Identification of tetraploid regenerants from cotyledons of diploid watermelon cultured in vitro

Summary Adventitious shoots were obtained from the diploid watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] cultivars Dixielee, Jubilee II, Mickylee, Minilee, and Royal Sweet by culturing excised cotyledons on shoot regeneration medium for six weeks. Tetraploid and diploid regenerants were identified by counting the number of chloroplasts per guard cell pair from leaves of regenerated plants. Cross fertilization of putative tetraploids with diploid pollinators and the production of triploid seed confirmed the efficacy of this approach. The mean number of chloroplasts for tetraploid regenerants was 19.1 whereas diploids averaged 11.2. These values were similar to tetraploid and diploid plants from seed. Ovary diameter, petal, and anther diameter of male flowers, and leaf length by width ratio were also good indicators of plant ploidy. Progeny obtained from self-fertile tetraploids of Mickylee were crossed with various diploid pollinators to produce triploid hybrid seed. All triploid plants from tissue culture-derived tetraploids produced fruit comparable in quality to fruit produced by currently-available triploid hybrids, demonstrating that in vitro tetraploid induction can be used to produce high quality tetraploid plants for use in triploid hybrid seed production.     

Polyploidy and sexual polyploidization in the genus Vaccinium

Genus Vaccinium,consisting of blueberries, cranberries, lingonberries and many related wild species, includes diploid, tetraploid, and hexaploid species. Most evidence indicates that the tetraploid species are autotetraploids, with non-preferential bivalent chromosome pairing. Although homoploid interspecific crosses usually produce numerous fertile hybrids if the parents are from the same section of the genus, inter sectional crosses at the diploid level normally produce no seedlings, weak seedlings, or seedlings that are very low in fertility. There is a strong but not complete triploid block within Vaccinium. Even with insections, tetraploid × diploid (and the reciprocal) crosses normally give only a few tetraploid and a few triploid hybrids. Hexaploid × diploid crosses within sections are very hard to make, and the few hybrids that have been obtained are pentaploid. The frequency of 2n gametes varies,both among genotypes within species and among species. Vaccinium pollen is normally shed in tetrads, and the frequency of large pollen grains shed in dyads can be used to estimate 2n gamete frequency. Cultivated blueberries occur at both the tetraploid and the hexaploid levels, and there are important genetic resources in the diploids. Unreduced gamete production has permitted transfer of genetic material from the diploid to the tetraploid level and from the diploids and tetraploids to the hexaploid level via triploid hybrids. Intersectional crosses can occasionally produce tetraploid Vaccinium hybrids that appear to behave as amphidiploids and have medium to high fertility. CommercialVaccinium cultivars are normally propagated by cuttings. Intersectional hybridization, chromosome doubling, and asexual propagation could permit the production of novel hybrid combinations with value as ornamentals or in fruit production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flow cytometric identification of sexually derived polyploids in hop (Humulus lupulus L.) and their use in hop breeding

The New Zealand hop breeding programme is based solely on the development of seedless triploid cultivars. This relies on the use of tetraploid parents. While a sexually derived tetraploid parents have been used successfully, sexually derived tetraploids offer a useful alternative. They may have a higher level of heterozygosity and are easier to obtain. Methods for the identification of tetraploids from seedling populations by flow cytometry are described. Two studies were conducted; one on field-grown plants, the other on plants grown in a glasshouse. Approximately 15%of seedlings in the two studies were identified as tetraploids (2n = 4x = 40). Between 70 and 80% were identified as either diploid (2n =2x = 20) or triploid (2n = 3x = 30). Remaining plants were haploid, pentaploid or probably aneuploid. Within triploid and tetraploid populations from 40to 50% of seedlings were female, 30 to40% were monoecious, with the remainder being males and non-flowering plants. Sexually derived tetraploid parents have been used successfully in the breeding programme, from which several promising triploid selections have subsequently been made. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polyploid and maternal effects on Rhizoctonia root rot resistance in sugarbeet

