Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.     

Production of interspecific hybrids between Alstroemeria pelegrina L. var. rosea and A. magenta Bayer by ovule culture

Interspecific hybrids were efficiently produced in the cross-incompatible combination between Alstroemeria pelegrina L. var. rosea and A. magenta Bayer by culturing immature ovules with placenta 7–14 days after pollination on 2 g/l Gelrite-solidified MS medium containing 3% (w/v)sucrose. The plants showed intermediate characteristics between the parents and their hybridity was confirmed by karyotype and DNA analyses. The mean number of chromosome association per PMC at metaphase I was 2.60I+6.70II, pollen stainability was20.8%, and they produced viable seeds after self-pollination. Furthermore, mature plants were obtained when the hybrids were backcrossed as male parents with both the parents. The backcross-progeny from A. pelegrina var. rosea × hybrids exhibited 3.8 to 79.7% pollen stainability and that from A. magenta × hybrids 78.8 to 98.3%. Almost all of these plants produced viable seeds after self-pollination, which implies that they can beutilized for breeding of novel cultivars of Alstroemeria. This revised version was published online in July 2006 with corrections to the Cover Date.     

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     

Interspecific hybridization of Brassica napus and B. juncea through ovary,ovule and embryo culture

Summary Employing in vitro culture of ovaries, ovules and embryos, interspecific hybrids have been obtained amongst two important oilseed crops, Brassica napus x B. juncea and their reciprocal. The test-tube hybrid plants have been transferred to the field, and reared to maturity. The F₁ seeds obtained from the hybrid ovaries showed normal germination, and the hybrid plants exhibited a range of variation of characters.     

Interspecific hybridisation of soybeans and perennial Glycine species indigenous to Australia via embryo culture

Summary Five sterile hybrids (2n=59) between a synthetic amphiploid of Glycine tomentella (2n=38) and G. caneseens (2n=40) as female and soybean cultivars Lincoln and Hark as males have been produced by embryo or ovule culture using transplanted endosperm. The hybrid plants are twining perennials like the female parent but possess a number of morphological characters which reflect the presence of the soybean genome. Indophenol oxidase isozymes from leaf extracts also provide good evidence of the hybrid nature of the cultured plants. These hybrids open the way for the exploitation of the diverse germplasm resources of the perennial Glycine species in soybean breeding.     

Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.     

Ovule and embryo culture to obtain hybrids from interspecific incompatible pollinations in chickpea

Many of the wild species of chickpea were not accessible to the improvement of chickpea due to cross incompatibility. In these interspecific incompatible crosses, fertilization takes place but the embryo aborts a few days later. The only way to obtain hybrid plants is by the application of growth regulators to pollinated pistils to prevent initial pod abscission and to save the aborting hybrid embryos by embryo rescue techniques. Although there are a few papers on regeneration from different explants of chickpea, information on embryo rescue techniques is not available. The paper summarises the embryo rescue techniques developed for chickpea, by the use of which hybrid plants between C. arietinum and C. pinnatifidum were produced. The paper also emphasises the effect of genotype to successfully obtain hybrids. The morphology of the hybrid plants resembled the male parent in leaf structure and growth habit. The colour of the flowers produced on the hybrid plant was pale violet, resembling the male parent whose flowers were violet in colour. The flower colour of the female parent was white. This revised version was published online in August 2006 with corrections to the Cover Date.     

Unique meiotic behaviourin F1 plants from a cross between a non-tuberous and a tuberous Solanum species in section Petota

Uniformly abnormal meiotic behaviour was observed in 12 F₁-plants from a cross between Solanum etuberosum (non-tuberous) and S. pinnatisectum (tuberous). Per pollen mother cell at MI an average was found of 3.64 bivalents (all rodshaped, 1–2 per cell, heteromorphic), 16.64 univalents (scattered haphazardly on a continuous bipolar spindle) and 0.03 trivalents (all Y-shaped). Lagging chromosomes and precocious division of univalents very frequently occurs, leading to unequal distribution of chromosomes, aneuploid gametes and male sterility. Heteromorphic bivalents at MI, loops in bivalents at pachytene and non-disjunction in one hybrid plant, point to a highly abnormal meiotic behaviour. The occurrence of few trivalents is discussed.Considering that according to the literature nearly normal pairing was observed in the intergeneric F₁-hybrids Lycopersicon esculentum × Solanum lycopersicoides and L. esculentum x S. pennellii, the lack of chromosome pairing in an interspecific F₁-hybrid, of which both Solanum parents belong to the same section, is paradoxical to a plant breeder and might even be conspicuous to a taxonomist.     

