The discovery of a common genetic denominator governing the impact of stress on pollen opens the way for researchers to develop a single heritable trait with the potential to stabilise yields under even the most challenging weather stresses.
Dr Rudy Dolferus, CSIRO Plant Industry.
PHOTO: Brad Collis
- The GRDC is supporting research to deliver to breeders a single trait that protects wheat crops at flowering from multiple climatic stresses, including frost and drought
- Improved germplasm is already finding its way to breeders even as DNA markers are being developed
A single wheat trait has been consistently allowing CSIRO scientists to make gains in plant tolerance to multiple stresses – including drought and frost – raising hopes for a plant breeding breakthrough that could deliver an all-in-one defence against climate extremes.
Researchers have found pollen sterility (which hits yield because fewer grains are produced on the head) to be a common factor with all of the main environmental stresses.
This is expected to open up a new area of plant breeding possibilities for climate-proofing cereal crops.
With GRDC support, Dr Rudy Dolferus from CSIRO Plant Industry has been screening wheat varieties to analyse the variation in plants’ ability to maintain fertile pollen under environmental stresses.
Dr Dolferus explains: “When you have a wheat field that has been drought-stressed, for example, you get a much higher frequency of out-crossing. This tells you that while drought has undermined the fertility of the plant’s own pollen, the female part of the flower is tolerating the stress and remains receptive to pollination from other plants.”
Dr Dolferus says this applies to drought and shading stress, and cold and heat stress.
Following the initial discovery of the multiple-stress-tolerance trait, improved germplasm was quickly identified and made available to breeders, but Dr Dolferus still worried that the conventional selection procedures would be too onerous for breeders and this would delay inclusion of the development into commercial varieties. (It is hard enough for breeders in their selection process to mimic a single stress, such as frost, let alone several at once.)
Thinking about this, it occurred to him that cereals were unlikely to be using different molecular pathways to signal the plant to shut down pollen formation in response to various stresses.
“To me that suggested a biological mechanism that is common to different stresses,” he says. “Otherwise the sheer dimensions and biochemistry of the plant would be ridiculously complex.”
To simplify selection, he decided to seek out the genes responsible for determining why, under the same stress conditions, some plants remain able to make fertile pollen and some are not.
The genetic mapping that arose from this is now making it possible to develop DNA markers to select for the particular gene variant that plants need to inherit to acquire multiple-stress tolerance.
Once this is done the markers will make it comparatively simple to breed this multiple-stress tolerance into new varieties.
The hydroponic osmotic stress screening facility used by CSIRO Plant Industry to identify markers associated with multiple-stress tolerance in a Cranbrook–Halberd wheat population.
To identify genetic factors contributing to the pollen sterility trait, Dr Dolferus needed to subject large populations of wheat plants to various types of stress.
To facilitate screening in the project’s early stages he developed biologically relevant surrogate stresses able to be applied under controlled environment conditions.
For example, osmotic stress in hydroponic growth systems was used to prevent water uptake by plants in a way that simulates drought. Low light was used to mimic shading stress.
He was able to screen a broad range of Australian bread wheat cultivars and identify cultivars with extreme levels of stress tolerance – the statistical ‘outliers’. One of the most tolerant was Halberd and one of the more susceptible was Cranbrook.
“Halberd has acquired a reputation as a variety that stands up to all kinds of bad weather conditions, so growers like it for that,” Dr Dolferus says. “It is known to be cold, heat and frost tolerant and we have shown it to also be drought tolerant.
“So it was a good candidate line to detect genetic control of the pollen sterility response to see if there was a common genetic factor we could pursue that might provide multiple-stress tolerance.”
That pursuit is now underway in earnest, with the odds looking favourable that such a desirable genetic factor and associated DNA markers – for multiple-stress tolerance – do exist.
Tracking it down decisively involves crossing Cranbrook and Halberd to produce a wheat population in which the susceptible and tolerant genomes have mixed and recombined in different configurations.
A special class of DNA markers are then developed that can distinguish between Cranbrook and Halberd DNA at various sites all along each chromosome. This ‘physical’ map is then compared and matched with the inheritance of stress tolerance and susceptibility, respectively.
It is slow, meticulous work but it can identify DNA regions – called quantitative trait loci (QTLs) – that are always inherited and bring with them some degree of stress tolerance.
For drought and shading stress tolerance, CSIRO has identified seven QTLs, with four proving to be similar between the two types of stresses. This is what is expected if the same genetic factors have a role in responding to various stresses.
The QTL with the strongest influence on stress tolerance levels has been found on chromosome 3 of bread wheat’s B genome.
For the first time, in 2013, the most tolerant and sensitive of the Halberd–Cranbrook lines (the so-called ‘tail lines’) were field-tested at the GRDC’s managed environment facility at Yanco, New South Wales. The lines were subjected to drought conditions through late sowing and the use of rainout shelters.
“The lines that contain the QTLs for drought and shading tolerance will now be tested in future trials for tolerance to other stresses such as heat stress, to confirm that the tolerance trait is shared by different stresses,” Dr Dolferus says.
Frost, humidity link
Dr Dolferus’s parallel work is on frost, a stress that in 2008 cost Australian grain growers $109 million.