Summary Three sugarbeet breeding lines partially resistant to the root-rotting fungus, Rhizoctonia solani, were converted to the tetraploid condition without selection. These three diploid and tetraploid lines were crossed with three diploid male-sterile lines to produce equivalent diploid and triploid hybrids. The triploid hybrids were significantly more resistant to Rhizoctonia than were the diploid hybrids. However, the tetraploid resistant limes were no different than their diploid equivalent lines. Reciprocal crosses provided no evidence of maternal effect on resistance. Cytoplasm that included the male-sterility factor had no influence on resistance. Triploid hybrids, where the resistant parent is tetraploid, should be advantageous in the breeding of rhizoctonia-resistant hybrid varieties.Joint contribution of the Agricultural Research Service, U.S. Department of Agriculture, the Colorado State University Experiment Station, and the Beet Sugar Development Foundation. Published with the approval of the Director of the Colorado State University Experiment Station as Scientific Paper Series No. 2072.     

Controlled pollination transfer of a nuclear male-sterile gene from a diploid to a tetraploid watermelon line

Summary A recessive male-sterility (ms) gene was transferred from a diploid to a tetraploid watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai) line through a series of controlled pollinations. Progeny testing and morphological characteristics confirmed the development of tetraploid lines containing the ms gene. The possible use of this gene transfer system in the improvement of tetraploid watermelon cultivars and the use of male-sterile tetraploids in the production of seedless triploid watermelons is discussed.     

The breeding of the cultivated potato species solanum x juzepczukii and S. x curtilobum II. The resynthesis of S. x juzepczukii and S. x curtilobum

Summary The possibility of combining anew the genomes of wild and cultivated progenitors of triploid S. x juzepczukii and pentaploid S. x curtilobum by following the known evolutionary pathway of these species was investigated.The resynthesis of S. x juzepczukii was easy, and a wide range of synthetic forms was bred. Among these were forms with higher frost resistance (-5°C) than has been found in natural S. x juzepczukii. The total tuber glycoalkaloid content of several synthetic hybrids was lower than or as low as that of natural clones. Most synthetic hybrids were more vigorous than natural S. x juzepczukii and produced about the same types of tubers as are found in the natural range of variation. The best diploid parents were found in the species S. goniocalyx.The attempt to resynthesize pentaploid S. x curtilobum has not been successful but tetraploid plants were obtained in the process. An explanation for the occurrence of tetraploids resulting from triploid x tetraploid and/or diploid crosses is offered.The newly bred tetraploids contain at least one genome from S. acaule (possibly two) and hybridize easily with ssp. andigena. They thus provide a means for the transfer of S. acaule germ plasm into the tetraploid cultivated gene pool which would profit from the frost resistance of S. acaule.     

Salinity tolerant performance and genetic diversity of four rice varieties

Nuclear DNA content in 84 Hosta cultivars was measuredby flow cytometry. As an internal standard we used the closely related Agave americana, the latter was calibrated against other reference species. Propidium iodide values were 20%higher than DAPI values, indicating a lower AT/GC ratio for Hosta. Ten new tetraploid plants were found in addition to the only tetraploid known so far; H. ventricosa.Small forms of the tetraploid H. ventricosa turned out tobe triploid hybrids. Three other triploids were discovered. Threesupposed tetraploids turned out to be diploid. In five plantsthe ploidy levels of the germ layers L1 and L3 appeared to be different. In these cases thick leaves coincides with a tetraploid DNA content. The DNA contents as measured in theseploidy chimeras coincide with differences in leaf colour, thusgiving an opportunity to analyze the build-up of Hosta plants.In general, analysis of the nuclear DNA content can be used as a simple means to increase the possibilities for creating new cultivars in this economically important perennial.     

Interspecific hybridization in the genus Arachis through embryo culture

Summary The embryos of a cultivated tetraploid peanut (Arachis hypogaea), a wild diploid species (A. villosa), and their hybrid embryos, which generally abort in nature, were cultured in vitro and the plants have been successfully transferred to the soil. The hybrids showed triploid chromosome number (3x=30). The significance of wide hybridization in peanut-improvement programs is discussed.     

Reproductive diversity and strategies for breeding in St. John's wort (Hypericum perforatum L.)

