Speed Breeding

The generation time of an organism varies from organism to organism. One of the defining features of model species is their short generation times, allowing fast analysis of, for example, gene mutations on phenotype (think of mice, zebrafish and the fruit fly). The shorter the generation time, the greater the number of experiments and analysis which can be performed within a set period of time.

In the world of plant science, Arabidopsis thaliana is generally considered the model organism and many papers on genetics and gene technologies use A. thaliana as the subject plant from which study results are drawn.

But conclusions drawn from a model organism doesn’t always translate to similar results in another organism – think of promising medical studies in mice that haven’t had the same effect in humans. Accordingly, model organisms may not always be appropriate for use to draw conclusions about other organisms.

Such is the case with the translation of studies in A. thaliana in some important crops such as wheat and barley. The problem with studying genetic traits in such crops is that the generation time of the plant may be long, significantly impeding important research.

Introducing Speed Breeding

Given the problem with generation times, a small army of researchers led by Lee Hickey of the Queensland Alliance for Agriculture and Food Innovation (and recent recipient of the Queensland Young Tall Poppy Scientist of the Year – congratulations Lee) developed a new protocol for speed breeding and tested a number of important crop plants for their response to the new protocol.

The Protocol

The protocol is pretty simple. Instead of using a standard photoperiod used when growing plants under study, the researchers increased the day length to 22 hours and reduced the length of time the plant is in darkness to 2 hours.

In fact, the paper specifies three protocols that all use this photoperiod, each of the protocols having a slight variation in the other growth conditions including the third protocol which is meant to be a less expensive set up able to generate similar results to that found in laboratory conditions.

Testing the Protocol

The first testing of the protocol took place in Norwich, United Kingdom, and compared the growth rates of bread wheat, durum wheat, barley and a model grass Brachypodium distachyon when grown using the speed breeding protocol or grown under usual greenhouse conditions with no supplemental lighting or heating.

The crops grown in the modified setting consistently flowered earlier than the control plants, taking approximately half the time to begin flowering with B. distachyon taking an average of 26 days and wheat taking on average 37 to 39 days. A significant change in the amount of time required to reproduce a target number of generations.

To ensure that the faster flowering plants didn’t suffer from loss of seed viability, the researchers tested whether the speed-bred plants were able to generate the following generation of plants. The researchers quantified effects on three phentoypic variables related to viability, including:

  • wheat seed counts per spike – a decrease compared to control;
  • spikes per plant – healthy number in the speed-bred plants; and
  • viable seed production – no difference.

Moving back from Norwich to Queensland, the researchers tested a second but very similar protocol with spring wheat, barley, canola and chickpea cultivars. This protocol used high pressure sodium lamps to produce the 22-hour day for these plants and compared the effect of this protocol to the same plants grown in glass house with a 12 hour day/night cycle. This time, both treatment and control groups were subject to the same temperatures for the day and night portions.

Similar to the first test, the time to anthesis was significantly less under the speed breeding conditions, but this time the wheat plants produced more spikes, grain number didn’t show the same reduced effect as it did the first protocol and the flowering times of the different crop species were more uniform than in the control. The researchers point out that this last effect, crops flowering at the same time, is advantageous when genotype crossing is to be performed. Seed viability was retained even when harvested 14 days post-anthesis and subsequently subjected to cold treatment for 4 days, indicating that further time savings can be made in the production of generation times.

In canola and chickpea there was no significant difference in seed production between the treatment and control groups.

Speed Breeding

Figure 1 from article demonstrating the photoperiod and temperature ranges under the speed breeding (left) and control (right) conditions and the number of generations which can be produced in wheat, barley, chickpea and canola under each condition.

Overall, the number of generations that can be produced under speed breeding conditions in a year were:

  • wheat – 5.7 generations;
  • barley – 5.4 generations;
  • canola – 3.8 generations;
  • chickpea – 4.5 generations.

The possibility of using the speed breeding technique in conjunction with single seed descent breeding and research programs was the basis of a seed viability analysis in wheat and barley. Using 100-cell trays, seed viability was confirmed for both genotypes at 80% two weeks after anthesis and 100% at four week post-anthesis. Accordingly, single seed descent programs can also be used in conjunction with speed breeding.

A low-cost speed-breeding protocol was also tested. In this set-up, LEDs were used exclusively in conjunction with a conventional split-system air conditioner to create speed breeding conditions and garnered similar results.

What about effects other than generation times?

Although faster generation times are extremely useful, if the effect of the speed breeding is to alter the crops’ phenotype, research programs would be plagued by unwanted confounding factors and breeding programs would be rendered ineffective.

The research team therefore mutated the awn supressor B1 locus and the Reduced height (Rhd) genes in wheat and obtained the expected phenotypes under the faster generated crops.

Testing any variation in disease responses was performed by inoculating wheat spikes with the bacteria which causes fusarium head blight, one set of spikes belonging to a susceptible cultivar and the second belonging to a head blight-resistant cultivar. The speed-bred cultivars showed the expected resistant and susceptible phenotypes.

Finally, the use of plant breeding in genetic engineering programs was addressed by looking at transformation efficiency of barley seeds grown under the protocol and, again, found no difference when compared to conventionally grown transformed plants.


Hickey’s team has successfully tested a protocol that will greatly assist research efforts into these studied plants and whose results will likely be replicable in many other plants. They cite sunflower, pepper and radish as plants that have shown similar responses to being grown in extended daylight conditions.

The ability quickly test, for example, the effect of knocking out or over-expressing a particular gene will certainly lead to more efficient test results and, hopefully will be another stepping stone to overcoming some of the limits to efficient research we currently endure.


C4 Photosynthesis – Testing Candidate Maize Genes in Rice

It would be no surprise to regular readers of the Legume Laboratory that we hold significant interest in the possibility of engineering C4 photosynthesis into C3 crops. Projects such as the C4 Rice Project are dedicating their efforts to this goal and its promise of more efficient crop growth and improved food security in the face of a changing climate.

Scientific Reports recently published an article funded through the C4 Rice Project which was looking to take the next step in procuring this biological engineering feat.

Maize is an interesting plant as it contains the two forms of photosynthesis, utilising C4 photosynthesis in foliar leaves while husk leaves use C3 photosynthesis. Previous studies have compared the transcriptomes of the two types of maize leaves to identify differences in gene expression. Given both leaves contain the same genome, hidden within the 283 differences in gene expression should be those differences which convert a leaf from using C3 photosynthesis to using C4 photosynthesis.

Working from this base, the researchers selected 60 of these genes to analyse further. The choice to look at 60 genes instead of the 283 differently regulated genes identified was born of the lack of high throughput technology needed to test gene functions efficiently enough for all to be analysed. The 60 genes tested were selected as they were predominantly transcription factors or leucine rich repeat receptor like kinases. A maize ubiquitin promoter was ligated upstream of the each individual gene resulting in 60 gene constructs that were individually transformed into rice plants.

In order to identify whether the genes made any change to transgenic rice plants, the researchers compared the leaf anatomy of the transformed leaves to the wild-type leaves. Regression analysis of vein number and leaf width in rice plants found a linear relationship between the two (a wider leaf will have a greater number of veins proportional to the change in width when compared to a narrower leaf). Given the difference in cells surrounding veins in C4 photosynthetic plants compared to C3 plants, there is a difference in the space between veins in the two plant types. The hope was to identify changes in the ratio of vein number to leaf width in transformed plants, an indication of a possible change in the method of photosynthesis.


