Rick Jellen and Dr. Richard W. Lucas, Agronomy and Horticulture

Most living organisms have two sets of chromosomes, one set from each parent. Some vascular plants, however, have been shown to possess more than just two sets of chromosomes. Such plants are referred to as being polyploid. The number of sets of chromosomes an organism has is called its ploidy level. Ploidy level is actually a major mechanism of speciation in the plant kingdom (1). Typically, polyploid species have been better able to survive in unstable or harsh environments than diploid species (species containing only two sets of chromosomes). It is believed this is so due to the greater variety of allelles available to the polyploid species (2,3).

Atriplex, or saltbrush, is a drought tolerant genus widely distributed throughout western North America. Thirty miles east of Wendover, NV right along I-80 is a freeway exit called “Knolls”. Here grow interesting populations of Atriplex (hereon referred to as A.). Hexaploid A. tridentata grows along side tetraploid A. canescens. Amongst these populations, a new hexaploid species of Atriplex is believed to have arisen. Little is currently known about this species other than it is believed to have hybridized from A. tridentata and A. canescens. It has morphologically been described by Stutz and Sanderson as a variety of A. canescens (4). My project was to gather data on this putative species and demonstrate that it is indeed a new species of Atriplex.

I had planned on using C-banding and genomic in situ hybridization (GISH) to show that this putative species is genuine. Both procedures require that I find cells in which the chromosomes are visible. C-banding involves sequential treatments of chromosomes with hydrochloric acid, barium hydroxide, saline solution, and a Romanowsky eosin methylene blue stain, in this case Giemsaf (5). GISH analysis involves biotin-labelling the total DNA from one ancestral species and adding it to unlabeled DNA from the other candidate ancestral species. The mixture is then hybridized to a microscope slide carrying chromosomes from both genomes. Hybridization of biotinylated DNA to the homologous genome is detected using strepfavidin-peroxidase (POD) and a POD color change assay involving diaminobenzidine and urea-peroxidase (6).

Root tips are some of the fastest growing tissue in a plant, so the required meiotic cells were going to be obtained from root tip squashes. The root tips would be harvested from seed germinated in the greenhouse or laboratory. Seed from the three populations was gathered November 1997 and subsequently planted in potting soil in December of the same year. Unfortunately, sufficient root tips were never obtained. Squashes were prepared in February and it was determined that the plants were not mature enough to yield adequate root tips. I periodically checked the young Atriplex plants, but never found any good root tips to make squashes from. I waited until June when I could more intensely work on this project to try to harvest more root tips. By this time, all the roots had become woody and were completely inadequate to prepare squashes from.

I tried planting more seed in June to get some to germinate so that I might be able to get some workable root tips. Atriplex commonly grows in sandy soil, so this time I planted the seed in sand that I obtained from the Knolls site. None of the seed germinated this time. I also went out to Knolls on an other occasion to dig up some seedlings that had germinated and begun to grow out there. Unfortunately, I was still unable to collect any root tips suitable for preparing squashes. The soil was very hard and dry, despite the recent. spring rainfall that was had in that area. The tap roots penetrated very deep into the soil and I was unable to dig out any seedlings without breaking off most of the tertiary roots. I was not sure how resilient Atriplex is. But I hoped that I might be able to get the seedlings to grow again if I brought them back to the lab and planted them. I hoped that they would be able to sprout new roots and that I might obtain some good root tips this way. So far the seedlings that I transplanted to the lab have not done very well. I don’t expect that I will be able to harvest any root tips from them either. Without root tips, I was unable to complete my project as I had proposed.

I plan on once again trying to germinate some Atriplex seed. This time it will be in rich potting soil that holds the moisture better than sand. Instead of using the three inch pans, I will use some long and narrow seed containers. I will try again to get usable root tips this way. If this fails, then I will collect some more seed again in the fall and begin with new seed.

I am disappointed that I have not been able to do as much on this project as I had anticipated, but am very glad for the opportunity to take part in such a project. This has been a valuable learning experience for me. I will continue to learn as I finish this project.

References

1. Stebins, G. L. 1971. Chromosomal evolution in higher plants. Addison-Wesley, Reading, Mass.
2. Song, K., et al. 1995. Proclamation of the National Academy of Science, USA. 92: 7719- 7723.
3.  Allard, R.W., et al. 1993. Genetics 135: 1125-1139.
4.  Stutz, H. C. and S. C. Sanderson. 1979. Amerian Journal of Botany 66(10): 1181-1193.
5.  Jellen, E. N. and H. W. Rines. 1993. Genome 36: 1129-1137.
6. Jellen, E. N., B. S. Gill and T. S. Cox. 1994. Genome 37: 613-618.