St. John's wort (Hypericum perforatumL.) is a medicinal crop plant that reproduces by apomixis. To study the reproductive diversity within the initial breeding material, 656 plants from 92 accessions of the species were analysed by flow cytometric seed screening (FCSS) and compared to 66 plants of cv. ‘Topaz’ as a control. Among plants from the 92 accessions, 16 were obligate sexuals, nine were obligate apomicts and all remaining ones were facultative apomicts. Among controls, there were six obligate and 60 facultative apomicts. Tests of reproductive pathways indicated that, in the investigated collection, all apomictic plants were tetraploid and all sexual plants were diploid. A strategy for a cross breeding program in St. John's wort is illustrated by crossing one diploid obligately sexual mother plant with six tetraploid facultative apomicts. All 91 F₁ progeny obtained were triploid with 58% being obligate sexuals and 42% facultative apomicts. To quantify the different routes of seed formation within facultatively apomictic individuals, the reproductive pathways were reconstructed from about 30 single seeds from each of five tetraploid and two triploid plants using FCSS. The frequency of seed formed by apomixis ranged from 54% to 67% among four tetraploid genotypes. One triploid and two tetraploids formed 23% to 73% of the so-called B_III hybrids (fertilization of unreduced eggs) and one triploid was obligately sexual. The results show that while the mode of reproduction in H. perforatum is highly variable, tetraploid obligate apomicts as well as diploid and triploid obligate sexuals can be selected as starting material for breeding purposes. This revised version was published online in July 2006 with corrections to the Cover Date.     

The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Summary To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments geve rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

Unreduced pollen frequencies versus hybrid production in diploid-tetraploid Vaccinium crosses

Summary Reciprocal crosses were made between highbush Vaccinium corymbosum L. cultivars (4X) and high, medium and low frequency diplandroid pollen producing diploid clones of V. elliotti (Chapm.) and diploid pubescent forms of V. corymbosum. Pollination of 3,342 flowers produced 15 hybrids-11 tetraploids and 4 triploids. All V. elliottii hybrids were tetraploid while each successful diploid-tetraploid V. corymbosum cross produced at least one triploid. The ratio of tetraploid hybrids to 100 pollinations (H: 100P) was considered to be the best criterion for assessing crossability. Vaccinium elliottii produced a total of 8 4X hybrids while 2X V. corymbosum only produced 3. Vaccinium elliottii was more successful as a female parent and diploid V. corymbosum only produced hybrids as male parents. High frequency diplandroid clones showed a lower mean crossability as pollen parents (H: 100P=0.2) than medium frequency diplandroid clones (H: 100P=0.8). As predicted, 0% diplandroid clones wer unsuccessful as pollen parents. Unreduced pollen production was essential for tetraploid hybrid production in 4X×2X crosses. There appeared to be no consistent, inherent pattern between 2n pollen and 2n egg production in a clone nor was a tetraploid genotype effect on crossability consistently observed.Paper No. 10781 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC., 27695-7601     

Production of 2n pollen of Asiatic hybrid lilies by nitrous oxide treatment

Tetraploid varieties of lilies have superior agronomic traits such as large flowers and resistance to physiological disorders. In the present study, we attempted to induce 2n pollen of Asiatic hybrid lilies by arresting the meiotic process with nitrous oxide (N₂O) gas. To determine which meiotic stage is optimal for induction of 2n pollen, plants with attached buds at different meiotic stages were treated with N₂O for 24 h in a pressure-tolerant cylinder. A few 2n pollen grains were induced using plants with anthers in prophase I, whereas mixed pollen grains of differing size were produced using plants undergoing meiotic metaphase predominantly in anthers. Although normal lily pollen grains are elliptical, nitrous oxide exposure induced giant pollen grains that appeared spherical. Flow cytometry analysis showed that the giant pollen grains were diploid. When mixed pollen that included normal and giant pollen was crossed to tetraploid cultivars, the resulting seedlings were tetraploid and aneuploid, indicating that the giant pollen grains were diploids that could generate tetraploid seedlings through fusion to diploid eggs supplied from a tetraploid female parent. Thus, treatment with N₂O is useful for the production of 2n lily pollen and may provide a new approach for tetraploid lily breeding.     

