20.1 Dissotis Rotundifolia

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by Susan M. Hawkins, Department of Horticulture, University of Georgia, Athens, GA. Institutional sponsor is Dr. John Ruter, Allan Armitage Endowed Professor of Horticulture, University of Georgia.


Abstract

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.


Introduction

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).

Figure 1 Dissotis rotundifolia flower and immature fruits.


Figure 2 Dissotis rotundifolia foliage.


Figure 3 Dissotis rotundifolia on the greenhouse bench showing creeping growth habit. This population had been potted up from cuttings approximately six months previous to the time this picture was taken. Each plant had already been trimmed back to the edge of the pot once during that time period.


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.


Floral Morphology and Breeding Ecology

According to Renner (1989), Dissotis rotundifolia, like all the Melastomaceae, is buzz-pollinated by bees in its native range. Species in the Melastomaceae have poricidal anthers and pollen dehisces from the anthers through one or two pores; the number of the pores depends on the species. Pollen does not dehisce in species with poricidal anthers unless the anthers are manipulated in some way, usually by an insect. Pollen has also been observed to be dispersed when the plant is shaken by rain or high winds; this occurrence is rare. The vibration imparted to the anthers by bees is at least 420 Hz, making it unlikely that pollen will dehisce accidently from the anthers. Therefore, pollination in breeding must be performed in a similar fashion to what is done in solanaceous crops in the greenhouse such as tomatoes and peppers.

Melastomaceae flowers are perfect, having both pistils and stamens. Luo et al. (2008) state that many species in the family, including Dissotis rotundifolia, have two kinds of anthers: feeding anthers and pollination anthers. The feeding anthers are shorter than the pollination anthers and are yellow, a color which attracts bees. When the bees alight on the flower to gather pollen, they concentrate on the feeding anthers. While the bee sonificates the feeding anthers, it also sonificates the pollination anthers, causing pollen to dehisce from both sets of anthers. The pollen that dehisces from the pollination anthers is deposited on the body of the bee in places that the bee cannot reach while grooming. When the bee lights on the next flower, this pollen is deposited on the stigma of the flower and pollination occurs.

Renner (1989) reported that Melastomaceae flowers are herkogamous, which means that their stamens and pistils are of different lengths. In the Melastomaceae the style is often longer than the stamen. The herkogamy of the flower combined with their poricidal anthers promotes outcrossing. Before anthesis, anthers are folded up, with the ends tucked away from the stigma, an arrangement which makes accidental self-pollination very unlikely. However, if the end of the anthers touch the stigma as the anthers unfold and the flower is shaken or moved in some way that causes pollen to dehisce, it is possible to get self-pollination. Melastomaceae are often self-compatible. For example, Dissotis rotundifolia will set seed in a greenhouse without being pollinated, indicating that it is probably self-compatible (Ruter, personal communication, 2012). However, it is possible that Dissotis rotundifolia could set seed by agamospermy, as this has been observed in other species within Dissotis (Renner, 1989).


Breeding Techniques and Strategies

Different pollination techniques have been used with crops with poricidal anthers, such as solanaceous crops. One technique is to use insect pollinators, such as honey bees or bumble bees; another is to use a mechanical pollinating wand. Both techniques have been used with success in solanaceous crops, although fruit set and quality has been shown to be significantly better in insect-pollinated crops (Ahmad Al-abbadi, 2009). Much labor is saved by using insect pollination, since each flower does not need to be manipulated directly to collect pollen. However, in breeding D. rotundifolia, two problems ensue from using insect pollination: lack of control over pollinations and the failure of Melastomaceae flowers to attract certain pollinators. Melastomaceae flowers typically only offer pollen, and not nectar, which makes them unattractive to honeybees (Renner, 1989). Also, their herkogamy causes them to be difficult to negotiate for honey bees and other small bees (Almeda, personal communication, 2012). Even if larger bees, such as bumble-bees, are used, there is no way to tell which flowers have been pollinated or from which plants the pollen came.

Use of a pollination wand is common in greenhouse crops requiring buzz pollination, such as tomatoes. The standard technique is to touch the pollination wand to the pedicel of the flower late in the morning; it is a very effective way of causing pollen to dehisce from the anthers of the flower (Hogendoorn et al., 2010). The wands are readily available from greenhouse supply companies and require only a supply of batteries to keep them operational. However, due to the herkogamy of the D. rotundifolia flowers, a pollination wand would not be an effective way of causing the flower to self-pollinate (for example, for seed production). The pollen would still have to be captured in a container and applied to the stigma of the flower to be pollinated.

Dissotis pollen may also be gathered by using a tuning fork in the key of E to mimic the sonification of the flowers by bees (Ruter, personal communication, 2012). The tuning fork is struck upon a hard surface and held to the stamens; a container is needed to catch the pollen as it exits the anthers. (Renner, 1989) (Figure 4). Renner (1989) reported that a copious amount of pollen could be collected using this technique; since the pollen is binucleate, it can be stored by freezing for use at a later time, if necessary.


