**6. Early flowering by combination between** *FT* **expression and** *TFL1* **suppression in apple**

As already described, early flowering of horticultural plants can be achieved by expression of highly active *FT* genes such as *AtFT* with ALSV vectors (**Figures 4**–**6**). AtFT induces the early flowering of apple plants at a rate of 30% when expressed by the ALSV vector, but MdFT1 does not induce early flowering in any apple plant [10]. This result reconfirms that highly active FT homologs should be expressed by ALSV vectors to achieve early flowering, rather than endogenous but weakly active FT homologs. Consistent with this idea, other highly active FT homologs (AtTSF and GtFT1) also induced early flowering in apple plants [11]. Unlike herbaceous plants such as tobacco and soybean, the early-flowering rate of apple plants was as low as 30%, even with the most highly active *FT* genes. This difference between plant species may be related to their natural intervals from germination to flowering (months in herbs and years in trees).

flowering [37]. Thus, the balance between FT and TFL1 expressions have important roles in determining the time of flowering, and determining the parts of plant shoots where the flowers are generated. For example, the mutant of strawberry *FvKSN* gene and the mutant of rose *RoKSN* gene (the TFL1 family genes) can both flower in any growing season (continuous flowering), whereas ordinary strawberry and rose plants generate flowers only at one specific season of the year (seasonal flowering) [38]. The subgroup CEN is represented by the snapdragon *AmCEN* gene. This subgroup does not regulate flowering time, but does regulate the architecture of inflorescence (flower clusters at shoot apices). Snapdragon usually generates 'indeterminate' inflorescence, where many flowers are repeatedly generated on the side of the inflorescence, and the inflorescence continues to elongate without generating a flower at the very apex of the inflorescence. On the other hand, a snapdragon mutant in the *AmCEN* gene has 'determinate' inflorescence, where a limited number of flowers are generated on the side of the inflorescence, and the inflorescence stops elongating with a flower generated at the very apex of the inflorescence (called terminal flower). Thus, CEN negatively regulates flowering

at the apex of the inflorescence to generate indeterminate inflorescence [39].

**Figure 8.** Phylogenetic tree of FT/TFL1 family genes. Five subfamilies are indicated by sectors.

82 Plant Engineering

As apparent in the phylogenetic tree (**Figure 8**), apple has two copies of each five FT/TFL1 subgroup genes. The functions of these apple genes are not completely clear, but there are several reports on their activities and expression patterns. *MdFT1* and *MdFT2* are both expressed in Early flowering of plants may be also achieved through suppression of the *TFL1* gene, the negative regulator of flowering. *MdTFL1-1* was silenced in apple plants with ALSV vector [13]. Early flowering was observed, but in only 10% of the infected apple plants. To further improve the early-flowering rate of apple plants, simultaneous expression of *AtFT* and suppression of *MdTFL1* homologs were performed [11]. The early-flowering rate was not increased when *MdTFL1-2* was suppressed simultaneously with the expression of *AtFT*, but the earlyflowering rate increased to 90% when *MdTFL1-1* was suppressed simultaneously with the expression of *AtFT*. Thus, early flowering of apple plants was successful at high rates with the combination between *AtFT* expression and *MdTFL1-1* suppression. As well as early flowering at high rates, part of the early-flowering apple plants obtained by *AtFT* expression/*MdTFL1-1* suppression continuously generates flowers on branches, whereas the early-flowering apple plants obtained by only *AtFT* expression generate flowers only once. This difference in flowering traits may be also caused by negative regulation of flowering by *MdTFL1-1* in young apple plants. An example of early-flowering apple plants obtained by simultaneous *AtFT* expression and *MdTFL1-1* suppression is shown in **Figure 9**. Early-flowering apple plants set fruits and seeds after pollination with the pollen gathered from other compatible cultivars. Apple fruits typically mature at about 6 months after flowering. The next-generation seedlings germinated within 1 year, counting from the germination of the mother plant [11].

**Figure 9.** Early-flowering apple plant. (A) ALSV vector used in this analysis. 'MdTFL1-1-201' represents a 201-base fragment of the *MdTFL1-1* gene. (B) Early-flowering apple plant (seedling of Orin progeny: 'progeny' means 'nextgeneration'). Photograph was taken 67 days after inoculation of viral RNA to germinated seed.

#### **7. Conclusion**

ALSV will be among the most useful viral vectors for genetic engineering of horticultural plants, although ALSV is not presently applicable to cereals. Like other viral vectors, inserted sequences into the ALSV cloning sites are easily deleted upon infection to plants, depending on the sizes and sequences of the inserted sequences. What is more, ALSV vectors with insertions sometimes do not infect the plants. However, these problems can be managed by technical efforts and the selection of the insertion sequences. Nucleotide fragments larger than 1 kb can even be introduced into the XSB site of the ALSV vector and infected to plants. We hope that this chapter furthered general understanding of the structure and function of the ALSV vector, and promote its use in both basic and applied studies.

Early flowering through infection of ALSV vectors shortens the generation times of horticultural plants. This technique is expected to promote breeding of horticultural plants. It may not be a popular concept, but there seems to be strongly active *FT* genes (such as *AtFT*) and weakly active *FT* genes (such as *MdFT1*) according to our experiments. The analysis of chimeric *FT* genes in ALSV vector indicated that the difference in the activities of *AtFT* and *MdFT1* is determined by the C terminus, rather than the N terminus. Both types of *FT* genes will have ecological advantages in specific plants species, but highly active types of FTs are useful for early flowering of horticultural plants with ALSV vectors. In addition, simultaneous expression of *FT* and suppression of *TFL1* is beneficial for high rate of early flowering and continuous flowering. ALSV is not transferred to most of the next-generation plants, so the next-generation plants are neither transgenic nor infected by ALSV. Therefore, ALSV can be used as a new plant breeding technique (NPBT).
