Many generations are required to obtain a suite of desirable traits while eliminating non-desirable traits. It would be much easier to improve an existing rootstock that possesses many desirable characteristics by inserting one or more genes that will enhance rooting in a stool bed or infer resistance to fireblight or some other desirable trait. Imagine the benefits of a clone of M.9 with good rootability, and resistance to fireblight, collar rot, and low winter temperatures. Or consider the advantages of a dwarfing version of MM.111 or Robusta #5. This may be possible with genetic engineering, which is the genetic manipulation of DNA to alter the characteristics of an organism.
I am not a plant breeder or a molecular biologist, but while reading some of the scientific literature, I was impressed by the way breeders are using new techniques to better understand how rootstocks influence tree physiology and growth, and how these new techniques can be used to study rootstock function. You will probably be hearing more about this in the future, so in this article, I will try to summarize some of the recent work concerning the genetic manipulation of rootstocks and how these modified rootstocks can be used to study tree physiology.
Why Develop Genetically Modified Plants?
A transgenic plant is a plant that has been genetically engineered by inserting a gene into a chromosome of a plant. Genetically engineered organisms can be transgenic as well as nontransgenic (genetically modified plants using target genes from the same or closely related species). The need to regulate nontransgenic plants is currently being debated by government agencies and by the public. The biggest advantage of transformation may be that genetic improvements can be made that would otherwise be impossible with traditional breeding techniques because genes for the desirable trait may not exist within a species.
Although technology exists for transforming plants, commercialization of transgenic fruits and vegetables has been slow. Regulatory issues still exist, and there may be issues related to consumer acceptance. These issues may be circumvented by using genetically modified rootstocks in combination with non-transgenic scion cultivars if the introduced genetic material is not found in the scion or fruit. This is referred to as “transgrafting.”
In some plants, certain types of genetic material, such as RNAs and proteins may cross the graft union. When non-transgenic grapes were grafted onto a transgenic rootstock expressing a certain peptide (a compound consisting of 2 or more amino acids linked in a chain), the peptide was found in the xylem sap of the scion (Dutt et al. 2007). However, studies with other species showed that genetic material did not cross the graft union. So, regulation of transgrafted plants may depend on the plant species and the specific genes that are inserted into the plant. Therefore, some researchers are developing genetically modified rootstocks in hopes that they will someday be commercialized. Even if these rootstocks are never commercialized, they can be used to study various aspects of rootstock physiology.
Commercialized Genetically Modified Crops
Commercial cultivation of genetically modified plants (GMPs) has rapidly increased worldwide in the past 20 years. Roundup Ready soybeans were first grown in 1996. By 2013, 27 countries planted 432 million acres of GM crops, mostly soybean, corn, canola, cotton and sugar beet, that were developed with herbicide- or insect-resistance. Of the 30 crops listed on the International Service for the Acquisition of Agri-Biotech applications web site, only three genetically engineered fruit crops (papaya, plum, and ‘Arctic’ apple) are listed. The most successful genetically modified fruit crop is probably transgenic cultivars of papaya that are resistant to papaya ringspot virus. There is no known natural source of resistance, and the introduction of these new cultivars saved the Hawaiian papaya industry.
Pennsylvania peach growers are very aware of the potentially catastrophic introductions of pests, such as the plum pox virus (PPV) that causes sharka disease. More than 20 years ago, Dr. Ralph Scorza and his colleagues at the USDA developed the first genetically engineered PPV-resistant plum, ‘HoneySweet’ (Scorza et al., 1994). After 20 years of testing, regulatory agencies finally cleared ‘HoneySweet’ for cultivation in the US. ‘HoneySweet’ has not been widely planted because PPV was eradicated. However, if the need should arise in the future, we now know how to develop similar resistant cultivars, and we have a better understanding of how to commercialize them. Additionally, hybrids derived from ‘HoneySweet’ crosses will not require further regulatory approval. Considering predicted climatic changes and the periodic introduction of invasive pests, there will likely be increased need for rootstocks and cultivars with enhanced resistance to various biotic and abiotic stresses.
Honeysweet Plum Trees A Transgenic Answer to the Plum Pox Problem (article, USDA-ARS)
Photo: These transgenic plums contain a gene that makes them highly resistant to plum pox virus. Photo by Scott Bauer.