However, researchers have found it particularly difficult to screen for improved levels of frost tolerance. Dr Dolferus believes part of the problem is the complexity of frost events.
“There are lines that are tolerant in Western Australia but not in South Australia and are completely different again in Queensland,” Dr Dolferus says.
“So there is obviously a difference in frost events that affects a plant’s response. This lack in consistency is a problem for the breeding work because which frost data do you believe?”
He takes these complications into account by having a multi-pronged approach. Included is a novel strategy in which frost’s impact on wheat is broken down into various components that contribute to frost tolerance.
He explains that prior to frost events, plants sense a range of changes in their environment – drops in temperature and the resulting increase in humidity in the canopy or the increase in far-red light that occurs at sunset – and they start adapting and preparing to protect themselves.
“Chilling conditions by themselves can cause yield loss due to pollen sterility,” Dr Dolferus says. “It is a plant’s lack of capacity to adapt to chilling that then causes problems with ensuing frost conditions. Plants make all kinds of compounds under cooling conditions that protect their tissues from freezing.”
In WA, Dr Ben Biddulph of the Department of Agriculture and Food, WA, has come across this effect in field trial work on frost tolerance. He has noted the occurrence of pollen sterility under conditions where there was chilling but no frost.
The two researchers are now collaborating, with Dr Biddulph providing lines from the National Frost Initiative for Dr Dolferus, expanding the capacity to work with the pollen trait.
“Most field trials only concentrate on frost events and frost damage, but it is actually the much milder cold periods that need more attention to solve the problem, because that is where the plants get ready for worse frost conditions,” Dr Dolferus says.
“This shows the complexity of the cold-frost problem and why we need to break down these events into all the components that are involved.
“It is basically a race against time to get all the protection in place for frost, and some lines are better at this than others,” Dr Dolferus says.
He has worked up techniques to detect these early physiological changes and has shown that pollen sterility is much higher when there is a combination of cold and high humidity. This may explain the variability between the different field environments in Australia.
Some varieties are tolerant to both cold and the combination of cold and humidity while others are tolerant only to cold but not high humidity.
“This illustrates that there is different genetic control, underlining the complexity and the need for a stepwise improvement strategy for frost tolerance,” Dr Dolferus says.
“We are working with two lines – Wyalkatchem and Young – that show consistent differences in their tolerance to cooling down in both WA and in our controlled environment experiments in Canberra. This material is therefore ideal for getting a better understanding about what is happening ‘under the bonnet’.”
The goal is to identify variation in the adaptation response that builds up the plant’s ability to survive frost. Tolerant plants are expected to adapt better than a sensitive variety and the researchers want to understand those biological differences.
Pollen sterility seems to be a key and similarities with the mechanisms that make pollen drought, shading and heat-tolerant are going to be examined to determine whether markers for the maintenance of pollen fertility under those stress conditions will also lead to grain production/yield being maintained under chilling conditions.
Since Halberd is known to be chilling and frost-tolerant, the Cranbrook–Halberd mapping population used in the drought tolerance work is being used to check for potential overlaps with chilling and frost tolerance.
However, Dr Dolferus warns that due to the complexity of this, improving chilling-frost tolerance may require a cumulative breeding approach where different traits – the maintenance of pollen fertility, humidity tolerance, capacity to adapt to cold and frost conditions – may need to be added in a step-by-step breeding process.
For Dr Dolferus, the end goal is to replace growers’ stress-avoidance tactics with stress tolerance.
“There is just a small window of opportunity in Australian climates to optimise wheat yields and growers capitalise by manipulating sowing time,” Dr Dolferus says.
“That amounts to a stress-avoidance, but growers still can’t control the weather, so unexpected events routinely cause big
“To get around that you need to do something about tolerance, as opposed to avoidance. So that is what we are focusing on with our pollen fertility work.”
Pollen sterility trait fertile grounds for science prize
The pollen sterility induced in wheat by drought was the theme of a scientific paper that was awarded the 2012 ASPS–FPB Best Paper Award by the Australian Society of Plant Scientists and CSIRO Publishing. The award is issued annually to early career scientists for papers published in the journal Functional Plant Biology. It recognises both an outstanding discovery in plant biology and excellence in scientific writing.
The 2012 winner was GRDC scholar Hollie Webster who is in the final stages of her PhD at the Australia–China Joint Research Centre for Wheat Improvement (ACCWI) at Murdoch University in Western Australia.
Ms Webster’s winning paper, ‘Genome-level identification of cell wall invertase genes in wheat for the study of drought tolerance’, describes how a family of five invertase (INV1) genes were identified in a genome-wide search of the wheat genome. The genes were characterised in ways that clarify their role in inducing pollen sterility in response to drought.
The work drew on the recently available wheat genome survey sequence by the International Wheat Genome Sequencing Consortium.
“I am honored that I and my ACCWI colleagues received this recognition,” Ms Webster said. “I am particularly proud that our work in wheat genomics is highlighting how strongly Murdoch University supports excellence in crop genomics research to advance global food security and sustainable food production locally.”
Dr Rudy Dolferus
02 6246 5010
For more on frost, see Ground Cover Supplement included with this issue.
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GRDC Project Code
CSP00143, CSP00175, GRS10028
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