Stem cuttings and micropropagation protocols for Bitters rootstock large scale production

In the last years citriculture needed to face several biotic and abiotic constraints. In particular, the spread of citrus tristeza virus (CTV) and more recently the outbreak of the Huanglongbing makes breeding and selection of new rootstocks of outmost importance. Among the CTV-resistant rootstocks, Bitters (C22), a hybrid of Citrus sunki and Poncirus trifoliata orange, has shown promising performance in terms of yield efficiency, productivity and tolerance to the soil and climate conditions typical of southern Italy. Most of the commercially used citrus rootstocks are propagated through nucellar seed; however, this is a time consuming process and the polyembryonic trait, that is not present in all genotypes, made the development of alternative propagation methods such as stem cuttings and tissue culture, both guaranteeing large-scale propagation in shorter times, a necessity. In this study we describe two alternative propagation protocols developed, in the frame of a research program financed by the PSR Sicilia, mis. 16.1, project VIVAICITRUS, for this genotype in comparison with the traditional seed propagation method. Our optimized in vitro micropropagation procedure, starting from 140 internodes of 1 cm in length obtained from the sowing of 20 seeds of Bitters, allows the production of more than 300 shoots in one month allowing the set-up of further multiplication for large scale production; the entire micropropagation process (germination, shoot multiplication, elongation, rooting and acclimatization) can be performed within 5-6 months plus the time needed for plant elongation in nursery (15 months) compared to 2 months of traditional seed germination (plus nursery plant elongation). In case of low seed availability, the use of cuttings allows to obtain a high number of plants propagated from limited source material. The method required a simple mist bench and jiffy pods with standard potting mix, as the one used for citrus seedlings. Shoot and root growth rapidly occurred at 4 weeks after cutting plantation. More than 70% of Bitters cuttings rooted and after 6 months from the starting of the protocol, plants were transferred in plastic pots achieving an adequate size for grafting. On the other side, starting from 20 seeds, nucellar propagation allowed the obtainment of 25 plants that reached the size for grafting after 18 months from sowing. On the whole, the use of micropropagation and microcutting protocols represents a valuable alternative to sowing in order to propagate new citrus rootstocks for which seeds availability is a limiting factor.