Interspecific and Intergeneric Hybridization in Dissotis and Tibouchina

in HortScience

Four species of Dissotis and three species of Tibouchina, two genera of the Melastomataceae family, were crossed in an attempt to create interspecific and intergeneric hybrids. Intergeneric crosses set seed at a rate of 18.1% and interspecific crosses had a 32.3% rate of seed set. Germination was extremely poor, with only four crosses having germinated seed. Crosses produced 31 seedlings. Three of the seedlings were from intergeneric crosses between Dissotis canescens and Tibouchina lepidota. Interspecific crosses produced 25 seedlings from crosses between Dissotis princeps and Dissotis rotundifolia and three seedlings from crosses between D. canescens and D. princeps. The prognosis for conventional breeding for species in Dissotis and Tibouchina is poor due to low seed set, poor germination, and slow growth of progeny.

Abstract

Four species of Dissotis and three species of Tibouchina, two genera of the Melastomataceae family, were crossed in an attempt to create interspecific and intergeneric hybrids. Intergeneric crosses set seed at a rate of 18.1% and interspecific crosses had a 32.3% rate of seed set. Germination was extremely poor, with only four crosses having germinated seed. Crosses produced 31 seedlings. Three of the seedlings were from intergeneric crosses between Dissotis canescens and Tibouchina lepidota. Interspecific crosses produced 25 seedlings from crosses between Dissotis princeps and Dissotis rotundifolia and three seedlings from crosses between D. canescens and D. princeps. The prognosis for conventional breeding for species in Dissotis and Tibouchina is poor due to low seed set, poor germination, and slow growth of progeny.

The Melastomataceae family comprises between 185 and 190 genera containing 5000 species (Almeda and Chuang, 1992). Melastomataceae species are found in Asia, Africa, North and South America, and Australia; South America contains the majority of species (Michelangeli et al., 2013; Renner and Meyer, 2001). Some species, such as D. rotundifolia (Sm.) Triana, have naturalized in places with tropical climates such as Indonesia, Brazil, Puerto Rico, and Hawaii (Liogier and Martorell, 1982; Renner and Meyer, 2001).

Species in the Melastomataceae vary widely in ploidy and chromosome count. Tibouchina chromosome numbers are based on x = 9, ranging from n = 9 to n = 63 (Almeda, 1997; Almeda and Chuang, 1992). Many species of Tibouchina are polyploid. Although tetraploidy is the most common polyploid level, ploidy level varies (Almeda, 1997). Tibouchina granulosa has a chromosome count of n = 18 and is tetraploid (Solt and Wurdack, 1980). Tibouchina lepidota is n = ≈62 or 2n = ≈122 and is thought to be 14-ploid (Almeda, 1997). Dissotis has been variously reported by Solt and Wurdack as having a chromosome count of n = 15 and by Almeda as having a chromosome count of n = 10 (Almeda, 1997; Solt and Wurdack, 1980). Dissotis rotundifolia is reported to be a diploid with n = 15 (Solt and Wurdack, 1980). Dissotis canescens, formerly treated as Heterotis canescens, has been reported to be n = 17 and is possibly polyploid (Almeda, 1997).

Most Melastomataceae species have poricidal anthers and exhibit herkogamy (Renner, 1989). Herkogamy and poricidal anthers promote outcrossing in Melastomataceae. The poricidal anthers require manipulation for the pollen to disperse. Pollen disperses when the pollinator vibrates the anthers with its flight muscles, a process called buzz pollination (Luo et al., 2008; Renner, 1989). To obtain pollen for use in hybridization, plant breeders mimic buzz pollination by using a tuning fork in the key of E to get pollen to dehisce (Renner, 1989).