Interspecific hybrids of Cyclamen persicum Mill. and C. purpurascens Mill. produced by ovule culture

Summary Interspecific crosses were made to introduce the scent of flowers of C. purpurascens into C. persicum cultivars and ovule culture was used to rescue the abortive hybrid embryos. Cultivars of C. persicum diploid (CPD, 2n=2×=48) and C. persicum tetraploid (CPT, 2n=4×=96) were the pistillate parents and wild species of C. purpurascens (CP, 2n=34) were staminate parents. After pollination, crossed ovaries were collected periodically and examined using paraffin sections. Histological observations suggested that both hybrid ovules of CPD x CP and CPT x CP should be transferred to culture medium 35 days after pollination. Based up on this observation, crossed ovaries were collected 28 days after pollination and ovules with placenta were transferred to MS (1962) medium containing 3% sucrose. These ovules were cultured in the dark at 25° C. The hybrids (2n=41) derived from CPD x CP had the scent of C. purpurascens, whereas the hybrids (2n=65) derived from CPT x CP had the scent of C. persicum. Although both hybrids had complete genomes from the parents and produced a few viable pollen grains, they failed to yield viable seeds by self- and cross-pollination with fertile pollen grains of C. persicum cultivars.Abbreviations CPD C. persicum diploid - CPT C. persicum tetraploid - CP C. purpurascens     

Genome classification of banana cultivars from South India using IRAP markers

Summary The banana cultivars are originated from the intra- and inter-specific hybridization of two wild diploid species, Musa acuminata Colla and Musa balbisiana Colla, contributing the A and B genomes, respectively. They are classified into genomic groups by scoring morphological features. Molecular markers provide a quick and reliable system of genome characterization and manipulation in breeding lines. In the present study a PCR based molecular marker specific for B genomes is been reported. The IRAP primer, designed based on the LTR sequence of banana Ty3-gypsy-like retroelement (Musa acuminata Monkey retrotransposon, AF 143332), was used to identify the B genome in the banana cultivars. Further a primer pair designed from B specific bands of Musa balbisiana `Pisang Gala' was used to classify AAB and ABB cultivars in the collection. Among the 36 cultivars tested with this primer, the B specific band was absent in the AA and AAA cultivars (except in one AAA and AAB cultivar) but present in all other AB, AAB and ABB cultivars. Among the triploid AAB/ABB, the PCR products with B specific primers showed restriction pattern polymorphism with AluI. In ABB genomes the band intensity was high whereas low intensity band observed in AAB genomes. Four cultivars reported to have the ABB genome showed a pattern similar to AAB, and one cultivar reported to have AAA genome showed a pattern similar to ABB genome, suggesting missampling or misidentification. The primers used in this study are useful to identify the presence of B genome in banana cultivars, and band intensity may be a preliminary indicator of ploidy level of the B genome but needs further studies with competitive PCR for clarification. These authors contributed equally in this paper.     

Generation and characterisation of colchicine-induced autotetraploid Lavandula angustifolia

Lavandula angustifolia (lavender) is a small woody perennial grown for essential oil, which is steam distilled from flowers. To potentially improve size of flowers and oil yield we produced and characterised autotetraploid plants. L. angustifolia seed germinated in the presence of the mitotic spindle inhibitor colchicine at concentrations of 125 mg l⁻¹ or less resulted in plants carrying sports with larger flowers. Propagation of two sports gave rise to putative polyploid cultivars C3/2 and C6/24. Direct chromosome counts in root tip cells of seedlings from four common cultivars of L. angustifolia and the seed lot from which C3/2 and C6/24 were derived was 50 whereas C3/2 and C6/24 had greater than 90 chromosomes indicating they were autotetraploid. Ploidy level assessed by flow cytometry (FCM) of nuclei showed that 12 cultivars of L. angustifolia had similar nuclear DNA content whereas C3/2 and C6/24 had double the amount of DNA confirming autotetraploidy. The genome size (1C-value) of a diploid L. angustifolia cultivar was estimated by FCM to be 0.90 (±0.07) pg. Morphological characteristics were measured in autotetraploid and control plants. Autotetraploids had thicker peduncles, larger flowers and larger seeds than diploids. Scanning electron microscopy revealed peltate glandular trichomes were larger in the tetraploids relative to diploids. Both tetraploid and diploid cultivars had complex non-glandular trichomes on leaves and sepals and two different types of capitate glandular trichomes were identified on leaves. Autotetraploid lavenders represent useful germplasm both for commercial oil production and future breeding.     

