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.

Keywords: citrus vector; early flowering in juvenile citrus plant; speed up genetic studies.

Figure 1

Outline of the clbvlNpr‐AtFT or clbvlNpr‐CitFT vectors generated by cloning the FT gene from Arabidopsis. thaliana or Valencia late sweet orange (Citrus sinensis (L.) Osb.), respectively, in the ClbvINpr vector. Triangle represents the duplicated CP sgRNA promoter. White box represents the FT gene introduced.

Figure 2

Flowering of a juvenile C. excelsa (Wester) plant (a), and a triploid hybrid recovered from a hybridization between a tetraploid Fina clementine (C. clementina Hort. ex Tan.) and a diploid tangor hybrid (C. clementina × C. sinensis) (c) inoculated with the ClbvINpr‐AtFT viral vector, respect to the same genotype inoculated with the wild‐type CLBV virus (Figure 2b,d, respectively) propagated on Rough lemon (Citrus jambhiri Lush) rootstock.

Figure 3

Graft inoculation procedures used to infect citrus plants with the clbvINpr‐FT viral vector to induce early flowering: (a) graft inoculation of seedling plants of the candidate genotype with two infected bark patches and then decapitation of the plant leaving one bud above the inocula, (b) propagation of a scion bud of the candidate genotype onto a Rough lemon rootstock with simultaneous graft inoculation of the rootstock with two bark patches from a plant infected with the viral vector, and (c) elimination of the root of a small plantlet of the candidate genotype and top‐working the upper part on a Rough lemon rootstock with simultaneous graft inoculation of the viral vector as in (b).

Figure 4

Plant architecture of (a) a noninoculated adult C. excelsa plant and (b) a juvenile C. excelsa plant inoculated with the clbvINpr‐AtFT viral vector. Both plants were prepared by propagation of healthy C. excelsa buds onto Rough lemon rootstocks.

Figure 5

Germination of pollen from a juvenile C. excelsa plant inoculated with the clbvINpr‐AtFT viral vector.

Figure 6

Fruit development in a triploid hybrid obtained by hybridization between a tetraploid Fina clementine and a diploid tangor. A triploid bud was propagated onto a Rough lemon rootstock inoculated with the clbvINpr‐AtFT viral vector.

Figure 7

Electropherograms obtained using the MEST191 SSR locus for genetic analysis of a diploid Honey hybrid mandarin (Citrus nobilis × Citrus deliciosa), the P‐46 diploid tangor hybrid [(Citrus reticulata × Citrus sinensis) × Citrus sinensis)] and the triploid hybrid SA.14.187.1.6, displaying the alleles of both parents. The numbers indicate the size of alleles in nucleotides for each SSR locus.

Figure 8

Relative mRNA expression of the endogenous APETALA (AP1) and citrus FLOWERING LOCUS T (CiFT) genes in plants of C. excelsa propagated on Rough lemon rootstock and inoculated with the clbvINpr‐AtFT vector or with WT‐CLBV. The mRNA levels were estimated by RT quantitative real‐time PCR. The average expression in control plants infected with WT‐CLBV was considered 100, and the expression in the clbvINpr‐AtFT‐infected plants was relative to the average of the control plants. Bars represent standard deviation values.