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AOBPreview published online on April 18, 2006
Annals of Botany, doi:10.1093/aob/mcl072
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© The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received November 4, 2005
Revised December 8, 2005
Accepted February 11, 2006

Technical Article

Isolation of Individual Egg Cells and Zygotes in Alstroemeria Followed by Manual Selection with a Microcapillary-connected Micropump

YOICHIRO HOSHINO 1 *, NAHO MURATA 2, and KOICHI SHINODA 2

1 Field Science Center for Northern Biosphere, Hokkaido University, Kita 11, Nishi 10, Kita-Ku, Sapporo 060-0811, Japan; Division of Innovative Research, Creative Research Initiative ‘Sousei’ (CRIS), Hokkaido University, Kita 21, Nishi 10, Kita-Ku, Sapporo 001-0021, Japan
2 National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-Ku, Sapporo 062-8555, Japan

* To whom correspondence should be addressed.
YOICHIRO HOSHINO, E-mail: hoshino{at}exfarm.agr.hokudai.ac.jp


  Abstract

• Aims To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules.

• Scope An attempt was made to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these ovule sections were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillaries connected to a micropump. For zygote isolation, ovules were excised from ovaries 24 h after self-pollination. By treating excised ovules with an enzyme solution and subsequently dissecting them using a glass needle, zygotes were successfully isolated from the ovules and collected with a microcapillary. The isolated zygotes were associated with pollen tubes and one of the synergids. Egg cells and zygotes were viable for up to 2 h following isolation, as determined by fluorescein diacetate staining.

• Conclusions The procedures for isolating egg cells and zygotes in Alstroemeria were established, and each egg cell and zygote was captured with a microcapillary.

Keywords: Alstroemeria aurea, egg cell, enzyme treatment, microdissection, zygote.
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