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Growing Arabidopsis In Vitro: Cell Suspensions, In Vitro Culture, and Regeneration
Abstract and Figures
An understanding of basic methods in Arabidopsis tissue culture is beneficial for any laboratory working on this model plant. Tissue culture refers to the aseptic growth of cells, organs, or plants in a controlled environment, in which physical, nutrient, and hormonal conditions can all be easily manipulated and monitored. The methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Techniques, including callus production, cell suspension cultures, and plant regeneration, are all indispensable tools for the study of cellular biochemical and molecular processes. Plant regeneration is a key technology for successful stable plant transformation, while cell suspension cultures can be exploited for metabolite profiling and mining. In this chapter we report methods for the successful and highly efficient in vitro regeneration of plants and production of stable cell suspension lines from leaf explants of both Arabidopsis thaliana and Arabidopsis halleri.
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... CCA1::LUC, LHY::LUC, and FKF1::LUC seeds were sterilized in 50% (v/v) bleach and 0.02% (v/v) Triton X-100 and placed on a callus induction medium. We used two recipes of callus induction media reported in Barkla, Vera-Estrella, and Pantoja (2014) and Sello et al. (2017). The Barkla medium was MS medium supplemented with 1-μg/mL 2,4-D, 0.05-μg/mL BA, 3% (w/v) sucrose, and 0.8% (w/v) agar (pH 5.7) (Barkla, Vera-Estrella, and Pantoja 2014). ...
... We used two recipes of callus induction media reported in Barkla, Vera-Estrella, and Pantoja (2014) and Sello et al. (2017). The Barkla medium was MS medium supplemented with 1-μg/mL 2,4-D, 0.05-μg/mL BA, 3% (w/v) sucrose, and 0.8% (w/v) agar (pH 5.7) (Barkla, Vera-Estrella, and Pantoja 2014). The Sello medium was MS medium supplemented with 0.5-μg/mL 2,4-D, 0.25-μg/mL benzylaminopurine (BA), 3% (w/v) sucrose, and 0.8% plant agar (pH 5.5) (Sello et al. 2017). ...
... Seeds were stratified at 4°C for 3 days and then moved to a growth chamber with the condition described above. Hypocotyls, first leaves, and roots were isolated from 7-day-old seedlings and placed on Barkla callus induction medium (Barkla, Vera-Estrella, and Pantoja 2014). Calli induced from those tissues were subcultured to fresh medium every 2 weeks. ...
Callus and cell suspension culture techniques are valuable tools in plant biotechnology and are widely used in fundamental and applied research. For studies in callus and cell suspension cultures to be relevant, it is essential to know if the underlying biochemistry is similar to intact plants. This study examined the expression of core circadian genes in Arabidopsis callus from the cell suspension named AT2 and found that the circadian rhythms were impaired. The circadian waveforms were like intact plants in the light/dark cycles, but the circadian expression in the AT2 callus became weaker in the free‐running, constant light conditions. Temperature cycles could drive the rhythmic expression in constant conditions, but there were novel peaks at the point of temperature transitions unique to each clock gene. We found that callus freshly induced from seedlings had normal oscillations, like intact plants, suggesting that the loss of the circadian oscillation in the AT2 callus was specific to this callus. We determined that neither the media composition nor the source of the AT2 callus caused this disruption. We observed that ELF3 expression was not differentially expressed between dawn and dusk in both entrained, light–dark cycles and constant light conditions. Overexpression of AtELF3 in the AT2 callus partially recovers the circadian oscillation in the AT2 callus. This work shows that while callus and cell suspension cultures can be valuable tools for investigating plant responses, careful evaluation of their phenotype is important. Moreover, the altered circadian rhythms under constant light and temperature cycles in the AT2 callus could be useful backgrounds to understand the connections driving circadian oscillators and light and temperature sensing at the cellular level.
... Plant hormones play an important regulatory role in the processes of plant bud induction, proliferation and rooting [30,31]. There are obvious differences in the growth effects of different genotypes of poplar using different hormone types and ratios [32]. Plant hormones, such as 2,4-dichlorophenoxyacetic acid (2,4-D), indole butyric acid (IBA) and naphthylacetic acid (NAA), are commonly used as auxins in poplar tissue culture, which can promote cell division and elongation [26,30,33]. ...
