Artificial Chromosomes in Rice (Oryza sativa)

Chunhui Xu1, Weichang Yu2

1 School of Life Sciences, Shandong University, Jinan, Shandong, 2 Shenzhen Research Institute of The Chinese University of Hong Kong, Shenzhen
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20008
Online Posting Date:  May, 2016
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Chromosomes are the carriers of genetic material in biological organisms. Each chromosome has three essential components: a centromere, telomeres, and origins of replication. The understanding of the essential structural and functional organization of chromosomes has made it possible to produce artificial chromosomes (ACs), which are human‐engineered minichromosomes. There are two approaches to make ACs: de novo assembly (bottom‐up) and truncation of existing chromosomes (top‐down). Rice (Oryza sativa) ACs are produced by telomere‐mediated chromosome truncation, and may have many applications, such as genetic engineering to stack and express multiple genes in rice to combat diseases caused by bacteria, fungi, and viruses, to enhance tolerance of rice to environmental stresses such as drought, heat, and salinity, and to improve yield and quality. © 2016 by John Wiley & Sons, Inc.

Keywords: artificial chromosome; genetic engineering; minichromosome; telomere truncation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Telomere‐Mediated Chromosome Truncation of Rice
  • Support Protocol 1: Preparation of Gold Particles for Rice Transformation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Telomere‐Mediated Chromosome Truncation of Rice

  • Rice seeds
  • 70% (v/v) ethanol
  • 50% (v/v) bleach, diluted from household bleach
  • Sterile water
  • Plates containing rice callus induction medium (see recipe)
  • Osmotic treatment plate (see recipe)
  • 0.6‐μm gold particles (Bio‐Rad)
  • Plasmid DNAs
  • 70% (v/v) isopropanol
  • Helium tank
  • Selection medium (see recipe)
  • 50 mg/ml hygromycin
  • Regeneration medium (see recipe)
  • Rooting medium (see recipe)
  • Yoshida medium (see recipe)
  • Soil
  • 100‐ml conical flasks
  • Rotary shaker
  • Sterile blotting papers
  • 28°C incubator
  • Gene gun (Bio‐Rad)
    • Macrocarriers
    • Rupture disks (650 psi)
    • Stopping screen (sterilized by autoclaving)
  • Clean hood
  • Sterile filter paper
  • Vortex
  • Vacuum pump
  • Growth chamber

Support Protocol 1: Preparation of Gold Particles for Rice Transformation

  • 0.6‐μm gold particles
  • 100% ice‐cold ethanol, −20°C
  • ddH 2O, autoclaved, incubate on ice before use
  • 2.5 M CaCl 2, filter‐sterilized, 50‐μl volumes, and stored at −20°C
  • 0.1 M spermidine (Sigma), filter‐sterilized, 20‐μl volumes, and stored at −20°C
  • Plasmid DNAs (see protocol 1Basic Protocol)
  • 1.5‐ml sterile microcentrifuge tubes (siliconized to reduce adhesion of gold particles to walls)
  • Ultrasonic cleaner
  • Bench‐top microcentrifuge
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Literature Cited

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