The two genera from the Melastomataceae family that were chosen for this project are Dissotis and Tibouchina. Dissotis is native to Africa and Tibouchina to Mexico as well as Central and South America (Almeda and Chuang, 1992; Renner and Meyer, 2001; Todzia, 1999). Tibouchina contains species of importance in the ornamental horticulture trade, such as Tibouchina ‘Athens Blue’, whereas Dissotis sp. have not been widely used as ornamentals. However, some Dissotis sp., such as D. rotundifolia, have excellent ornamental qualities, such as soft, fuzzy leaves, or attractive pink or purple flowers, as well as being easy to cultivate and propagate. Hybridizing Dissotis and Tibouchina could create unique combinations of growth form and flower color and produce novel cultivars for the ornamental horticulture trade. The overall goal of this project was to investigate the feasibility of hybridization within and between various species of Dissotis and Tibouchina to create interspecific and intergeneric hybrids of these genera.

Materials and Methods

Several species of Dissotis and Tibouchina were placed in a greenhouse in Athens, GA in Feb. 2012 (Table 1). Stock plants were each clones of one genotype of each species. Cuttings of each species were taken, treated with a 5-s dip of potassium salt of indole-3-butyric acid (K-IBA) at 1000 mg·L−1, and stuck in Fafard Germination Mix containing processed pine bark (40%), Canadian sphagnum peat, perlite, and vermiculite (Sun Grow Horticulture, Agawam, MA), and placed under intermittent mist at 8 s every 10 min and 50% shade. After 6 weeks, cuttings were potted up in Fafard 3B Mix consisting of Canadian sphagnum peat, processed pine bark, perlite, and vermiculite in 2.8-L (15.2 cm diameter) trade containers.

Table 1.

Melastomataceae species used as parents in breeding program.

Table 1.

Dissotis sp. started blooming in July 2012. Tibouchina sp. started blooming in Aug. 2012. Interspecific and intergeneric crosses were made as species came into bloom beginning in July 2012 and ending in Mar. 2013. Flowers were emasculated to prevent self-pollination. Emasculations were carried out before anthers had completely unfolded when possible. Pollen was extracted from flowers of male parents by vibrating anthers with a tuning fork (key of E). Pollen was captured in glass containers and applied to the stigmas of flowers of the female parents using either a Q-tip or a small paintbrush. A total of 1036 crosses were made, 588 crosses were interspecific and 448 crosses were intergeneric.

Fruits were harvested when ripe. Since all species produced capsular fruit, fruits were determined to be ripe when they were light to medium brown and hard. Seed was extracted from the ripe fruits and examined under a dissecting microscope to assess probable viability. Seed that was shriveled and flattened was deemed to be nonviable. Seed determined to be of probable viability was sown in 0.56-L (10.2 cm diameter) pots on top of Fafard Germination Mix under fluorescent lighting at a set temperature of 21 °C.

Two different methods of germination were used. The first method, used from Sept. to Nov. 2012, was a humidity tent. Seeds were sown on top of moist substrate. The pots were covered with clear plastic and substrate was misted periodically to maintain a high level of humidity. Beginning in Jan. 2013, harvested seeds were sown on top of substrate and placed under mist in a greenhouse using natural lighting only.

Seedlings that germinated were transplanted into separate 0.56-L (10.2 cm diameter) pots containing Fafard Germination Mix and placed in a greenhouse under 50% shade. Once seedlings had grown to at least 5.08 to 7.62 cm tall, they were transplanted into 2.8-L (15.2 cm diameter) trade containers filled with a pine bark substrate with micronutrients and removed from shade.

Seed set from initial crosses was low, so pollen germination and pollen tube growth through the styles were evaluated to determine whether barriers to fertilization existed between the species used as parents in the study. Interspecific and intergeneric crosses (Table 2) were performed during Jan. and Feb. 2013. The number of repetitions of each cross was dependent on the number of flowers available. Some species were less floriferous in the winter, such as D. princeps, T. granulosa ‘Gibraltar’, and T. lepidota, so fewer crosses were performed using those species. Styles were harvested 24 h after pollination and placed in ethanol:acetic acid (1:2 w/v) for 1 to 24 h. They were transferred to 65% ethanol for 20 min, autoclaved in 0.8 mol·L−1 NaOH at 120 °C for 20 min, and stained with 0.1% aniline blue in 0.1 m K3PO4 (Bo et al., 2009; Ledesma and Sugiyama, 2005). The styles were kept in the aniline blue for 4 to 24 h, then mounted on slides and examined under an Olympus BX51 fluorescent microscope (Olympus Corp., Waltham, MA). Pictures were taken with an Olympus D70 microscope camera and Olympus DP Controller software.