Inheritance pattern for pod lenghth and ovule number in wide crosses among Cajanus and Atylosia species

Summary Six interspecific/intergeneric F₂ populations involving two cultivars of C. cajan and five Atylosia species were scored for segregation patterns for pod length and ovule number. It was observed that for both characters the F₂ means and the ranges were correspondingly lower than the midparent values and the spread of the two parents. However, the intrageneric cross of A. lineata and A. scarabaeoides showed transgressive segregation for pod length and the F₂ mean and range were correspondingly larger than the midparent value and spread of the two parents, suggesting the usefulness of combining ability test for choice of suitable parents.     

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Summary Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F₁ hybrids. The F₁ hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F₂, F₃ and F₄ progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F₀ parental cross.As a route of introgression, diploid F₁ hybrids were backcrossed with cultivated pigeonpea and BC₁ progeny obtained by in vitro culture of aborting embryos. BC₂ plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F₁ and BC₁ plants, it was possible to produce BC₂ and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.Special project assistant till December, 2003.     

Cryopreservation of Delphinium pollen at –30 °C

Pollen of garden varieties and species of Delphinium that was cryopreserved at –30 ^°C after 3 hours of air drying and storage on silica gel, had high germination and pollen tube growth in vitroeven after 180-day storage. On the other hand, pollen stored at 25 ^°C showed a marked decrease in the germination rate within 10 days. The best in vitro germination of Delphinium pollen was on a 1% water agar containing 15% sucrose and 0.005 to 0.01% boric acid and at 15 to 20 ^°C. Field pollination with the cryopreserved pollen showed higher fruit and seed set than pollination with pollen stored at 25 ^°C, and was not significantly different from fresh pollen. This revised version was published online in August 2006 with corrections to the Cover Date.     

Relationships between African species of the genus Cucumis L. Estimated by the production,vigour and fertility of F1 hybrids

Summary The production, vigour, and fertility of F₁ hybrids between nine African species of the genus Cucumis L. were studied as a measure of the relationships between the species. Hybrid plants were obtained from 29 out of the 72 possible cross combinations. Two F₁ hybrids died as seedlings, and 27 hybrids were raised to maturity. Pollen production and stainability varied greatly amongst these hybrids, as did fruit and seed set following self pollination and backcrossing with either parental species. The fruit shape of the hybrids was always intermediate between that of both parental species.Two species appeared to be closely related: C. prophetarum L. and C. anguria var. longipes A. Meeuse. Most other species produced highly to moderately fertile F₁ hybrids with at least one other species. C. metuliferus Naud. produced only sterile hybrids with C. zeyheri Sond. 2x. The results sustain the recent taxonomic classification of the genus (Jeffrey, 1980).     

Interspecific hybrids between three eggplant (Solanum melongena L.) cultivars and two wild species (Solanum torvum Sw. and Solanum sisymbriifolium Lam.)

Three Greek eggplant cultivars, ‘Langada’, ‘Tsakoniki’ and ‘Emi’ (2n= 24), were crossed with two wild species (Solanum torvum Sw., 2n= 24 and Solanum sisymbriifolium Lam., 2n= 24). Ovules isolated 15-27 days after pollination were cultured in a modified MS medium at 24°C and a 16h photoperiod. Fifty days later, the ovules were dissected and the interspecific embryos were cultured in the same medium. Interspecific hybrids were achieved only from crosses between the eggplant cultivars and S. torvum. The hybridity of the putative interspecific F¹ hybrid (Solanum melongena×S. torvum) was confirmed by using morphological and biochemical (isozyme isocitrate dehydrogenase A, phosphoglucomutase A, phosphoglucose isomerase B, 6-phosphogluconate dehydrogenase A, 6-phosphogluconate dehydrogenase B) markers. The F¹ plants (‘Langada’×S. torvum) were selfpollinated and backcrossed to both parents. Fruits, however, were produced only when the F¹ hybrid was backcrossed as female with the eggplant cultivar ‘Langada’.     

