20.1 Dissotis Rotundifolia
by Susan M. Hawkins, Department of Horticulture, University of Georgia
Although little is known about Dissotis rotundifolia, it is a species with great potential for ornamental use in the Southeast and as a specialty crop for medicinal uses. D. rotundifolia also has possibilities as a parent in interspecific crosses to produce new hybrids for ornamental use. Challenges in breeding D. rotundifolia include pollen collection, limitations to growth range due to lack of cold-hardiness, and difficulties in making interspecific crosses. Dissotis species are crossed in the greenhouse unless the nursery facility is in a tropical climate. Pollination is done by hand, using a tuning fork to cause pollen to dehisce and collecting the pollen in a vessel for use in pollinations. The crosses are evaluated for abortion rate and seed germination rate, with crossing strategies adjusted for crosses with high abortion rates. Once viable seed is obtained, it is sown on top of slightly acidic potting media and maintained under mist until germination. The resulting progeny are phenotyped and evaluated for ploidy using flow cytometry. The main traits of importance in phenotyping the hybrids are size of flowers, length of flowering period, and vigor of growth. Once the progeny is phenotyped, the best of the progeny for ornamental use are used in further crosses, or propagated vegetatively to increase the numbers of the new hybrid.
Dissotis rotundifolia, commonly called Spanish Shawl or Pink Lady, is not well known as an ornamental outside tropical and subtropical regions of the United States; however, it has the potential to be more widely used in the landscape (Ruter, personal communication, 2012). D. rotundifolia is a short lived perennial plant with bright pink flowers and ovate, fleshy leaves, prickly fruits, and a trailing or creeping growth habit (Abere et al., 2010, Porembski et al., 1996). (Fig. 1, 2, and 3). D. rotundifolia is an excellent ground-cover, although it would be an annual in zones that are not tropical or subtropical (Ruter, personal communication, 2012). The trailing habit of this species lends itself well to use in a hanging basket (Ruter, personal communication, 2012). The species is resistant to drought and very easy to propagate vegetatively (Ruter, personal communication, 2012; personal observation).
Dissotis rotundifolia also has the potential to be used as a specialty crop for medicinal purposes. In its native range in Africa, D. rotundifolia is used as a medicinal plant to treat several illnesses such as dysentery, rheumatism, circulatory problems, conjunctivitis, venereal disease, and hookworm infestation; it is also used to prevent miscarriages (Abere et al., 2010, Abere et al., 2009). An extract of D. rotundifolia was shown to contain compounds that are effective in killing Trypanosoma brucei, the parasite that causes African sleeping sickness (Mann et al., 2009). Phytochemical screening of D. rotundifolia found anti-microbial activity that could effectively be used to treat dysentery, diarrhea, stomach ailments, and venereal disease (Abere et al., 2010). More pharmacological studies of the species are needed to determine its full potential.
Dissotis rotundifolia is a member of the Melastomaceae family, which is comprised of tropical and subtropical plants. There are 185 to 190 genera and approximately 5,000 species in the Melastomaceae to date (Almeda and Chuang, 1992). The genus Dissotis contains 140 species, all native to Africa; D. rotundifolia is native to tropical West Africa (Abere et al., 2009). According to Porembski et al. (1996), D. rotundifolia is found on rock outcrops and in the bottom of sandy depressions; and the species also grows as a weed alongside roads and in waste spaces in its native range.
D. rotundifolia is one of the few Dissotis species for which the chromosome count is known. Solt and Wurdack (1980) report that D. rotundifolia has a chromosome count of 2n = 2x = 30; since the base number of the genus Dissotis is n=15, the species is diploid.
Breeding Strategies: Triploid (Seedless) Watermelon
Seedless watermelon fruit is produced by stimulative parthenogenesis when sterile hybrid triploid watermelon plants are pollinated by diploid plants. The development of these triploid hybrid watermelon cultivars include the following steps (Figure 3): (i) production of tetraploids from diploids using colchicine or dinitroaniline (ii) development of stable inbred tetraploid lines and (iii) develop hybrid triploid cultivars.Currently a limiting factor in seedless watermelon production is the susceptibility of triploid cultivars to Fusarium wilt. Resistance to Fusarium wilt races 0 and 1 is common in diploid (seeded) cultivars.
