Origin of Multicellular Pollen and Pollen Embryos in Cultured Anthers of Pepper (Capsicum Annuum)

Anthers of Capsicum annuum L. were cultured on Murashige and Skoog (MS) medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ kinetin. Inoculated anthers were subjected to 31 °C and development of microspores in anthers of varying stages was observed cytologically using 4′-6-diamidino-2-phenylindol-2HCl (DAPI). Pepper was characterized by a strong asynchrony of pollen development within a single anther. Percentage of pollen at different stages changed with the culture period, and the proportion of dead pollen increased drastically from day 2 after culture. Microspores that were cultured at the late-uninucleate stage followed one of two developmental pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be a typical bicellular pollen. Embryogenic pollen was formed by repeated divisions of the vegetative nucleus. In the second pathway, which occurred in fewer microspores, the first division was symmetric and both nuclei divided repeatedly to form embryogenic pollen. In early-bicellular pollen, sporophytic pollen was produced through division of the vegetative nucleus. In mid-bicellular pollen, the generative nucleus may undergo division to produce two or more sperm-like nuclei. However, division of the generative nucleus alone to form the embryo was never observed. The anther stage optimal for embryo production contained a large proportion (>75%) of early-binucleate pollen. Associations were found among the percentage of early-binucleate pollen, the frequency of embryogenic multinucleate pollen, and the yield of pollen embryos.     

High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.)

We developed an efficient culture system for producing cotyledonary embryos from isolated microspores of hot pepper (Capsicum annuum L.) and analyzed the ploidy levels of regenerated plants. Three culture protocols were studied: liquid, double-layer, and two-step culture. In the double-layer culture, cotyledonary embryos were produced more efficiently when the same medium composition was used for the liquid upper-layer and the solid under-layer. The two-step culture system, in which microspores were first incubated on liquid medium and then subcultured on double-layer medium, was most effective for producing cotyledonary embryos. Cotyledonary embryos were produced more efficiently when the isolated microspores were cultured in liquid medium for 1 week in 60 × 15-mm plates at a density of 8–10 × 10⁴/mL and microspore suspensions from two liquid culture plates were combined into a single 100 × 20-mm plate containing solid medium, and the culture was continued for an additional 3 weeks. When cotyledonary embryos obtained from this two-step culture were transplanted into regeneration medium, more than 95 % developed into plants. Only 31 of the 190 analyzed plants (16.3 %) generated by this method were spontaneous doubled haploids. This two-step culture system outperforms all previously reported culture protocols for isolated microspores of hot pepper, and appears to be a promising tool for the production of haploid plants for hot pepper breeding.     

Embryogenesis and plant regeneration of hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) through isolated microspore culture

We report high frequencies of embryo production and plant regeneration through isolated microspore culture of hot pepper (Capsicum annuum L.). Microspores cultured in modified NLN medium (NLNS) divided and developed to embryos. Globular and heart-shaped embryos were observed from 3 weeks after the beginning of culture, and many embryos reached the cotyledonary stage after 4 weeks of culture. These cotyledonary embryos developed to plantlets after transfer to solid B5 basal medium. We also optimized conditions for embryo production by varying the pretreatment media, the carbon sources, and culture densities. Heat shock treatment in sucrose-starvation medium was more effective than in B5 medium. Direct comparisons of sucrose and maltose as carbon sources clearly demonstrated the superiority of sucrose compared to maltose, with the highest frequency of embryo production being obtained in 9% (w/v) sucrose. Microspore plating density was critical for efficient embryonic induction and development, with an optimal plating density of 8 × 10⁴–10 × 10⁴/ml. Under our optimized culture conditions, we obtained over 54 embryos, and an average of 5.5 cotyledonary embryos when 10 × 10⁴ microspores were grown on an individual plate.