Figure 4 Pollen collection from Dissotis rotundifolia using a tuning fork.


Breeding Objectives and Strategies

The main objective in breeding Dissotis rotundifolia is to make interspecific crosses with other Dissotis species, such as D. princeps or D. canescens (Ruter, personal communication, 2012). The goal of the crosses is to bring the best features of the Dissotis species being crossed into the hybrid and to induce polyploidy (Ruter, personal communication, 2012). Polyploidy can cause plants to produce larger flowers and a more vigorous growth habit and can cause the plants to evince heterosis. Although diploid plants also exhibit heterosis, the effect may be more pronounced in polyploids, as was shown by a study of diploid and triploid hybrids of maize (Auger et al., 2005; Abel and Becker, 2007). Homozygous recessives can be masked, increasing the instance of heterozygosity in polyploids (Stadler, 1929). Heterozygosity is an advantage if the crop is being bred to improve a quantitative trait, such as number of flowers, rate of growth, size of flowers, and some types of disease resistance (Stillwell et al., 2003). In a study of red foliage color in flowering dogwood (Cornus florida), plants with alleles inherited from both parents showed a marked increase in red foliage (Wadl et al., 2011). Heterozygosity in Alstroemeria influenced both leaf length and width (Han et al., 2002).

Plants exhibiting polyploidy, especially interspecific hybrids, may be sterile. Evidence of difficulty in completing normal meiosis was found in a study of allohexaploid wheat by Sears (1976). However, inducing polyploidy may restore fertility to sterile diploid hybrids. Treatment of diploid and triploid roses with oryzalin, an herbicide that acts as a mitotic spindle inhibiter, was shown to induce polyploidy, and led to an increase in pollen viability, increasing the fertility of the species (Kermani et al., 2003). If the progeny of an interspecific cross of Dissotis rotundifolia with another Dissotis species is found to be sterile, oryzalin treatment is applied to attempt to restore fertility. Treatment with oryzalin is done by applying a single drop of 50 umol L-1 in a solution made with 5.5 g L-1 agar to seedling meristems. The number of applications will vary from one to three, depending on the interspecific cross; each application must be separated by three days (Jones et al., 2008). At least twenty seedlings of the progeny of each cross should be used for each treatment, as the treatment will not induce polyploidy in each seedling, and the greater number of seedlings that are used, the better the chances of inducing polyploidy are. Seedlings should then be grown in the greenhouse for three months, after which time their probable status as polyploid should be determined by examining the morphology of the plants, including leaf thickness and stomatal size. However, since ploidy may not be definitively determined by the examination of plant morphology, flow cytometry should be used to verify the ploidy of the probable polyploids (Contreras et al., 2009). The optimum dosage of oryzalin will likely be different for each interspecific cross. Once the optimum dosage of oryzalin to induce polyploidy in the progeny of each cross is determined, this dosage may be applied to all future progeny.


Hybridization

Since Dissotis rotundifolia is native to a tropical area, all hybridization must occur in the greenhouse if breeding is done in a temperate area. Even in sub-tropical or tropical areas, it is advisable to make crosses in the greenhouse if possible in order to better control pollination. Control of the growth and spreading of the plants is much easier in a greenhouse, also. This is essential with D. rotundifolia as they root very easily at the nodes (Figure 5). In the field, this growth habit could cause one population to very easily grow out of its plot and intermingle with another population.


Figure 5 Roots at node of Dissotis rotundifolia. Picture taken approximately four months after plant was potted up from a cutting.


Reciprocal crosses should be made between plants in each population if possible (Fehr et al., 1987). Flowers to be used as the female parent should be emasculated before the anthers unfold completely to prevent accidental self-pollination (Figure 6). Once the pollen is collected from the flowers of the male plant, it is applied to the stigma by paintbrush or other implement (Figure 7). If pollinations are made in the greenhouse, it is not necessary to bag the pollinated flowers. However, if pollinations are made in the field, bagging should be done as a precaution against accidental pollination by insects. The pollinated flowers are then tagged with the name of the parents in the cross and the date of the cross noted on the tag.

Figure 6 Emasculation of Dissotis rotundifolia flower to be used as male.


Figure 7 Pollination of Dissotis rotundifolia female flower.


Seed Extraction and Germination

Dissotis rotundifolia fruits take one to two months to mature (personal observation). Fruit maturation time in interspecific hybrids will vary depending upon the other parent in the cross. The fruit is a dry capsule when mature (Ruter, personal communication, 2012) (Figure 8). The capsule may be cut open or crushed in order to extract seeds. Seeds are extremely small and not able to be easily seen by the naked eye (Solt and Wurdack, 1980). Therefore, it is necessary to evaluate the seeds under a microscope in order to evaluate the number and quality of seed (personal observation) (Figure 9 and 10).