Controlled Pollinations: Controlled Pollinations in the Greenhouse
The ideal greenhouse temperatures for watermelon breeding are between 70-85 ºF. During the winter months artificial light should be supplied (14 hours light: 10 h darkness). Precautions should be taken to exclude pollinators (e.g. bees) from the greenhouse. Preventative spray programs for diseases, especially powdery mildew are essential. Using a shade cloth in the greenhouse will promote development of powdery mildew, but is essential to keep greenhouse temperatures down in summer. Insects such as whiteflies should also be carefully controlled.
Mix one 2.8 cu ft bag of Fafard 3B soil mix and 178.4 g of Osmocote classic (14-14-14). Fill seedling trays (cell dimensions: 3.1 x 3.1 x 2.3 in) with soil mix. Prepare plant labels with the date and cultivar/line name. Plant seeds approximately ½ inch deep and water well. Seeds should be watered lightly once a day, be careful not to overwater, especially wild germplasm. It will take between 4 days and 2 weeks for seeds to germinate (Figure 4). Germination of wild germplasm is more varied than for cultivars and it might take several weeks for all seeds to germinate.
At 3 - 4 weeks after sowing, plants should be transplanted to 12 inch pots. It is important to transplant the seedlings before they start flowering. Make sure that every plant is labeled with the date, cultivars/accession name as well as the number of the specific plant. Depending on the purpose of the cross, it is often necessary to be able to tell the specific plant in a cross, not just the cultivar/line. The most space-efficient way to grow watermelon in the greenhouse is to trail them vertically using stings. However, it should be kept in mind that it is very labor intensive to maintain plants grown in this way
Once the seedlings start growing after transplanting, the main shoot should be manually trailed along the string. Side-shoots growing from the bottom can be removed. As the plant grows more side shoots can be removed, but don’t remove all the side-shoots – just enough to make it possible to trail the plant easily and keep it from growing into adjacent plants. This trailing and removing shoots should be done several times a week. If the plant reaches the top of the string it should be trailed back down.
Crossing of plants will be easier if the plants you plan to cross are close to each other in the greenhouse. Keep in mind that once you have started trailing the plants, they cannot be moved. Wild accessions often take longer to flower and when crossing a wild accession with a cultivar, it might be necessary to sow the seeds of the wild accession earlier in order for flowering to overlap. This is usually not needed when crossing cultivars. All pollinations should be completed before 11 am.
Female watermelon flowers generally stay open for only a single day and if the flowers are not pollinated, you will have to wait for the next female flower to open. It is therefore imperative that plants be inspected every day. When the petals of the bud turn yellow, you can expect the flower to open the following day (Figure 6).
Male flowers stay open longer, but it is important to use only fresh pollen. Do not use flowers if the anthers have started to turn brown. The freshest flower will usually be the flower closest to the tip of the vine. Pick the desired male flower, fold the petals back, and carefully use the anthers as a brush to transfer the pollen to the stigma of the selected female flower (Figure 7). It is imperative to not touch or damage any part of the female flower. Breaking the petals or touching the ovary will lead to abortion of the fruit. Make sure to cover the entire surface of the stigma with pollen. It might be necessary to use more than one male flower in order to have enough pollen. A lack of pollen will lead to deformed fruit (Figure 8).
Use a paper jewelry tag to identify the cross (Figure 9). The tag should be attached to the main stem, NOT the fruit petiole. The information on the tag should include the date the cross was made, as well as the identity of the parents.
Once a plant has been pollinated, all other female flowers should be removed on a daily basis. This process must be continued until the fruit is approximately baseball size. If the other female flowers are not removed, the fruit will abort. When fruit reach approximately softball size, the fruit should be bagged. The bag should be secured in such a way that it supports the entire fruit weight instead of the fruit hanging from the petiole (Figure 10). Plants should be continually trailed and trimmed until fruit is ready for harvest.
Controlled Pollinations in the Field
Approximately 2-3 weeks after sowing seedling should be hardened off for 5-7 days before transplanting in the field. Follow recommendations in the current, local extension publications, e.g. South Eastern U.S. 2012 Vegetable Crop Handbook (http://www.thegrower.com/south-east-vegetable-guide/) .