The constitutive expression of 47 of the 60 genes made no observable difference to the ratio of veins to width of leaf normally seen in rice plants and the plants exhibited similar phenotypes to the wild type plants.

Three genes resulted in the transformed plantlets being unable to regenerate. The authors explain that rice tissue culture requires an excess of cytokinin to auxin in order for shoot growth to be promoted. Research into the three genes involved (ZmIDD16, ZmbHLH106 and ZmHCA2) revealed homologous genes in Arabidopsis that show evidence of effects on auxin biosynthesis. As a result, it was hypothesised that each of the three genes have resulted in an increase in auxin biosynthesis, throwing out the ratio of cytokinin to auxin needed for regeneration to occur, although further research was noted as necessary to confirm this hypothesis.

A different set of three genes disrupted shoot and root development, again believed to be due to alterations in the cytokinin/auxin ratio. The YUCCA gene seemingly caused the biosynthesis of excessive amounts of auxin, resulting in characteristic curled leaflets and hairy roots. A gene encoding a SMALL AUXIN UPREGULATED RNA (SAUR) disrupted the formation of lateral roots, with homologous genes demonstrating a disruption in normal auxin transport, reducing auxin-related root elongation and retarding initiation of lateral roots. The third gene (ZmSACL3) affected cytokinin, inhibiting its synthesis and resulting in similar phenotypes to that caused by the YUCCA gene due to the altered auxin/cytokinin ratio.

The overexpression of transcription factor R2R3 MYB caused intermediate veins to lose sclerenchyma cell connections to the abaxial epidermis. The loss of strengthening cells resulted in curled leaves. A different and novel gene resulted in the opposite effect on sclerenchyma formation, causing an increase in the proliferation of this cell type and resultant thicker walls than in wild type plants. Further, a couple of larger than usual bundle sheath cells around each vascular bundle formed. The result of the overexpression of this unnamed gene were infertile plants with short shoots and roots.

Figre 4 from C4 OX study

Figure 4 from article showing the effects of over expression of an as-yet unidentified gene. Figure b shows the reduced root growth while figure c shows the reduced shoot formation. Panel g shows normal sclerenchyma in wild type rice plants while panels h and l show the increased cell wall thickness described above.

ZmWRKY12, a gene which inhibited callus formation and therefore had to be inserted behind an inducible promoter to allow phenotypic analysis, caused dwarfing and reduced root growth in transgenic plants. The vein number to leaf width ratio was unaffected by the transgene but the normally lobed mesophyll cells in rice plants were lost in the transgenic lines. The orthologous gene in Arabidopsis is known to be involved in the formation of secondary cell walls, but these researchers have discovered that it also seems to play a role in cell lobing, a phenomenon for which the genetic basis was previously unknown.

Figure 5 from C4 OX study

Figure 5 from article showing the effects of ZmWRKY12 expressed in rice plants. The most interesting phenotype change was the change in mesophyll cell lobing from wild-type (panels d, f, and h) to the lack of lobing in the transgenic lines (panels e, g, and i).

Finally, three closely related Zinc finger nuclease genes showed normal vein spacing but caused spindly plants with drooping stems and leaves. It was theorised that the cause of the phenotype was due to alterations in the production gibberllic acid in the plants.


This paper is a great example of the incremental advancement of knowledge that builds upon earlier increments and is built upon by later increments. Those increments fill gaps in knowledge, or rule out particular cause/effect relationships, which help to direct and propel research towards its particular end (although, of course, research and enquiry need not always have a particular end to be directed at).

In the case of this research, the experiment resulted in some interesting and novel results, even though it didn’t yield any specific advance towards understanding the difference in gene regulation between the two forms of photosynthesis. That being said, we know that constitutive expression of any of 60 genes isn’t enough to trigger C4 photosynthesis. That leaves the remaining 223 genes to test, or perhaps a temporally regulated expression of one or more of the candidate genes, or some other combination of gene expression not yet contemplated.

The possible causes of C4 photosynthesis are numerous. This study also highlighted the deficiency in technology related to the efficient transformation, validation and phenotyping of transgenic plants. At present we lack the ability to quickly test over-expression of individual genes, and this is a relatively simple analysis to perform. The possibility that differential expression of a number of the genes being required to establish C4 photosynthesis requires significant advances in both wet lab and computation biology if we are to quickly make significant leaps in this type of research.

Great work by a great group of committed scientists.

Synbio in Space!

The challenges of surviving in space (with or without hostile enemies intent on dominating the universe) has made and will continue for a long time to make for a good sci-fi story. The resourcefulness required to survive in the vast emptiness between (and on) planets also forces us think deeply about better ways of propelling, feeding, housing and medicating ourselves. The cost of launching extra weight into space plus the limited lifespan of necessities such as food and medicine leaves us with complex questions about how to sustain ourselves as we look to explore more of our solar system and, hopefully one day, beyond it.

A 2015 review in the Journal of the Royal Society Interface considered a number of space missions and analysed how synthetic biology may assist those missions in the four resource areas mentioned above; propellant, food, habitat and medication. Of most interest, given the significance of the challenges presented, is a six-person, 916 day return voyage to Mars which would include 420 days of traveling there and back plus a 496 day stay on the red planet.

What are we going to have to do to be real-life Martians?


Although one of the shorter sections of the paper, how we will advance food production being the focus of this blog means that we will put the section of food production in space up-front and centre.

Based on astronauts living on the International Space Station, living in space requires 1.83kg of food per crew member per day. As a result, the six person Mars mission requires around 10 tonnes of food that needs to be shipped in order to sustain a mission if all of it was shipped from Earth although more accurate assessments of food amounts required to be produced whilst on mission have a lower estimate of 4.5 to 5 tonnes.

So the ability to produce food using available elements and compounds whilst away from the Earth is a significant problem. And the most accessible food production option is to use photosynthesizing bacteria and plants. Producing food using bacteria has the most significant weight savings, with Spirulina (a nutritional biomass made from either Arthrospira platensis or Arthrospira maximum) being promoted in earlier papers as a suitable option to producing food in space compared to attempting to grow and harvest plants. According to those previous papers, it is possible to produce 5.271kg of Spirulina per day using three 2,000 litre bioreactors. This rate of production would sustain the crew for the entirety of their stay on Mars and for the return to Earth and save a significant amount of launch mass.


Spirulina powder.

So how can synthetic biology help if Spirulina is already producible in adequate amounts to sustain the majority of a Mars trip? If you can imagine eating the same substance every meal for 706 days straight, one problem is easy to identify. It is suggested in the paper that synbio efforts could be directed to diversifying the textures and the tastes produced by the bacteria so that a variety of ‘foods’ can be consumed by those on the Mars mission.

Increasing the rate that the bacteria is able to reproduce will also aid the weight reductions at lift off and increase the redundancy built into the rations of food for the trip. There are some reported increases in production rates reported in the paper, but, for example, whether increased photosynthetic efficiency can be engineered into the bacteria to increase production is a question common to current research efforts in earth-bound food production.

Finally, being able to increase the nutrient content in the food produced to aid general health and space-specific health problems will increase the chances of a successful mission.


The shelf-life of pharmaceuticals are shortened in space travel and at present the issues caused by this are solved by  sending more supply missions. Given the time and distance involved in a trip to Mars, such a solution has little practical use.

