Journal of Taiwan Agricultural Research

Author:Chi-Ni Hsia*, Uei-Chern Chen, Chin-Yi Tsao, and Yi-Mei Chou

Abstract:

    Brassica oleracea var. italica is an important vegetable in the world with rich nutrition for human health. The objective of this study was to establish a microspore culture system using elite broccoli F₁ hybrids from Taiwan to produce doubled-haploid (DH) lines for breeding purpose. Microspores taken from 5 commercial F₁ hybrids were surveyed for embryogenesis. Microspores were inoculated with 4 × 10⁴ microspores/mL into NLN-13 medium in combination with a heat shock treatment at 32℃ for 1 d before moving to 25℃ for culturing. Embryo induction was found derived from 3 out of 5 cultivars, among them ‘Chinglong B-45’ had the highest embryogenesis rate. However, two cultivars were found without any embryogenesis. Microspores of ‘Known-you Tender green’ were taken from two different bud lengths for embryogenesis. A higher embryo induction rate (17.1 embryos/petri dish) was obtained from 4.0–5.0 mm length of bud than that of from 3.0–3.9 mm length of bud (7.0 embryos/petri dish). An experiment using two culture media in combination with three durations at 32℃ was conducted using ‘Chinglong B-45’. Among six treatments, NLN medium with 1-d culturing at 32℃ had the highest embryogenesis with 6.9 embryos/petri dish. Various stress treatments were conducted using cotyledonary embryos derived from ‘Chinglong B-75’ to improve germination. Treated embryos were precultured on the hormone containing medium for 1-week followed by 4 wk culturing on a hormone-free medium for shoot germination. A low shoot germination rate of 1.7% was obtained from the control which was without any stress treatment. In contrast, shoot germination rate of 46.7% and 48.3% were obtained from filter paper dry treatments at either 25℃ for 1 d or at 4℃ for 5 d, respectively. Ploidy levels were analyzed using flow cytometry for a total of 23 microspores derived plants of ‘Chinglong B-75’. Rates of 34.8% and 60.9% were obtained for haploid and diploid, respectively. A high DH production rate of 60% derived from the microspore culture would facilitate breeding program in practice.

Key words:Broccoli, Embryogenesis, Doubled haploid

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