Controlled pollinations in the field - video
Phenotyping Fruit Traits
Traits like yield, fruit weight, fruit shape and rind thickness are usually collected. In addition, sugar content (°Brix) is usually measure with a refractometer and flesh firmness with a penetrometer (0.8 cm probe). Flesh color is usually visually evaluated.
Seed size and color are important traits that should be evaluated.
Fusarium Wilt (Fusarium oxysporum fsp. Niveum; FON)
A disease severity rating scale (0-5) is commonly used to evaluate resistance to FON race 1 and race 2 (Figure 11). Data should be collected every 7 days for 4 weeks after inoculation. Data analysis is done by comparison of mean-disease severity of the genotypes tested. If appropriate mock inoculated controls are used to standardize among cultivars/lines, plant dry weight, 4 weeks after inoculation can be used as a objective quantitative measurement.
Experiments are conducted to select grass genotypes that could later be released as a variety. Most of these trials involve screening for a particular trait of Paspalum There are three potyviruses that infect watermelon, Zucchini yellow mosaic virus (ZYMV); Papaya ringspot virus – watermelon strain (PRSV-W) and Watermelon mosaic virus-2 (WMV-2). Watermelon seedlings can be easily inoculated using mechanical inoculation (Fig 12) with infected squash or zucchini leaves ground up in 0.02 M phosphate buffer.
Plants can be visually scored as infected/not infected starting 1 week after inoculation, but weekly evaluations should continue for at least 4 weeks (Figure 13). Enzyme-linked immunosorbent assays (ELISA) should be carried out to confirm visual evaluation and with proper controls can be used to obtain quantitative resistance data.
Once fruit is mature it can be removed from the vine and the seed harvested. The number of days from pollination to harvesting fruit will depend on the cultivars/accessions used as parents and need to be determined empirically. Before extracting the seed record all the required fruit data (section 7)
Seed can be extracted by cutting up the fruit and picking out the seed by hand. Seeds are then washed and rinsed using a sieve. Make sure all flesh is removed and seeds are clean. Surface sterilize the seeds for 10 minutes in 10% bleach, followed by rinsing in water and then spread seeds out on a piece of paper to dry (~24 hours). The tag should accompany the seeds throughout the process. Once seeds are dry, put them into clearly labeled paper envelopes or bags (Figure 14). Seed can be stored in a cooler with humidity control (%RH + ºF <100).
A germination test should be done for all seedlots before the parental plants are discarded. Plant 8 seeds in a Speedling tray and record germination percentage (Fig 15). If the germination percentage is not satisfactory the cross should be repeated.
In some cases, e.g. mapping studies, plant material need to be collected for DNA extraction. Label 15 ml tubes with the plant ID and the date of collection. Collect young, healthy leaves in the tube and freeze immediately in liquid nitrogen and transfer to - 80ºC (Figure 16).
For each cross the following data should be collected:
- Seedlot ID
- Maternal and paternal plant number
- Planting date
- Pollination date
- Fruit harvest date
- Number of days from pollination to harvest
- Fruit size (Weight, length & width)
- Cut fruit longitudinal and take a picture. The picture should include a card with the identity of the cross and a ruler for size estimation (Figure 17).
- Fruit shape
- Flesh color
- Brix (Refractometer)
- Rind thickness
- Rind color
- Seed extraction date
- Date seed put in storage
- Number of seed harvested
- Seed treatment (if any)
- Seed germination percentage
- Any other comments.
All data should be added to a database for easy access.
Abel, S. and H.C. Becker. 2007. The effect of autopolyploidy on biomass production in homozygous lines of Brassica rapa and Brassica oleracea. Plant Breed. 126: 642-643.
Abere, T.A., P.E. Okoto, and F.O. Agoreyo. 2010. Antidiarrhoea and toxicological evaluation of the leaf extract of Dissotis rotundifolia Triana (Melastomataceae). BMC Compl. and Alt. Med. 10: (17 November 2010)-(2017 November 2010).
Abere, T.A., D.N. Onwukaeme, and C.J. Eboka. 2009. Pharmacognostic evaluation of the leaves of Dissotis rotundifolia Triana (Melastomataceae). Afric. J. Biotech. 8: 113-115.
Ahmad Al-abbadi, S.Y. 2009. Efficiency of different pollination treatments on Solanaceae yields grown in plastic house. J. Bio. Sci. 9: 464-469.