Synthetic biology has the ability to play a role in readily producing pharmaceutical substances during a mission to Mars. For example, acetaminophen (paracetamol) can be produced using a modified chromisate pathway in E. coli. Unfortunately, space travel doesn’t suit E.coli as the compounds it requires to survive are not readily available. But if an autotroph can be manipulated to contain the same pathway and produce the same substance, we may have a new way to produce the pharmaceutical with the restricted inputs. The cyanobacterium Synechocystis sp. PCC 6803 has been identified as a possible candidate that will survive on the inputs available in space and could host the appropriate pathway to produce acetaminophen.


Figure 8 from article demonstrating the proposed pathway to create acetaminophen from Synechocystis sp. PCC 6803.

It can be imagined that with enough research we should be able to produce a range of pharmaceuticals or at least their components from the limited inputs available in space.


The further we go and the more weight we carry, the greater the fuel required. Complicating the matter further, the more fuel we carry into space, the more fuel we require to lift the extra fuel off the ground. The problem of needing more fuel to lift even more fuel to be able to propel more weight into space is one that permeates through the problems of supplying enough food and equipment to astronauts traveling for long periods of time.

The possibility of producing fuel in space is limited, according to this paper, to using methanogens to create methane. The type of methanogen used in bioreactors effects the quality of methane gas created and the rate of production. Further, a number of methanogens are able to use the compounds, particularly CO2, created by the crew of a space mission.

Synthetic biology uses to address the problem of fuel production in space will likely focus on the production of methane by methanogens. Particularly, the ability to produce methane using the least amount of inputs and with the greatest nutrient recycling efficiencies will be key. Similarly, modification of cyanobacterial production of ethylene, an alternative propellant, for greater production efficiency is another possible use of synthetic biology to aid space flight.

Habitat Construction

Lastly, the ability of erect a habitat on Mars will require either the supply of material from Earth and/or the use of material available on Mars with equipment supplied from Earth. The proposed use of 3D printing to construct habitat allows the use of finer materials in an additive manufacturing process. But the possibility of biologically produced materials (biopolymers) provides a method of producing the base materials using inputs available away from the Earth and significantly reducing the launch mass of materials dedicated to habitat construction.


Figure 7 from article demonstrating the shipping mass saved for habitat construction in missions to Mars (left) and the Moon (right).

Synthetic biology efforts focused on increasing the synthesis of biopolymer material per unit of bacteria will result in less equipment in the form of bioreactors being required to travel with the crew with the time for construction of the habitat being reduced as well.

Synbio in Space

At its most basic level, the quest to man a mission to Mars creates problems that must solved using the most efficient means possible. As a result, the use of synthetic biology to solve problems in the four areas listed above looks little further than what inputs are available in space and how they can be used to solve the problems spaceflight presents.

However, advances in modifying organisms to meet these challenges will likely lead to flow-on effects in the same areas here on a better-resourced planet. For example, should it be possible to create different textures and tastes in Spirulina in the confines of space then one would expect that it will be possible to use different inputs and different genetic circuits to create a variety of foods. Further, food produced should be more efficient, sustainable and economical than traditionally generated food.

Genetic Underpinnings of Heat Resistant Potatoes

Identifying the genetic basis of a particular trait is the building block on which molecular and transgenic breeding strategies build upon. How we connect the dots between a genotype and trait is the basis of a past post “How do we find genes related to traits? A review of bulked sample analysis”.

Despite only being behind rice and wheat as the most important crop worldwide, quantitative trait loci (QTLs) for heat tolerance in potatoes have not, until recently, been identified.

Potatoes are particularly sensitive to heat, with temperatures above the ideal maximum of 22°C resulting in significant decreases in growth and yield. The higher temperatures have a number of effects:

  1. Strongly suppresses tuberisation;
  2. Reduces the proportion of carbon assimilated for tuber starch;
  3. Reduces chlorophyll levels and CO2 fixation rates;
  4. Halts tuber dormancy resulting in early sprouting and secondary growths.

A recent study in the Plant Biotechnology Journal sought to identify QTLs for heat tolerance by comparing differences in gene expression and tuber yield between progeny of a biparental diploid potato (06H1) when grown at 22°C and 28°C.


Temperature response analysis and gene identification

Single node cuttings were used as a method to quickly induce tuberisation of subject potato plants, allowing a faster assessment of tuber yield (see this article discussing its use – paywalled unfortunately). 170 genotypes from the 06H1 population were analysed using 6 nodal cuttings from each to assess tuber yield under 22°C and another 6 for each genotype assessed at 28°C. On average, tuber yield at the lower temperature (1.017 grams) was greater than for nodes grown at the higher temperature (0.285 grams). The difference in performance was used in combination with single nucleotide polymorphism (SNP) maps of the 06H1 genome to identify genetic markers which may be connected to the genes which alter expression rates at the two temperatures.

fresh weight tuber yield nodal cutting assay

Figure 1 from article. Comparison of tuber fresh weight yield when grown at 22°C and 28°C.

Statistical analysis of the differences in expression levels at each marker found 3 markers which may be connected to the difference in phenotype. Using the Potato Genome Browser the researchers browsed the list of genes connected to the markers for suitable gene candidates for further research. Of the three markers, one marker contained a possible gene candidate, that encoding Heat Shock Cognate 70, a gene that has been suggested as involved in the heat tolerance in cabbage and potato.

With a candidate gene identified, the gene was isolated at amplified for analysis of allelic differences within the gene, of which 4 were identified and named A1, A2, A3 and A4. Nodes of potatoes containing each the four alleles were assessed for tuber yield, with allele A2 having the greatest fresh weight at both 22°C and 28°C and combined A2A3 genotypes having the highest yield. Gene transcript levels were assessed in plants grown at both temperatures which showed that the HSc70 gene was upregulated in A2A3 plants but not in the A1A4 plants, while the HSc70 allele up-regulated in plants grown at the warmer temperature were all from the A2 allele.

Analysis of the amino acids of each of the alleles themselves found that the A2 and A3 proteins contained an additional C-terminal sequence of KIEEVD (thought to be involved in interactions with co-chaperones), while no other variations were unique in a single allele.

What was unique to the A2 allele was a difference in the promoter sequence where a run of ten TA repeats was found as opposed to a run of four TA repeats in the promoters of the other repeats. The researchers later hypothesize that the greater number of TA repeats may result in enhanced expression levels, a hypothesis based on previous research in yeast, although the specific effect of the repeat TA sequence is not known.

Confirming the effects of the HSc70 A2 allele

To test the A2 allele and its promoter sequence, the gene and upstream component was transferred into Nicotiana benthamiana plants and subjected to 45°C heat in a growth chamber. After 24 hours the expression of the HSc70 gene was 7 times greater than in the control plants and cell membrane injury in the transgenic plants were significantly lower.

The unique promoter was then targeted for further analysis. The A2 allele was put downstream of a series of promoters with TA repeats varying between four and ten times. After being exposed to the higher temperatures the level of HSc70 transcription was measured. Alleles with eight to ten TA repeats in the promoter showed statistically significant elevated transcript levels compared to alleles with four to six TA repeats in the promoter.

HSc70 expression levels

Figure 6 from article. a) HSc70 expression levels for promoters containing 4, 6, 8 and 10 TA repeats and b) cell membrane injury percentage for plants containing the same numbers of TA repeats.

