Dataset Information

Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum).

ABSTRACT:

Background

Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols.

Results

To establish efficient transformation system of ice plants, three types of ice plant materials, hypocotyl-derived callus, aseptically-grown seedlings and pot-grown juvenile plants, were used to develop Agrobacterium-mediated transformation protocols. The highest transient transformation efficiency was with 5-day-old ice plant callus co-incubated with an Agrobacterium tumefaciens at 2.5?×?10⁹ cells mL^-1 for 48 h. The 3-day-old ice plant seedlings with root tip removed were successfully infected with A. tumefaciens or A. rhizogenes, and obtained 85% and 33-100% transient transformation rates, respectively. The transient transformation assays in ice plant callus and seedlings demonstrated that the concentrations of Agrobacteria, the durations of co-incubation time, and the plant growth stages were three important factors affecting the transient transformation efficiencies. Additionally, pot-grown juvenile plants were syringe-injected with two A. rhizogenes strains A8196 and NCPPB 1855, to establish transformed roots. After infections, ice plants were grown hydroponically and showed GUS expressions in transformed roots for 8 consecutive weeks.

Conclusions

Our Agrobacterium-mediated transformation protocols utilized hypocotyl-derived callus and seedlings as plant materials, which can be easily obtained in large quantity. The average successful transient transformation rates were about 2.4-3.0% with callus and 33.3-100.0% with seedlings. We also developed a rapid and efficient protocol to generate transgenic roots by A. rhizogenes infections without laborious and challenging tissue culture techniques. This protocol to establish composite ice plant system demonstrates excellent improvements in efficiency, efficacy, and ease of use over previous ice plant transformation protocols. These Agrobacterium-mediated transformation protocols can be versatile and efficient tools for exploring gene functions at cellular and organ levels of ice plants.

SUBMITTER: Hwang HH

PROVIDER: S-EPMC6323063 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum).

Hwang Hau-Hsuan HH Wang Chih-Hao CH Chen Hsiao-Huei HH Ho Jia-Fang JF Chi Shin-Fei SF Huang Fan-Chen FC Yen Hungchen Emilie HE

Botanical studies 20190107 1

<h4>Background</h4>Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols.<h4>Results</h4>To establish efficient transformation system of ice plants, three types of ice plant materials, hypocot ...[more]

PMID: 30617933

Similar Datasets

Project description:The halophyte Mesembryanthemum crystallinum (common or crystalline ice plant) is a useful model for studying molecular mechanisms of salt tolerance. The morphology, physiology, metabolism, and gene expression of ice plant have been studied and large-scale analyses of gene expression profiling have drawn an outline of salt tolerance in ice plant. A rapid root growth to a sudden increase in salinity was observed in ice plant seedlings. Using a fluorescent dye to detect Na(+), we found that ice plant roots respond to an increased flux of Na(+) by either secreting or storing Na(+) in specialized cells. High-throughput sequencing was used to identify small RNA profiles in 3-day-old seedlings treated with or without 200 mM NaCl. In total, 135 conserved miRNAs belonging to 21 families were found. The hairpin precursor of 19 conserved mcr-miRNAs and 12 novel mcr-miRNAs were identified. After 6 h of salt stress, the expression of most mcr-miRNAs showed decreased relative abundance, whereas the expression of their corresponding target genes showed increased mRNA relative abundance. The cognate target genes are involved in a broad range of biological processes: transcription factors that regulate growth and development, enzymes that catalyze miRNA biogenesis for the most conserved mcr-miRNA, and proteins that are involved in ion homeostasis and drought-stress responses for some novel mcr-miRNAs. Analyses of the functions of target genes revealed that cellular processes, including growth and development, metabolism, and ion transport activity are likely to be enhanced in roots under salt stress. The expression of eleven conserved miRNAs and two novel miRNAs were correlated reciprocally with predicted targets within hours after salt stress exposure. Several conserved miRNAs have been known to regulate root elongation, root apical meristem activity, and lateral root formation. Based upon the expression pattern of miRNA and target genes in combination with the observation of Na(+) distribution, ice plant likely responds to increased salinity by using Na(+) as an osmoticum for cell expansion and guard cell opening. Excessive Na(+) could either be secreted through the root epidermis or stored in specialized leaf epidermal cells. These responses are regulated in part at the miRNA-mediated post-transcriptional level.

