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From Plough to Pipette – Tools for Crop Development

In part 2 of our plant synthetic biology series we teamed up with Cameron Tout of the Legume Laboratory blog to introduce some of the tools of plant synbio and how these are being applied to agriculture.

Over 9000 years ago the first domesticated varieties of wheat were created in South West Asia. What was remarkable about these plants is that they were selected by humans to retain their seeds rather than dispersing them by wind. This meant that wheat became dependent on farmers for propagation, but allowed people to harvest grain without the pods shattering in their hands.


Source: Wheat by Sleepy Claus (


Since then, humans have been modifying plants in ever more sophisticated ways, the 20th century saw the introduction of mutation breeding and hybrid technology, resulting in massive gains in crop yields.


Unfortunately there is strong evidence for a plateauing of yield increases in intensive cropping systems (such as wheat in Northwest Europe and rice in East Asia) which is suggested as evidence that farmers are begin to reach the biophysical limits of production. In other regions low or stagnating yield increases are caused by a lack of access to agricultural inputs.




When reading the introduction to any recently published research on crop yield, disease resistance, environmental stress tolerance and any number of similar issues relating to food production, forefront are the related concerns of an increasing global population, limited agricultural land and the expected detrimental effects of climate change on food security. Combined social, economic, political and scientific efforts will be necessary to deliver the sustainable intensification required meet the demand without destroying the environment.


In short, we need to grow more food, on the same amount of land, with less input.


The recent NAS report ‘Genetically Engineered Crops: Experiences and Prospects’ found GMOs to be safe, but suggested that it was unclear to what extent some of the current technologies had increased yields. At present, commercially available genetically modified foods are limited to a few staple crops and their transformations have been limited to specific pest or herbicide tolerances.




However, as the research and technology development in the field of genetic manipulation has grown, so has the possibility of using these advances to either increase food production or to main yield levels despite the increase in such problems as drought, salinity and nitrogen deficiency.

 A great overview of topic is provided by Devang Mehta where he talks about approaches for ‘rewriting our food supply’ ranging from maintaining and generating biological diversity to who will own the innovations, and the most recent advances in plant synthetic biology were presented at the recent International Conference on Arabidopsis, which was enthusiastically reported on by Mary Williams of Plant Science Today and she has supplied overviews and links on the advances in genome engineering as presented by some of the leading plant scientists.


Where are we now? Top-down” Synbio and its application to plants


“Top-down” synthetic biology is the use of existing genomes and modifying the genes within it. It is the common approach to genetic engineering, particularly plants, at present. An example of this is the addition of a gene from Bacillus thuringiensis, a bacteria that has the contains a protein that causes death in many pest insects when ingested but which does not affect humans. Instead of spraying Bacillus thuringiensis over crops as has been done in the past, a crop with this particular gene added to its genome produces the toxin itself and confers the same resistance.




Previously (and in many cases still to this day), the location of incorporation of a new gene into an existing genome was random. The randomness made transformation events hard to come by as the new gene may not insert into the genome at all or it may be introduced within another vital gene, a fatal event for the host plant.


Breaking DNA at a specific location has the added advantage that the nucleotide sequences one each side of the break can be predicted. With this knowledge, one of two methods, depending on the type of break made, can be used to precisely insert a gene at the site of the break. The most useful cut-and-paste method available to researchers is to create a break that leaves one or more nucleotides overhanging on one strand of the double stranded DNA at each side of the break, commonly called ‘sticky ends’. A piece of DNA containing the gene to be inserted into the broken DNA with nucleotides on either side that complement the nucleotides of the sticky ends will anneal with each, a process called homologous recombination. The result is a new gene inserted with precision in the existing genome.

Image: Overview of homologous recombination and nonhomologous end joining. Source: Wikipedia.


With the site-specific methods now available, the addition of genes can be directed with much more precision, although further improvements are needed to reduce “off-target” mutations. 


One aim of current research is to ‘stack’ multiple traits in a crop. Adding multiple genes into an organism haphazardly has the likely result that not all of the traits will be passed onto the next generation. However, using site-specific nucleases two or more genes can potentially be transformed into a plant located close together with a resultant increase in the prospect of being passed to subsequent generations, a must for food crops. Such a transformation was made recently in a study using a zinc-finger nuclease to stack multiple genes in a precise location within the genome and close enough together for transmission to subsequent generations.

Zinc finger DNA binding domain dimer. By Boghog at en.wikipedia – Own work (Original caption: “Created myself using PyMol”)Transferred from en.wikipedia by SreeBot, Public Domain,


Performing this research without the available tools was formerly a time consuming, labour intensive endeavour. The advent, implementation and refining of precision editing is leading the way to us accumulating and applying our knowledge of the plant genome to begin addressing our food security concerns.


Where are we headed?


Long-term future – Synthetic plants from scratch


But the future of plant synthetic biology will no doubt develop along the same lines as bacteria based research, being the design and construction of new genomes from scratch in a ‘bottom up’ approach. The same aims as those of Craig Venter’s ‘Synthia 3.0’, reducing an organism to its essential components with the removal of redundant genes (genes in multiple copies in the genome), are reflected in the aims of scientists looking to develop plants stripped down to the bare necessities with the added ability to insert genes of choice.


Plant Tissue Culture. Source


The current deficiencies that stand in the way of us producing ‘bottom-up’ designed plants are multi-sided and there are a number of recent review pieces in leading journals discussing how the science is likely to advance.


Aligning plant synthetic biology to engineering


At present, we are still developing the required knowledge to meet the principles developed in traditional engineering that have resulted in the complex mechanical and electronic instruments we use today. The ability to decouple complex biological problems into smaller, manageable tasks is still limited by the holes in our knowledge and will come with time.


