By Wayne Handlos

Over the past year, the news media have picked up a scientific report about some genetically modified geraniums (Pelargonium sp.). I have included some of this information in our newsletttr.(Nov. 2012) with additional comments. The changes which had been induced in various geraniums resulted in the plants being unable to produce viable pollen. The news media touted this change as a boon to asthma and hay fever sufferers. Since none of the members of the geranium family (Geraniaceae) produce wind borne/blown pollen, they are unlikely to be the cause of anyone suffering. [If anyone has a medical problem with these plants, it will more likely be due to the fragrant oils produced by the glandular hairs on the leaves and stems.] This seemed like a classic case of a journalist knowing this was an important piece of research but totally missing its true significance. This sent me off to find and read the original scientific paper reporting on this research.

The title of the report "Production of engineered long-life and male sterile Pelargonium plants" seems straight forward enough � the fact that the paper has nine authors (Bego�a Garcia-Sogo, Benito Pineda, Edlin Roque, Teresa Ant�n, Alejandro Atar�s, Maris� Borja, Jos� Pio Beltr�n, Vicente Moreno, Luis Antonio Ca�as) might be a hint that this could be a hard read. In fact, that is true. There is a lot of scientific terminology and many procedures that most of us are not familiar with. I hope that this article will explain this in terms that will be understandable.

The results of the scientific work are simple enough in concept. Plants were produced whose leaves were longer lived than normal (unmodified or control) plants. The stems were more highly branched and the stems (internodes or the area between successive leaves on a stem) were shorter than normal. The flowers were more intensely colored. Commercially, this work produced plants which would have been appreciated by consumers, i.e., short, well branched, long-lasting plants with brightly colored flowers. Who wouldn�t like that?

The other major change resulted in plants whose anthers (the pollen bearing structures of the flower) did not complete development and did not produce any pollen � rendering the plant male sterile. For the average consumer, this change has no importance. But from a commercial stand-point this is a useful modification for producing hybrid seed. The plants retain their ability to produce seeds but the pollen must come from another plant � that is, there is no self-pollination. When producing hybrid seed, you cross two different line (genetic lineages) of plants. To do this by hand is very time consuming and expensive to remove the anthers of one plant to prevent self- pollination and then bring pollen from another plant to effect pollination, fertilization and finally seed production. If one of the lines is male sterile, then you can grow the different parental plants side by side and let the bees do the pollen transfer for you. Then you just collect the seeds from the male sterile parent and you have your hybrid seed.

Male sterility has another ecological effect that is worth noting. Since these plants do not produce pollen, the genetic changes which have been induced cannot be spread to other plants. (Or if these plant shed pollen into the wind [which no Pelargonium does], they could not cause hay fever [the irrelevant fact that journalists picked up on].)

At this point, this is an unremarkable story. Pelargonium plants were "engineered"; i.e. changed in ways that have commercial applications as well as aesthetic and ecological significance. Now you should say, "Why are we following what superficially looks like an ordinary study of minimal importance?"

The plot thickens. We have not used two trigger phrases which engage (enrage?) people; that is, "genetic engineering" and "GMO � genetically modified organism." The latter term, GMO, triggers an emotional response in many people because it engenders many negative views of tampering with nature. [In a future newsletter, I will cover more aspects of "genetic engineering" and look at some GMO�s.]

<Normal anther �370�   <Sterile anther �370�

< (1)                                                                                                                                                                                                  < (2)     The Garcia-Sogo paper, which I have been summarizing up to this point, was published in 2012 and, as noted, was picked up by the news media. What was the nature of the genetic engineering carried out by this group?  They used two different cultivars of Pelargonium: P. peltatum cv. �Aranjuez�(1) and P. x hortorum cv. �370�(2). Using various techniques of tissue culture and micropropagation, they produced surface-sterilized "clean" plants.

<Gall caused by A. tumefaciens   <Agrobacterium tumefaciens growing on a plant cell.

From these clean plants they removed pieces of leaf which were exposed to cells of different strains of the bacterium Agrobacterium tumefaciens. By various selection and elimination procedures, they obtained cells in which genetic material (DNA) carried by the bacterium was transferred to the Pelargonium leaf cells. 