A series of tissue culture regeneration protocols were conducted on gray poplar (P. tremula × P. alba) to select the most efficient callus induction medium, adventitious shoot differentiation medium, shoot elongation medium and rooting medium, which laid the foundation for the optimization of genetic transformation technology for gray poplar. The results showed that the Woody Plant Medium (WPM) supplemented with 0.10 mg L−1 kinetin (KT) and 1.00 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) was the most suitable medium for callus induction. The callus induction rates of different tissues were greater than 85.7%. The optimal adventitious shoot differentiation medium was the WPM supplemented with 0.02 mg L−1 thidiazuron (TDZ), and the adventitious shoot differentiation rates of young tissues were 22.2–41.9%. The optimal direct differentiation medium was the Murashige and Skoog (MS) medium supplemented with 0.20 mg L−1 6-benzylaminopurine (6-BA), 0.10 mg L−1 indole butyric acid (IBA) and 0.001 mg L−1 TDZ, and the differentiation rate of adventitious shoots was greater than 94%. The best shoot elongation medium for adventitious shoots was the MS medium with 0.10 mg L−1 naphthylacetic acid (NAA). After 45 days of cultivation in the MS medium with 0.10 mg L−1 NAA, the average plant height was 1.8 cm, and the average number of elongated adventitious shoots was 11 per explant. The 1/2 MS medium with 0.10 mg L−1 NAA showed the best performance for rooting, and later, shoot growth. The direct shoot induction pathway can induce adventitious shoots much faster than the indirect adventitious shoot induction pathway can, and the time cost via the direct adventitious shoot induction pathway can be shortened by 2–6 weeks compared to that of the indirect shoot induction pathway.
... Tissue culture is commonly used to mass produce ornamental plants such as African Violets (Stewart 1999), to improve and conserve horticultural crops such as blueberries and avocados (Sharma 2014;Hiti-Bandaralage et al. 2017), and to produce plants for research into plant function such as Arabidopsis thaliana (Barkla et al. 2015). It can also be used to support ex situ conservation for species that are highly threatened, do not produce viable seeds, or do not produce seeds suitable for banking ). ...
Full text can be downloaded at:
https://www.anpc.asn.au/plant-germplasm/
The Germplasm Guidelines are focused on conservation of common and threatened plant species, including many species that are endemic to Australia. The Guidelines provide an evidence-based best practice guide for the management of ex situ (off site) collections of seeds, plant tissues, or plants in nurseries and living collections. The guidelines aim to provide access to current research findings and practice to maximise the value of every seed or plant collection.
... Tissue culture is commonly used to mass produce ornamental plants such as African Violets (Stewart 1999), to improve and conserve horticultural crops such as blueberries and avocados (Sharma 2014;Hiti-Bandaralage et al. 2017), and to produce plants for research into plant function such as Arabidopsis thaliana (Barkla et al. 2015). It can also be used to support ex situ conservation for species that are highly threatened, do not produce viable seeds, or do not produce seeds suitable for banking ). ...
... Tissue culture is commonly used to mass produce ornamental plants such as African Violets (Stewart 1999), to improve and conserve horticultural crops such as blueberries and avocados (Sharma 2014;Hiti-Bandaralage et al. 2017), and to produce plants for research into plant function such as Arabidopsis thaliana (Barkla et al. 2015). It can also be used to support ex situ conservation for species that are highly threatened, do not produce viable seeds, or do not produce seeds suitable for banking ). ...
... Tissue culture is commonly used to mass produce ornamental plants such as African Violets (Stewart 1999), to improve and conserve horticultural crops such as blueberries and avocados (Sharma 2014;Hiti-Bandaralage et al. 2017), and to produce plants for research into plant function such as Arabidopsis thaliana (Barkla et al. 2015). It can also be used to support ex situ conservation for species that are highly threatened, do not produce viable seeds, or do not produce seeds suitable for banking ). ...
This chapter gives an overview of tissue culture and the main steps involved culturing individual plant species. The chapter gives examples of Australian species in culture, and case studies showing application of the technique to generate material for translocation and restoration. The chapter can be downloaded free of charge from the Australian Network for Plant Conservation at https://www.anpc.asn.au/wp-content/uploads/2021/09/GermplasmGuidelinesThirdEdition_FINAL_210902.pdf
... Tissue culture is commonly used to mass produce ornamental plants such as African Violets (Stewart 1999), to improve and conserve horticultural crops such as blueberries and avocados (Sharma 2014;Hiti-Bandaralage et al. 2017), and to produce plants for research into plant function such as Arabidopsis thaliana (Barkla et al. 2015). It can also be used to support ex situ conservation for species that are highly threatened, do not produce viable seeds, or do not produce seeds suitable for banking ). ...