Embryo-rescue in the genus Tulipa L.; successful direct transfer of T. kaufmanniana Regel germplasm into T. gesneriana L.

Summary Embryo-rescue was studied as a means to overcome post-fertilization barriers in interspecific crosses in the genus Tulipa. With compatible T. gesneriana L. cultivar crosses, ovule culture was found to be superior to isolated embryo culture. Complete plantlet formation was possible from an embryo size of about 0.5 mm onwards.In the interspecific cross T. gesneriana x T. kaufmanniana, which is hampered by embryo breakdown, successful rescue of abortive embryos was demonstrated. Optimal embryo-rescue was achieved in cultures started seven to nine weeks after pollination. With cultures initiated at a later time, the rate of success decreased. A low number of germinative seeds were obtained after normal ripening of the seed pods, but by using ovule culture the efficiency of seedling formation could be increased dramatically.The ovule culture procedure will allow novel crosses and will offer new possibilities for the introduction of desirable genes into tulip cultivars.     

Production of intergeneric hybrids between a C3-C4 intermediate species Moricandia arvensis and a C3 species Brassica oleracea through ovary culture

Summary Intergeneric hybrids between Moricandia arvensis (a C₃-C₄ intermediate species) and Brassica oleracea (a C₃ species) were obtained through ovary culture. Many hybrid embryos (2.71 per pollination) were produced in the M. arvensis × B. oleracea cross, but none were produced from the reciprocal cross. Though most embryos failed to develop into plantlets directly, plants were obtained by inducing shoots from hypocotyl explants. The hybrid plants were morphologically intermediate between the parents except for the petal color. Cytogenetic observations indicated that partial homology existed between the two genomes. Ovary culture is an efficient technique for gene transfer from M. arvensis to B. oleracea.     

High-performance liquid chromatographic analysis of anthocyanins in Japanese garden iris and its wild forms

Summary Following bud pollination of tartary buckwheat (Fagopyrum tataricum) with pollens from common buckwheat (F. esculentum), cv. Mancan, 31% of the ovaries began to grow but all turned brown and withered after 6 to 14 days. Fluorescence microscopy of the growing ovaries showed the pollen-tube entering the embryo-sac. The ovules from the growing ovaries failed to produce any embryo in the culture medium in which the immature embryos from the self-pollinated (compatible) tartary flowers were able to mature and germinate. No embryo development was observed after cross-pollination. The interspecific incompatibility is attributed to the failure of gametes to fuse.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid     

Interspecific Hybridization in the Genus Lens Applying in Vitro Embryo Rescue

Summary Four different hybridization experiments were carried out to obtain interspecific hybrids with Spanish cultivars of lentil (Lens culinaris M.). In hybridization experiments I and II, undertaken with only pollination and pollination with the addition of gibberellic acid after fertilization, respectively, no lentil hybrids were recovered. A single interspecific hybrid with L. odemensis was obtained in experiment III using the embryo rescue protocol of Cohen et al. (1984), in this case, a crossing efficiency of 0.11% and a rescue efficiency of 2.5% were obtained. Hybridization experiment IV used a specific embryo rescue protocol developed in this study. In this experiment, ovule-embryos of 18 DAP were cultured on MS salts with 1% sucrose and 1 μ M IAA + 0.8 μ M KN; after two weeks, embryos were released from the ovular integuments and cultured on the same medium for another two weeks in upright position. Afterwards, the embryos were transferred to test tubes containing the same medium and one month later plantlets were obtained. Using the above protocol, out of a total of 1707 pollinations, 6 interspecific hybrids with L. odemensis, 2 with L. nigricans and one with L. ervoides were recovered, yielding on average a crossing efficiency of 0.53% and an average rescue efficiency of 8.26%. Taking into consideration only the interspecific crossing blocks in which hybrids were recovered, the crossing efficiency with L. odemensis was 9%, while with both L. nigircans and L. ervoides the crossing efficiencies were 3%. Rescue efficiencies based on hybrids recovered per number of ovules cultured ranged between 50–100%.     