A2 allele-containing plants with significantly increased expression under 28°C heat were selected for analysis under 40°C conditions for 24 hours in comparison to wild-type plants. Extracts of plants after 4 hours at this temperature showed HSc70 expression being enhanced by up to 50 times more than in control plants. However, after 24 hours the transcript levels fell back to that seen prior to being subjected to the extreme heat. The A2 allele-containing lines has significantly less cell membrane damage compared to the wild-type.

Finally, the ability of the A2 allele to protect tuber yield at elevated temperatures was tested. Using the same nodal cutting tuber yield assay as before, A2 allele containing plants were compared with wild Desiree potato plants when grown at 20°C showed little difference in tuber yield. However, at 28°C the Desiree plants reduced yield by 75% compared to yield at 22°C. By comparison, transgenic lines had significantly lower yield reductions, with some lines have twice the fresh and dry weight yield as the wild type.


The identification of the heat shock cognate protein 70 within the potato genome and the quantification of the effects of the specific A2 allele demonstrates the significance of quality genome research, genome annotation and the ability of powerful molecular tools to seek out expression variances that allow us to pinpoint the basis of variances in phenotype. Coupled with well prepared and executed experiments, the development of these tools will continue to enable us to extract information that will assist us to assist crops and food production.

Whether it is through transgenic technology or molecular breeding, the identification of genes such as the one discovered in this study will assist food production meet the challenges of a changing climate.

Identifying the specific allele of the HSc70 protein that contributes to its resistance to the negative effects of elevated temperature is surely a big step into future-proofing the security of this important crop.

C4 Photosynthesis: Discovery, Understanding and Future Development

“The C4 pathway is one of the most complicated biochemical pathways likely to be placed in front of a university undergraduate, and attempts at C4 engineering have only served to reveal additional uncertainties about how the pathway operates, how it is regulated at the level of gene expression and post-translationally, and some of the physical properties of the anatomical specialization of Kranz C4 leaves.”

Robert Furbank (2016) “Walking the C4 pathway: past, present, and future”, Journal of Experimental Botany, vol. 67, no. 14,  pp. 4057 – 4066

Whilst writing our post titled “C4 Photosynthesis Evolution: Why some intermediates may not assist our understanding” we came across a 2016 paper written by Professor Furbank covering the discovery of C4 photosynthesis, the research and stories behind our understanding of this important photosynthetic trait, the gaps in our knowledge that remain to be filled and where the research may lead moving forward.

Readers of this blog may recall that Professor Furbank generously gave us the time to explain the problems in a research article proposing that C4 photosynthesis already existed in wheat which greatly assisted our updated post on the issue. He has also recently been a guest on the Australian Broadcasting Corporation’s “The Science Show” talking about C4 photosynthesis and the positive effects it could have on our food security (listen here).

Discovery of an important trait

Prior to the biochemical description of C4 photosynthesis detailed by Hal Hatch and Roger Slack in 1966, the special properties of particular plants that were later understood to use C4 photosynthesis had been documented since the turn of the 20th century. Professor Furbank’s review lists the important research articles which led the way to our current understanding, such as the discovery of Kranz anatomy, plants increased water use efficiency and increased photosynthetic rates compared other plants.

The separation between the collection and concentration of CO2 in mesophyll cells before pumping the CO2 to bundle sheath cells, where it is reduced and decarboxylated, is the most important and distinguishing trait used to differentiate C4 photosynthesis from other forms of photosynthesis. The concentration of CO2 allows Rubsico to operate at greater efficiency, reducing the competition for the active site of the enzyme that normally plays out between CO2 and O2 in non-C4 species, benefiting the growth rate and resource efficiency of the plant.

Furbank - C4 diagram

Figure from article demonstrating the Kranz anatomy (ring of purple bundle sheath (BS) cells and mesophyll cells (M) within the plant tissue. The numbers on the right of the figure denote; 1. carbonic anhydrase, 2. PEP carboxylase, 3. malate dehydrogenase, 4. NADP-malic enzyme, 5. pyruvate orthophosphate dikinase.

Unpublished data, criticisms of legitimate research findings and unnoticed publications of important research in obscure or foreign-language journals hampered early progress in discovering C4 photosynthesis until the seminal work of Hatch and Slack (who we are proud to learn are Australian scientists) in 1966.

Hatch and Slack’s 1966 research paper used 14C-labelled CO2 to study the phenomenon, applying a pulse of labeled CO2 and then illuminating the subject leaf to follow the progression of the labeled CO2 as it made its way through the photosynthetic cycle. This method is commonly known as a ‘pulse-chase’ analysis. The idea for the experiment came from similar earlier experiments conducted previously by other researchers but which had provided results that lacked certainty in the identification of the labeled compound. Whilst having chatting over a beer during the Australian Biochemical Society conference held in Hobart in 1965, Hatch and Slack decided to try their hand at replicating these earlier experiments in a manner that would reduce or remove the uncertainty contained within the earlier results and shed further light on the strange data that was being produced in these earlier works.

In something reminiscent of the oversight of Gregor Mendel’s early research on genetics in pea plants, it was discovered in the late 1960’s that a Russian researcher named Yuri Karpilov had reported similar findings from his research as that obtained by Hatch and Slack in an article he published in 1960 in a little known journal.

What wasn’t grasped at the time was that this newly discovered pathway resulted in a pool of CO2 concentrated in some cells while Rubsico, an enzyme difficult to extract, was present and operating in the separate bundle sheath cells. This discovery was slowly uncovered in a series of papers in the late 1960s and early 1970s, as was the importance of the concentration of CO2 in reducing photorespiration and its link to improved plant performance.

As our methods of isolating and probing at different cells have improved, so has our knowledge of the variety of biochemical pathways that exist within the C4 pathway. However, we are still grasping at which of these pathways provides the greatest flux of metabolites. When we acquire this understanding it will focus our attention to those pathways likely to result in the greatest improvements in plant performance and harness our attempts to engineer C4 photosynthesis in important crops.

Recent research

Since the significant increase in our knowledge of this important photosynthetic pathway around the time of Hatch and Slack’s 1966 paper, genetic transformation of plants and the increase in our ability to extract and subject plant data to bioinformatics techniques has continued to push research forward. The use of green foxtail millet as a C4 model grass by the biofuels community has allowed for the development of transformation protocols to help probe deeper into the genetics, transriptomics and protein products that form the basis of this system. Further, this information is crucial to our engineering attempts aimed at improving crop productivity.

Similarly, next-generation sequencing (NGS) has revolutionized research probing the evolution of C4 photosynthesis and how they may have evolved from their C3 ancestors. Probing the differences between the genetics and gene transcripts between the differentiated cells is steadily helping us understand and pinpoint which genes are absolutely necessary for a functioning C4 pathway. Analysis of C4 meristem tissue as it grows and the adjustment of gene transcription through this growth is also assisting in deciphering what gene regulation is required if we are to successfully install C4 photosynthesis into important C3 crops.

Where to now?

There is still important research to be done and discoveries to be made in C4 photosynthesis research.

Quantifying the amount of fixed CO2 that is leaked from mesophyll cells, under what conditions greater or lesser amounts of CO2 is leaked and what physical properties of the bundle sheath cells may change the rate of flux are all questions that remain to be answered if we are to successfully engineer the system into C3 crops. As with many areas of research, new tools to extract the information and avoid the uncertainty that plagues the more indirect methods of investigation we are currently limited to will greatly assist the research.

