AtWus overexpression results in abnormal development of somatic embryos.

<p><b>A:</b> Many abnormal somatic embryos were produced in 35S:WUS lines, and the somatic embryos were inflated and lacked cotyledons. <b>B:</b> Formation of normal somatic embryos in CK lines at different stages. Scanning electron microscopy: Holistic perspective of somatic embryos in 35S:WUS lines (<b>C</b>) and CK lines (<b>D</b>). <b>E</b>–<b>J:</b> Normal somatic embryos at different stages. <b>E</b>, <b>F:</b> globular embryo. <b>G:</b> heart-shape embryo. <b>H</b>–<b>J:</b> cotyledonary embryo. <b>K</b>–<b>P:</b> Abnormal somatic embryos having various appearance. <b>O:</b> leaf-like embryo. <b>P:</b> multiple-cotyledon embryo. Bar in <b>A</b> or <b>B,</b> 1 cm.</p

<i>AtWuschel</i> Promotes Formation of the Embryogenic Callus in <i>Gossypium hirsutum</i>

<div><p>Upland cotton (<i>Gossypium hirsutum</i>) is one of the most recalcitrant species for <i>in vitro</i> plant regeneration through somatic embryogenesis. Callus from only a few cultivars can produce embryogenic callus (EC), but the mechanism is not well elucidated. Here we screened a cultivar, CRI24, with high efficiency of EC produce. The expression of genes relevant to EC production was analyzed between the materials easy to or difficult to produce EC. Quantitative PCR showed that CRI24, which had a 100% EC differentiation rate, had the highest expression of the genes <i>GhLEC1</i>, <i>GhLEC2</i>, and <i>GhFUS3</i>. Three other cultivars, CRI12, CRI41, and Lu28 that formed few ECs expressed these genes only at low levels. Each of the genes involved in auxin transport (<i>GhPIN7</i>) and signaling (<i>GhSHY2</i>) was most highly expressed in CRI24, with low levels in the other three cultivars. WUSCHEL (WUS) is a homeodomain transcription factor that promotes the vegetative-to-embryogenic transition. We thus obtained the calli that ectopically expressed <i>Arabidopsis thaliana Wus</i> (<i>AtWus</i>) in <i>G. hirsutum</i> cultivar CRI12, with a consequent increase of 47.75% in EC differentiation rate compared with 0.61% for the control. Ectopic expression of <i>AtWus</i> in CRI12 resulted in upregulation of <i>GhPIN7</i>, <i>GhSHY2</i>, <i>GhLEC1</i>, <i>GhLEC2</i>, and <i>GhFUS3</i>. <i>AtWus</i> may therefore increase the differentiation potential of cotton callus by triggering the auxin transport and signaling pathways.</p></div

SAM structure of somatic embryos in transgenic lines observed with scanning electron microscopy.

<p><b>A:</b> Abnormal somatic embryo of 35S:WUS lines. <b>B:</b> Normal somatic embryo of CK lines. <b>C:</b> Enlarged image of SAM in <b>A</b>. <b>D:</b> Enlarged image of SAM in <b>B</b>.</p

Somatic embryo weight in CRI12.

*<p>p<0.05.</p

Cotton cultivars have different differentiation rates in EC.

<p>After 90 days of culture in NEIM, all calli of CRI24 produced EC, whereas calli of CRI12, CRI41, and Lu28 were dark green and tight and unable to differentiate into EC. Bar, 1</p

<i>AtWus</i> expression in calli of CRI12.

<p><i>AtWus</i> expression in calli of CRI12.</p

Analysis of gene expression in the transgenic calli carrying <i>AtWus</i>.

<p>Analysis of gene expression in the transgenic calli carrying <i>AtWus</i>.</p