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<p>Embryogenic cultures were initiated from haploid and diploid seedlings of maize inbred LH244 and monitored for ploidy stability by flow cytometry over the course of six mo. Both culture types were transformed with three different constructs using <i>Agrobacterium tumefaciens</i> to compare transformation and gene editing frequencies. Paromomycin resistant cell lines were split prior to plant regeneration, and half of the tissue was treated with colchicine to induce chromosome doubling (a “mock” treatment without colchicine was used for the diploid target tissues). Ploidy measurements were taken on transgenic cell lines immediately prior to the doubling treatment and again approximately six wk later prior to plant regeneration. T0 plants were sampled and assessed for ploidy, transgene copy number, and Cas12a (LbCpf1) mediated gene editing. Chromosome stability of haploid cultures was excellent within three mo of initiation, and cultures remained predominantly haploid even after six mo in culture. Transformation frequencies of haploid and diploid cultures were comparable. A simple colchicine treatment of haploid callus was reasonably effective for doubling chromosome number to produce dihaploid plants. Chlorophyll deficient T0 plants were observed only from the haploid cultures, an indication of the immediate phenotypic display of recessive mutations. Editing frequency at the <i>ch3_reg1_1.1</i> gRNA target site was very high for both haploid and diploid cultures. As expected, diploid plants derived from the haploid cultures were primarily homoallelic, and those from diploid cultures primarily biallelic. The results provided a framework for future editing work or mutational studies in maize using haploid embryogenic callus as the starting material.</p>

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Haploid embryogenic cultures of maize (Zea mays L.): stability, doubling efficiency, and use in gene editing

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月12日

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  • Charles Armstrong,
  • Andrei Kouranov,
  • Michelle Gasper,
  • Sam Yang,
  • Daniel Karcher,
  • Huai Wang,
  • Nicholas Homa,
  • Steven Beach,
  • Yuanji Zhang,
  • Larry Gilbertson

摘要

Embryogenic cultures were initiated from haploid and diploid seedlings of maize inbred LH244 and monitored for ploidy stability by flow cytometry over the course of six mo. Both culture types were transformed with three different constructs using Agrobacterium tumefaciens to compare transformation and gene editing frequencies. Paromomycin resistant cell lines were split prior to plant regeneration, and half of the tissue was treated with colchicine to induce chromosome doubling (a “mock” treatment without colchicine was used for the diploid target tissues). Ploidy measurements were taken on transgenic cell lines immediately prior to the doubling treatment and again approximately six wk later prior to plant regeneration. T0 plants were sampled and assessed for ploidy, transgene copy number, and Cas12a (LbCpf1) mediated gene editing. Chromosome stability of haploid cultures was excellent within three mo of initiation, and cultures remained predominantly haploid even after six mo in culture. Transformation frequencies of haploid and diploid cultures were comparable. A simple colchicine treatment of haploid callus was reasonably effective for doubling chromosome number to produce dihaploid plants. Chlorophyll deficient T0 plants were observed only from the haploid cultures, an indication of the immediate phenotypic display of recessive mutations. Editing frequency at the ch3_reg1_1.1 gRNA target site was very high for both haploid and diploid cultures. As expected, diploid plants derived from the haploid cultures were primarily homoallelic, and those from diploid cultures primarily biallelic. The results provided a framework for future editing work or mutational studies in maize using haploid embryogenic callus as the starting material.