Further information about the positioning of important organelles within the cells and the ratios of bundle sheath cells to mesophyll cells are yet to be understood and which could prove to hamper engineering efforts.

Finally, despite the significant leap in the genetic and transciptomic knowledge we have seen of recent times, there is still only a developing understanding of gene regulation in the growing plant tissue to ensure the separation of the two parts of the carbon fixation and decarboxylation process within the tissue. Important transcription elements specific to this demarcation of activity between the cells are still being investigated and isolated.

The future

Significant research effort is being funded to enable the discovery of these important missing pieces of information and into attempting to transfer C4 photosynthesis into important C3 crops. The C4 Rice Project, funded by the Bill and Melinda Gates Foundation, is one such effort that looks very promising.

So, can we supercharge a VW to perform like a Porsche? The progress being made and the dedication to the problem must lead us to a sense of optimism that it will happen within our lifetimes.

Successful Field Testing of Drought-Resistant Transgenic Rice

Two points of concern facing all who are focused on future food security are a growing population and the disruption of food production caused by the effects of climate change. One effect of climate change predicted is that of longer and dryer periods of drought. Drought stress has a significant impact on crop productivity, further impeding the ability to feed a growing population.

The quest to build tolerance to such stresses as drought into important crops is a significant area of study. We have previously written on developing crop tolerance to biotic and abiotic stresses and new studies in the area are regularly being reported on in biotechnology journals.

We write here about a new study in the Plant Biotechnology Journal (Open Access) which field trialled two modified species of rice, an extremely important crop, for increased drought resistance.

The Study

The article starts by citing a number of studies that have found that over-expressing particular genes has resulted in increased resistance to drought stress, although usually such tests are performed under laboratory conditions. The difference noted is that of improved grain yield in the transgenic crops compared to the non-transgenic control plants when subjected to drought stress.

The adaptation of plants to drought stress and cellular injury results from the accumulation of metabolites within the plant cells, a phenomenon identified by the accumulation of soluble sugars in response to such a stress. The raffinose family of oligosaccharides, which includes the raffinose and galactinol sugars, are one such family of sugars found to accumulate in plants under drought stress. The raffinose sugars are part of a metabolic pathway that is induced by a number of genes named the GolS genes. One of these genes, the GolS2, is induced only by water availability problems due to drought and salinity.

Using this knowledge, the researchers sought to over-express the GolS2 gene in rice plants grown under field conditions to assess whether the test plants performed better when stressed compared to their non-transgenic precursor.


Creating the transgenic lines

After creating a gene construct containing the GolS2 gene taken from Arabidopsis thaliana and controlling its expression with the constitutive maize ubiquitin promoter, the constructs were transferred into two varieties of rice crop (Curinga and NERICA4) and plants confirmed to have been transformed with once copy of the gene construct were selected for study.

The modified plants were analysed for galactinol accumulation without any stress. The modified plants were shown to have considerably higher galactinol content than non-transformed plants. It was also tested whether markers for other drought inducible genes had seen an increase in transcription which would be an alternative explanation for the increase in galactinol content, but no inducement was found. Therefore, it appears likely that any difference in drought tolerance that may be seen would be related to the over-expression of the GolS2 gene.

Fig1 AtGolS2

Figure 1 from article. GolS2 transcription levels (top) and galactinol levels (bottom) in transgenic and non-transgenic lines (top).

Before beginning testing, any difference in agronomic quality between the transgenic and non-transgenic lines was also assessed with no obvious difference between them.

Testing the Transgenic Lines

Curinga lines were then tested with 3 weeks of drought stress at the vegetative stage of development (3 weeks old plants) with the following differences noted between the transgenic and non-transgenic lines:

  1. Transgenic lines maintained their height compared to the non-transgenic plants;
  2. Drought-induced leaf rolling occurred earlier in the non-transgenic plants;
  3. Dry biomass after re-watering were significantly higher in all but one line of the transgenic plants compared to the non-transgenic lines.

Testing was then carried by applying drought conditions to plants at the reproductive stage in field trials conducted in triplicate over 3 growing seasons. Grain yield in the transgenic lines was found to be significantly greater than in the non-transgenic lines. Two lines in particular were identified as consistently outperforming their non-transgenic cousins under severe drought conditions, including that they:

  1. Had a significantly greater number of panicles and of greater length;
  2. Had a lower incidence of leaf rolling;
  3. Recovered from the drought conditions faster;
  4. Flowered earlier; and
  5. Had greater grain fertility.

Following these tests five of the best performing transgenic lines were selected for further physiological and gene expression analysis. The analysis included testing of relative water content before and after stress, the photochemical efficiency of photosystem II and the chlorophyll content.

Relative water content between the test and control before drought showed no difference but after drought stress being imposed for a week the non-transgenic lines showed a decrease in relative water content with little change noted in the transgenic lines. Three weeks of drought resulted in transgenic lines losing 18% to 22% relative water content. The same length of drought reduced relative water content in non-transgenic lines by 30% in comparison.

Photochemical efficiency was tested by comparing the variable fluorescence of leaves with the maximum fluorescence. Unstressed and after one week of stress, the efficiency of both sets of crops were comparable. After three weeks of stress, transgenic lines retained greater photosynthetic efficiency compared to non-transgenic lines.

Chlorophyll content testing showed similar results; no difference between the two sets when unstressed, but chlorophyll content reduced significantly after three weeks of drought stress in the non-transgenic lines while such a drastic reduction was not seen in the non-transgenic lines.

Finally, the Curinga lines were tested in rain-fed water systems at a trial site in Columbia in three sets of tests between 2012 and 2015. Based on rainfall data taken over the previous 10 years, drought events occur at this site at a time the coincides with rice reproductive stages. When severe drought conditions arose during these trial periods, two transgenic lines in particular retained higher numbers of panicles and greater grain yield compared to non-transgenic lines.

Fig 6 AtGolS2

Figure 6 from article. Grain Yield in transgenic and non-transgenic lines in three experiments (a to c) and photo of transgenic line 2580 compared to non-transgenic Curina lines.

To test whether the positive effects of the gene construct on drought tolerance observed was transferable to a rice crop from a difference background, the NERICA4 lines were transformed and tested. Before drought conditions were imposed there was no phenotype difference between the two sets of crop. However, nine days of drought followed by a seven day recovery period resulted in most non-transgenic lines failing to recover (88.5%) compared to four of seven transgenic lines recovering with rates of survival being between 26.2 and 34.5%.

These lines were also tested for grain yield during the second and third field trials that the Curinga lines were tested in. The transgenic lines held higher grain yields than the non-transgenic lines.

The Correlation between Grain Yield and Galactinol Content

This section of the paper leaves something further to be researched.

The galactinol content of the plants increased in line with the increase in the transcription of the GolS2 gene. However, a dose effect of galactinol level on grain yield wasn’t always observed, leaving a question mark over the how the over-expression of the GolS2 and subsequent galactinol accumulation resulted in the observations reported by the researchers.


The study supports the proposition that over-expression of the GolS2 gene results in greater drought resistance and that such a result is seen in both laboratory and field testing. It also shows that over-expression of the gene doesn’t result in any unwanted phenotype changes in the crops that would affect their use in times of good water availability levels.

Why there was a lack of correlation between the galactinol levels and plant performance is a bit peculiar. It may be that there is no dose effect beyond a certain concentration of galactinol, but the question is worthy of further study.