Symbiotic mutualisms between plants and fungi or plants and rhizobia are often essential for their growth and survival in the wild. In particular, the Orchidaceae and Fabaceae (the second and third largest plant families in the world) are highly reliant on their symbioses with mycorrhizal fungi and rhizobia, respectively. In Australia, a number of unique and endemic species in these two plant families are at risk of extinction, with three species of each already becoming extinct since European settlement. For these species, conservation of the symbionts is critical for the long-term conservation and eventual rewilding of these plants. This chapter will therefore focus on techniques for collecting,
growing and conserving symbionts, as well as seeds, to support conservation of threatened species in the Orchidaceae and Fabaceae. The chapter is available for download free of charge from the Australian Network for Plant Conservation - https://www.anpc.asn.au/wp-content/uploads/2021/09/GermplasmGuidelinesThirdEdition_FINAL_210902.pdf
... Tissue culture is commonly used to mass produce ornamental plants such as African Violets (Stewart 1999), to improve and conserve horticultural crops such as blueberries and avocados (Sharma 2014;Hiti-Bandaralage et al. 2017), and to produce plants for research into plant function such as Arabidopsis thaliana (Barkla et al. 2015). It can also be used to support ex situ conservation for species that are highly threatened, do not produce viable seeds, or do not produce seeds suitable for banking ). ...
This chapter describes the different types of non-orthodox seeds (i.e. seeds not suitable for standard seed banking), direct and indirect methods for identifying them, and options for conserving them. It is available for free download from the Australian Network for Plant Conservation via this link - https://www.anpc.asn.au/wp-content/uploads/2021/09/GermplasmGuidelinesThirdEdition_FINAL_210902.pdf
... Tissue culture is commonly used to mass produce ornamental plants such as African Violets (Stewart 1999), to improve and conserve horticultural crops such as blueberries and avocados (Sharma 2014;Hiti-Bandaralage et al. 2017), and to produce plants for research into plant function such as Arabidopsis thaliana (Barkla et al. 2015). It can also be used to support ex situ conservation for species that are highly threatened, do not produce viable seeds, or do not produce seeds suitable for banking ). ...
Introduction
Cryopreservation involves the storage of germplasm (e.g., seeds, embryo axes or tissue cultured shoot tips) at very low temperatures, typically utilising liquid nitrogen (LN) (-196 °C), or its vapour (-130 to -192 °C), to preserve living tissue in a state of suspended animation. Cryopreservation has become a viable long-term conservation tool for many threatened and economically valuable species (Walters and Pence 2020).
Cryopreservation provides a relatively low maintenance and space efficient conservation option for very long-term storage (i.e., >25 years), particularly for germplasm that is not suitable for conventional dry storage at -20 °C that must be maintained as living collections (Dulloo et al. 2009). This chapter provides guidelines on when to use cryopreservation, and what tissues to use. It also provides methods that may be used to establish a cryogenic germplasm bank of Australian plant species.
Callus and cell suspension culture techniques are valuable tools in plant biotechnology and are widely used in fundamental and applied research. For studies in callus and cell suspension cultures to be relevant, it is essential to know if the underlying biochemistry is similar to intact plants. This study examined the expression of core circadian genes in Arabidopsis callus from the cell suspension named AT2 and found that the circadian rhythms were impaired. The circadian waveforms are similar to intact plants in the light/dark cycles, but the circadian expression in the AT2 callus stopped in the free-running, constant light conditions. Temperature cycles could drive the rhythmic expression in constant conditions, but there were novel peaks at the point of temperature transitions unique to each clock gene. We found that callus freshly induced from seedlings had normal oscillations, like intact plants, suggesting that the loss of the circadian oscillation in the AT2 callus was specific to this callus. We determined that neither the media composition nor the source of the AT2 callus caused this disruption. We observed that ELF3 expression was not differentially expressed between dawn and dusk in both entrained, light-dark cycles and constant light conditions. Overexpression of ELF3 in the AT2 callus partially recovers the circadian oscillation in the AT2 callus. This work shows that while callus and cell suspension cultures can be valuable tools for investigating plant responses, careful evaluation of their phenotype is important. Moreover, the altered circadian rhythms under constant light and temperature cycles in the AT2 callus could be useful backgrounds to understand the connections driving circadian oscillators and light and temperature sensing at the cellular level.
The aseptic culture of plant cells and tissues as technique is now well established. Successful development of tissue culture was necessitated by a physiological problem which clearly demanded for its solution some extreme form of isolation of the tissues being studied. Although real success first came with animal tissues, the botanist Gottlieb Haberlandt (1854-1945) (Fig.1) clearly set forth the purposes and potentialities of cell culture after having attempted culture of plant cells. Haberlandt was not entirely successful but foresaw the use of cell culture as an elegant means of studying physiological and morphological problems.
A cell culture system providing a suitable source of free cells for in vitro genetic manipulations is presented. The system is based on the correlation between dissociation rate and growth pattern in suspension cultures. This correlation is used to control the balance between cell division and cell dissociation. 2,4-Dichlorophenoxyacetic acid (2,4-D) at a concentration of 1 mg/l is estimated to be the most efficient dissociating factor. The culture is permanently maintained in the late log phase by adding fresh medium to the biomass at a well-defined ratio; thus, cells are kept growing and cell dissociation is favoured. This makes it possible to use large cell aggregates as a continuous source of single cells and of small cell aggregates. A yield of 0.8 to 1.8·106 viable cells per ml is obtained.