By Bill Bertram – Bill Bertram, CC BY-SA 3.0,


Standardisation of gene sequences and how they are to be connected is an area in active development. Standardisation of the different parts that make up a genome requires us to research and identify genes, transcription factors, untranslated regions and terminator sequences that we can reliably use to generate the same product under the same conditions in a variety of hosts. Modular units that can be looked up in a catalogue and ordered with confidence that it will connect with other units and work as described is the major hurdle standing in the way of reliably creating new genomes from scratch. PhytoBricks are closest we have to a lego set of plant biological parts. At present the catalog is limited to a small number of pieces when compared to the parts in the BioBricks catalogue of genetic parts for synthetic bacterial systems.


SBOL visual standards for BioBrick assembly. By Jacob Beal and the SBOL visual working group –, Public Domain,


Standardisation also requires that the way we incorporate genes into a genome follows a set standard that can be reproduced in other laboratories. A number of different frameworks for assembly frameworks have been suggested in the academic literature. GoldenBraid 2.0  is one such framework that has been suggested which has been built upon an earlier version of GoldenBraid and incorporating the MoClo Modular Cloning System (3.0 is also in development).


GoldenGate assembly method. Source:


The effort to standardised has even recently been introduced into the journal ACS Synthetic Biology in the form of a specific method for researchers to depict their genetic circuits instead of each researcher having his or her own method for displaying this information. The idea behind the standardisation is to make understanding and replicating successful creations of synthetic circuits will serve to streamline our research and knowledge-building efforts.


Faster testing cycles


One current limitation to plant synthetic biology is the time required to confirm that the plant has been transformed by the desired particular piece or segment of DNA. As opposed to conventional models such as Escherichia coli and other bacteria which have quick generation times and resultant quick confirmation of transformation, genetic modification of plants requires the plants growth and reproduction which takes a significantly longer time. The result is a rate limiting step to progress in plant synthetic biology and plant science generally.  Further developments are needed in methods of quickly determining whether the desired traits have been successfully inserted into the genome of a plant without waiting for the plant to grow and reproduce.


Plant tissue cultures, in the Population, Genetics and Genomics laboratory, at the Agriculture Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP), in Ft. Collins, CO, during the visit of U.S. Department of Agriculture (USDA) Secretary Tom Vilsack visits on Wednesday, Nov. 6, 2013. NCGRP is the largest agricultural genebank facility in the United States, and one of the largest in the world. The NCGRP staff conducts groundbreaking research to develop more efficient and effective means for preserving plant and animal germplasm, and for better understanding the genetic structure of these invaluable materials. The research findings, preservation techniques, and specialized technology developed by the NCGRP have been adopted by genebanks around the world: many international scientists travel to the NCGRP for research and training. The NCGRP staff conducts groundbreaking research to develop more efficient and effective means for preserving plant and animal germplasm, and for better understanding the genetic structure of these invaluable materials. The research findings, preservation techniques, and specialized technology developed by the NCGRP have been adopted by genebanks around the world: many international scientists travel to the NCGRP for research and training. The NCGRP staff includes 34 full-time USDA/ARS employees (7 of whom are Ph. D.–level scientists), plus 8 part-time and 16 student employees. The FY13 budget for the center is $4.7 million. For more information, see USDA photo by Lance Cheung
Plant Tissue Cultures. By USDA, Lance Cheung – Flickr, CC BY 2.0,


Protoplasts, plant cells that have had their cell wall removed and which allow easier transformation with site-specific nucleases, are the most likely candidate for synthetic genomes to be tested in with the ability to quickly verify the transformation event has occurred. Very recently an article was published reporting on an automated system of DNA insertion and screening of plant protoplasts.


By Lab of Ralf Reski – University of Freiburg, Lab of Ralf Reski (, CC BY-SA 3.0,


Orlando de Lange of the New Leaf Blog provides an overview of an article from the Plant Cell which reviewed the current techniques in plant transformations and, particularly, the bottleneck that currently exists when a researcher gets to the point of trying to transform a gene circuit they know works in a plant model into a crop.


Developing new parts


Possibly one of the more exciting possibilities is the development of molecular switches that can be quickly used to switch on and off particular genes in response to particular inputs such as a certain wavelength of light. Binary digital parts similar to logic gates with AND, OR and NOR functions could result in particular, definable responses in gene expression being couple to complex inputs such as light or water stress.


Logic Gates. Source:



Building on the idea of creating plants with completely novel abilities is the development of plants as biosensors. An example of the research in the area can be found in this Harvard Gazette piece which details the development of a biosensor in Arabidopsis thaliana that starts fluorescing when it detects digoxin by fusing the fluorescent signal component to a ligand-binding domain that specifically binds digoxin. The potential application to agriculture, environmental monitoring or even its use as a confirmatory tool in the lab would be significant. A similar review on the development novel traits and abilities being transformed into plants was published in Nature Plants recently.


Credit: Wyss Institute at Harvard University


Computer modelling


When the development of orthogonal parts and a standardised method of engineering the parts into a functional, predictable piece of DNA is achieved, the ability to build models of these systems in silico will result in the quick and simple assessment of whether a genetic construct will have the desired output to a given input. In much the same way as a multitude of computer aided design programs can be used to piece together multiple, simple parts into an elaborate virtual circuit board for circuit testing, so will plant and crop scientist be able to take simple parts and existing complex designs to toy with different ideas and prove concepts as either workable or not.


Couple quick and simple modelling with more reliable wet lab techniques, which are likely to be roboticized for speed, accuracy and reliability, and the future of plant synthetic biology holds significant promise in addressing the threats to food security.


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