Name: a robotic platform for high-throughput protoplast isolation and transformation
Uploaded: 2016-09-27T13:00:00
Description: Watch this Scientific Journal Video about A Robotic Platform for High-throughput Protoplast Isolation and Transformation at JoVE.com

This research was supported by Advanced Research Projects Agency - Energy (ARPA-E) Award No. DE-AR0000313.

1. Establishment of Suspension Cell Cultures
<ol>
	<li>Prepare liquid BY-2 media by adding 4.43 g Linsmaier &#38; Skoog Basal media, 30 g of sucrose, 200 mg KH2PO4, and 200 &#181;g of 2,4-dichlorophenoxyacetic acid (2,4-D) to 900 ml of distilled water and pH to 5.8 with 0.1 M KOH. After adjusting pH, adjust final volume to 1,000 ml with distilled water and autoclave. Media can be stored up to 2 weeks at 4 &#176;C.</li>
	<li>Inoculate a 250 ml Erlenmeyer flask with 100 ml of liquid BY-2 media and a...

The protocol described above has been successfully validated for protoplast isolation, enumeration, and transformation using the BY-2 tobacco suspension cell culture; however, the protocol could easily be extended to any plant suspension culture. At present, protoplast isolation and transformation has been achieved in numerous plants, including maize (Zea mays)10, carrot (Daucus carota)32, poplar (Populus euphratica)33, grape (Vitis vinifera)34</su...

In recent years there has been significant impetus placed on the design of transgenic crops to overcome various diseases1, endow herbicide resistance2, confer drought3,4 and salt tolerance5, prevent herbivory6, increase biomass yield7, and decrease cell wall recalcitrance8. This trend has been aided by the development of new molecular tools for generating transgenic plants, including genome-editing using CRISPR and TALENs9, and gene silencing through dsRNA10, miRNA11, and siRNA12. While these technologies have simplified the generation of transgenic plants, they have also created a bottleneck, where the sheer number of transgenic plants generated cannot be screened using traditional systems that rely on plant regeneration. Related to this bottleneck, while silencing and genome-editing constructs can be rapidly inserted into plants, many of the targeted traits fail to produce the desired effect, which is often not discovered until plants are analyzed in the greenhouse. In this work, we have developed a method for rapid, automated, high-throughput screening of plant protoplasts, specifically to address the current bottleneck in early screening of large numbers of genome-editing and gene silencing targets.
The use of protoplasts, as opposed to intact plant cells, has several advantages for the development of an automated platform. First, protoplasts are isolated after digestion of the plant cell wall, and with this barrier no longer present, transformation efficiency is increased13. In intact plant cells there are only two well established methods for transformation, biolistics14 and Agrobacterium-mediated transformation15. Neither of these methods can be easily translated to liquid handling platforms, as biolistics requires specialized equipment for transformation, whereas Agrobacterium-mediated transformation requires co-culture and subsequent removal of the bacteria. Neither are amenable for high throughput methods. In the case of protoplasts, transformation is routinely conducted using polyethylene glycol (PEG)-mediated transfection16, which only requires several solution exchanges, and is ideally suited for liquid handling platforms. Second, protoplasts, by definition, are single-cell cultures, and thus the problems associated with clumping and chain formation in plant cell cultures, are not observed in protoplasts. In terms of rapid screening using a plate-based spectrophotometer, clumping of cells, or cells in multiple planes will lead to difficulty in acquiring consistent measurements. Since protoplasts are also denser than their culture media, they sediment to the bottom of wells, forming a monolayer, which is conducive for plate-based spectrophotometry. Finally, while plant cell suspension cultures are primarily derived from callus17, protoplasts can be harvested from a number of plant tissues, leading to the ability to identify tissue-specific expression. For example, the ability to analyze root- or leaf-specific expression of a gene can be very important to phenotype prediction. For these reasons, the protocols developed in this work were validated using protoplasts isolated from the widely-used tobacco (Nicotiana tabacum L.) &#39;Bright Yellow&#39; 2 (BY-2) suspension culture.
The BY-2 suspension culture has been described as the &#34;HeLa&#34; cell of higher plants, owing to its ubiquitous use in molecular analysis of plant cells18. Recently, BY-2 cells have been used to study the effects of plant stressors19-22, intracellular protein localization23,24, and basic cell biology25-27 demonstrating the broad utility of these cultures in plant biology. An additional advantage of BY-2 cultures is the ability to synchronize the cultures with aphidicolin, which can lead to enhanced reproducibility for gene expression studies28. Furthermore, methods have been developed for the extraction of BY-2 protoplasts using low-cost enzymes29,30, as enzymes traditionally used for generating protoplasts are cost prohibitive for high-throughput systems. As such, the protocol described below has been validated using the BY-2 suspension culture, but it should be amendable to any plant cell suspension culture. Proof-of-concept experiments are performed using an orange fluorescence protein (OFP) reporter gene (pporRFP) from the hard coral Porites porites31 under the control of the CAMV 35S promoter.

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a robotic platform for high-throughput protoplast isolation and transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

In the current study, the doubling rate of BY-2 varied from 14-18 hr dependent on the temperature at which the cultures were incubated, consistent with previous reports of a mean cell cycle length of 15 hr. With this doubling rate, a 1:100 starting inoculum was used to initiate cultures, leading to cultures with a packed cell volume (PCV) of 50% in 5-7 days. In the current protocol, in which cultures were grown in 200 ml of media, a PCV of 100 ml was generated in 7 days, which provided en...

Watch this Scientific Journal Video about A Robotic Platform for High-throughput Protoplast Isolation and Transformation at JoVE.com

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

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