Figure 8 Mature Dissotis rotundifolia fruits, with ruler to show size of fruit.


Figure 9 Opened Dissotis rotundifolia fruit under microscope to evaluate seed.


Figure 10 Closer view of Dissotis rotundifolia seed under microscope. The larger objects under the microscope are parts of the fruit that was opened.


Seed is germinated by sowing directly on the surface of moist potting media in a flat or a pot and placing under mist (Figure 11). The potting media should be slightly acidic, such as a peat mix, since most species in the Melastomaceae family prefer acidic soil. The seeds should germinate in approximately two weeks. Once the seeds have germinated and have at least two true leaves, they may be transferred into individual pots. Seedlings are slow-growing for the first several months and grow more quickly thereafter (Solt and Wurdack, 1980). Seedlings may be fertilized at 50 ppm after they have been transferred to individual pots. The rate of fertilization may be gradually increased to 100 ppm after two weeks at 50 ppm.

Figure 11 Pots with seed of interspecific crosses of Dissotis rotundifolia with D. princeps, D. canescens, and D. debilis on the mist bench. Each pot in the first flat contains the seed of one fruit. Pots with individual seedlings of crosses in the second flat.


Greenhouse Block Design

Different populations of Dissotis rotundifolia should be clearly labeled in order to minimize errors when crossing. Each pot should be labeled with the species name, origin of the plant, and date potted. Each population should be arranged on the benches in a randomized complete block design in order to minimize environmental effects and other extraneous variability on the plants being crossed (Dowdy et al, 1991). As D. rotundifolia grows very quickly (Ruter, personal communication, 2012), care must be taken to periodically trim the plants back so that they do not grow into each other. A plant growth regulator may be applied to slow the rate of growth and avoid having to periodically prune back the plants. Paclobutrazol applied as a drench works well for this purpose.

If the crosses to be made are interspecific crosses, each population of the other Dissotis species to be crossed with D. rotundifolia should also be arranged on the benches in a randomized complete block design, with at least one replicate of each Dissotis species in each block. More than one replicate of each species is desirable (Dowdy et al, 1991).


Phenotyping

As Dissotis rotundifolia has little variability within the species, phenotyping of crosses between different populations of D. rotundifolia will mainly consist of finding progeny with larger flowers, longer flowering time, and greater numbers of flowers than either parent. However, phenotyping for interspecific crosses is much more complicated. For example, D. rotundifolia has waxy, fleshy leaves, while many other Dissotis species have leaves with trichomes (personal observation). Growth habits also differ between Dissotis species, varying from creeping in D. rotundifolia to upright in D. princeps and D. canescens. Interspecific hybrids may have to be evaluated on an individual plant basis to assess their ornamental potential. Progeny should also be evaluated to see if they are intermediate to the parents for any trait.

Data Collection

A record of each cross should be made, listing the parents (including the population the parents belong to) and the date the cross is made. Plants should be evaluated daily for ripened or aborted fruit. Ripened fruit should be collected. Both ripened and aborted fruit should be recorded. From this data the abortion rate can be calculated. According to Fehr et al. (1987), this is especially important in interspecific crosses, as it is likely that one species will be more suited to being the female parent than the other; this will usually be the species with the higher chromosome number. If a cross using one species as a female parent results in a high percentage of abortions, then the strategy of using reciprocal crosses may be abandoned and the plant with the higher chromosome number should be used as the female in every cross to maximize the likelihood of successful crosses.

Once ripe fruit has been obtained, the seed should be germinated and the germination percentage should be recorded. In interspecific crosses, it is possible that germination will not occur due to incompatibility between the endosperm and the embryo of the seed (Ng et al., 2012).

Once viable seed has been obtained and germinated, the progeny should be grown out and phenotyped. In crosses between different populations of Dissotis rotundifolia, the flower size, flower color, flowering rate, and length of blooming period should be evaluated and compared to that of the parents to calculate genetic gain. In interspecific crosses, morphology of the progeny should be compared to that of the parents. Flower size, flower color, flowering rate, and length of blooming period should again be evaluated and compared to that of the parents to calculate genetic gain, if any.


Propagation and Increase

Once a hybrid is obtained with desirable characteristics, it should be clonally propagated in order to increase it. Cuttings should be made, approximately three inches long, and treated with a five-second dip of 1,000 ppm potassium indole-3-butyric acid (K-IBA). The cuttings should then be struck into a flat filled with a mixture of half-and-half peat and perlite. The flats should be placed upon a mist bench and the mist set at ten seconds of mist every six minutes. It is important not to mist the cuttings too heavily, as they will rot if given too much water. Rooting should take four to six weeks. Once the cuttings are well rooted, they may be potted up into one- or two-gallon pots and placed on the bench in a greenhouse. Growth of Dissotis rotundifolia propagated from cuttings is very rapid; plants propagated from cuttings will fill a one-gallon pot in two to three months (personal observation).

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