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Incorporation of Genes of Potential Interest






The availability of genetic transformation systems for many different citrus genotypes is allowing us to incorporate transgenes of potential agricultural interest into citrus rootstocks and varieties.

Soil salinity signifi cantly limits citrus production in many areas worldwide. Carrizo citrange, considered an excellent citrus rootstock, is very sensitive to salt stress, which restricts its use in salty soils. We have successfully transformed plants of Carrizo citrange with the halotolerance gene HAL2 (Cervera et al., 2000b), origi- nally isolated from yeast and implicated in salt tolerance mechanisms (Murguí a et al., 1995). Plants showing higher transcription levels in northern analyses were chosen to undergo in vivo salt stress tolerance assays, by using transgenic plants as rootstocks for a sensitive citrus variety. These tests are currently being performed at our Institute.

Phytophthora citrophthora is the most widespread fungus over all the citrus-grow- ing areas and represents one of the major causes of crop losses. Constitutive overex- pression of proteins involved in the plant defence mechanism against disease is one of the strategies proposed to increase plant tol- erance to fungal pathogens. p23 is a 23 kDa


pathogenesis-related protein induced in tomato (Lycopersicon esculetum Mill. cv. Rutgers) plants when these are infected with citrus exocortis viroid, and its antifungal activity has been shown in in vitro assays (Rodrigo et al., 1993). We have successfully produced transgenic Pineapple sweet orange plants with a chimeric gene construct com- prising the coding region of the p23 gene. Transgenic lines constitutively expressing the PR protein were challenged with P. cit- rophthora and one of them achieved plant survival rates higher than the control when whole transgenic plants were inoculated with fungal cultures. These results provide evidence for the antifungal activity in vivo of the p23 pathogenesis-related protein against

P. citrophthora and suggest that this may be employed as a strategy aimed at the engi- neering of Phytophthora disease resistance in citrus (Fagoaga et al., 2001).

CTV is the causal agent of the most important virus disease of citrus in the world. Several strategies have been used to engineer plant resistance to viral pathogens. Most are based on the concept of pathogen-derived resistance (Sanford and Johnston, 1985), which proposes that the introduction and expression in plants of viral sequences could interfere with the life cycle of the same or a closely related chal- lenging virus, thus providing resistance to infection. This approach has proved to be applicable to a range of more than 40 virus species in different transgenic hosts. We are introducing coding and non-coding ver- sions of the major coat protein gene of the virus in lime, which is very susceptible to CTV, to evaluate pathogen-derived resist- ance. More than 40 transgenic lines expressing the coat protein gene of the virus have already been produced (Domí nguez et al., 2000). When plants propagated from each transgenic line were graft or aphid inoculated with CTV, two types of response to viral challenge were observed: some lines developed CTV symptoms similar to those of non-transgenic controls, whereas others exhibited protection against the virus (Fig. 15.3A). This protection con- sisted of a proportion of plants, ranging


 

 

 

Fig. 15.3. Incorporation of genes of potential interest. (A and B) Engineering citrus resistance to CTV. (A) Non-transgenic control (left) and CP (p25) transgenic (right) lime plants infected with CTV. The transgenic CP plant showed resistance to the virus. (B) Leaves from a non-transgenic control lime plant inoculated with CTV (top) and from a p23-transgenic lime plant (bottom). Expression of p23 in citrus plants causes the appearance of CTV-like symptoms. (C and D) Overexpression of APETALA1 and LEAFY genes from Arabidopsis in Carrizo citrange transgenic plants. (C) Transgenic AP1 plants fl owering six months after sowing (four plants on the right) compared with a control non-transformed plant (plant on the left). (D) Transgenic LFY plant that fl owered after being grafted on a vigorous rootstock in the greenhouse.

Transgenic LFY plants generally showed developmental aberrations.

 


 

from 10 to 33%, that were resistant to CTV, and the remainder showed a signifi cant delay in virus accumulation and symptom onset (Domí nguez et al., 2002). This is the first demonstration of pathogen-derived resistance in transgenic plants against a Closterovirus member in its natural host. The major coat protein gene of CTV has been also inserted into sour orange, and transgenic plants either expressing or not expressing the transgene have been gener- ated (Ghorbel et al., 2000).

The 3’-terminal gene of CTV codes for a

23 kDa protein (p23), which is an RNA- binding protein that contains a motif rich in cysteine and histidine residues. For this reason, a regulatory role in CTV replication or gene expression has been suggested for p23. To explore whether overexpression in transgenic plants of this protein could affect the normal CTV infectious process, we have produced transgenic lime plants carrying the p23 transgene, or a truncated version thereof. Constitutive expression of p23


 

induced phenotypic aberrations resembling symptoms caused by CTV in non-transgenic lime plants (Fig. 15.3B), whereas transgenic plants expressing the truncated version of p23 were normal. The onset of CTV-like symptoms in p23-transgenic plants was associated with the expression of p23, and its accumulation level paralleled symptom intensity (Ghorbel et al., 2001b). This demonstrates that p23 is involved in symp- tom development and that it probably plays a key role in CTV pathogenesis. This fi nding also delimits a small region of the large CTV genome for future mapping of specifi c path- ogenic determinants.

Results of genetic improvement pro- grammes for citrus are almost non-existent, due, among other reasons, to the extremely long juvenile phases of the trees which pro- long the time required to analyse late traits such as fruit features. With the aim of accel- erating their fl owering time, we have trans- formed juvenile Carrizo citrange seedlings to constitutively express the Arabidopsis



LEAFY or APETALA1 genes, that are suffi - cient to promote flower initiation in Arabidopsis (Mandel and Yanofsky, 1995; Weigel and Nilsson, 1995). Both types of transgenic citrus plants produced fertile fl owers and fruit as early as the fi rst year, notably through a mechanism involving a dramatic shortening of their juvenile phase (Fig. 15.3C and D). This phenomenon had not been observed in Arabidopsis. Furthermore, expression of APETALA1, being as effi cient as LEAFY in the initiation of fl owers, did not produce any severe devel- opmental abnormality. Both types of trans- genic trees fl owered again in consecutive years and their fl owering response was under environmental control. In addition, sexual and nucellar derived transgenic seedlings had a very short juvenile phase and fl owered in their fi rst spring, demon- strating the stability and inheritance of this trait (Peñ a et al., 2001). This opens up the possibility of using independent APETALA1 transgenic plants as parents in crosses with non-transformed genotypes that would yield 50% of the progeny fl owering and setting fruit in 1–2 years, thus providing the oppor- tunity to evaluate fruit features very early and to advance generations rapidly. On the other hand, by retransformation of APETALA1 transgenic citrus plants, it could be possible to test the effect of the expression of certain transgenes rapidly under fl ower organ- or fruit-specifi c promoters as a system to look for biotechnological strategies to develop seedless varieties, modify fruit colour or favour easy-peeling characteristics.

 

 


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