Overall, this study reports an exciting development that could assist to secure our food supplies in areas increasingly affected by drought.

C4 Photosynthesis Evolution: why some intermediates may not assist our understanding

C4 photosynthesis, a form of photosynthesis which converts CO2 with greater efficiency than the more common C3 variant, is widely seen as a promising trait which could be used to improve crop growth. This is especially so with the looming concerns over the effects of climate change on food production.

We have previously written about C4 photosynthesis, how plant anatomy and chemistry differs between the two forms of photosynthesis and the possibility of being able to engineer C4 photosynthesis into crop plants.

Further, we have written about an article that suggested that certain parts of wheat plants may already perform C4 photosynthesis and, in a subsequent piece, we wrote about two rebuttals to that assertion.

In the aforementioned article regarding engineering C4 photosynthesis, the use of intermediate crops as a means to assist us to discover how some C3 crops have been able to evolve into C4 crops has been suggested as a means of furthering our knowledge of C4 evolution and to assist us to convert important C3 crops into more efficient light converters.

The article we are writing about this week warns researchers that the use of C3-C4 intermediate crops for such a purpose may not hold the answers to the questions we are asking.

C3-C4 Intermediates

This report recently published in the New Phytologist starts with a short description of C4 photosynthesis and its discovery. Two of these early papers on C4 photosynthesis evolution suggested that plants containing a photosynthetic system which appears to be an intermediate between C3 and C4 photosynthesis may be a proxy for the evolution of C4 photosynthesis.

However, the authors of this paper also point out that it is possible that these intermediates may, instead of depicting an plant in the throws of an evolutionary conversion, be a hybrid plant between two closely related species where one parent uses C3 photosynthesis and the other uses C4 photosynthesis.

The problem this raises is that research of C4 evolution may be misdirected if the intermediate under study is not truly undergoing this process.

To examine this possibility, the authors of the paper looked at the evidence of both natural and experimentally generated C3-C4 hybrids and described the categories of intermediates that have been discovered.

Categories of Intermediates

Intermediates of photosynthesis were categorised in a previous article by Sage et al (2014) which distinguished four categories of intermediate:

  1. Proto-Kranz: the plant contains bundle sheath cells or Kranz-like cells which have some similar traits to C4 species such as being larger and orientated in a centripetal position.
  2. C2 type 1: contains the same characteristics as proto-Kranz cells, but which also shuttles glycine produced in the mesophyll cells during photorespiration to the Kranz-like cells where its is decarboxylated and the photorespired CO2 is concentrated and partially refixed by Rubisco. This refixation of CO2 that would otherwise be lost in photorespiration in C3 and proto-Kranz photorespiratory cells improves efficiency.
  3. C2 type 2: the same as C3 type 1 save that the refixation of CO2 appears to rely largely on enzymes normally found in C4 photosynthesis, although their expression is lower than that found in C4 and C4-like species. C2 type 1 cells, by comparison, rely largely on a glycine shuttle and C4 enzymes are much less active.
  4. C4-like: a rarely found intermediate that shows increased activity of enzymes used in the C4 photosynthetic cycle and reduced reliance on C3-active enzymes.

Adapted from the Sage article is a table which contains a comprehensive list of plant species that fit within each criteria and under which category they sit. The Sage article also provides the below diagram illustrating the evolution from C3 to C4 photosynthesis.

C3 evolution

Figure from Sage et al (2014) showing the differences between C3, C4 and each intermediate category of photosynthesis. The figure in the article contains a lengthy description of the illustration (and the article is freely available).

The possibility of photosynthetic hybrids

Hybrid intermediates were experimentally attempted prior to the discovery of naturally occurring hybrids and have since been successfully created experimentally in a number of closely related species. Experimentally created hybrids have resulted in a mix of intermediate photosynthetic systems that were scattered throughout the spectrum between C3 and C4 photosynthesis. Intermediates generated would contain some components that related to C4 photosynthetic systems but would retain some C3 properties and similar properties would be passed in varying degrees to the segregating populations generated from the experimental hybrid.

In all, the previous research demonstrated the hybrids (C3 x C4, C3 x C3-C4, and C4 x C3-C4) could be classified in accordance with the criteria contained in the Sage paper to classify naturally occurring hybrids. The problem this causes, the authors suggest, is that the hybrids cannot be differentiated from true evolutionary intermediates on the basis of the Sage criteria.

The Naturally Occurring Hybrids

Naturally occurring photosynthetic hybrids have been considered likely in a number of papers where lineages have contained C3, C4 and some intermediate species. The article provides examples of a number of papers which observed or considered likely that hybridisation is the cause of the intermediate species.

The concern raised by the authors is that studies looking at phylogenetic trees of such lineages rarely look specifically for hybridisation as a cause of the photosynthetic intermediates. Therefore, the cause of the identified intermediates may be mistaken as being due to evolutionary changes rather than being due to hybridisation.


On the basis of the similarity between experimentally created photosynthetic hybrids and natural intermediates and their inability to be distinguished from each other using the Sage criteria, the authors believe that greater care should be taken when considering which intermediate is to be used to study C4 evolution. Taking the point further, actively excluding the possibility of the intermediate under study being of hybrid rather than evolutionary basis would be best practice before embarking on its use for the purpose of studying how C4 may have evolved and linking it to research of how we may create a similar evolution.

Finally, although hybrids may not be good candidates for evolutionary study, such hybrids aren’t without any worth. The hybrids experimentally created and discussed in the papers cited by these authors could be themselves be the basis of further research that could assist us to improve photosynthetic efficiency in food crops.

Abscisic Acid and Plant Stress Tolerance – A Mini Review

Frontiers in Plant Science recently collaborated with two other journals in putting together a number of articles related to the research topic “Mechanisms of abiotic stress responses and tolerance in plants: physiological, biochemical and molecular interventions”. As part of the topic a mini review was published reviewing what we know about abscisic acid signalling and abiotic stress tolerance, what further information we need to acquire and what we may be able to do with the knowledge for future food production.


Biological chemistry is mind-blowingly complex yet crucial to developing our understanding how organisms grow, reproduce, protect themselves and die, amongst others. How plants, stuck in one spot and forced to deal with whatever conditions may befall them, have the ability to respond to biotic and abiotic stress is important to know if we are going to be able to adapt crops to changing environmental conditions and improve food production security and efficiency.

Abscisic Acid (“ABA”) is one of a small number of phytohormones that play a significant role in how plants develop and grow. The amount of ABA produced by a plant is known to be affected by extracellular stresses and is considered important in assisting plants to adapt to and withstand abiotic stresses when they arise as well as being involved in such processes as seed development.


Abscisic Acid phytohormone. Credit: Wikipedia

ABA is produced in all parts of plants but accumulate in roots and terminal buds of growing plants, forming the basis of the hypotheses that they play a role in communication between the two ends of the plant during periods of stress. In the context of stress response, it plays a crucial role in assisting the plant in times of dehydration, thermal stress, strong UV absorption and uptake of heavy metals. The paper cites previous studies which have shown that mutant plants lacking ABA biosynthesis show decreased tolerance to fluctuations in their environments compared to the wild-type, while overexpressing plants have increased resilience.

ABA table 1

Table 1 from article. ABA regulation of stress effects on plants.