Table 2.

Interspecific and intergeneric crosses in Dissotis and Tibouchina performed to assess pollen tube germination.

Table 2.

Pollen tube data were taken on the number of pollinated styles in which pollen tubes reached the end of the style. Data were taken on number of crosses producing fruit with seed of probable viability and number of crosses germinating. Percentages of seed-producing crosses and germinating crosses were calculated from this data. Number of seedlings resulting from these crosses was counted.

Results

Pollen tubes were able to grow to the end of the style in every cross tested (Table 2; Figs. 1 and 2). Intergeneric crosses and interspecific crosses showed the same pattern, indicating that prezygotic barriers to fertilization due to pollen and pistil incompatibility did not exist in these crosses.

Fig. 1.
Fig. 1.

(A) Pollen germinating on end of stigma and beginning of pollen tubes in Dissotis debilis × Tibouchina lepidota cross. (B) Pollen tubes growing to end of style in D. debilis × T. lepidota cross.

Citation: HortScience horts 51, 4; 10.21273/HORTSCI.51.4.325

Fig. 2.
Fig. 2.

(A) Pollen germinating on end of stigma and beginning of pollen tubes in Dissotis rotundifolia × Dissotis debilis cross. (B) Pollen tubes growing to end of style in D. rotundifolia × D. debilis cross.

Citation: HortScience horts 51, 4; 10.21273/HORTSCI.51.4.325

Seed set from both interspecific and intergeneric crosses was low and showed great variability among parents. For interspecific crosses, D. princeps and D. rotundifolia had the highest rate of seed set at 42.7% and 42.9%, respectively (Table 3). Species with the lowest rate of seed set were Tibouchina fothergillae ×pilosa at 0.0%, and T. lepidota at 4.3% (Table 3). Seed set among parents in intergeneric crosses was also highly variable. Dissotis canescens had the highest rate of seed set at 31.5% (Table 4). Tibouchina fothergillae ×pilosa and T. lepidota again had the lowest rate of seed set at 3.5% and 4.0%, respectively (Table 4).

Table 3.

Crossings performed and seed set results for interspecific Melastomataceae crosses.

Table 3.
Table 4.

Crossings performed and seed set results for intergeneric Melastomataceae crosses.

Table 4.

The first method of propagation, germinating seed under high humidity, produced no seed germination after 90 d. Germination was obtained only from placing seeds under intermittent mist in the greenhouse. Germination time ranged from 2 to 9 weeks. Crossing results for seeds germinated on the mist bench is presented in Tables 5 and 6. Germination rates for the few crosses that did germinate were still very low.

Table 5.

Summary of results for interspecific Melastomataceae crosses—mist bench propagation only.

Table 5.
Table 6.

Summary of results for intergeneric Melastomataceae crosses—mist bench propagation only.

Table 6.

Seeds from only three crosses germinated, one of the crosses was intergeneric and two were interspecific (Table 7). Dissotis canescens by D. princeps produced only three seedlings, whereas D. princeps × D. rotundifolia produced 25 seedlings. Three seedlings were obtained from the cross of D. canescens × T. lepidota. Morphological examination of the seedlings from all crosses showed that seedlings resembled female parents in growth habit, leaf shape, and leaf size. Although several of the D. princeps × D. rotundifolia putative hybrids eventually bloomed, all plants except one had lavender-colored flowers like the female parent. One D. princeps × D. rotundifolia putative hybrid produced some pink flowers as well as lavender flowers, but did not survive. One seedling of the D. canescens × T. lepidota cross bloomed and the flower was like that of the female parent in size, shape, petal number, and color. Although seedlings strongly resembled the female parent, they were weak and did not grow vigorously. Seedling performance was poorer than that of either parent, suggesting that they could be hybrids. Seedling mortality was high. Of the 31 seedlings produced, none survived to Oct. 2015. Detailed data and results for crosses are presented in Table 8.

