In 1954, John Gladstones commenced a post-graduate study to investigate the potential of sweet lupins, formative work to initiate the WA sweet lupin industry. Now new scientific understanding and tools are being deployed by a post-graduate student to develop a 'bittersweet' lupin.

The presence of bitter compounds plays two important roles in narrow-leaf lupins (NLL). Bitter tasting and toxic quinolizidine alkaloids (QAs) provide important pest resistance and prevent the plants being eaten.

However, NLL do contain healthy compounds, so if the bitterness can be lowered in the grain, while maintaining the natural pest defence of the rest of the plant, market opportunities could be expanded for the crop.

This is the concept of ‘bittersweet’ NLL which Chunsheng Xiao is exploring as a GRDC-supported PhD student under the guidance of Professor Chengdao Li, the director of the Western Crop Genetics Alliance, which is a joint research centre of Murdoch University and the Western Australian Department of Primary Industry and Regional Development (DPIRD).

“Narrow-leaf lupin is a leguminous crop; as nitrogen fixers they play a significant role in Australian cropping rotations,” Ms Xiao says.

“They contain 30 to 40 per cent protein in the seed, 40 per cent carbohydrates – mostly dietary fibres – and are rich in minerals and vitamins, making NLL a crop with significant health potential and an excellent option for replacement of both animal protein and gluten-containing products.

However, quinolizidine alkaloids in NLL seeds have curtailed its wider use as food. Traditionally, grains of NLL have been prepared for human dietary purposes after being de-bittered by boiling.

This de-bittering process can remove the toxins, but a considerable portion of nutrients such as soluble proteins, minerals, flavonoids, monosaccharides and sucrose are also eliminated by boiling the seeds, in addition to the cost of the process.

Therefore, traditional plant breeding efforts focused on developing alkaloid-free lupins. A successful program led by John Gladstones ensured that Australia became the main producer of these ‘sweet’ lupins in the 1960s.

However, lupin production in Australia has been on the decline since the late 1990s. One of the main reasons for this is the grain from ‘sweet’ lupins is not completely free of QAs and, depending on harvest year or location, could exceed industry thresholds (0.05 per cent of dry seed weight for animal feed and 0.02 per cent of dry seed weight for human consumption).

Additionally, ‘sweet’ NLL are not only low in seed levels of QA but also in the plant vegetative tissues, making them more susceptible to insects and herbivores and more costly to produce due to the increase in chemicals required to manage these pests. Interactions between QA content of NLL cultivars and environment are known to be complex and also cultivar-specific.

Basis of PhD study

“My study is focusing on the comprehensive characterisation of molecular mechanisms regulating QA content and determining the basis of the transportation of alkaloids through the plant,” Ms Xiao says.

Published studies tend to support that QA synthesis occurs primarily in the green parts of NLL, showing that most QAs might indeed be translocated to the seed in NLL. Considering this, breeding a ‘bittersweet’ NLL variety could be feasible through genetic engineering to block QA transportation from vegetative tissues to seeds.

Specifically, I am aiming to firstly identify specific regions known as quantitative trait loci (QTL) in the NLL genome that regulate QA content in vegetative tissue and seeds.

“(I will) then move on to functionally characterise key genes within these specific regions regulating QA accumulation in vegetative tissues and seeds, including regulators and transporters. And, finally, to enhance our knowledge of QA synthesis and transportation mechanism and explore the feasibility of breeding ‘bittersweet’ NLL through genetic approaches.”

Ms Xiao is two years into her PhD study, supported by a Murdoch International Postgraduate Scholarship, along with a GRDC Research Scholarship. She brings with her experience from completing a master’s degree majoring in crop breeding in China in 2019.

She established contact with Professor Li in 2018 and worked with him as a visiting student to Australia on barley improvement for six months. This experience inspired her return to Australia to pursue further study.

Application of innovative technology

“The complexity of the challenge we are looking to solve for NLL requires the deployment of several cutting-edge technologies,” Ms Xiao says.

Chromogenic reaction studies have been used to identify QA content in plant tissue samples of young leaves, old leaves, stems and roots of a population derived from crosses between domesticated ‘sweet’ and wild type ‘bitter’ NLL. Young leaves from the different NLL types exhibited the most significant colour disparity, suggesting their suitability as the primary tissues for assessment of QA level.

“Together with polymerase chain reaction, a technique to amplify small segments of DNA, I was then able to identify near-isogenic lines (NILs) within this population differing only in QA content. NILs are a fundamental tool for genetic analysis and comparison.”

QTL mapping and fine mapping was then carried out to identify the location of QA genes within the NLL genome and map them to other known and described regions within the genome.

This means that a significant QTL or region of the NLL genome has been identified governing QA content in vegetative tissues of NLL, with up to 26 genes identified.

“Drawing insights from known alkaloid transporters in model plant species such as Arabidopsis, tobacco and Medicago, and the recently GRDC-supported sequenced NLL genome, 23 transporter genes for QA have been identified in NLL.”

The research paves the way for future activities within Ms Xiao’s PhD study, including detailed gene function analyses, to validate the role of candidate genes in QA content regulation and transportation for NLL.

Specifically, more-precise gene expression analysis of candidate regulators or transporters will be completed.

Sub-cellular and cellular studies will be conducted for the genes of interest to determine their location and function. For instance, for QA regulators functioning as transcription factors, their expected location would be within the nucleus. In the case of QA transporters, their anticipated locations could be within the cell membrane, cytoplasm, vacuole and other relevant cellular compartments.

A major bottleneck in NLL research to characterise the identified candidate genes has been the inability to use genetic transformation methods applied by Agrobacterium. This is because NLL is a difficult-to-regenerate species via this tissue culture mediated method. Ms Xiao will use a technique known as virus-induced gene silencing (VIGS) to determine whether the genes of interest change QA content when they are transiently silenced. Apple latent spherical virus (ALSV) will be used in this phase of the project, which can infect NLL without causing severe symptoms. This VIGS system is a much faster and more-efficient tool for NLL to confirm gene function.

This means that we will be able to identify a mutant with low QA in a natural population, enabling breeders to develop new NLL lines with low QA in their seeds but retaining the bitter QA component in their vegetative tissues – the ‘bittersweet’ NLL.

Once tangible outputs have been generated from the project, communication will be instigated with the national breeder of NLL, Australian Grain Technologies.

Find out more about GRDC Grains Research Scholarships.

More information: Chengdao Li, c.li@murdoch.edu.au, chunsheng.xiao@murdoch.edu.au