Potential and Prospects of MMCT in Citrus

MMCT in citrus is still in its developmental stages; however, it is possible to envisage its potential for citrus breeding. The trans- fer of a limited number of chromosomes from one citrus species or relative to another citrus could greatly impact the development of new cultivars. For sweet

orange, grapefruit, lemon and limes, which are highly polyembryonic, this would be especially important, since so far natural or induced mutation has been the primary method for cultivar development for these groups. In mammalian cells, as previously discussed, microcell-mediated chromo- some transfer is still one of the most power- ful tools for gene mapping, analysis of gene function and the molecular cloning of defi ned chromosome regions (Jakobs et al., 1999). Most of the 22 human chromosomes are currently available in monochromoso- mal cell hybrids, produced using microcell- mediated chromosome transfer (Cuthbert et al., 1955; Murakami et al., 2000; Inoue et al., 2001). For citrus, MMCT could also become an important tool for cultivar improvement and for others genetic and molecular analysis. Theoretically, several different individuals can be produced in a single microprotoplast fusion experiment. Considering that the haploid chromosome number of citrus is nine and that a single chromosome is transferred from a donor to a recipient species, at least nine different hybrids may be produced. Since the micro- protoplast suspension contains a mixture of donor chromosomes, the fusion with the recipient genome will occur in a com- pletely random way. Therefore, if two chro- mosomes are transferred, the number of different hybrids would theoretically be the combination of nine chromosomes two by two. More than two chromo- somes can be transferred, which further increases the probability of different indi- viduals per fusion. We have been regenerat- ing plants with 2 n + 1 to 2 n + 6 chromosomes so far.

Additionally, more than one donor can be used per single recipient species. Recently we fused microprotoplasts from grapefruit plus mandarin as donor, with sweet orange as recipient. We have so far obtained four plantlets from this combina- tion, which have been micro-grafted into sour orange and will later be analysed cyto- logically and molecularly. Additionally, if delay of the DNA replication of the donor chromosomes in the immediate cell cycle

after fusion occurs, the progression of the recipient genome to metaphase may induce pre-chromosome condensation (PCC) of the S-phase donor chromosome, leading to donor DNA or chromosome breakage. The

released DNA may be integrated into the recipient genome through the transforma- tion and repair process (Ramulu et al. 1955), further increasing the possibilities for new hybrid genotypes.