Highly Efficient CRISPR/Cas9 Mediated Gene Editing in Ocimum basilicum 'FT Italiko' to Induce Resistance to Peronospora belbahrii.

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of "Genovese-type" basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum 'Italiko', the élite cultivar used to produce "Pesto Genovese D.O.P", was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of 'FT Italiko'. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

Genetic Improvement of Arundo donax L.: Opportunities and Challenges.

Arundo donax L., the giant reed-being a long-duration, low-cost, non-food energy crop able to grow in marginal lands-has emerged as a potential alternative to produce biomass for both energy production, with low carbon emissions, and industrial bioproducts. In recent years, pioneering efforts have been made to genetically improve this very promising energy crop. This review analyses the recent advances and challenges encountered in using clonal selection, mutagenesis/somaclonal variation and transgenesis/genome editing. Attempts to improve crop yield, in vitro propagation efficiency, salt and heavy metal tolerance by clonal selection were carried out, although limited by the species' low genetic diversity and availability of mutants. Mutagenesis and somaclonal variation have also been attempted on this species; however, since Arundo donax is polyploid, it is very difficult to induce and select promising mutations. In more recent years, genomics and transcriptomics data are becoming available in Arundo, closing the gap to make possible the genetic manipulation of this energy crop in the near future. The challenge will regard the functional characterization of the genes/sequences generated by genomic sequencing and transcriptomic analysis in a complex polyploid genome.

Pollen Grain Preservation and Fertility in Valuable Commercial Rose Cultivars.

In the cut flower market, traditional breeding is still the best way to achieve new rose cultivars. The geographical delocalization of cultivar constitution (generally made in Europe and North America) and plant cultivation (large areas in Africa and South America) represents a limit point for crossing and selection. Rose breeders often need to overcome geographical distances, resulting in asynchrony in flowering among crossing parents, by storing and sending pollen. Hence, a key aspect in breeding programs is linked to pollen availability and conservation, jointly with the identification of parameters related to pollen fertility. In this study we present the results of three different trials. In the first, pollen diameter and pollen viability were chosen as fertility predictors of 10 Rosa hybrida commercial cultivars. In the second trial, aliquots of dried pollen grains of six R. hybrida cultivar were stored under two different temperatures (freezer at T = -20 °C and deep freezer at T = -80 °C) and after a wide range of conservation period, their viability was measured. In the third trial, the effective fertilization capacity of frozen pollen of 19 pollen donor cultivars was evaluated during 2015 crossing breeding plan, performing 44 hybridizations and correlating the number of seeds and the ratio seeds/crossing, obtained by each cultivar, with in vitro pollen germination ability.

In vitro propagation of Hydrangea spp.

Hydrangea (Hortensia) is a highly popular ornamental plant for garden decoration, and now it is commercially produced for cut flower branches. For in vitro culture, Murashige and Skoog medium supplemented with BA (0.25 mg/L) and sucrose (30 g/L) was used. Culture conditions were 23 ± 1°C of temperature, light intensity of 35 µmol/m(2)/s P.P.F.D., and 16/8 h day/night photoperiod. Following shoot proliferation, the in vitro rooting frequency was 100% on a medium containing NAA 0.5 mg/L. However, 95% direct in vivo rooting was achieved by dipping microcuttings in a 5,000 ppm K-IBA solution which were transferred afterward to a glasshouse for acclimatization. After 21 days, fully acclimatized and well-established plants were obtained, suitable for commercialization. Furthermore, leaf fragments derived from in vitro plantlets were cultured for callus induction and adventitious shoot regeneration.

In vitro propagation of ornamental myrtus (Myrtus communis).

The Myrtle (Myrtus communis L.) is an evergreen shrub typical of the Mediterranean area; it is an interesting plant with multipurpose use. The ornamental use takes into account the production of green cut branches for indoor decoration and production of pot plants for gardening. In this species, there is a great variability in the natural germplasm around the Mediterranean coasts for type and size of fruit, plant architecture, leaf size and internode length. Selected genotypes have been successfully sterilized and cultured in vitro. The shoots were multiplied on MS (16) salts and vitamins, with 0.5 mg/L BA and 0.2 mg/L IAA. Clones showed variation of multiplication rate and rooting percentage. IAA or IBA at 0.5 mg/L increased the rooting percentage and noticed differences in root number and length. The sucrose concentration can affect rooting, such as light intensity during the in vitro rooting phase can modulate biomass production and chlorophyll content. The combination of these factors enhanced the frequency rate of acclimatization.