dCas9-3xSRDX-mediated transcriptional repression in sugarcane.

KEY MESSAGE: Targeting dCas9 fused with the 3xSRDX effector to the 5'UTR leads to strong repression of magnesium chelatase in highly polyploid sugarcane.

Anther Culture and Chromosome Doubling in Mediterranean Japonica Rice.

Anther culture is the most used technique to produce doubled haploid lines in rice. This technique is well developed in a wide range of indica rice genotypes. However, in japonica type, and more specifically, the Mediterranean japonica, the protocols are yet to be optimized. Japonica and indica have different androgenic response, as well as different induction and regeneration rates, albinism ratios and chromosome doubling competence. The step-by-step anther culture protocol presented in this chapter allows to regenerate doubled haploid rice plantlets from anther microspores in 8 months. We also include an in vitro chromosome doubling protocol to induce doubled haploids from haploid plantlets by immersion in a colchicine solution. This chromosome doubling protocol complements the anther culture by taking advantage of the regenerated haploid plantlets.

Doubled Haploid Parthenogenetic Production of Melon 'Piel de Sapo'.

Parthenogenesis is the main technique to produce doubled haploid lines in melon. Although parthenogenesis is a genotype-dependent process and melon has a huge genotypic diversity, we developed a successful protocol for haploid embryo production via pollination with irradiated pollen and a protocol for chromosome doubling of haploid plants of 'Piel de Sapo' genotypes. 'Piel de sapo' genotype has lower efficiencies during the process in comparison with other genotypes, for instance, of the agrestis subspecies. Nevertheless, the doubled haploid lines produced have a great potential as pure parentals for hybrid seed production.

Chromosome doubling methods in doubled haploid and haploid inducer-mediated genome-editing systems in major crops.

The doubled haploid technique aims to generate pure inbred lines for basic research and as commercial cultivars. The doubled haploid technique first generates haploid plants and is followed by chromosome doubling, which can be separated in time or overlapped, depending the procedure for each species. For a long time, much effort has been focused on haploid production via androgenesis, gynogenesis, or parthenogenesis. The obtention of haploid plants has frequently required more optimization and has lagged behind research and improvements in chromosome doubling methods. Nevertheless, chromosome doubling has recently been of renewed interest to increase the rates and efficiency of doubled haploid plant production through trialing and optimizing of different procedures. New antimitotic compounds and application methods are being studied to ensure the success of chromosome doubling once haploid material has been regenerated. Moreover, a haploid inducer-mediated CRISPR/Cas9 genome-editing system is a breakthrough method in the production of haploid plant material and could be of great importance for species where traditional haploid regeneration methods have not been successful, or for recalcitrant species. In all cases, the new deployment of this system will demand a suitable chromosome doubling protocol. In this review, we explore the existing doubled haploid and chromosome doubling methods to identify opportunities to enhance the breeding process in major crops.

In situ Parthenogenetic Doubled Haploid Production in Melon "Piel de Sapo" for Breeding Purposes.

Doubled haploids in cucurbit species are produced through in situ parthenogenesis via pollination with irradiated pollen for further use as parental lines for hybrid F1 production. In this study, seven genotypes of melon "Piel de Sapo" were appraised for agronomic traits and pathogen resistances to evaluate its commercial value and used as donor plant material for the parthenogenetic process. Then, in situ parthenogenetic capacity of melon "Piel de Sapo" germplasm was evaluated and optimized. Several steps of the parthenogenetic process were assessed in this study such as melon fruit set after pollination with irradiated pollen, haploid embryo obtention, in vitro germination and growth of parthenogenetic embryos and plantlets, in vitro and in vivo chromosome doubling with colchicine or oryzalin and fruit set of doubled haploid lines. Parthenogenetic efficiencies of "Piel de Sapo" genotypes showed a high genotypic dependency during the whole process. Three different methods were assayed for parthenogenetic embryo detection: one-by-one, X-ray and liquid medium. X-ray radiography of seeds was four times faster than one-by-one method and jeopardized eight times less parthenogenetic embryo obtention than liquid medium. One third of melon fruits set after pollination with irradiated pollen contained at least one parthenogenetic embryo. The 50.94% of the embryos rescued did not develop into plantlets because failed to germinate or plantlet died at the first stages of development because of deleterious gene combination in haploid homozygosity. The distribution of the ploidy-level of the 26 parthenogenetic plantlets obtained was: 73.08% haploid, 23.08% spontaneous doubled haploid and 3.84% mixoploid. Two in vitro chromosome doubling methods, with colchicine or oryzalin, were compared with a third in vivo colchicine method. In vivo immersion of apical meristems in a colchicine solution for 2 h showed the highest results of plant survival, 57.33%, and chromosome doubling, 9.30% mixoploids and 20.93% doubled haploids. Fruit set and seed recovery of doubled haploids lines was achieved. In this study, doubled haploid lines were produced from seven donor genotypes of melon "Piel de Sapo," however, further improvements are need in order to increase the parthenogenetic efficiency.

Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon.

CRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42-45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.

An improved anther culture procedure for obtaining new commercial Mediterranean temperate japonica rice (Oryza sativa) genotypes.

Rice is one of the greatest calorie supply for the world population, especially since its production is almost entirely destined to direct human consumption and its demand will increase along with the world population. There are efforts worldwide to increase rice yields by obtaining new improved and stabilized rice lines. The rice anther culture, a fast and cheap technique, allows to obtain double haploid lines in less than one year. We report its application with an improved protocol in four Mediterranean japonica rice genotypes at F2 generation. We performed a screening test for cold-pretreatment at 5.0±0.1°C and concluded that the optimum duration was 9 days as it produced the higher rate of anther-derived callus induction. This revised protocol was successfully applied to the four genotypes, obtaining good results in all the procedure's steps. At the end, more than 100 of double haploid green plants were generated. Moreover, 9 lines obtained from the anther culture procedure showed good qualities for the Spanish market at the growing, farming and grain production level during the field assays. Therefore, we report an improved anther culture procedure for obtaining double haploid lines from temperate japonica rice genotypes showing high commercialization expectance.