Project description:Common ice plant (Mesembryanthemum crystallinum L.) is a novel edible plant with a succulent and savory flavor. The plants display prominent epidermal bladder cells (EBCs) on the surface of the leaves that store water and sodium chloride (NaCl). The plants have high nutritional value and are adapted to saline soils. Previous research has determined the impact of NaCl on the growth and mineral content of ice plant, but as NaCl has an impact on a food's sensory properties, an interesting question is whether saline growth media can affect the plant's taste and texture, and if this alters consumers' sensory response to ice plant. The objective of this study was to evaluate the sensory aspects of ice plant, as well as consumer liking in response to increasing NaCl concentration in hydroponic nutrient solution. Four-week-old seedlings of ice plant were transplanted into deep water culture (DWC) hydroponic systems and treated with five NaCl concentrations (0 M [control], 0.05 M, 0.10 M, 0.20 M, and 0.40 M NaCl). Eight-week-old plants (after four weeks of NaCl treatment) were harvested, and the middle leaves of each plant were sampled for consumer testing. A total of 115 participants evaluated various flavor, texture, and appearance aspects of ice plant and provided their liking ratings. The consumers were able to discriminate differences in salt intensity from the plants based on NaCl treatment in the hydroponic nutrient solution. Low NaCl concentrations (0.05-0.10 M) did not have obvious adverse effect on consumer liking, which aligns with the result of previous research that 0.05-0.10 M NaCl could largely stimulate the growth of ice plant. NaCl concentrations higher than 0.20 M are not recommended from both a production and consumer perspective. With increased NaCl level in plant samples, the consumers detected more saltiness, sourness, and fishiness, less green flavor, and similar levels of bitterness and sweetness. NaCl treatment had no effects on leaf appearance and texture, and the consumers' overall liking was mainly determined by flavor. Overall, ice plant presents some unique attributes (salty and juicy) compared to other edible salad greens; however, consumer awareness of ice plant is very low, and purchase intent is relatively low as well. Consumers picture ice plant being used mainly in salads and in restaurants.

Project description:Background: The halophyte Mesembryanthemum crystallinum (ice plant) is a model for studying salt tolerance. The morphology, physiology, metabolism, and gene expression of ice plant have been studied for over 40 years. Although the complete genome sequence has not been revealed, large-scale analyses of gene expression profiling have drawn an outline of salt tolerance in ice plant. Despite ample information in the transcriptome, miRNA information has not been documented. Results: We examined responses to a sudden increase in salinity in ice plant seedlings. Using a fluorescent dye to detect Na+, we found that ice plant roots respond to an increased flux of Na+ by either secreting or storing Na+ in specialized cells. High-throughput sequencing was used to identify small RNA profiles in three-day-old seedlings treated with or without 200 mM NaCl. Totally 132 conserved miRNAs belonging to 22 families were found. The hairpin precursor of 19 conserved mcr-miRNAs and 12 novel mcr-miRNAs were identified. Target genes are involved in a broad range of biological processes: transcription factors that regulate growth and development, enzymes that catalyze miRNA biogenesis for the most conserved mcr-miRNA, and proteins that are involved in ion homeostasis and drought-stress responses for some novel mcr-miRNAs. After 6 h of salt stress, the expressions of most mcr-miRNAs were down-regulated, whereas the expressions of their corresponding target genes were up-regulated. Analyses of the functions of target genes revealed that cellular processes, including growth and development, metabolism, and ion transport activity were up-regulated in roots under salt stress. Conclusions: Analyses of small RNA profile of ice plant seedlings identified many conserved miRNA families and several novel miRNAs. The expression of ten conserved miRNAs and three novel miRNAs were reciprocally correlated to predicted targets hourly after salt stress. Based on the expression pattern of miRNA and target genes in combination with the observation of Na+ distribution, we suggest that ice plant roots respond effectively to increased salinity by using Na+ as an osmoticum for cell expansion and guard cell opening. Excessive Na+ could either be secreted through root epidermis or stored in specialized leaf epidermal cells. These responses are partially regulated at the miRNA-mediated post-transcriptional level.

Dataset Information

Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum).

Background

Results

Conclusions

Publications

Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum).

Similar Datasets

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