By Troy Weeks, Jingsong Ye, and Caius Rommens

It may not be entirely surprising that the release of a glyphosate tolerant
alfalfa crop was received as highly controversial. Roundup ReadyŽ alfalfa is
different from other GM 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 alfalfa1. Although preparation
of an environmental impact statement, in 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
issues associated with the original GM 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 crop2. 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 ß-glucuronidase
(gus) gene. Unique methods were then used to enhance the 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 ß-glucuronidase (gus) activity. Results from this experiment
indicated that infection results 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
developed into rooted seedlings that were planted in soil and transferred to
the greenhouse. Subsequent analyses of the upper new leaves of five-week old
plants demonstrated that 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 the 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, any 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 the need for selectable marker genes.

The 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
value3.

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 DNA4. 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% to contain 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.
Studies performed by others have already shown that these reduced lignin
levels enhance the value of alfalfa as feed for dairy cattle5.

The 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 GM crops that should be cleared through the
regulatory process in a timely and cost-effective manner
(http://pewagbiotech.org/events/0602). Based on various surveys, the
voluntary exclusion of foreign DNA also increases consumer support for GM
crops from 20% to about 80% in the United States6,7.

References
1. Fox JL (2007) US Courts thwart GM alfalfa and turf grass. Nat Biotechnol
25: 367
2. Weeks JT, Ye J, Rommens CM (2007) Development of an in planta method for
transformation of alfalfa (Medicago sativa). Transgenic Res DOI:
10.1007/s11248-007-9132-9
3. Samac DA, Austin-Phillips S (2006) Alfalfa (Medicago sativa L.). Methods
Mol Biol 343: 301-311
4. Rommens CM, Bougri O, Yan H, Humara JM, Owen J, Swords K, Ye J (2005)
Plant-derived transfer DNAs. Plant Physiol 139:1338-1349
5. Guo D, Chen F, Wheeler J, Winder J, Selman S, Peterson M, Dixon RA (2001)
Improvement of in-rumen digestibility of alfalfa forage by genetic
manipulation of lignin O-methyltransferases. Transgenic Res 10: 457-464
6. Lusk JL, Sullivan P (2002) Consumer acceptance of genetically modified
foods. Food Technology 56: 32-377. Lusk JL, Rozan A (2006) Consumer
acceptance of ingenic foods. Biotechnol J 1: 1-2
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