Wednesday, July 25, 2012

Gene transformation and chromosomal translocation – A plant breeder’s vista

K.K.Vinod, Neway Mengistu, Nicholas Adam Crowley and Jeffery Ryan Sullivan

(This was the subject of a threaded discussion at University of Nebraska-Lincoln)

When a breeder is looking to incorporate “alien” genes into a line, two good choices he has are translocation and transformation (transgenic events). With a little luck, time, and effort their results can show great benefit to commercial crops. The two methods are similar because you are adding DNA segments to an existing genome without conventional breeding methods. Both of these methods are used to add a desired gene(s) to a crop which lacks the gene of interest. Chromosome translocation is caused by the interchange of parts between non-homologous chromosomes. Transgenic inserts add new, although more controllable segments, to an existing genome. The two methods require selfing and selection to be successful.

Translocation mostly gives a successful result in polyploid crops. It has been tried in wheat and rye to transfer disease resistant genes from their wild relatives. Moreover, translocation lines are more acceptable in polyploidy systems, wherein other chromosomes in the genome can compensate for a lost arm/part of chromosome eventuate in translocation events. Chromosomal translocations are random events. Translocations therefore differ from crossing over by randomness of insertion points. Any chromosomal segment can get attached to any arm of another chromosome. Classical examples are random translocations caused by transposable elements. When a chromosomal fragment carrying a desirable gene is getting translocated to a cultivated variety, the event may result in transferring of some other unknown alleles along with the gene of interest. For example, if a chromosomal segment from a wild relative carrying a disease resistance gene is translocated into a cultivated variety, it may also carry some undesirable wild traits into the cultivar. In the earlier days translocation was not much utilized in crop improvement due to this impediment. Nowadays, by use of transposable elements translocations and insertions are being utilized for site directed mutagenesis and random transfer of genetic elements.

Transformation techniques use biolistics/particle bombardment or Agrobacterium tumefaciens to insert a gene. These transformation techniques are “quick” means to introduce a gene into the plant of interest. Transformation events, try to incorporate new resistance, tolerance, or quality traits. Examples of transgene insert performed in soybean (RoundupReady® soyaben), cotton (Bt-cotton), rice (golden rice - enriched with vitamin A) etc., give a good reasoning to suggest that it can work. However, most of the traits are novel, which produce a function beneficial to humankind/cropping systems and the genes responsible are not found in that specific crop or any of its relatives. Transferring a trait using transgene insert can be manipulated by genetic engineering techniques to isolate the gene and manipulate it through cloning. In the case of a transgene, the insertion is a random event and can occur any where in the genome. This will result in hemizygosity for the transgene.

Although the advantages of these two methods are a great benefit to breeders, both have their own considerations when using them for adding desirable genes to a crop. The translocated disease resistant gene would contain surrounding chromosome segments from the wild relative. The surrounding genome would likely be undesirable since it is being donated by a distant relative to the crop. Moreover, it may take longer years to find a stable line that contain the disease resistant gene. In theory, transgenic events seem simple, and scientists/geneticists have found ways to make it as easy as it sounds, but when breeders work with translocation lines, it is hard to get everything seem simple. One case in which the breeders/scientists have used to make this technique easier is the Ph mutant in wheat. This mutation allows homoeologous pairing and crossing over between alien chromosome and its crop homoeologue, allowing transfer of chromosome segment containing the alien gene. And also if the crop is a kind of polyploid chromosomal translocation may be preferred than transgene insert - because of better tolerance of the new chromosome fragment in polyploids. On the other hand, many diseases are controlled by a single gene resistance which may not justify transfer of a chromosome fragment. A transgene insert may accomplish the job very well. However, transgene methods are still questionable by some and this must be measured when developing transgenic crops. Transgenic methods must also be isolated and sequenced for the desired gene.

Notwithstanding the fact that these methods have similarities and differences, both techniques can be used for transferring desirable traits like disease resistance. The similarity between the translocation and transgene lies in the hemizygosity it produced. Since corresponding allele(s) from the translocated fragment are not found in the homologous pair, or transgene is attached only to one chromosome, hemizygosity produces a situation where only one allele is present in excess on one chromosome, while it is totally absent on its pair. As breeders, hemizygosity is not a desirable situation as we have the threat of missing the event upto 50% among gametes. The best solution is to self the plants to generate a homozygous line for the transferred gene. This homozygous line can be used for further breeding programmes. Another similarity can be from incorporation of many novel genes from translocation and transformation. Translocation between species can provide more than one beneficial gene. This is also the case in transformation; a good example is YieldGuard® plus corn hybrids. These incorporate herbicide resistance, and various insecticide genetic events to produce a corn hybrid that is beneficial to the farmer.

One way, in which these methods differ, lie in the mode of prediction of the gene transformation event itself, i.e. marker. In the case of transformation, an antibiotic resistance, herbicide tolerant or gus gene is added for easy phenotypic identification in early stages of development. In translocation crosses, phenotypic markers that may be by chance linked to the gene being transferred need to be looked to identify the plant with the alien translocation.

The major difference between a transgene insert and the translocation event is that, we know the number and nature of the genes inserted in the transgenic event, while we are unsure of the number and nature of alleles inserted through a translocation event. Shorter the translocation insert more stable will be the translocated event, while the question of stability of the transgene is still not resolved. Depending upon the length of the translocated fragments, the number of alleles will vary which may comprise of many introns and exons. Transgene insert usually has gene of interest along with antibiotic or herbicide resistance markers and/or gus markers plus the promoter regions and plasmid fractions.

When using the translocation technique in a breeding program, a breeder must consider the difficult task of achieving fertilization from the parents and possible seed abortion and using embryo rescue. In the case of transformation this is not a problem. Transformation can cause problems when the gene of interest is placed in an existing gene for interest and produces a mutant or lethal plant.

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