Table 7.

Number of seedlings produced for Melastomataceae crosses with germinating seed showing number of seedlings produced.

Table 7.
Table 8.

Detailed data for Melastomataceae crosses. Germination data are for crosses propagated on the mist bench only.

Table 8.

Discussion

Pollen tubes grew through the styles of all crosses tested, indicating that pollen–pistil incompatibility did not cause low fertility. Low rates of seed set could have been partly due to lack of fertilization or embryo abortion. Incompatibilities between parents in an interspecific or intergeneric cross may lead to poor seed set. In crosses between Chimonanthus praecox and Chimonanthus nitens, even though successful fertilization occurred in 30.4% of pollinations, 100% of embryos aborted by 25 d after pollination due to the low number of viable embryos that developed and the complete failure of the endosperm to develop (Wang et al., 2014).

We visually examined the seed to determine probable viability and deemed seed that was flat instead of rounded to be nonviable. Wiens et al. (1987) found that flattish, collapsed seed was a sign of aborted embryos in Epilobium angustifolium L.; it is probable that flat seed in this study also contained aborted embryos. Prezygotic barriers to fertilization appeared to be low based on pollen tube germination tests. Postzygotic barriers could have been caused by parent species having different chromosome numbers, as the species with known chromosome counts had widely varying numbers of chromosomes. For instance, differing endosperm balance numbers in parental species or the incompatibility of parental genomes could have caused postzygotic barriers (Talluri, 2012). In addition, chromosome elimination in hybrid seed during initial mitosis after fertilization may have resulted in the weakness of the hybrids and contributed to the nonviability of seed (Hancock et al., 2015). Use of species with the same numbers of chromosomes as parents could result in successful hybridization. As well, ovule culture is a possibility for increasing the number of progeny from future crosses if the failure of endosperm to develop is the cause of embryo abortion.

Even though the Melastomataceae seed used for germination appeared to be fully mature, germination of both interspecific and intergeneric crosses was extremely poor. Incompatibility between the embryos and the endosperm could account for poor germination (Wiens et al., 1987). The propagation protocol used affected the germination rate. No germination resulted when seeds were kept under a humidity tent, although this method had worked for other researchers (Solt and Wurdack, 1980). Even seed of D. rotundifolia that was obtained from B & T World Seeds, Paguignan, France, failed to germinate under the humidity tent, but did germinate once placed on the mist bench. The mist may have mimicked the rainy season of the regions where the species in this study originated, providing the necessary amount of moisture to germinate the seeds and wash away any phenolic compounds that could have inhibited germination. Our recommended protocol for germination is to sow seed on top of media and place under intermittent mist until seed germinates.

Low germination of seed from crosses and subsequent slow growth of the seedlings indicates that the species used in this study are poor prospects for a conventional breeding program. Seedlings took several months from germination to grow more than 5 cm tall. Once seedlings started to become potbound in the 0.56-L (10.2 cm diameter) pots into which they were initially transplanted, the seedlings grew rapidly and commenced flowering. Establishing a root system that is substantial in size relative to the aerial parts of the seedling may be a survival strategy for these species, which evolved in environments with dry and rainy seasons. Low seed set, poor germination, and slow progeny growth from the initial crosses were the main factors in the decision not to continue the study past the first generation of crosses.

