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Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding.

ABSTRACT: The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus-based vector (clbvINpr-AtFT and clbvINpr-CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4-6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr-AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.

SUBMITTER: Velazquez K

PROVIDER: S-EPMC5043495 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding.

Velázquez Karelia K Agüero Jesús J Vives María C MC Aleza Pablo P Pina José A JA Moreno Pedro P Navarro Luis L Guerri José J

Plant biotechnology journal 20160325 10

The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus-based vector (clbvINpr-AtFT and clbvINpr-CiFT, respectively). Citrus plants of differe ...[more]

PMID: 26920394

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Project description:Background and aimsIn woody species, the juvenile period maintains the axillary meristems in a vegetative stage, unable to flower, for several years. However, in adult trees, some 1-year-old meristems flower whereas others remain vegetative to ensure a polycarpic growth habit. Both types of trees, therefore, have non-flowering meristems, and we hypothesize that the molecular mechanism regulating flower inhibition in juvenile trees is different from that in adult trees.MethodsIn adult Citrus trees, the main endogenous factor inhibiting flower induction is the growing fruit. Thus, we studied the expression of the main flowering time, identity and patterning genes of trees with heavy fruit load (not-flowering adult trees) compared to that of 6-month-old trees (not-flowering juvenile trees). Adult trees without fruits (flowering trees) were used as a control. Second, we studied the expression of the same genes in the meristems of 6-month, and 1-, 3-, 5- and 7-year-old juvenile trees compared to 10-year-old flowering trees.Key resultsThe axillary meristems of juvenile trees are unable to transcribe flowering time and patterning genes during the period of induction, although they are able to transcribe the FLOWERING LOCUS T citrus orthologue (CiFT2) in leaves. By contrast, meristems of not-flowering adult trees are able to transcribe the flowering network genes but fail to achieve the transcription threshold required to flower, due to CiFT2 repression by the fruit. Juvenile meristems progressively achieve gene expression, with age-dependent differences from 6 months to 7 years, FD-like and CsLFY being the last genes to be expressed.ConclusionsDuring the juvenile period the mechanism inhibiting flowering is determined in the immature bud, so that it progressively acquires flowering ability at the gene expression level of the flowering time programme, whereas in the adult tree it is determined in the leaf, where repression of CiFT2 gene expression occurs.

Project description:BACKGROUND:Global warming will expand the range of new and invasive pathogens in orchards, and subsequently increase the risk of disease epidemics and economic losses. The development of new resistant plant varieties can help to reduce the impact of pathogens, however, the breeding speed can be extremely slow, due to the growth rates of the plants, and the availability of resistance genes. Citrus trees are suffering immense damage from serious diseases such as citrus canker (XCC), huanglongbing (HLB), and citrus tristeza virus (CTV). A fast-track breeding system, that aimed at shortening the duration for disease resistance breeding by incorporating the resistance genes from related species to commercial varieties, has been developed using the integration of precocious transgenic trifoliate orange with the overexpression of CiFT and MAS. It was applied here to incorporate CTV resistance of trifoliate orange into citrus germplasm. RESULTS:One generation of backcrossed breeding, that would normally take at least 5?years, was achieved in a single year by fast-track breeding system. Linkage analysis using the corresponding DNA markers revealed that CTV resistance and T-DNA integrated regions were found in different linkage groups, and they were independently segregated in the BC progenies. The CTV resistant null segregants, in which the T-DNA integrated region was removed from their genome, were feasibly obtained by MAS in each generation of the BC progenies, and their CTV resistance was confirmed by immunological analysis. Several BC3 null segregants, whose genetic backgrounds had been substituted into citrus germplasm, except for the haplotype block of CTV resistance, were successfully obtained. CGH and NGS analyses revealed that the T-DNA integrated region was safely segregated out in null segregants. CONCLUSION:Fast-track breeding systems are expected to shorten the required breeding time by more than one-fifth in comparison with conventional cross breeding techniques. Using this system, we obtained BC3-8, whose genetic background was successfully substituted except for the CTV resistance locus, and could be a novel mandarin breeding material. The fast-track breeding system will be useful to introduce important traits from related species to citrus germplasm while also drastically reducing the time required for breeding.

Project description:Plant viral vectors are superior tools for genetic manipulation, allowing rapid induction or suppression of expression of a target gene in plants. This is a particularly effective technology for use in breeding fruit trees, which are difficult to manipulate using recombinant DNA technologies. We reported previously that if apple seed embryos (cotyledons) are infected with an Apple latent spherical virus (ALSV) vector (ALSV-AtFT/MdTFL1) concurrently expressing the Arabidopsis thaliana florigen (AtFT) gene and suppressing the expression of the apple MdTFL1-1 gene, the period prior to initial flowering (generally lasts 5-12 years) will be reduced to about 2 months. In this study, we examined whether or not ALSV vector technology can be used to promote flowering in pear, which undergoes a very long juvenile period (germination to flowering) similar to that of apple. The MdTFL1 sequence in ALSV-AtFT/MdTFL1 was replaced with a portion of the pear PcTFL1-1 gene. The resulting virus (ALSV-AtFT/PcTFL1) and ALSV-AtFT/MdTFL1 were used individually for inoculation to pear cotyledons immediately after germination in two inoculation groups. Those inoculated with ALSV-AtFT/MdTFL1 and ALSV-AtFT/PcTFL1 then initiated flower bud formation starting one to 3 months after inoculation, and subsequently exhibited continuous flowering and fruition by pollination. Conversely, Japanese pear exhibited extremely low systemic infection rates when inoculated with ALSV-AtFT/MdTFL1, and failed to exhibit any induction of flowering. We also developed a simple method for eliminating ALSV vectors from infected plants. An evaluation of the method for eliminating the ALSV vectors from infected apple and pear seedlings revealed that a 4-week high-temperature (37°C) incubation of ALSV-infected apples and pears disabled the movement of ALSV to new growing tissues. This demonstrates that only high-temperature treatment can easily eliminate ALSV from infected fruit trees. A method combining the promotion of flowering in apple and pear by ALSV vector with an ALSV elimination technique is expected to see future application as a new plant breeding technique that can significantly shorten the breeding periods of apple and pear.

Dataset Information

Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding.

Publications

Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding.

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