Almeda, F. and T.I. Chuang. 1992. Chromosome numbers and their systematic significance in some Mexican Melastomataceae. Syst. Bot. 17: 583-593.
Auger, D.L., E.H. Coe, Jr., J.A. Birchler, A. Kato, A.D. Gray, and T.S. Ream. 2005. Nonadditive gene expression in diploid and triploid hybrids of maize. Genetics 169: 389-397.
Contreras, R.N., W.W. Hanna, and J.M. Ruter. 2009. An oryzalin-induced autoallooctoploid of Hibiscus acetosella ‘Panama Red’. J. Amer. Soc. for Hort. Sci. 134: 553-559.
Dowdy, S.M. and S. Wearden. 1991. Statistics for research / Shirley Dowdy and Stanley Wearden. Wiley series in probability and mathematical statistics. Applied probability and statistics. New York : Wiley, c1991.
Fehr, W.R., E.L. Fehr, and H.J. Jessen. 1987. Principles of cultivar development / Walter R. Fehr ; with the assistance of Elinor L. Fehr and Holly J. Jessen. New York : Macmillan ; London : Collier Macmillan, c1987.
Han, T., H.J.v. Eck, M.J.d. Jeu, and E. Jacobsen. 2002. Mapping of quantitative trait loci involved in ornamental traits in Alstroemeria. HortScience 37: 585-592.
Hogendoorn, K., M.A. Keller, and F. Bartholomaeus. 2010. Chemical and sensory comparison of tomatoes pollinated by bees and by a pollination wand [electronic resource]. J. Econ. Entomol. 103: 1286-1292. doi:http://dx.doi.org/10.1603/EC09393.
Jones, J.R., T.G. Ranney, and T.A. Eaker. 2008. A novel method for inducing polyploidy in Rhododendron seedlings. American Rhododendron Society Journal. Am. Rhododen. Soc., Niagara Falls; USA. p. 130-135.
Kermani, M.J., V. Sarasan, A.V. Roberts, K. Yokoya, J. Wentworth, and V.K. Sieber. 2003. Oryzalin-induced chromosome doubling in Rosa and its effect on plant morphology and pollen viability. Theor. Appl. Gen. 107: 1195-1200.
Luo, Z., D. Zhang, and S.S. Renner. 2008. Why two kinds of stamens in buzz-pollinated flowers? Experimental support for Darwin's division-of-labour hypothesis. Func. Ecol. 22: 794-800.
Mann, A., E.C. Egwim, B. Banji, N.U. Abdukadir, M. Gbate, and J.T. Ekanem. 2009. Efficacy of Dissotis rotundifolia on Trypanosoma brucei brucei infection in rats. Afric. J. Biochem. Res. (AJBR) 3: 5-8.
Ng, D.W.K., J. Lu, and Z.J. Chen. 2012. Big roles for small RNAs in polyploidy, hybrid vigor, and hybrid incompatibility. Curr. Opin. Plant Bio. 15: 154-161.
Porembski, S., J. Szarzynski, J.P. Mund, and W. Barthlott. 1996. Biodiversity and vegetation of small-sized inselbergs in a West African rain forest (Taï, Ivory Coast). J. Biog. 23: 47-55.
Renner, S.S. 1989. A survey of reproductive biology in neotropical Melastomataceae and Memecylaceae. Ann. Missouri Bot. Gard. 76: 496-518.
Sears, E.R. 1976. Genetic control of chromosome pairing in wheat. Ann. Rev. Gen. 10: 31-51.
Solt, M.L. and J.J. Wurdack. 1980. Chromosome numbers in the Melastomataceae. Phytologia 47: 199-220.
Stadler, L.J. 1929. Chromosome number and the mutation rate in avena and triticum. Proc. Natl. Acad. Sci. USA 15: 876-881. doi:10.1073/pnas.15.12.876.
Stillwell, K.L., H.M. Wilbur, C.R. Werth, and D.R. Taylor. 2003. Heterozygote advantage in the American chestnut, Castanea dentata (Fagaceae). Amer. J. Bot. 90: 207-213.
Wadl, P.A., A.M. Saxton, X.W. Wang, V.R. Pantalone, T.A. Rinehart, and R.N. Trigiano. 2011. Quantitative trait loci associated with red foliage in Cornus florida L. Mol. Breed. 27: 409-416.