Production and Cytogenetical Analysis of a Rye-Cytoplasmic Tetraploid Triticale

A rye-cytoplasmic tetraploid triticale was found in F_s progenies of crosses between tetraploid rye‘No 1323’and hexaploid triticale‘KT 77′. In the tetraploid triticale, two complete rye genomes and two mixed wheat genomes, consisting of the chromosomes 1A. 2A, 4A, 7A, 3B, 5B, and 6B are present. The rye cytoplasm did not affect stability of rye chromosome pairing during metaphase 1, since rye chromosomes participated in pairing irregularities just as did wheat chramosomes, even on a larger scale. The fertility of F₀, plants ranged from 0 to 75.6 %, always associated with high grain shrivelling. The analyzed pairing behaviour of induced triploid hybrids from crosses between the tetraploid triticale and diploid rye indicates the presence of at least one wheat-rye translocation in one of the investigated triploid plants.     

Crossability relationships of andean potato varieties of three ploidy levels

Summary The formation of triploids in 4x×2x crosses was studied in primitive potato varieties from the Andes of Peru and Bolivia. Triploid frequencies were as high as 80–100% in some crosses, although the actual number of seeds was low. The triploid block varied according to the tetraploid parent, and was never entirely absent. The likelihood of triploid formation was studied in 4x×2x crosses where pollen from both tetraploids and diploids of known genotype was used. Even though the diploids were chosen for their non-production of 2n gametes, as detected in pollen grain size, 66% of the hybrids were tetraploid. The amount and probability of gene-flow between the tetraploid and diploid gene-pools, either directly through 2n gametes or through triploid bridges was examined. Most of the 20 cultivars of S. x chaucha produced seed in crosses with tetraploids and diploids, although the fertility of the latter crosses was four times higher. The female fertility of some of the triploids thus allows them to exchange genes with the tetraploids and diploids, although gene-flow occurs more rapidly through direct transfer from diploids to tetraploids via 2n gametes.     

Seed formation in triploid loquat (Eriobotrya japonica) through cross-hybridization with pollen of diploid cultivars

As the fruits of loquat (Eriobotrya japonica, 2n = 2x = 34) carry large seeds, the breeding of seedless loquat has long been a goal. The recent creation of triploid cultivars (2n = 3x = 51) and the application of gibberellins allow commercial production of seedless loquat, but the possibility of seed formation in triploid loquats has not been carefully investigated. Through crossing experiments and cytological observations of meiosis and pollen tube growth, we found that the triploid line 3N-N28 was essentially self-sterile, but developed seeds on pollination with pollen from diploid cultivars at rates of up to 5.5%. Almost half of the seedlings survived to 5 months, and carried diploid (2n = 34), tetraploid (2n = 68), or aneuploid chromosome numbers. Our results suggest that triploid loquat cultivars might retain the risk of seed formation. Protection from pollination by diploid cultivars or the development of new triploid cultivars will be necessary to ensure the production of seedless loquat fruits.     

Crosses between Hordeum vulgare L. and H. bulbosum L. I. Production,morphology and meiosis of hybrids,haploids and dihaploids

Summary From crosses between diploid and autotetraploid cytotypes of Hordeum vulgare L. (cultivated barley) and H. bulbosum L. (bulbous barley grass) diploid, triploid and tetraploid interspecific hybrids were produced. Both directly and after vegetative segregation crosses in either direction also gave rise to haploids and dihaploids resembling H. vulgare. The use of embryo culture was necessary. Plant morphology of the hybrids was much like that of H. bulbosum, although the hybrid plants were less vigorous. Meiosis in the hybrids was more or less disturbed, and this seemed to be the main cause of the high level of sterility.