Drought Stress

Water deficiency in plants results in an osmotic stress in plant cells which cause cell desiccation and a resistance to water uptake. ABA levels rise in these conditions with one study showing that the levels of ABA under drought stress can be 40 times higher than when the plant isn’t stressed. Studies involving transgenic plants overexpressing ABA genes suggest that the hormone assists plants by maintaining membrane stability and causing changes in metabolite accumulation when water availability is low. Further, ABA is believed to be involved in stomatal closure during drought periods when water retention is of paramount importance to the plant. Studies which have applied ABA exogenously have also been shown to result in increased resistance to drought stress.

Drought stress can also result in the production of ethylene, a chemical which is commonly used in agricultural industries to bring on fruit ripening due to its effect of bringing on early senescence. However, early senescence in crops not yet at the stage of being useful for food production is problematic. Studies have shown that increased ABA production reduced ethylene production associated with this type of stress and others such as UV stress (see below).

Whilst ABA acts to protect the plant from water stress, it is generally agreed that the protective mechanisms initiated also result in decreased plant growth above that caused by the water stress itself. Plants subjected to drought stress with purposefully lowered ABA levels were demonstrated to have reduced growth. Further, plant shoot growth may be inhibited by ABA even when appropriately watered.

Heavy Metal Stress

Heavy metals such as cadmium, iron, mercury, copper and chromium, which are generated as a result of human activities and which can pollute agricultural land, can be taken up by plants leading to a toxicity caused by the reactivity of these metals. Reacting with cellular components results in energy loss, lowered photosynthetic capacity, reduced growth and early death.

Heavy metal absorbance results in increases in ABA levels. Testing of cadmium-tolerant and cadmium-susceptible rice cultivars has shown that the ABA level of the tolerant cultivar exceeded that of the susceptible one. Further, the application of exogenous ABA to the susceptible cultivar increased resistance to cadmium.

Chromium and copper can cause intracellular stress and resultant production of oxidants and free radicals. The uptake of these metals in plants has also been shown to result in ABA biosynthesis. Conversely, reduced intake of heavy metals leads to a reduction in the levels of ABA, indicating that ABA plays some role in the stress response.

UV Stress

Ultraviolet radiation absorbed by plants can result in the creation of reactive oxygen species within the plant, resulting in damage to cellular components which leads to retardation in plant development and growth.

Ethylene production increases with UV-B radiation exposure, causing early senescence in plant tissues. The presence of ABA in the plant tissue reduces ethylene production and thereby reduces the detrimental effects of the radiation exposure, a discussed above in relation to drought stress. In fact, research has demonstrated that a plant under one form of stress will also have an increased resistance to other forms of stress such as in the case drought stress and ultraviolet radiation stress.

The importance of Abscisic Acid

As can be taken from the examples provided, the production of ABA has been linked to the response of plants to such stresses as radiation, drought and heavy metal uptake. In some cases, research has linked stress tolerance to the presence of ABA and susceptibility to the lack of ABA present. However, the precise mechanism of action is not well understood.

The article also provides some background into the production of ABA and hints that, while we know many of the genes and proteins involved in its pathway to production we are still missing a complete understand of a number of steps and how the many branches of the ABA reaction link and/or work together.


This mini-review helpfully outlines some of the research surrounding ABA involvement in stress tolerance and the importance of understanding this if we are to enhance our crops.

It must be said that the review was in parts difficult to read and understand, perhaps due to to some issues with a translation to English, and that there was some unnecessary repetitiveness through the piece.

However, it is a good starting point for further research into abiotic stress tolerance and future food production.

A New Method to Accurately Measure Transgene Copy Number in Crops

A new technical article in The Plant Journal describes a method developed by the authors to ascertain the number of transgenes inserted into a variety of important crops.

Knowing the number of genes successfully inserted into an organism as a result of a transformation event is useful information. Although there are simpler methods to determine whether a plant has been successfully transformed, being able to determine whether there is one or more transgenes present assists understanding the effect of one gene being inserted on plant phenotype and its segregation through following generations. It also allows researchers to eliminate the possibility of multiple copies of the gene being inserted and resulting in silencing of the gene.

The researchers outline the current problem with attempting to determine the number of genes inserted into an organism. Southern blot analysis is used to confirm the insertion of target genes, but even the most skillful analysis can struggle to identify when multiple transformations have occurred in the same segment of the dissected genome. Further, the analysis is expensive, labour intensive and requires the use of radioactive materials, in turn requiring special permits and handling procedures.

Quantitative Polymerase Chain Reaction, although rapid, isn’t able to provide the accurate measurement data obtained from Southern blot analysis and distinguishing between one and two transgenes is difficult even with extensive optimisation.

A More Accurate, Faster Method

The researchers made use of a newer PCR method, droplet digital PCR. Here is a short video covering some of the basics, but there are more detailed videos covering the technology:

For further reading, here is a 2012 Nature review of the technology that explains the process and its advantages really well as well as pointing out the differences with qPCR, and here is another from the IDT website.

The importance of this method lies in the use of the small droplets into which the reaction mixture is divided and in which the PCR amplification takes place. The division of the fractionated genome into the droplets results in either one or no DNA segments being contained within any particular droplet. With each droplet containing primers for the gene of interest for PCR amplification and a labeled probe for identification, subsequent analysis of the amplified droplets can detect the number of droplets containing the sequence of interest.


Figure from Nature review of dPCR illustrating the division of DNA between droplets and subsequent amplification.

Using the number of positive and negative reactions combined with the volume of the sample, the number droplets used and a Poisson statistical analysis to determine the likelihood of more than one segment of DNA being contained within a single droplet, a precise determination of the number of times the gene of interest is present in the genome can be performed. It is even possible to determine hemizygous or homozygous presence of the gene.

In order to determine the number of transgenes inserted into a genome, a known single-copy gene within the same genome must be selected for identification and comparison.

Making it Work for Plants

The purpose of the research was to optimise the use of droplet digital PCR for use in a number important crops, particularly rice, citrus, potato, tomato, maize and wheat, and to test the results of their protocols against Southern blot analysis results of the same plants for validation. The result, they hope, is to provide a cheaper, more efficient method to determine transgene number in plants that can then be used confidently in further studies.

To perform the study, the researchers picked common transgenic target sequences and single copy genes in each of the subjects to serve as a reference and ran a duplex analysis of each of the transformed food crops.

For each of the 6 subjects, the paper identifies the both the transgene and the reference gene used, whether the analysis was conducted in the transformed adult or progeny plant and how successful the analysis was in determining transgene copy number when compared with the Southern blot analysis.

Even with the varying sizes of the genomes tested, the results of the digital droplet PCR in each crop were comparable to the Southern blot analysis, providing confidence that the method could be used in transgenic research.

Some things to consider

Although the assessments were successful, the researchers note a few observations they made to assist anyone else using the technique, including:

  • there is the possibility of obtaining ‘rainy’ droplets, where the droplet was found to be positive for the gene of interest but for which the signal wasn’t as strong as it was for the majority of the positive droplets. Despite the observation, the ‘rainy’ droplets didn’t appear to affect the result;
  • plants with larger genomes require a larger quantity of genomic DNA for analysis in order to obtain an accurate analysis;
  • sing either T0 plants or plants of known zygosity is important for an accurate analysis of transgene copy number using a single reaction;
  • quality, accurately measured DNA must be used for analysis to avoid poor quality droplets;
  • there is a range of optimal amounts of genomic DNA to be used dependent on the genome size in order that the confidence levels derived from the Poisson statistics are high enough that the results can be relied upon;
  • fractionation methods chosen for use are important. Complete digestion to allow for random segregation in the droplets of fragments is required;
  • if too few droplets are created the sample has been inadequately partitioned and results should be looked at closely; and
  • copy numbers that are midway between integers indicate a problem with the reaction.


