PLANT-KBBE - Food & Feed: crop yiels and nutrition security in the context of clinate change

Yield enhancement in oilseed rape – SEEDS

SEEDS: Yield enhancement in Oilseed rape

Cytokinin has come into focus as a regulator of biomass formation and seed yield. Free University of Berlin works on CKX genes and their role in plant development and yield formation for several years already. They could show in Arabidopsis that simultaneous mutation of the CKX3 gene and at least one other CKX gene (either CKX2, 4, 5 or 6) causes enlargement of the inflorescence meristem which leads in turn to the formation of more and larger flowers giving rise to ca. 40% more siliques.

The goal is to verify whether the grain yield of oilseed rape can be increased by transferring the knowledge generated for Arabidopsis regarding the role of cytokinins on plant and grain yield.

The main objective to increase yield of OSR is addressed in 2 work packages:<br />WP1: Increase of seed yield in OSR by GM approach.<br />WP1 includes the analysis of transgenic lines by FUB and the cloning of new cassettes and subsequent creation/analysis of new lines with reduced expression of cytokinin-related genes. <br />(i) Expression of an effective antiCKX3/5 amiRNA will be mostly limited to the apical meristem by driving their expression by specific promoters. The goal is to restrict locally down-regulation of CKX genes and to explore the potential to avoid side effects due to systemic expression of the antiCKX amiRNAs. <br />(ii) Work in Arabidopsis has shown that also the combination of ckx2, 4 or 6 mutant alleles with a CKX3 knockout caused the formation of more siliques although to a lesser extent than in the ckx3, 5 mutant combination. In order to study this observation in a systematic fashion, different transgenic events addressing the downregulation of CKX3 in combination with CKX2, 4 or 6 in a constitutive way will be generated. <br />(iii) In order to assess the relevance of AHP6 to regulate yield in OSR a construct harboring an hairpin targeting AHP6 under control of the constitutive (will be used to transform wildtype OSR as well as TILLING mutants for ckx3, 5 and the combination of ckx3 and ckx5. <br />WP2 : Increase of seed yield in OSR by a non-transgenic approach. <br />In WP2 gene variants and their combination that mimic the transgene effect will be searched in mutated OSR populations. Single and combinations of ckx gene mutations will be tested systematically as well as their combination. Mutant identification from TILLING populations will be performed by BCS. FUB will contribute information about functionally essential amino acid positions in CKX genes as well as to the mutant phenotyping. As part of a BCS program, stop-codon inducing mutations are available for several members of the CKX gene families and stacking and phenotyping program has been started. <br />

All gene sequence needed for construction of plant transformation vector were identified and cloned. Subsequently plant transformation vector for the different work packages were constructed and subsequently transformed to Agrobacterium strains suitable for OSR transformation. First Agrobacterium strains were already used to create transgenic events, which were selected for the molecular profile, so that only events with a certain copy number were transferred to Free university of Berlin for further cultivation and seed production.

WP1: Increase of seed yield in OSR by GM approach.
1) Three hairpin for down-regulation of ckx3, 5 under control of the 35S, STM (SAM specific) and KLUH (SAM-specific) promoters.
Vector for 35S and KLUH promoters are ready, and event creation will be started in October 2015.

2) Three hairpin constructs for constitutive down-regulation of ckx3 in combination with ckx2, ckx4 and ckx6.
Events have been generated and ca. 20 single copy event for each vector were transferred to FUB for seed production.

Two hairpin constructs for down-regulation of AHP6 under control of 35S and STM promoters. These constructs will be transformed in OSR as well as in TILLING mutants for ckx3, 5 and the combination of ckx3/ckx5 to study the effect of combined down-regulation of AHP6.
Vector containing AHP6 hairpin under control of 35S-promoter is ready for transformation. Transformation in 4 different backgrounds (wt, ckx3, ckx5 and ckx3/5) will be started in October. Aim is to create at least 15 single copy events per vector.

WP2: Increase of seed yield in OSR by a non-transgenic approach.
Focus was on identification of mutants for CKX3 and CKX5. Genome analysis of Brassica shows, that there are 4 gene copies (A1, A2, C1, C2) for CKX3 and 2 gene copies (A1, C1) for CKX5 in the Brassica genome. This requires the identification of mutants in 6 different alleles and subsequent stacking of the different alleles.
Mutants (stop codons) could be identified in all targeted gene copies.
Mutants lines containing stop codons in all alleles of CKX3 as well as for CKX5 were created and mutations for both genes were combined to create a complete ckx3/ckx5 knock out line.
All lines and the corresponding wildtype segregants were planted to field trials in Belgium (3 locations, 4 replica/location) and Canada (4 locations, 3 replica/location).
Field trials in Belgium are harvested and data analysis is ongoing.
Field trials in Canada were harvested in September for further analysis.

