An alternative approach to genetic engineered alfalfa

It may not be entirely surprising that the release of a glyphosate tolerant alfalfa crop has been received as highly controversial. Roundup Ready® alfalfa is different from other GE crops in that its pollen is easily dispersed by honey bees over distances that can exceed three kilometers. Transgenic pollen is likely to fertilize flowers of untransformed alfalfa plants that grow on an estimated 20 million acres throughout the United States.

The resulting contamination of seed with foreign DNA may compromise the perceived quality, sale, and export market of alfalfa. Furthermore, introduction of yet another Roundup Ready® large-acreage crop results in an inevitable increase in glyphosate usage while encouraging the further establishment of glyphosate resistant weed populations.

All these issues resulted in a recently imposed injunction, in effect revoking the 2005 approval of Roundup Ready® alfalfa. Although preparation of an environmental impact statement, to be released about a year from now, may convince the court to issue permanent injunctive relief, there will continue to be concerns about the production of Roundup Ready® alfalfa. It seems, therefore, imperative to develop alternative approaches to genetic engineering that make it possible to genetically modify crops while addressing most of the controversial issues associated with the original GE alfalfa crop.

A recent study published in Transgenic Research demonstrates that the quality of alfalfa can be enhanced without incorporating marker genes or other types of foreign DNA into the crop. One aspect of this new approach is the employment of a new marker-free transformation procedure. This method was developed by first incubating two-day old seedlings for 16 hr at 4°C. After excision of cotyledons at the apical nodes comprising meristematic tissues, the resulting explants were infected with a highly virulent Agrobacterium C58/pMP90 donor strain carrying the reporter β- glucuronidase (gus) gene. Unique methods were then used to enhance contact between acceptor and donor cells. Instead of applying a conventional agitation or vacuum infiltration step, emerged seedlings were vigorously vortexed for an extensive period of time (~30 min). This procedure did not cause any irreversible damage to treated seedlings. Indications for the extent of transient DNA transfer were obtained by assaying for β-glucuronidase (gus) activity.

Infection from Agrobacterium resulted in high levels of transient transformation. The cut and vortex-infected seedlings were inserted vertically into hormone-free media for a short recovery time, during which new shoots arose from the cut surfaces of about 60% of explants. After 14 days, the explants had developed into rooted seedlings that were planted in soil and transferred to the greenhouse. Subsequent analyses of upper new leaves of five-week old plants demonstrated 7% of these leaves stably expressed the gus gene in most or all cells ("all-blue" leaves), whereas an additional 17.5% expressed this gene in part of the tissue.

Extensive gus expression in upper leaves was expected to be indicative for transformation of meristematic and germ line cells. This theory was confirmed by allowing transformed plants to mature and set seed in the greenhouse. Subsequent analyses of T1 progenies demonstrated successful transgene transmission. Although segregation ratios in some cases deviated from the 3:1 ratio expected for fully transformed T0 plants, all of the independent families tested contained at least some siblings expressing the gus gene. DNA gel blot analyses of randomly-chosen T1 plants confirmed the integrity of transmitted T-DNAs, and estimated the average copy number of this element at 1.6. Collectively, the data demonstrated that alfalfa can be transformed without selectable marker genes.

This new method provides several additional advantages in addition to avoiding the stable integration of bacterial selectable marker genes. First, it limits the time, materials, and resources required for complex in vitro manipulations, while also eliminating the risk of somaclonal variation that is associated with both hormone treatment and callus formation. Second, the method substantially reduces the amount of time from transformation to seed set from about seven weeks for conventional systems5 to five weeks. Third, the in planta transformation method has been applied successfully to a commercial variety, whereas the conventional methods require very specific highly regenerable genotypes such as RegenSY that have little commercial value.

To demonstrate that the new transformation method could be used for production of intragenic plants displaying an enhanced quality trait, a silencing construct targeting the native caffeic acid o-methyltransferase (Comt) gene was positioned within an alfalfa-derived transfer DNA.4 Alfalfa plants were transformed as described above and allowed to mature in the greenhouse. Polymerase chain reaction (PCR)-based genotyping of 1,000 five-week old plants identified 2.4% that contained the modified P-DNA. Stem sections were isolated from intragenic progeny plants derived from eight randomly chosen original transformants and assayed for lignin accumulation. This analysis demonstrated reduced lignin levels in three of eight cases and. Studies performed by others have already shown that these reduced lignin levels enhance the value of alfalfa as feed for dairy cattle.

This new method is likely to be used to improve alfalfa with enhanced traits that are of interest to alfalfa producers and dairy farmers. Resulting plants may represent low-risk GE crops that should be cleared through the regulatory process in a timely and cost-effective manner.

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An alternative approach to genetic engineered alfalfa