by Lisa Klima Johnson, Department of Horticulture, University of Georgia
Muscadines (Vitis rotundifolia Michx.) are a unique minor fruit crop native to the southeastern United States, related to the bunch grape, but with a musky flavor and several distinctive characteristics. They are eaten fresh, as well as processed into juice, wine, and preserves. Muscadines have become more popular recently due to an awareness of their high polyphenolics content. There are three species in the Muscadinia subgenus, and they are sometimes used for increasing genetic variation in the bunch grape subgenera, the Euvitis. Muscadines have 40 chromosomes (2n = 2x = 40), which differs from the 38 chromosomes in bunch grapes, and can cause graft incompatibility and difficulty in hybridization. Male muscadine vines are no longer used in commercial vineyards. Breeding programs focus on providing female or self-fertile improved varieties. Muscadines have several desirable qualities breeders try to incorporate: large berry size, self-fertile flowers, seedlessness, good flavor, dry picking scar, early maturity, thin or palatable skin, high soluble solids, firmness, eye appeal, vigorous growth, and resistant to disease. Traditional breeding is being used to develop new cultivars, but some molecular breeding techniques are proving to be useful, especially involving disease resistance. Muscadine vines are evaluated over a period of several years, and selection can be complicated due to the large range of characteristics desired in a new cultivar. The primary challenge in breeding muscadines is that disease resistance and vigor decrease as the fruit quality increases, which provides an excellent subject for a lifetime of fruit breeding work.
Muscadines (Vitis rotundifolia Michx.) have a distinct flavor from bunch grapes, a thick skin, and large seeds. They are considered to be more fruity and musky (Morris and Brady, 2004). Most often, muscadines are eaten fresh, or used for producing wine and preserves. Wine and juice products have become more popular in recent years, and the fresh fruit has always been a regional favorite of the southeast (Conner, 2009). Also, interest has grown in using dried muscadine pomace as a functional food or nutritional supplement, due to the high levels of polyphenolics in the skins and seeds (Morris and Brady, 2004; Vasanthaiah et al., 2011; Vashisth et al., 2011). Pomace is the skins, seeds, and pulp left over from wine or juice processing. The breeding of muscadines is complex since it requires selection of several important traits at once, and a significant amount of time is required to develop new cultivars. Both molecular and traditional breeding techniques are being used in the advancement of this popular minor fruit crop (Conner, personal communication, 2012).
Species, Origin, and Genetics
Muscadines are a member of the genus Vitis, which is divided into two subgenera: Euvitis and Muscadinia. The area between the Black Sea and Caspian Sea is thought to be the center of origin of Vitis (Basiouny and Himelrick, 2001). Euvitis consists of the more common bunch grapes, Vitis vinifera, as well as several other species. Muscadinia consists of three species which are native to the southeastern part of the United States, and parts of South America. Vitis rotundifolia Michx. (muscadine) is the species which is grown commercially and found commonly in the wild, and will be discussed here. The two others, Vitis munsoniana and Vitis popenoeii, are found in very limited ranges, and are used occasionally to increase variation, but are not commercially important species (Conner, personal communication, 2012). Muscadines have been observed in North America since the 1500s in North Carolina (Ison, 1988; Morris and Brady, 2004). Wild and cultivated muscadines are found in southeastern states north to Virginia, Tennessee, and Arkansas, and west to east Texas (Basiouny and Himelrick, 2001). The first self-fertile muscadine was noted in 1910, also in North Carolina (Ison, 1988). Muscadines are a familiar crop in this region, but not nationally. The 'Scuppernong' is commonly considered to be a different fruit than a muscadine, but it is simply a cultivar. Many bronze-colored cultivars are usually called 'Scuppernong', as it is the most famous and oldest cultivar with that characteristic. However, there have been several bronze releases like 'Tara' (Lane, 1993) and 'Florida Fry' (Mortensen et al., 1994a) (Figure 1). The Muscadinia subgenera has 40 chromosomes (2n = 2x = 40), in contrast to the Euvitis subgenera, which has 38. This causes difficulty in hybridization between the two groups (Janick and Moore, 1975), and they are graft-incompatible as well (Olien, 1990). Often, the Muscadinia is viewed as a source for potential genetic variation for the Euvitis group in spite of these difficulties (Janick and Moore, 1996). This family is tolerant to a wide variety of diseases, is vigorous, and adapted to hot climates. Seven V. rotundifolia accessions, as well as three hybrids with V. rotundifolia as one of the parents, were found to be resistant to downy mildew, a commercially important grape disease (Staudt and Kassemeyer, 1995). Three V. rotundifolia accessions, as well as seven hybrids with V. rotundifolia as one of the parents, were found to be resistant to powdery mildew, another serious grape disease (Staudt, 1997). Disease resistance from muscadines is a coveted trait to grape breeders, but the flavor profile does not match Euvitis standards for fresh market fruit or wine production (Riaz et al., 2009).
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.
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.
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.
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.
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).
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.
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).
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.
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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