Work package 1 (WP1): Increase of seed yield in oilseed rape by a transgenic approach.
1) Three hairpin constructs for down-regulation of ckx3/ckx5 under control of the 35S-promoters (constitutive), STM promoter (SAM specific) and KLUH-promoter (SAM-specific).
Next steps:
- complete cloning of STM-promoter vector (Q4 2015)
- Start transformation for 35S- and KLUH-promoter vectors (Q4 2015)
- Transfer transgenic events for all vectors to FUB (Q1-Q2 2016)

2) Three hairpin constructs for constitutive down-regulation of ckx3 in combination with ckx2, ckx4 and ckx6.
Next steps:
- perform backcrossing of transgenic events for 2nd and 3rd vector (Q4 2015)
- Grow T1 plants to produce T1S1 seeds for all transgenic events of the three vectors (Q2 2016)
- Grow T1S1 plants and select azygous and homozygous plants for first phenotyping and seed
production (Q4 2016)

Two hairpin constructs for down-regulation of AHP6 under control of 35S-promoter and optional STM-promoter. These constructs will be transformed in wildtype oilseed rape as well as in TILLING mutants for ckx3, ckx5 and the combination of ckx3/ckx5 to study the effect of combined down-regulation of AHP6.
Next steps:
- Creation of transgenic events for first vector (35S-promoter) in all 4 backgrounds (Q1/2 2016)
- Transfer of transgenic events for first vector in all 4 backgrounds to FUB. (Q2 2016)
- Cultivation of transgenic events in the greenhouse and performing of backcross (Q3 2016)

Work package 2 (WP2): Increase of seed yield in oilseed rape by a non-transgenic approach.
Next steps:
- Data analysis and interpretation from field trials (Q1 2016)
- Planning of Field trials for 2016 (Q1 2016)
- Seed production for Field trials 2016 (Q1 2016)
- Field trials 2016 in Belgium and Canada (Q4 2016)

Field trial data from 2015 trial for ckx3/ckx5 double mutant will be used to support invention of Thomas Schmülling as well as to protect the mutations identified in Brassica napus

The main goal of the SEEDS project is to transfer knowledge on seed yield-regulating genes obtained in
Arabidopsis thaliana to its close relative, the crop plant oilseed rape (Brassica napus), and use it for
practical breeding. Oilseed rape is one of the most recently domesticated major crop species, and due
to intensive breeding has become the most important oilseed crop in Europe. Germany and France are
the main producers and cover together more than 50% of the European production. Yield per hectare
varies in different countries, reflecting different input levels and production efficiency. Rapeseed is thus
likely to respond strongly to programs aimed at selectively enhancing genetic variation for key economic
input and output traits. Indeed, its future sustainable use requires a substantial increase in productivity.
The objective of this proposal is to increase productivity of commercial varieties of oilseed rape by
enhancing the capacity to form seeds which is currently limited by the activity, size and number of the
reproductive structures. Increased productivity will be achieved primarily by increasing the size of the
inflorescence and floral meristems, which determines pod number. The activity of the ovule-forming
placenta tissue will be enhanced to increase the seed number per pod. Finally, research will be
undertaken to increase the seed size which is expected to enhance the sink capacity.
Recent research has shown that the hormone cytokinin has a central role in regulating these traits
quantitatively. The genes involved have been identified and various tools to change the cytokinin status
of plants in a targeted fashion have been developed. Proof-of-concept has been obtained in model
species as well as in oilseed rape and further work to extend the knowledge on cytokinin-related genes
involved in regulating seed yield in oilseed rape is planned by transgenic and non-transgenic
approaches. The availability of the B. napus genome sequence and highly efficient TiLLING procedures
are prerequisites to implement these research results in practical plant breeding as a contribution to a
sustainable use of oilseed rape. The collaboration between a research laboratory that has discovered
the cytokinin yield genes in Arabidopsis and an international commercial oilseed rape breeding company
is ideal for the commercial exploitation of the results obtained by the SEEDS project. The consortium is
completed by one SME performing oilseed rape transformation and analysis.

Project coordination

Bernard Pelissier (Bayer SAS)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


Bayer SAS Bayer SAS

Help of the ANR 52,000 euros
Beginning and duration of the scientific project: March 2014 - 36 Months

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