Features Agronomy Genetics/Traits
Technology yields new treatment methods

In a world demanding greater advancements in less time, research at the USDA
Agricultural Research Service Corn and Soybean Research Unit in Wooster, Ohio,
has succeeded in doing just that. Work that began in 1991 is removing much of
the uncertainty associated with the viral inoculation of corn and it is being
done much faster.

Vascular Puncture Inoculation (VPI) is the culmination of work originated by
Dr. Ray Louie. Using VPI, researchers can transmit viruses to 90 percent or
more of inoculated corn plants in some cases, and can even transmit viruses
that previously were only transmitted by vectors such as insects. Relying on
insect vectors for transmission can lead to problems. Sometimes the vector species
is unknown, so further research on the virus is blocked. Sometimes, it is difficult
to raise enough vectors to do the experiments and, in many cases, the level
of transmission is too low to test plants for virus resistance. Moreover, insect
evaluation systems take a month or more to complete. VPI halves the time and
doubles the capacity. Furthermore, using VPI, researchers can transmit unknown
viruses without knowing anything about them. Or how they are naturally transmitted.

In VPI, tiny insect-mounting pins affixed to a flattened piece of copper wire
are vibrated forward and backward in an engraving tool. The pins carry virus
particles into vascular tissues in the seed, near the plant embryo.

Transmission by natural vectors can be unpredictable
There are different means to transmit plant viruses, according to Dr. Roy Gingery,
who has continued the work since Louie's retirement. "Viruses can be divided
into two groups: those that are mechanically transmissible by rubbing leaves,
and those that aren't," explains Gingery. "The ones that are can usually
infect the cells on the surface of the leaf and the ones that aren't, usually
require an insect to probe through the surface and take the virus to cells in
the interior of the leaf."

Since relying on vectoring insects in a controlled setting can be time-consuming
and inefficient, different methods have been studied to introduce viruses into
the plants. One method is to use a knife that has been coated with the solution
of a virus and cut the tissue to infect the plant. Others have used pins to
pierce the leaves, or stems, but with varying degrees of success.

Researchers also have used embryonic slashing, which Gingery concedes can be
somewhat problematic. Given the size of a corn seed and the proximity of the
embryo to the surface, the method should work. "It has worked with some
of these viruses, but when you cut the embryo, you damage the plant and get
low survival and fairly low transmission," he says. "Louie's idea
was to introduce the viral solution into the scutellum behind the embryo, where
vascular elements that channel food from the seed to the embryo could also carry
virus particles and so cause infection without damaging the embryo."

New VPI enhancements
Gingery emphasizes the uniqueness of this technique is its ability to transmit
unknown, vector-limited viruses. "Before, if we had a tissue sample from
something that appeared to have a viral disease and we were unable to rub-inoculate
or use insects to transmit it to another plant, that was pretty much the end
of research," he says. "With this technique, if we get a plant that's
infected with a virus that we don't know, we can use VPI to transmit it to other
plants and so culture it for further study."

The original design inserted tiny pins at a 45 degree angle to access the vascular
network. However, affixing the pins to a vibrating engraving tool was the key
to high rates of transmission. "There are a lot of non-mechanically transmissible
maize viruses and VPI has worked with every one," says Gingery.

The original VPI technique depended greatly on an operator's steady eye-hand
co-ordination and dexterity to achieve consistent levels of transmission. These
limitations prompted Louie to design a second generation VPI apparatus that
more-easily controls the vibration frequency, depth and angle of penetration,
duration of inoculation and placement of the pins during inoculation.

Other uses
In addition to facilitating research on identification and characterization
of vector-limited viruses, VPI will be a valuable tool for determining host
plant resistance. "Normally, when researchers breed for resistance to vector-limited
viruses, breeding populations are screened for resistance in the field,"
says Gingery. There are many disadvantages to that approach, the most important
being the level of infection may be low, making it difficult to tell a resistant
plant from one that merely escaped infection. "With VPI, we can screen
on a timely basis with specific viruses, without having to maintain vector colonies,
while at the same time minimizing disease escapes. You just inoculate the seeds
with VPI and get the results."

The technology has been used for corn and soybeans, and although it has worked,
the seed sizes in wheat, rice, sorghum and canola pose a challenge to VPI technology.