Scientists at the Delhi-based National Institute of Plant Genome Research (NIPGR) have made a significant breakthrough in rice biotechnology.
They used CRISPR-Cas9 gene editing technology to enhance phosphate uptake and transport in japonica rice varieties, demonstrating the potential for improving genetic diversity in rice germplasm.
The gene edited rice lines showed increased seed and panicle numbers, leading to higher grain yield and improved stress tolerance.
Importantly, the seed quality, including protein content and resistant starch levels in the endosperm, remained unaffected despite the increased yield.
The research was conducted under greenhouse conditions, marking a step forward in sustainable rice production and precision agriculture techniques.
Significance of the Research
Phosphorus is vital for plant growth, cell division, and development; its deficiency leads to a drastic drop in crop productivity.
Phosphate fertilizers are commonly used, but plants absorb only 15–20% of the applied fertilizer; the rest is lost through leaching or runoff.
The gene edited rice lines demonstrated a significant improvement in phosphorus-use efficiency through enhanced gene expression of key transporters.
When the recommended dose of phosphate fertilizer was applied, the grain yield increased by 20% in the gene edited rice lines.
Remarkably, even with just 10% of the recommended fertilizer dose, the gene edited lines showed a 40% higher yield compared to the control, indicating improved stress tolerance and potentially enhanced immune system response.
These findings were published in the Plant Biotechnology Journal by Dr. Jitender Giri, the corresponding author from NIPGR, potentially leading to valuable intellectual property in the field of agricultural biotechnology.
Highlights from the Research
According to Dr. Jitender Giri, the experiment aimed to demonstrate the resilience of gene edited rice lines even under extreme phosphorus deficiency, which is crucial for developing stress-tolerant varieties.
Even with only 10% of the recommended phosphate fertilizer, the gene edited plants achieved 40% higher yield compared to the control group, which showed a sharp yield decline.
Dr. Giri emphasized that even if phosphate supply is reduced by 10–30%, the gene edited lines are still expected to outperform normal plants, showcasing improved salt tolerance and overall stress tolerance.
In rice, phosphate is absorbed by the roots and transported to the shoots via specialized inorganic phosphate transporters, a process regulated by various transcription factors.
The NIPGR team focused specifically on the OsPHO1;2 transporter, responsible for moving phosphate from root to shoot.
Enhancing the activity of OsPHO1;2 increases the phosphate demand in shoots, which in turn signals the roots to uptake more phosphate from the soil.
While the presence of a negative regulator of this transporter is known in Arabidopsis, this is the first time such regulation is being understood in rice, contributing to our knowledge of gene expression in different plant species.
Identification and Role of the Repressor
Using in silico and DNA-protein interaction studies, NIPGR scientists identified OsWRKY6 as the repressor of the phosphate transporter gene OsPHO1;2.
They demonstrated that OsWRKY6, a transcription factor, physically binds to the promoter region of the transporter gene, thereby reducing its expression.
To test its role, researchers used CRISPR technology to knock out the OsWRKY6 gene, aiming to lift the repression and alter gene expression patterns.
As a result, the expression of OsPHO1;2 increased significantly, enhancing phosphate movement from root to shoot.
However, contrary to expectations, the gene edited rice lines performed poorly in terms of yield compared to the control group.
Dr. Giri explained that OsWRKY6 also regulates other essential plant functions, and completely removing it impaired overall plant performance, including processes like tillering and embryo development.
This revealed the complex role of regulatory genes and the importance of balanced genetic modification for sustainable crop improvement.
Breakthrough Strategy: Removing the Repressor Binding Site
Researchers identified a 30 base pair region in the promoter of OsPHO1;2 where the repressor OsWRKY6 binds.
Using CRISPR-Cas9, they removed only the repressor’s binding site—not the repressor itself—allowing the repressor to retain its other vital functions in the plant.
This precise genome editing ensured that other regulatory proteins could still bind to the promoter and control the gene’s expression.
Dr. Giri compared this strategy to “minimally invasive surgery” in genetic engineering, highlighting its potential in creating mutant lines with specific traits.
The process involved careful manipulation of coding sequences to achieve the desired outcome without disrupting other essential functions.
