“The vision of CGIAR breeding is to have world-class breeding programs to have CGIAR-and National Agricultural Research Systems (NARS) breeding networks that generate genetic gains above 1.5% per annum. And the average area-weighted age of varieties in farmers’ fields to be less than 10 years,” Dr Michael Quinn, Director, CGIAR Excellence in Breeding Platform (EiB), said outlining the intent for using new technologies like gene editing.
Dr Quinn stated that EiB sets targets, standards and helps with breeding teams to achieve them. Stressing that the current rate of production gain in major cereal crops will not meet projected demand and increase in legumes, roots, tubers and banana production is essential to meet SDG goals in the face of climate change and increasing population. He went on to explain how traditional breeding pipelines are reconceptualized in their modern avatars as cyclic models, where new technologies significantly help in shortening the breeding cycle time.
“The opportunities from gene editing are huge. It can make for genetic gains in ways and for traits that conventional breeding cannot. The collective impact is greater for breeding technologies and gene editing will likely be no different,” he said while emphasizing that we need to understand how to use gene editing synergistically with other technologies.
Molecular biologists at multiple CGIAR centers shared information about gene editing work underway at the centers and at partnering organizations. Most of the gene editing work shared is being done using knock-out or knock-down approaches without introducing foreign genes.
Maize
Dr Kanwarpal Dhugga, who is leading the Agriculture Biotechnology program at CIMMYT, and CIMMYT molecular biologist Dr Zhengyu Wen, explained their work to develop maize resistant to Maize Lethal Necrosis (MLN).
The first approach involves multiple edits in a fine-mapped QTL of about 100 kb to identify the causal allele for resistance. The edits were done in elite lines,
Dr Dhugga stressed. The QTL for MLN resistance was identified after observing strong resistance to MLN, usually caused by a combined infection with Maize Chlorotic Mottle virus and other Potyviruses like Sugarcane Mosaic Virus, in an elite line in Thailand. Currently, the edited plants are being screened in a greenhouse.
Dr Wen described the second approach that involves knocking out genes responsible for proteins involved in mRNA translation. These proteins, called Eukaryotic Translation Initiation Factors (eIF), are also the plant’s natural defense mechanism against viruses; mutations in eIF genes have helped plants develop durable resistance to viruses, Dr Wen explained.
In the case of maize, four eIF genes are known in the genome. With a high degree of efficiency, the team knocked out each of these individually and in combination through gene editing in two elite lines to get plants to resist MLN.
Rice
Mentioning the use of gene editing to study function, including modifying a popular vector to develop a versatile plasmid DNA, Dr Inez Slamet-Loedin, Cluster Leader of Trait and Genome Engineering at IRRI, highlighted the gene function studies underway at IRRI and CIAT. These include understanding genes responsible for potential trait development of hybrid rice, incompatibility barrier between rice and its wild relatives, resistance to Rice Hoja Blanca virus and study of genes related to grain quality.
Emphasizing the importance of using gene editing to introduce essential agronomic traits like the resistance to Bacterial Leaf Blight, Dr Slamet-Loedin described the development of “Sweet” variants with broad spectrum resistance to multiple strains of Xanthomonas oryzae that causes BLB. These variants were field tested under controlled conditions at IRRI and CIAT.
“Recently, regulators in both Colombia and the US have declared resistant varieties as conventional breed cultivators,” Dr Slamet-Loedin said after demonstrating resistance in the new lines vs control plants during tests. Gene editing to biofortify polished rice with increase zinc concentration, for drought tolerance, male sterility, resistance to striga and to tolerate cadmium are other traits for which work is underway, she added.
Dryland cereals and legumes
Outlining gene editing work underway in multiple crops at ICRISAT and Common Bean in CIAT, Dr Pooja Bhatnagar-Mathur, Theme Leader Cell Molecular Biology and Genetic Engineering at ICRISAT, stated that several genetic and genomic resources and tools to enable technologies like gene editing have been developed for dryland cereals and legumes in collaboration with a number of global partners, including vectors for quick transformation, gene discovery and mining.
Dr Bhatnagar-Mathur explained the editing work in pigeonpea at ICRISAT to target photoreceptors and flowering time genes which are correlated to photoperiod sensitivity. This editing work is by knocking down of a single gene or two to three genes. As pigeonpea is a short-day crop and photoperiod-sensitive, it can only be grown in specific locations. Conventional breeding has not been able to take this high nutritious legume outside existing production areas.
In chickpea, ICRISAT is working on improving seed size and quality by editing a family of transcription regulators that result in increased seed size, which is beneficial as bigger seeds have a higher market value.
In Common Bean, CIAT has been targeting two genes that synthesize complex sugars that are not easily digested in humans and animals. The edited events are underway and the team is segregating the Cas9 gene for product development.
Aflatoxin is the biggest food safety issue in groundnut and many other crops, Dr Bhatnagar-Mathur said, while explaining the approach where comparative proteome profiling of near immune transgenic/ HIGS groundnuts and their wild type counterparts revealed susceptibility factors to aflatoxin and fungal infection. Using CRISPR, knock down of these susceptibility factors is being attempted to induce resistance without any insert.
In sorghum and pearl millet, a few candidate genes have been edited to reprogram strigolactones, a plant’s signaling hormones that have a role in its growth. The aim has been to block the signaling to striga, a parasitic weed, and prevent its germination from seed. Sorghum mutants with lost function on edits have been identified for pre-germination and post-attachment resistance and are being evaluated, she said.
Roots, Tubers and Banana
Dr Leena Tripathi, who leads the transgenic and genome editing research at IITA, shared updates from genome editing in banana and roots, tuber crops including work to understand gene function, disease resistance to bacterial wilt and fusarium wilt. She also informed that field trials for the gene edited resistant bananas are likely to begin early 2021.
She provided details from her work to develop resistance to Banana Streak Virus that involves targeting areas in the plant DNA where the virus is known to integrate. After infection, the plant remains asymptomatic until stressed. Following gene editing at loci where the viral genetic material is known to integrate, the edited mutants were subjected to water stress to observe improved outcomes.
“With the proof of concept of knocking out this integrated virus, we have integrated this technology into the breeding program. We are creating targeted mutations in the 4x hybrid and an improved diploid as these are crossed to get the improved plantain hybrid,” she said. Dr Tripathi also shared details about work to develop bacterial, fungal and downy mildew resistance in susceptible banana. Gene editing work in cassava at CIAT, potato and sweet potato at CIP and Yam at IITA was also presented by her.
Cocoa
Dr Paul Chavarriaga, who leads the Genetic Transformation and Gene Editing Platform at CIAT, described efforts to prevent uptake of heavy metals like cadmium by cocoa plant. He said several genes are involved in the process. Cadmium is toxic. He called for multiple approaches to tackle the problem.
“Plants regenerated from edited embryos are being tested using hydroponics to observe cadmium uptake. It is not easy as cocoa is highly tolerant to cadmium, which goes to the beans that are consumed. In the future, the edited plants will have to be tested for cadmium uptake in the field in CIAT as well,” he added. Collaborations