The paper outlines a method that other researchers can use to cheaply and accurately determine the number of transformation events in crop plants and should contribute to the efficiency of research in transgenic laboratories. The amount of reaction mixture, the primers and reporters used and the plant populations that the DNA are cultivated from can all be adjusted from the methods used in the paper and will hopefully lead to usable protocols in numerous plants.

* Feature image credit: Integrated DNA Technologies – http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2013/10/21/digital-pcr-(dpcr)-what-is-it-and-why-use-it-


Increasing Plant Pest Resistance with Bt + RNAi Pyramids

Lately we’ve written a few posts about how crop resistance may be increased in the future. Our last post covered the development of a material able to pass RNAi protection to crops, and the one prior to that covered the multitude of sustainable new methods that may play a role in disease management.

This month, the Plant Biotechnology Journal (which is open – thank you Wiley) published an article looking at a possible way of reducing the evolutionary pressure caused by transgenic crops with one specific method of protection (Bt cotton in this study) as well as improving protection against pests that have developed resistance.

Bt cotton, a transgenic crop which contains a gene for a toxin (Cry toxin) found in Bacillus thuringiensis, provides protection against a range of insects. Prior to this gene being inserted into cotton, corn and a number of other crops, the Baccilus thuringiensis bacteria would be sprayed onto the crops to provide the same protection.

As successful as Bt crops have been, the use of a singular method of pest control puts pressure on the pests to evolve a method of overcoming the effects of the control mechanism. In the case of Bt cotton crops, farmers have begun using lines of transgenic cotton containing a number of different Bt toxins designed to kill the same pest. However, these pyramids of multiple Bt toxin genes are not entirely effective due to the toxins interfering with each other or causing cross-resistance within the pests.

Pyramids of Bt toxins and RNAi

The research tested the possibility of constructing a cotton plant containing both a Bt toxin gene and one of two genes for a double stranded RNA aimed at interfering with two particular genes within the pest Helicoverpa armigera, a moth which develops by feeding on important crops such as cotton and corn.

To test whether the pyramids improved crop defence against H. armigera, cotton plants containing one of the two dsRNAs, the Bt toxin gene, a pyramid of Bt plus one of either of the RNAi constructs or a control plant were challenged with either a Bt susceptible or Bt resistant pest. Two essential genes which have previously been shown to effect pupation of H. armigera were the targets of the dsRNA constructs. The pest fed with an artificial diet containing either of the dsRNAs  was shown to result in an increase in pest mortality and those pests that did survive had a lower weight compared to those feeding on the control plant.

Cotton plants were then transformed via Agrobacterium tumefacnians-mediated transformation to create lines of cotton with either of the dsRNAs or a Green Fluorescence Protein as a control and then selfed the resulting lines to create lines homozygous for each of the genes. Resulting lines were shown through Southern blot analysis to contain only one of the inserted genes.

Susceptible pests fed either of the dsRNA transformed plants showed lower rates of transcription of the target genes.

Having created lines of cotton containing dsRNA able to effect pest growth, the researchers crossed the dsRNA lines with Bt lines in order create crops containing both traits and developed those lines into homozygous cotton plants with  the same number of copies of each of the genes.

Did the Pyramids make any difference?

Before testing the pyramided crops to compare their effect on Bt resistant pests to that of Bt-only crops, resistant H. armigera were fed Bt, transgenic GFP or non-transgenic cotton with no significant difference in their mortality or growth rates. Susceptible H. armigera pests fed the same diets showed the expected increased mortality when fed the Bt cotton diet.

When the resistant pests were fed the RNAi cotton crops, mortality rates were similar to that in susceptible pests, with both lines of pest having a mortality rate close to that of Bt susceptible pests when fed transgenic Bt plants. Of the two RNAi crops, there was no real difference in mortality rates and development between the two genes targeted by the RNAi constructs. Nor was there any significant difference between the effects of RNAi crops on resistant pest mortality and crops containing the Bt + RNAi construct, demonstrating that the pyramid did not have any effect on the pest save for the presence of the RNAi component of the pyramid.

Bt susceptible pests grown on the pyramid cotton crops did show an increase in mortality and days to development when compared to the RNAi crops alone. Using the index of multiplicative survival (“IMS” – comparing the mortality rates of the pyramid to the expected mortality rate of the pyramid which is calculated by multiplying the mortality rates of the pests when raised on crops containing only one or the other the pyramided genes) to determine whether the Bt and RNAi components were acting separately or in concert to cause the effect seen. Using this method of analysis it was thought that the two genes contained in the pyramid act independently against the susceptible pest.

Overall, the cotton crops containing the pyramids showed increased protection against both the susceptible and the resistant lines of pest.

bt and rnai pyramid figure 1

Figure 3b from article. Comparative mortality rates between susceptible and resistant H. armigera on wild type (W0), Green Fluorescence Protein transformed (GFP), Bt, two RNAi transformed crops (JHA and JHB) and Bt + RNAi pyramids. Astrix indicates statistically significant differences between the two lines of pest.

Just in case there is a possibility that Bt resistance resulted in a fitness cost to the H. armigera that may interfere with the analysis, resistant lines were fed on wild type cotton and the transgenic GFP cotton and their development rates monitored. Interestingly, resistance to Bt does come with a cost to development time, resistant pests having a 15 to 16% increase in development time compared to their susceptible cousins. Mortality between the two lines however did not show any difference.

What effect may use of the construct have in reducing resistance evolution?

Computer simulations were used to demonstrate what effect the use of pyramid cotton will have on resistance evolution in a number of scenarios with parameters taken from common growing conditions in northern China and varying levels of pest fitness cost of resistance and time for resistance development.

The amount of refuge land used in the scenarios had a significant impact on resistance development times. Using a refuge percentage of 50%, it was found that adding RNAi defence either in succession with Bt crops or in a pyramid crop increased the time to development of resistance against the defence when compared to Bt crops alone. Using pessimistic parameters for the development of resistance and fitness cost (faster evolution and little to no fitness cost associated with resistance development) and a refuge percentage of 50, it was demonstrated that the time to resistance increased by 5 years when RNAi was used in tandom with Bt crops while time resistance increased to 10 used when the two methods were used consecutively in a pyramid.

Bt plus RNAi Fig 2

Figure 5 from article. Simulations predicting years to resistance under a) realistic scenario, b) Optimistic scenario and c) Pessimistic Scenario with differing percentages of refuge area.


This may be the first time we have discussed a paper which experimented on something other than an important food crop. But Bt transformed food crops are in widespread use and the reliance on only one method of pest control results in the types of problems we are seeing today with the evolution of resistance. Therefore, developing the ability to provide more sustainable, longer term protection to crops could be fast-tracked using a technology like this where the gene targeted by the RNAi can be designed for a specific pest with minimal side effects on related species of insect.

The accuracy of the computer simulations is a little difficult to make out without a better knowledge of the underlying data but could be the basis of field tests and more sophisticated simulations.

The development of RNAi technology, from examples like this to the creation of crop protection technologies like BioClay, is impressive and seems likely to play significant role in the future protection of food production.