Outcomes of Binding Site Removal
This edit led to enhanced promoter activity in the roots, resulting in:
Increased phosphate transport from root to shoot
Greater shoot phosphate accumulation
Improved plant growth and grain yield
The root-bound phosphate transporters began to absorb more phosphate, turning the roots into a nutrient “sink”.
The gene edited plants showed a 20% increase in yield due to the production of more panicles (fruiting bodies bearing seeds), demonstrating improved stress tolerance and potentially enhanced rice blast resistance.
The modifications also led to changes in stomatal density, which could contribute to improved water use efficiency.
Impact on Seed and Fertilizer Use
Seed quality remained unaffected—no changes were found in seed size, shape, starch, or phosphate content. The protein content and resistant starch levels in the endosperm were also maintained.
Despite higher phosphate absorption, it doesn’t necessitate higher fertilizer use:
Only ~20% of applied phosphate fertilizer is absorbed by plants; the rest gets locked in the soil by forming insoluble complexes with calcium, magnesium (alkaline soil), or iron, aluminium (acidic soil).
Gene edited rice plants absorb phosphate faster, before it becomes insoluble, making them more phosphate-efficient and potentially reducing heavy metal accumulation in the soil.
The research also explored the potential impact on seed dormancy, an important trait for crop management and storage.
Validation in Japonica Rice and Future Scope
The japonica rice variety Nipponbare was used for initial gene-editing studies due to its ease of transformation and faster experimentation.
Indica rice varieties, common in India, are more challenging to work with and require more time to generate gene edited lines.
Dr. Giri emphasized that japonica lines help validate hypotheses quickly, which can then be replicated in indica varieties for practical application, expanding the genetic resources available for rice improvement.
Future research may explore the use of other CRISPR systems, such as CRISPR-Cas12a, to further refine gene editing techniques in rice.
Scientific and Agricultural Relevance
According to Dr. P.V. Shivaprasad (NCBS), the work is a major scientific advancement, especially relevant for phosphorus-deficient Indian soils.
Replicating these edits in indica rice lines could significantly reduce phosphate fertilizer usage without affecting yield—crucial for Indian agriculture and precision agriculture practices.
The research also opens up possibilities for improving other traits, such as cooking quality, which is an important factor in consumer acceptance of rice varieties.
Addressing Concerns Around Gene Editing
Off-target Effects
CRISPR-Cas9 can sometimes cause unintended edits (off-target events), raising biosafety concerns.
NIPGR scientists used predictive software and tested the top 10 potential off-target sites—no deletions were found.
Only plants with precise editing and no off-target events are allowed to reach the seed production stage.
Dr. Shivaprasad confirmed the availability of multiple in silico tools and Southern blot analysis for verifying precision and assessing mutation frequency.
The research team also considered the potential impact on DNA repair mechanisms, particularly non-homologous end joining, which is crucial in maintaining genome stability.
Foreign DNA Concerns
Cas9 enzyme comes from Streptococcus pyogenes, and vectors like Agrobacterium tumefaciens are used for gene delivery.
Dr. Giri stated that these foreign DNA components are removed in the second generation through Mendelian segregation (3:1 ratio).
Only foreign DNA-free lines are propagated for further use as breeding materials.
Dr. Shivaprasad affirmed that complete removal of bacterial DNA is feasible using established genetic techniques.
The team also explored base editing as an alternative approach that could potentially reduce concerns about foreign DNA insertion.
Broader Impact
India imports ~4.5 million tonnes of Diammonium Phosphate (DAP) annually to meet fertilizer demands.
Gene edited rice that uses phosphate more efficiently could reduce dependency on imported fertilizers.
If extended to Indian indica rice varieties, the technology could boost yield, cut costs, and promote sustainable agriculture.
This research contributes to food security and climate change adaptation by developing stress-tolerant rice varieties with improved water use efficiency and potentially enhanced bacterial blight resistance.
The success of these field trials paves the way for further agricultural biotechnology advancements and potential commercialization of genome-edited crops, pending regulatory approval.
Future research may focus on enhancing other aspects of rice quality, such as nutritional value through biofortification or improving cooking and processing quality.
As the global market demand for high-quality rice increases, this gene edited rice variety could play a crucial role in meeting those needs while addressing phosphorus deficiency issues and potentially reducing cadmium content in rice.
India's No.1 PU & Degree With Integrated IAS & IPS(UPSC/PSC) Training College.
