By Troy Weeks, Jingsong Ye, and Caius Rommens
It may not be entirely surprising that the release of a glyphosate
tolerant alfalfa crop has been received as highly controversial. Roundup
ReadyŽ 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 ReadyŽ 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 ReadyŽ 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 ReadyŽ 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 ?- 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 ?-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

[pewagbiotech.org]