The International Potato Centre (CIP, by its Spanish acronym) has been working for years on the development of frost­resistant varieties of potato. In many Andean agricultural systems, frost substantially hinders potato production. Crops have their specific temperature requirements and the geographic distribution of crops is linked to altitudinal belts. Temperatures below 0^oC imply crop loses for most cultivars, including potato, whose optimum soil temperature has been estimated to be as high as 20­23^oC.

CIP has been screening and selecting potato cultivars able to resist temperature shocks of ­4^oC. Up to 1991, 2,000 cultivars had been screened and 537 clones selected at CPI's research station in Cajamarca, Peru. Most ­if not all­ of this research has been done by means of conventional techniques. The process, however, has been slow and complicated.
CPI's interest in cold tolerance has led to an application of biotechniques aiming at the development of cold­tolerant potato cultivars through the introduction of a flounder (type of flat­fish) anti­freeze protein. This initiative was a co­operation between the University of Louisiana, the Central University of Venezuela and the Programa de Investigación en Papa (PROINPA) programme at the Instituto Boliviano de Tecnologia Agropecuaria (IBTA), one of the public agricultural research institutes in Bolivia, with the promotion and technical support of CIP and financing from the Andean Development Bank.
It has been known since the early eighties that a flounder, Pseudopleuronectes americanus, can survive temperatures of ­1.5^oC. Several genes that codify for different anti­freeze proteins (APs) have been isolated from this fish. These proteins are in the fish blood and protect it from freezing. The APs concentration in the fish's blood varies along the year. Their production starts in autumn, reaching the highest concentration during winter time, and descending again in springtime.
The group of the University of Louisiana successfully used genetic engineering techniques to find out whether these proteins would have the same protective effect in plant cells. Based on the Agrobacterium mediated genetic transformation technique, they constructed a plasmid containing a gene coding for one of these APs. The gene was placed between two marker genes permitting the selection of transformed plants.
The Venezuelan group introduced the plasmid supplied by the University of Louisiana group into strains of Agrobacterium rhizogenes. These bacteria are used as vectors to carry the genetic information of the selected AP into potato plant cells. Infecting potato plant tissue with these strains, the AP gene has been transferred to a group of potato clones.

After the regeneration of complete plants by the use of in vitro culture, molecular tests confirmed the presence of the anti­freeze gene in the regenerated plants. The efficiency of the transformation varied according to the genotype of the potato clones.
The transgenic potato clones were transferred to Bolivia, and multiplied. Since 1993, the PROINPA group is testing the new varieties in a contained green house. Preliminary results indicate that a level of cold tolerance has been achieved. The next step should be field tests, which require a biosafety evaluation and clearing by national regulatory authorities. Bolivia has some experience with field releases. National biosafety guidelines have been drafted, and transgenic cotton varieties have been tested.

The commercial introduction of these clones, in case the agronomic evaluation warrant such a step, faces interesting questions of intellectual property and commercial distribution. They will be an important challenge for Bolivia's (and perhaps also Venezuela's) legal and commercial systems related to biotechnology.
This co­operative project shows the potential of international co­operation in transferring generic technologies to developing countries. The Venezuelan group now has some incipient capabilities for plant transformation, using the Agrobacterium alternative. The group aims at the complement of this capability as soon as possible with genetic engineering expertise, e.g. constructing the plasmids.
Walter Jaffé/Miguel Rojas