Plant and Crop Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK.
Ann Bot. 2011 Oct;108(5):809-19. doi: 10.1093/aob/mcr197. Epub 2011 Aug 31.
The genus Nicotiana includes diploid and tetraploid species, with complementary ecological, agronomic and commercial characteristics. The species are of economic value for tobacco, as ornamentals, and for secondary plant-product biosynthesis. They show substantial differences in disease resistance because of their range of secondary products. In the last decade, sexual hybridization and transgenic technologies have tended to eclipse protoplast fusion for gene transfer. Somatic hybridization was exploited in the present investigation to generate a new hybrid combination involving two sexually incompatible tetraploid species. The somatic hybrid plants were characterized using molecular, molecular cytogenetic and phenotypic approaches.
Mesophyll protoplasts of the wild fungus-resistant species N. debneyi (2n = 4x = 48) were electrofused with those of the ornamental interspecific sexual hybrid N. × sanderae (2n = 2x = 18). From 1570 protoplast-derived cell colonies selected manually in five experiments, 580 tissues were sub-cultured to shoot regeneration medium. Regenerated plants were transferred to the glasshouse and screened for their morphology, chromosomal composition and disease resistance.
Eighty-nine regenerated plants flowered; five were confirmed as somatic hybrids by their intermediate morphology compared with parental plants, cytological constitution and DNA-marker analysis. Somatic hybrid plants had chromosome complements of 60 or 62. Chromosomes were identified to parental genomes by genomic in situ hybridization and included all 18 chromosomes from N. × sanderae, and 42 or 44 chromosomes from N. debneyi. Four or six chromosomes of one ancestral genome of N. debneyi were eliminated during culture of electrofusion-treated protoplasts and plant regeneration. Both chloroplasts and mitochondria of the somatic hybrid plants were probably derived from N. debneyi. All somatic hybrid plants were fertile. In contrast to parental plants of N. × sanderae, the seed progeny of somatic hybrid plants were resistant to infection by Peronospora tabacina, a trait introgressed from the wild parent, N. debneyi.
Sexual incompatibility between N. × sanderae and N. debneyi was circumvented by somatic hybridization involving protoplast fusion. Asymmetrical nuclear hybridity was seen in the hybrids with loss of chromosomes, although importantly, somatic hybrids were fertile and stable. Expression of fungal resistance makes these somatic hybrids extremely valuable germplasm in future breeding programmes in ornamental tobacco.
烟草属包括二倍体和四倍体物种，具有互补的生态、农艺和商业特性。这些物种因其次生代谢产物的范围而具有不同的经济价值，可用于烟草、观赏植物和次生植物产物的生物合成。由于其次生产物的差异，它们在抗病性方面表现出显著的差异。在过去的十年中，有性杂交和转基因技术倾向于使原生质体融合在基因转移中黯然失色。体细胞杂交被用于本研究中，以产生涉及两个有性不亲和的四倍体物种的新杂种组合。通过分子、分子细胞遗传学和表型方法对体细胞杂种植物进行了表征。
野生抗真菌种 N. debneyi（2n = 4x = 48）的叶肉原生质体与种间有性杂交种 N. × sanderae（2n = 2x = 18）的原生质体进行电融合。在五个实验中手动选择了 1570 个原生质体衍生细胞集落，从中选择了 580 个组织进行芽再生培养基的培养。再生植物被转移到温室中，并根据其形态、染色体组成和抗病性进行筛选。
89 株再生植物开花；与亲本植物相比，其中 5 株由于中间形态、细胞学组成和 DNA 标记分析被确认为体细胞杂种。体细胞杂种植物的染色体组成为 60 或 62 条。通过基因组原位杂交鉴定染色体来源于亲本基因组，包括 N. × sanderae 的全部 18 条染色体，以及 N. debneyi 的 42 或 44 条染色体。在电融合处理的原生质体培养和植物再生过程中，一个亲本 N. debneyi 的四条或六条染色体被消除。体细胞杂种植物的叶绿体和线粒体可能都来自 N. debneyi。所有体细胞杂种植物都是可育的。与 N. × sanderae 的亲本植物不同，体细胞杂种植物的种子后代对烟草霜霉病菌的感染具有抗性，这一特性是从野生亲本 N. debneyi 中导入的。
通过涉及原生质体融合的体细胞杂交，克服了 N. × sanderae 和 N. debneyi 之间的有性不亲和性。尽管重要的是，杂种植物具有不对称的核杂种性和染色体缺失，但仍具有可育性和稳定性。真菌抗性的表达使这些体细胞杂种成为未来观赏烟草育种计划中极具价值的种质资源。