Overcoming the barrier to interspecific hybridization of Fagopyrum esculentum with Fagopyrum tataricum

Summary Tartary buckwheat (Fagopyrum tataricum) was successfully hybridized with common buckwheat (F. esculentum), both diploid (2n=16), using the latter as male parent during bud pollination. The barrier normally encountered in such hybridization was overcome by enhancing the cross-compatibility of the two species, which was accomplished by synthesizing a unique genotype of common buckwheat. This novel plant was produced by selecting common buckwheat plants that exhibited, at isozyme loci PGM, SKDH and ADH, alleles with similar mobility to those found in tartary and then transferring these alleles to a single plant through six generations of breeding. Ovule culture was used to rescue the 7–10 day old embryos. On the rescue-culture medium 41% of the hybrid embryos formed calli larger than 200 m in diameter. Most ceased to grow before reaching 1500 m, but four out of 263 cultured ovules continued to grow as callus. One of these differentiated and formed callus with buds and shoots from which cloned plants were produced. The remaining embryos either did not grow at all or formed very small calli. When tartary was crossed with the original genotype of common buckwheat, only 22% of the hybrid embryos formed small calli and none differentiated. Hybridity of the calli and the plantlets was determined by RAPD and isozyme analysis, respectively. Flowers produced by the hybrid plants were of the same type (homomorphic) and size as those of tartary, but with white sepals like common buckwheat.Abbreviations CTAB cetyltrimethylammonium bromide - GA₃ gibberellic acid - IAA indoleacetic acid - IBA indolebutyric acid - PVP polyvinylpyrrolidone     

Interspecific hybridization in the genus Gossypium through embryo culture

Summary With embryo rescue and culture techniques interspecific hybrids have been obtained amongst diploid cultivated (Gossypium arboreum, G. herbaceum) and wild species (G. stocksii, G. anomalum) of cotton. The early abscission of the young bolls was prevented by repeated application of growth regulators followed by the culture of immature hybrid embryos (15 days after pollination). The best growth and development were obtained on modified Murashige and Skoog's medium with supplements. The plants were stouter when the cultures were first incubated in the dark for 15–25 days, followed by exposure to light. The hybrid plants transferred to soil developed further and matured, and were more or less intermediate between their respective parents.     

Breaking the crossability barriers between disomic tetraploid Solanum acaule and tetrasomic tetraploid S. tuberosum

Summary A combination of compatible second pollinations and embryo rescue was applied for systematic production of true tetraploid hybrids from crosses between disomic tetraploid Solanum acaule and tetrasomic tetraploid potato, S. tuberosum. Several genotypes of tetraploid potatoes were pollinated with S. acaule, and the compatible second pollinations were made on the following day, with a genotype of S. phureja, IvP 35 to promote fruit development. Embryo rescue was carried out in 21 families, 14 to 27 days after the first pollination. A total of eight plants were obtained from the embryo rescue and their chromosome numbers were counted in the root tips. Three of the eight plants were identified as tetraploid, and five others as diploid. Morphology, isozyme banding patterns, and pollen stainability, as well as potato spindle tuber viroid (PSTVd) resistance, indicated the hybrid nature of the three plants. This is the first report of successful tetraploid hybrid production between disomic tetraploid S. acaule (4x) and tetrasomic tetraploid potatoes. Seed set from the crosses between one of hybrids and diploid potatoes indicated workable levels of both male and female fertility for introgression of valuable genes from S. acaule into the cultivated potato gene pool. The methodology used may be applied to other disomic tetraploid tuber-bearing Solanum species and with some modifications also to distantly related solanaceous species and genera.