Cytological analysis of hybrid embryos of intergeneric crosses between Salix viminalis and Populus species

Intergeneric hybridisation between Salix viminalis L. as the female and four Populus species (Populus trichocarpa, P. tremula, P. × canadensis and P. simonii) as male pollen donors was performed by in vitro stigma pollination. To overcome postzygotic barriers, transfer of hybrid embryos to new medium is necessary. We carried out detailed ultrastructural analyses to establish: (i) at which stage of embryo development the first signs of programmed cell death (PCD) could be detected; and (ii) at which stage the lack of serious or irreversible changes guaranteed that advanced development of hybrid plants could occur after embryo rescue. Transmission electron microscopy and confocal laser scanning microscopy revealed the presence of both developing and degenerating embryos. Developing globular, heart-shaped, and early cotyledonary embryos contained cells of correct ultrastructure. The only sign of intergeneric hybridisation was a delay in development for a few days, in comparison with control embryos. The earliest indicators of embryo degeneration were noted at 9 days after pollination (DAP). The most common indicators were excessive embryo vacuolisation, which was characterised by a large number of vesicles and formation of small vacuoles, as well as enlarged central vacuoles. Extended plastid thylakoids, folding of the cell wall, and autophagosomes were observed. Our detailed investigation of PCD in hybrid embryos enabled us to conclude that the embryo rescue technique was most effective in intergeneric willow × poplar crosses if applied between 9 and 16 DAP.

Potential for using Vasconcellea parviflora as a bridging species in intergeneric hybridisation between V. pubescens and Carica papaya

There have been numerous attempts to transfer Papaya ringspot virus type P (PRSV-P) resistance from wild Vasconcellea relatives to Carica papaya L. Success has been limited by the high degree of genetic divergence and thus incompatibility between Vasconcellea spp. and C. papaya. In the present work, intergeneric hybrid populations of C. papaya × V. pubescens Lenne et Koch, C. papaya × V. parviflora A. DC. and interspecific populations of V. pubescens × V. parviflora were produced and evaluated for morphological characteristics and PRSV-P resistance. F2 and F3 populations were produced from the V. pubescens × V. parviflora F1 and PRSV-P resistant individuals with homozygous genotypes (RR) were selected with the aid of a co-dominant CAPS marker and backcrossed to V. parviflora or outcrossed to C. papaya. Leaf vein number (seven) from V. pubescens, flower colour (pink) from V. parviflora and petiole colour (red–green) and plant size from C. papaya were transferred as dominant traits in interspecific and intergeneric crosses. Other traits, e.g. flower shape and fruit size, were intermediate in hybrids. When the wild species or their hybrids were outcrossed to C. papaya, inheritance patterns did not always follow Mendelian ratios, suggesting abnormal pairing of chromosomes or preferential elimination of the Vasconcellea genes. For example, in crosses between C. papaya × V. pubescens, all flowers were female and in crosses between C. papaya × V. parviflora, the cream flower colour of C. papaya was dominant over the pink colour of V. parviflora. However, the PRSV-P resistance gene from V. pubescens was successfully backcrossed into V. parviflora from F2 hybrids, and in the F1 hybrids between C. papaya and V. parviflora, 45% pollen fertility was obtained thus V. parviflora is proposed as a potential bridging species between C. papaya and V. pubescens.

Reproductive biology and intergeneric breeding compatibility of ornamental Portulaca and Calandrinia (Portulacaceae)

Portulaca grandiflora Hook and P. umbraticola Kunth (Portulacaceae) are popular garden annuals, and have been bred for improved ornamental value. However, limited research has been published on hybridisation of Portulaca, with no reports on intergeneric hybridisation. Calandrinia balonensis Lindley and Calandrinia sp. nov. (not yet fully classified) are floriferous Australian Portulacaceae species, with potential as novel flowering pot plants, and are potential candidates for breeding with ornamental Portulaca. We studied the reproductive biology of these four species and breeding compatibility for reciprocal crosses of P. grandiflora × C. balonensis (2n = 18) and P. umbraticola × C. sp. nov. (2n = 24). All four species produced seeds for intraspecific outcrosses. P. grandiflora and C. sp. nov. are partially self-compatible whereas P. umbraticola and C. balonensis are highly self-incompatible. Autogamy was detected only for P. grandiflora. Reciprocal crosses of P. grandiflora × C. balonensis and P. umbraticola × C. sp. nov. with similar chromosome numbers did not produce seeds, primarily because of pollen–pistil incompatibility that prevents pollen-tube growth within the stigmata. Methods to overcome hybridisation barriers of these species combinations need to be established to create novel products for ornamental horticulture.