Genetically Modified Rootstocks
One of the first rootstock transformations was performed at Cornell University. In 1994 transgenic plants of M.26 and M.7 were obtained carrying the attacin E gene and were more resistant to fireblight under greenhouse conditions than the non-transformed plants (Norelli et al. 1994). The attacin E gene is an antimicrobial gene obtained from a cecropia moth. Enhanced resistance was confirmed in a 2-year field study, where one line was much more resistant against fireblight as compared to the control. After 12 years, field-grown ‘Galaxy’ trees grafted onto transgenic M.26 were still resistant to fireblight, and there was no effect on tree morphology, leaves or fruit size and quality (Borejsza- Wysocka et al., 2010). Some other antimicrobial genes of insects (cecropins SB-37 and Shiva-1), the lysozyme gene of hen eggwhite, and the bacteriophage T4 have also been transferred to M.26 rootstock (Norelli et al. 1994; Aldwinckle et al. 2003).
Some rootstocks root more easily than others. One reason that M.9 was selected more than 2,000 years ago is probably that it roots easier than most apples. Rootstocks such as Ottawa 3, G.16 and Jork.9 do not root as well. Through increased sensitivity to auxin, the rol gene promotes rooting in different plant species, such as kiwi, almond, walnut, and cherry. Transformed M.26 containing the rolA gene produced a more dwarf plant and including the rolB gene also enhanced rooting in Jork.9, M.26 and M.9 apple rootstocks and the poor-rooting pear rootstock BP10030 (Zhu et al., 2001). The percentage of cuttings that rooted increased by 90%. Some of these genetically modified plants produced seven times as many roots as non-transformed pants.
When several cultivars were grafted onto transformed M.9 or M.26 rootstocks, tree height, trunk diameter, and shoot length after five years was 3 to 25% smaller for transformed vs. non-transformed rootstocks. Trunk diameter was also reduced, but the number of shoots was usually increased. Flowering and fruit set were usually reduced; fruit size and firmness were little effected, but fruit red color was usually increased by as much as 40% (Smolka et al. 2010). They also found no introduced genetic material in the flowers or leaves of the scion, suggesting that these materials were not translocated from the rootstock to scion.
Two genes responsible for dwarfing in apple rootstocks have been identified. It may be possible that vigorous rootstocks with few problems, such as Robusta #5 or MM.111 may be made more dwarfing by inserting dwarfing genes. For example, M.26 stem length was reduced when it contained the phytochrome B (phyB) gene from an Arabidopsis plant.
Development of herbicide-resistant rootstocks might also be possible. Russian researchers transformed several rootstocks of apple and one pear with the bar gene. The bar gene conferred resistance to the herbicide bialaphos and was cloned from the bacterium Streptomyces hygroscopicus. Bialaphos is a natural herbicide and a metabolite of Streptomyces hygroscopicus. It was the first herbicide produced by fermentation.
Genetically engineered rootstocks and apple cultivars will likely not be commercially available soon. However, these transformed plants offer researchers new opportunities to study the interactive effects of scion/rootstock combinations. Some areas of research that may benefit from studying these plants include nutrient uptake by roots, flowering, and resistance to drought, cold, soil salinity, and pests. The information generated with these plants may even be used to modify orchard practices for commercially available trees to enhance the profitability and sustainability of the fruit industry.
Aldwinckle H.S, E.E. Borejsza-Wysocka, M. Malnoy, S.K. Brown, J.L. Norelli, S.V. Beer, X. Meng, S.Y. He, and Q.L. Jin. 2003. Development of fire blight resistant apple cultivars by genetic engineering. Acta Hort. 622: 105-111
Borejsza-Wysocka, et al. 2010. Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period. BMC Biotechnology 10:41.
Dutt, M. et al. 2007. Transgenic rootstock protein transmission in grapevines. Acta Hort. 738:749-753.
Norelli J.L., H.S. Aldwinckle, L. Destéfano-Beltrán, J.M. Jaynes. 1994. Transgenic ‘Malling 26’ apple expressing the attacin E gene has increased resistance to Erwinia amylovora. In: Schmidt H., Kellerhals M. (eds) Progress in Temperate Fruit Breeding. Developments in Plant Breeding, vol 1. Springer, Dordrecht
Scorza, R. M. Ravelonandro, A.M. Callahan, J.M. Cordts, M. Fuchs, J.Dunez, and D. Gonsalves. Transgenic plum (Prunus domestica L.) express the Plum pox virus coat protein gene. Plant Cell Rep. 1994, 14, 18–22.
Smolka, A. et al. 2010. Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in apple. Transgenic Res. 19:933-948.