on a large scale. In addition, various genetic transformation techniques, including microprojectile bombardment, mediated, Polyethylene glycol mediated mediated, and DNA microinjection, have been developed by optimizing various parameters for the efficient genetic transformation of oil palm. This review mainly emphasizes the methods established for in vitro propagation and genetic transformation of oil palm. Finally, we propose the application of the genome editing tool CRISPR/Cas9 to improve the various traits in this oil yielding crop.
Topics: Agrobacterium tumefaciens; Arecaceae; CRISPR-Cas Systems; Gene Editing; Microinjections; Palm Oil; Plant Somatic Embryogenesis Techniques; Polyethylene Glycols; Protoplasts; Tissue Culture Techniques; Transformation, Genetic
PubMed: 31661801
DOI: 10.3390/ijms20215353 -
Plants (Basel, Switzerland) Nov 2021Passion fruit () is an important fruit crop with high economic value. Genetic engineering plays an important role in crop improvement with desired traits and gene...
Passion fruit () is an important fruit crop with high economic value. Genetic engineering plays an important role in crop improvement with desired traits and gene functional studies. The lack of a simple, efficient, and stable transformation system for passion fruit has greatly limited gene functional studies. In this study, a simple and efficient -mediated transformation system for passion fruit was established, using virulent strain EHA105 harboring the binary vectors pCAMBIA1301 and pCAMBIA1302 with GUS and GFP reporter genes. The system requires less time and labor costs than conventional transformation systems, and no additional phytohormones and sterile conditions are required. Regeneration efficiency of 86% and transformation efficiency of 29% were achieved, when the wounds were wrapped with Parafilm and the plants were kept in darkness for 15 days. Approximately 75% of the regenerated plants had a single shoot and 26% multiple shoots. The transformation was confirmed at the DNA and RNA levels as well as by GUS staining and GFP fluorescent measurements. The developed protocol will contribute to the genetic improvement of passion fruit breeding.
PubMed: 34834821
DOI: 10.3390/plants10112459