Plant transformation accelerated by stacking genes using a new approach

In a discovery intended to accelerate the development of crops with the practical advantages of jet biofuels, scientists at the Department of Energy’s Oak Ridge National Laboratory have developed the ability to insert multiple genes into plants in a single step.

“As we try to achieve the goal of a carbon-neutral aviation industry by 2050 using sustainable fuels, the challenges are enormous – technically, economically and biologically,” said Jerry Toscan, CEO at the ORNL-led Center for Bioenergy Innovation. or CBI, which directed the research. “Having the ability to test multiple genes at once will speed up this process and increase the likelihood of achieving the nation’s goal of 100% displacement of petroleum-based jet fuel by 2050.”

Plant transformation technology, known as gene stacking, replaces the painstaking method of inserting one gene at a time into the DNA of a target plant and then sequencing the plant to ensure that the genes are in the right place and have the right orientation to trigger the desired physical traits.

Genes do not operate in a vacuum. The complex traits that researchers want, such as faster growth and drought tolerance, are often controlled by multiple genes. Conventional genetic engineering involves adding one gene and its associated biochemical machinery to plants, proving it effective, then taking that plant material and transforming it a second time with another gene and proving it effective, then a third gene and so on, in a complex, time consuming process.

“It would be more efficient if you could do all of that in one shift,” said Toscan, who was also involved in the research.

ORNL scientists have created a new delivery method using protein segments called inteins that have the natural ability to separate from large proteins and then stick back together to make new proteins. The researchers used inteins to create a split-selectable marker system that simultaneously introduces four genes into plants, including genes that “mark” or identify transformed cells, underpin their stability and make modifications detectable by biosensors.

The technique described in Communication biologyon tobacco, model plant Arabidopsis thaliana and poplar biomass stocks.

The resulting hybrids were screened using ORNL-developed biosensors, which indicated that new genes had been incorporated into the plant. The results were confirmed by examining the plant’s DNA.

ORNL project leader Xiaohan Yang said the project “is the result of years of research at CBI dedicated to creating robust bioenergy feedstocks grown under less-than-ideal conditions.” “We want to accumulate traits in the poplar that make the tree economically viable for processing jet fuel.” Yang said the new ability to stack genes is easily implemented into existing plant transformation pipelines while providing much faster results.

As part of its mission to develop a sustainable crop of non-food feedstocks for clean aviation fuels, CBI has identified genes that control plant traits such as higher yield and biomass composition that lend themselves more readily to processing into biofuels and drought tolerance.

Yang and his colleagues set out to replicate this technique of simultaneously inserting 12 genes–10 of which are associated with biological functions in poplars and two of which are markers. Yang said it is possible to improve the technology to support stacking of up to 20 genes.

“We have demonstrated that we can apply these new approaches to building plant facilities in a way that is predictable, more efficient, and requires less validation and testing,” Toscan said. The new capability “goes beyond incremental improvement in current technology and is the culmination of many people’s efforts, and represents an important step forward in our ability to transform plants.”