Clonal reproduction of plants via seed production that doesn't require pollen, known as apomixis, has the potential to change plant breeding technology in a way that returns empowerment to farmers and breeders.
CAMBIA's interest in apomixis is that it would allow perpetuation of the high yield gains from particular hybrids. This could provide the farmer with greater autonomy and choice in planting future generations and enable capture of the advantages of superior cultivars adapted to local conditions. Additionally, stock production of potatoes, cassava and other important vegetatively-propagated crops through disease-free, true breeding seed could facilitate international trade of improved varieties, and finding new markets and new growing environments (Jefferson R A, 1994, Apomixis: A social revolution for agriculture?, in Biotechnology and Development Monitor 19:14-16; Jefferson R A, Bicknell R, 1996, The potential impacts of apomixis: A molecular genetics approach, in The Impact of Plant Molecular Genetics. B.W.S. Sobral, ed., pp. 87-101, Birkhäuser, Boston)
To achieve such a dramatic impact in a way that is both environmentally sound and socially equitable we need to ensure that apomixis technologies are readily accessible by all. To find out more about apomixis, its uses and social implications, read the Bellagio Agreement.
An analysis of Genetic Use Restriction Technologies was the first U.N.-sponsored intellectual property and technical analysis of "Terminator" technologies, which sterilize or reduce the agronomic value of second generation seed. The full paper and background information on the meeting where this paper was tabled is available on the UN Convention on Biological Diversity site.
Because plant sexual reproduction is a complex pathway where many redundancies would be expected, there are a variety of different approaches to apomixis. Check this page for new links to additional material, and please take part in our Forum if you know of research findings in this area that would be of interest to other users of the BioForge.
Papers disclosing an Egg Apparatus-Specific Enhancer (EASE) have been recently published: Yang W, Jefferson R A, Huttner E, Moore J M, Gagliano W B, Grossniklaus U (2005) An Egg Apparatus-Specific Enhancer of Arabidopsis, Identified by Enhancer Detection, Plant Physiology 139:1421-1432. Download this paper. See also Brand L, Hörler M, Nüesch E, Vassalli S, Barrell P, Yang W, Jefferson RA, Grossniklaus U, Curtis MD. (2006) A Versatile and Reliable Two-component System for Tissue-specific Gene Induction in Arabidopsis. Plant Physiology 141:1194-1204 (abstract on Medline). Vectors containing the EASE will soon be available from CAMBIA under a BiOS License. Read more about EASE.
Additional CAMBIA-based work on DNA sequences that could be useful in apomixis research has been documented in the Ph.D. thesis of Satya Nugroho.
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An ovule showing EGFP
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EASE stands for Egg Apparatus-Specific Enhancer and is a DNA sequence that can direct gene expression in the egg apparatus in Arabidopsis. The relevant research was published in the journal Plant Physiology in November 2005.
Despite a central role in plant reproduction, few gametophyte-specific genes and promoters have been isolated, particularly for the inaccessible female gametophyte (embryo sac). Using the Ds-based enhancer detector line ET253, we have cloned an egg apparatus-specific enhancer (EASE) from Arabidopsis thaliana.
The genomic region flanking the Ds insertion site was further analyzed by examining its capability to control gusA and GFP reporter gene expression in the embryo sac in a transgenic context. Through analysis of a 5' and 3' deletion series in transgenic Arabidopsis, the sequence responsible for egg apparatus-specific expression was delineated to 77 bp.
Our data showed that this enhancer is unique in the Arabidopsis genome, is conserved among different accessions, and shows an unusual pattern of sequence variation. This egg apparatus-specific enhancer works independently of position and orientation in Arabidopsis but is probably not associated with any nearby gene, suggesting either that it acts over a large distance or that a cryptic element was detected. Embryo-specific ablation in Arabidopsis was achieved by transactivation of a diphtheria toxin gene under the control of the EASE.
The nucleotide region that confers specificity of expression is described in WO 01/21785. This region, an enhancer, may be used to control expression of a toxin gene to generate female-sterile transgenic plants, or to promote expression of an embryogenesis gene to allow apomixis.
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An open silique |
View the 'shrink wrap' Bios License agreement for EASE vector pWY-O63.1
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GUS expression directed by EASE.
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As part of the efforts towards the identification of genes associated with apomixis, CAMBIA initiated a project to isolate regulatory elements and/or genes involved in the process of female gametophyte development by using particular enhancer detector lines with spatially restricted GUS expression patterns.
The egg apparatus-specific enhancer (EASE) was isolated and identified from an Arabidopsis enhancer detector line ET253, which shows specific GUS expression mainly in the egg apparatus and no GUS expression in anthers and any other tissues. There is only one Ds insertion in ET253 and its flanking sequences were analyzed by 5' and 3' deletion tests. A 77 bp sequence (ccacgatgca aatatatcga taacgttatt aaaaaaagta accgcatgat atattctctt tcgtatgata ttaaggc, GenBank accession no. AX100536) was identified as possessing the egg apparatus-specific enhancer activity in Arabidopsis and therefore named as EASE.
The sequence bearing the EASE is located on chromosome IV in Arabidopsis in an intergenic region and not associated with genes directly next to it. Without the use of enhancer detection, it might never have been uncovered. The EASE has the following features:
Although the endogenous function of the EASE in the Arabidopsis genome is still unknown, our experimental results show that its capability to control gene expression in the egg apparatus and early embryo is clear. Without the minimal promoter, the EASE itself is not functional. However, when it is fused to the CaMV 35S minimal promoter, the EASE can direct gusA or GFP gene expression in a very specific and highly efficient manner.
The EASE has also been tested in a genetic ablation system based on cell specific expression of the diphtheria toxin A-chain gene for embryo-specific ablation. Arabidopsis lines expressing the hybrid Gal4-VP16 transcription activator under the control of the EASE plus the minimal CaMV 35S promoter were generated. By crossing these lines with an Arabidopsis line harbouring the DTA gene controlled by an artificial promoter consisting of multiple copies of the upstream activating sequence (UAS) recognized by GAL4, linked to the minimal CaMV 35S promoter, embryo-specific ablation was achieved in the hybrid seeds. In these experiments, the silique development of the crossed flowers showed no difference from that of the selfed flowers. However, the ovules of the crossed flowers had the embryos arrest at early globular stage while the integument and endosperm development remained normal.
This demonstrated that the EASE could be used as a tool to study and manipulate gene expression in the female gametophyte and during early embryo development.
The information contained in this page was believed to be correct at the time it was collated. New patents and patent applications, altered status of patents, and case law may have resulted in changes in the landscape. CAMBIA makes no warranty that it is correct or up to date at this time and accepts no liability for any use that might be made of it. Corrections or updates to the information are welcome, please send an email to info@bios.net.
