Improving Citrus Using Genetic Tools

By Manjul Dutt

Editor’s Note: This is the first in a series of two articles on plant improvement. Next month will feature the timeline for CRISPR-developed trees.

Traditional methods for improving citrus, such as crossbreeding different varieties, can be time-consuming due to the creation of a random mix of genes from both parents through a process called genetic introgression, leading to offspring with unpredictable characteristics. This process may require extensive screening and considerable time to identify a suitable offspring.

Newer plant improvement tools, such as genetic transformation and genome editing using CRISPR, enable scientists to alter a plant’s genetic makeup. Understanding the complexities of genetic transformation and CRISPR is essential for developing innovative solutions in biotechnology, such as creating genetically modified crops and implementing strategies against huanglongbing (HLB). This article discusses the processes of genetic transformation and genome editing using CRISPR and the timeframe to create genetically modified plants.

GMO vs. CRISPR

Genetic transformation results in the creation of genetically modified organisms (GM/GMOs), while CRISPR is a gene-editing technique used to alter the DNA of living organisms. Although both methods aim to modify genetic material, they differ in their approaches and outcomes.

GM plants are typically created by inserting genes from different species into an organism. For instance, a disease-resistance gene from tobacco can be introduced into citrus plants to make them tolerant to HLB. This process is less precise and may lead to random DNA insertions within the plant’s genome.

In contrast, CRISPR is a process that allows scientists to directly edit specific genes without necessarily adding foreign DNA. It functions like molecular scissors, precisely cutting DNA at a specific location to delete, repair or replace a gene. Although both techniques seek to improve traits such as disease resistance or crop yield, CRISPR is generally more accurate, as it can create changes that could also occur naturally.

Genetic transformation is the process of altering a plant’s DNA by introducing a new gene to enhance beneficial traits, such as disease resistance, drought tolerance or improved fruit quality. Typically, this process does not change the existing genetic characteristics of the plant variety being transformed. Genetic transformation enables scientists to add a specific trait to trees without altering the rest of their characteristics, making the process faster and more precise compared to traditional breeding, which mixes traits from two different trees. Elite citrus cultivars that undergo genetic transformation can acquire new traits while maintaining the other qualities that make them valuable.

CREATING A GMO PLANT

There are two main methods that scientists use to change the genes in citrus trees:

1. Using Bacteria (Agrobacterium Method)

This method involves using a natural soil bacterium called Agrobacterium tumefaciens, which can insert its DNA into plants. Scientists take advantage of this ability by removing the harmful components of the bacterium’s DNA and replacing them with beneficial genes.

To begin, scientists use either young citrus tissue (such as small, etiolated stems) or pieces of mature citrus stems. They then mix this tissue with the engineered bacteria, which can subsequently insert the new genes into the citrus plant’s DNA.

Once the DNA insertion is complete, scientists can grow the cells containing the modified DNA into new plants in a laboratory setting. These new plants are maintained in a greenhouse and tested to ensure that they possess the desired DNA modifications. The evaluation process may involve testing the modified plants in either a greenhouse or a field setting to assess tolerance to HLB.

2. Using Protoplasts (Direct DNA Insertion Method)

A protoplast is a plant cell that has had its outer cell wall removed, leaving only the inner part of the cell, which is surrounded by a thin membrane. When scientists want to introduce new DNA into a plant, using protoplasts simplifies the process because there is no tough cell wall to contend with.

To create protoplasts, scientists take plant cells and use special enzymes to dissolve the cell walls. Then, they mix the DNA containing the desired trait (such as a gene that may confer disease resistance) with the protoplasts and a chemical called polyethylene glycol. This chemical makes the cell membrane more flexible, allowing the DNA to enter the cell more easily. Once the DNA has successfully entered the protoplast, the cell is grown in a special nutrient-rich medium that enables it to rebuild its cell wall, divide and eventually develop into a new plant that incorporates the new DNA.

GMO DEVELOPMENT TIMELINE

1. Gene Identification, Gene Insertion and Lab Development (1–2 Years)

Scientists identify a trait of interest, such as tolerance to HLB. They identify and isolate a potential gene that could make the plant tolerant to HLB. This gene is inserted into citrus plants using Agrobacterium-mediated transformation or protoplast techniques. The modified plants, referred to as transformants, are then generated and screened. Laboratory testing is conducted to ensure that the gene is expressed correctly and is stably inherited in the citrus plants.

2. Greenhouse and Field Trials (3–7 years)

The best genetically modified lines of citrus trees are clonally propagated and screened for HLB tolerance in the greenhouse or tested in the field under endemic HLB conditions. Scientists also collect necessary data for regulatory submissions.

Citrus trees developed from juvenile tissues typically take five to seven years to flower and bear fruit, requiring field observations during this period. In contrast, trees created through the mature transformation process can flower and produce fruit within two years of transformation.

3. Regulatory Approval and Certification (2–3 years)

Once a specific plant line has been identified, the developer submits budwood to the Florida Department of Agriculture and Consumer Services Citrus Budwood Program to certify that the budwood is free from viruses and graft-transmissible diseases. The developer submits a comprehensive dossier containing scientific data required for evaluation by the U.S. Department of Agriculture, Environmental Protection Agency and Food and Drug Administration. This process typically includes public consultations and requests for additional data.

4. Commercialization (2–3 years)

Once approved, the GM line is officially released and clonally propagated for large-scale planting. Marketing and outreach to growers, which began earlier, is intensified, and consumers are informed about the new trees.

Manjul Dutt is an assistant professor at the University of Florida Institute of Food and Agricultural Sciences Citrus Research and Education Center in Lake Alfred.