Since the progeny of the crosses resembled the female parents, determination that they were truly hybrids was inconclusive. Apomixis has been reported in nine Melastomataceae genera (dos Santos et al., 2012; Mendes Rodrigues and Oliveira, 2012; Renner, 1989). Although none of the species we used in the study have been reported to be apomictic, apomixis cannot be ruled out as a factor due to the identical appearance of the progeny and the female parents. The main factor that indicated the hybrid status of the progeny was their extreme weakness. If the seedlings had been apomictic, we would have expected them to grow as vigorously as the female parent. Other factors, such as the loss of paternal chromosomes, could have led to the close resemblance to the female parents.

Species in Dissotis and Tibouchina have high ornamental value. Methods such as inducing mutations through ethyl methanesulfonate (EMS) or gamma radiation might create novel cultivars for the ornamental industry. Creating interspecific and intergeneric hybrids through conventional breeding could take too long to be economically or practically feasible for many breeding programs. Although hybridization between species in Dissotis and Tibouchina has the potential to create novel ornamental cultivars, practical barriers to creating such hybrids remain to be overcome.

Literature Cited

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    • Export Citation
  • AlmedaF.ChuangT.I.1992Chromosome-numbers and their systematic significance in some Mexican MelastomataceaeSyst. Bot.17583593

  • BoJ.ZonggenS.JinboS.DaY.XianyongS.HongfeiL.2009Germination and growth of sponge gourd (Luffa cylindrica) pollen tubes and FTIR analysis of the pollen tube wallSci. Hort.122638644

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  • dos SantosA.P.M.FracassoC.M.Luciene dos SantosM.RomeroR.SazimaM.OliveiraP.E.2012Reproductive biology and species geographical distribution in the Melastomataceae: A survey based on New World taxaAnn. Bot.110667679

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  • HancockW.KuraparthyV.KernodleS.LewisR.2015Identification of maternal haploids of Nicotiana tabacum aided by transgenic expression of green fluorescent protein: Evidence for chromosome elimination in the N. tabacum × N. africana interspecific crossMol. Breed.359179

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  • Mendes-RodriguesC.OliveiraP.E.2012Polyembryony in Melastomataceae from Brazilian Cerrado: Multiple embryos in a small worldPlant Biol.14845853

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  • MichelangeliF.A.GuimaraesP.J.F.PenneysD.S.AlmedaF.KriebelR.2013Phylogenetic relationships and distribution of New World Melastomeae (Melastomataceae)Bot. J. Linn. Soc.1713860

    • Search Google Scholar
    • Export Citation
  • RennerS.S.1989A Survey of reproductive biology in Neotropical Melastomataceae and MemecylaceaeAnn. Mo. Bot. Gard.76496518

  • RennerS.S.MeyerK.2001Melastomeae come full circle: Biogeographic reconstruction and molecular clock datingEvolution5513151324

  • SoltM.L.WurdackJ.J.1980Chromosome numbers in the MelastomataceaePhytologia47199220

  • TalluriR.2012Interploidy interspecific hybridization in FuchsiaJ. Genet.9117177

  • TodziaC.A.1999Ten new species of Tibouchina (Melastomataceae) from MexicoBrittonia3255279

  • WangW.ZhouL.HuangY.BaoZ.ZhaoH.2014Reproductive barriers in interspecific hybridizations among Chimonanthus praecox (L.) link, C. salicifolius SY Hu, and C. nitens Oliver from pollen–pistil interaction and hybrid embryo developmentSci. Hort.1778591

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  • WiensD.CalvinC.L.WilsonC.DavernC.FrankD.SeaveyS.1987Reproductive success, spontaneous embryo abortion, and genetic load in flowering plantsOecologia71501–509

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Contributor Notes

This paper is part of a thesis submitted by Susan M. Hawkins as part of the fulfillment of a Master’s Degree.

Corresponding author. E-mail: ruter@uga.edu.

Article Sections

Article Figures

  • View in gallery

    (A) Pollen germinating on end of stigma and beginning of pollen tubes in Dissotis debilis × Tibouchina lepidota cross. (B) Pollen tubes growing to end of style in D. debilis × T. lepidota cross.