Eventually, after several months, these transformed cells grew into plantlets which were ultimately removed from their sterile containers and planted in pots of peat moss and perlite (3:1). Their paper was basically a report on techniques used and their efficacy in converting or transforming existing cultivars without using classical plant breeding techniques.

< gfp in callus

The following genes in the bacterium were found to have been transferred: nptII, gfp, uidA, barnase-barstar, ipt. The ipt gene is involved in delaying senescence, increased branching, reduced intermodal length, smaller leaf size, reduced flower size, intensification of leaf and flower color. The barnase gene is related to male sterility through the collapse of cells in the anther interrupting the production of pollen. The nptII is a marker gene; the uidA gene is a reporter gene; the gfp (green fluorescent protein) gene is a marker gene also. [More about gfp in a future newsletter.]

Same age <Normal plant  <Long-life plant

<Normal stem  <Short internodes

 <(on left) WT=Normal leaf     (on right) ipt= Reduced leaf

<(on left) WT=Normal flowers   (on right) ipt=Reduced-size flowers

Now for some history. A survey of published reports indicates the following.

D.A. Tepfer et al. began working with the genetic transformation of Pelargonium using the bacterium Agrobacterium rhizogenes in the 1980�s. By 1994, he applied for a patent on a method for transferring genetic material and regenerating whole plants from transformed cells. His patent was granted in 1997. He worked on various cultivars of scented geraniums including �Lemon�, �Old Spice�, �Lime�, �Old Fashioned Rose�, �Peppermint� and �Apple�. The species used included P. x nervosum, P. graveolens, P. tomentosum, P. odoratissimum, P. capitatum, P. vitifolium and probably P. radens. Several of his species designation are not clear. He also transformed one numbered cultivar of P. x hortorum �370�. In general, the genes from the bacterium had the effect of reducing the size of the plants because of shorter internodes, increasing branching, increasing essential oil production, reducing the size of leaves and causing the leaves to be less wrinkled.

By 1995, M.-P. Robichon et al. reported on the transformation of P. x hortorum seedlings using Agrobacterium tumefaciens. [Henceforth, A. is the abbreviation for Agrobacterium.)

In 1996, A. Pellegrineschi and O. Davioli-Mariani, using A. rhizogenes transformed three species of scented geraniums (P. x fragrans, P. odoratissimum, P. quercifolium). These plants showed an increase in the proportions of desirable essential oils and darker green leaves on plants that had two to three times more branches than the normal (control/untransformed) plants.

M.R. Boase et al. by 1996 had effected transformation in the regal geranium �Dubonnet� (P. x domesticum �Dubonnet�) using A. tumefaciens. The resulting plants exhibited a dwarf or super dwarf stature, smaller leaves and petals but earlier flowering than the control plants.

By 1997 Y.M. Bi, S. KrisnaRaj and P.K. Saxena developed increased resistance to the disease Botrytis cinerea in the scented geranium �Frensham� using a form of the bacterium Agrobacterium which carried an antimicrobial gene from onions.

A commercial cultivar of the rose-scented geranium (P. sp. �Hamanti�) was transformed by 1997 using A. rhizogenes by G. Saxena et al. Some of the plants had increased concentrations of the desirable essential oils geraniol and geranyl esters. The transformed plants also showed increased branching, more leaves, shorter stature and leaves without wrinkles.

By 2005, T. Winkelmann et al. worked out a technique for shoot regeneration in transformed cultivars of P. x hortorum and P. peltatum using A. tumefaciens. They worked with nine cultivars of zonal geraniums and seven cultivars of ivy geraniums.

And this brings us to 2012 and the report of B. Garcia-Sogo et al. and the creation of long-life and male sterile plants of P. x hortorum and P. peltatum, using A. tumefaciens to introduce the desired genes.

In summary, we can see that techniques to create GMO�s or genetically modified plants in Pelargonium (zonal, scented, ivy and regal types) have been with us since the 1980�s. The early work perfected the techniques for using the two bacteria, Agrobacterium rhizogenes and A. tumefaciens to transfer alien genes into various species and cultivars of Pelargonium and methods for generating whole plants from individual transformed leaf, root and stem cells. This work has evolved to the present day with the introduction of specific genes to develop commercially desirable traits in the various cultivars of Pelargonium

Pelargonium illustrations from B. Garcia-Sogo et al.; bacterial images from and