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- Author or Editor: Haiyan Li x
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An improved three-stage protocol for plant regeneration via somatic embryogenesis of the horticulturally important plant Iris germanica L. was developed using shoot apex segments as explants. At the first stage of the experiment, 60% of callus was obtained from shoot apex segments of I. germanica on Murashige and Skoog’s (MS) medium supplemented with 4.52 μm 2,4-dichloropheoxyacetic acid (2,4-D) and 0.44 μm 6-benzyladenine (6-BA). When nonembryogenic calli were subcultured on MS medium with 11.31 μm 2,4-D and 0.44 μm 6-BA, maximum frequency of embryogenic callus (66.0%) was obtained. At the second stage, the treatment of 9% (w/v) sucrose resulted in the optimum somatic embryo (SE) formation (70.0%). More than 90.0% of SEs germinated with bipolar structure and regenerated into plantlets on plant growth regulator-(PGR)free MS medium during the third stage. Regenerated plantlets were successfully acclimatized in greenhouse environment with little somaclonal variation. Histological study showed that somatic embryogenesis stages were asynchronous and SEs developed from the surface and inner tissue of embryogenic calli.
The investigation of hybridization processes and embryogenesis of heterozygote is an effective approach for early hybrids’ identification, which could provide reliable information for successful crossbreeding. In this study, we reported the whole hybridization processes of the direct cross and reciprocal cross between Michelia yunnanensis Franch. ex Finet et Gagnep. and Michelia crassipes Law using fluorescence microscopy after aniline blue staining, with the pollen germination on stigmas, pollen tube growth in styles, and subsequent extension into the embryo sac as well as the double fertilization processes are documented in detail. The M. yunnanensis × M. crassipes combination displayed considerable cross-compatibility, and the heterozygote embryogenesis was further observed with an approach of modified cryosectioning technique. Besides, the whole formation processes of hybrid seeds from artificial pollination to maturation were successfully observed. However, in the reciprocal cross, we found incompatibility between pollen grains of M. yunnanensis and stigmas of M. crassipes for the reason of hysteretic identification, as well as the abnormal callose deposition which belongs to the prefertilization barriers. This is the first study in which the complete and clear hybridization processes in Michelia were reported. We inferred that unilateral incompatibility of M. crassipes detected in this study may also exist in some other Michelia species. In artificial hybridization practices, we suggest some special treatments for overcoming prefertilization barrier should be taken when treating M. crassipes as the maternal parent.
Bougainvillea Comm. ex Juss. (Nyctaginaceae; Bougainvillea) is a popular ornamental plant with vigorous growth, luxuriant blooming, colorful bracts, and a high tolerance to the stresses of temperature, drought, and soil pollution, and thus is widely cultivated in tropical and subtropical regions. However, the paucity of information on ploidy and the genomic constitution is a significant challenge to genome research and cultivar improvement. We present a flow cytometry method for ploidy detection in bougainvillea based on evaluating different lysates and tissues, identify the ploidy level of a batch of bougainvillea accessions, and infer the genome size of horticultural species Bougainvillea glabra, Bougainvillea spectobilis, and Bougainvillea peruviana. The results show that tender leaves and woody plant buffer (WPB) were optimal for flow cytometry analysis. The 2C nuclear DNA amounts in 176 bougainvillea accessions ranged from 4.66 ± 0.04 to 12.26 ± 0.1 pg, which represents 161 diploids, 13 triploids, 1 tetraploid, and 1 di-tetraploid mixoploid. For B. glabra, B. spectobilis, and B. peruviana, the mean 1C values were 3.201, 3.066, and 2.915 pg, respectively. The genome size of B. glabra was significantly larger than that of B. peruviana (P = 0.0004), but had no significant variation with that of B. spectobilis (P = 0.1061). These results reveal fundamental cytogenetic information for bougainvillea that are beneficial to whole-genome sequencing and hybrid breeding programs.