  • View in gallery

    (A) Pollen germinating on end of stigma and beginning of pollen tubes in Dissotis rotundifolia × Dissotis debilis cross. (B) Pollen tubes growing to end of style in D. rotundifolia × D. debilis cross.

Article References

  • AlmedaF.1997Chromosome numbers and their evolutionary significance in some neotropical and paleotropical MelastomataceaeBioLlania6167190

    • Search Google Scholar
    • Export Citation
  • AlmedaF.ChuangT.I.1992Chromosome-numbers and their systematic significance in some Mexican MelastomataceaeSyst. Bot.17583593

  • BoJ.ZonggenS.JinboS.DaY.XianyongS.HongfeiL.2009Germination and growth of sponge gourd (Luffa cylindrica) pollen tubes and FTIR analysis of the pollen tube wallSci. Hort.122638644

    • Search Google Scholar
    • Export Citation
  • dos SantosA.P.M.FracassoC.M.Luciene dos SantosM.RomeroR.SazimaM.OliveiraP.E.2012Reproductive biology and species geographical distribution in the Melastomataceae: A survey based on New World taxaAnn. Bot.110667679

    • Search Google Scholar
    • Export Citation
  • HancockW.KuraparthyV.KernodleS.LewisR.2015Identification of maternal haploids of Nicotiana tabacum aided by transgenic expression of green fluorescent protein: Evidence for chromosome elimination in the N. tabacum × N. africana interspecific crossMol. Breed.359179

    • Search Google Scholar
    • Export Citation
  • LedesmaN.SugiyamaN.2005Pollen quality and performance in strawberry plants exposed to high-temperature stressJ. Amer. Soc. Hort. Sci.130341347

    • Search Google Scholar
    • Export Citation
  • LiogierA.H.MartorellL.F.1982Flora of Puerto Rico and adjacent islands: A systematic synopsis. 1a ed. Editorial de la Universidad de Puerto Rico Río Piedras PR

  • LuoZ.ZhangD.RennerS.S.2008Why two kinds of stamens in buzz-pollinated flowers? Experimental support for Darwin’s division-of-labour hypothesisFunct. Ecol.22794800

    • Search Google Scholar
    • Export Citation
  • Mendes-RodriguesC.OliveiraP.E.2012Polyembryony in Melastomataceae from Brazilian Cerrado: Multiple embryos in a small worldPlant Biol.14845853

    • Search Google Scholar
    • Export Citation
  • MichelangeliF.A.GuimaraesP.J.F.PenneysD.S.AlmedaF.KriebelR.2013Phylogenetic relationships and distribution of New World Melastomeae (Melastomataceae)Bot. J. Linn. Soc.1713860

    • Search Google Scholar
    • Export Citation
  • RennerS.S.1989A Survey of reproductive biology in Neotropical Melastomataceae and MemecylaceaeAnn. Mo. Bot. Gard.76496518

  • RennerS.S.MeyerK.2001Melastomeae come full circle: Biogeographic reconstruction and molecular clock datingEvolution5513151324

  • SoltM.L.WurdackJ.J.1980Chromosome numbers in the MelastomataceaePhytologia47199220

  • TalluriR.2012Interploidy interspecific hybridization in FuchsiaJ. Genet.9117177

  • TodziaC.A.1999Ten new species of Tibouchina (Melastomataceae) from MexicoBrittonia3255279

  • WangW.ZhouL.HuangY.BaoZ.ZhaoH.2014Reproductive barriers in interspecific hybridizations among Chimonanthus praecox (L.) link, C. salicifolius SY Hu, and C. nitens Oliver from pollen–pistil interaction and hybrid embryo developmentSci. Hort.1778591

    • Search Google Scholar
    • Export Citation
  • WiensD.CalvinC.L.WilsonC.DavernC.FrankD.SeaveyS.1987Reproductive success, spontaneous embryo abortion, and genetic load in flowering plantsOecologia71501–509

    • Search Google Scholar
    • Export Citation

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