Table 1.

Breeding stages and their goals in the BGU program for Hylocereus cultivation

Stage	Strategy	Goal	Milestone
1-	Introduction	Germplasm collection	Collection of more than 200 genotypes from 10 Hylocereus species
		Establishment of a live gene bank	The largest Hylocereus live gene bank in the world
		Development of agro-techniques	Development of plant growth and orchard management techniques
2-	Pre-breeding research	Identification of the introduced germplasm	Taxonomical identification of all the genotypes collected
		Cytological studies	Determination of the ploidy level and pollen viability in Hylocereus species. Chromosomal aberrations were found in Hylocereus megalanthus
		Self-(in)compatibility mechanisms	Self-incompatibility was found in diploid species and diploid hybrids
		Mating system	Crosses between all the Hylocereus species were found to be feasible and resulted in fruit and seed set. Metaxenia was reported
		Genetic relationships	Autopolyploid origin was posited for the tetraploid H. megalanthus, and the species was classified into the Hylocereus genus
		Development of laboratory protocols	Protocols for DNA isolation, in situ autopolyploidization, androgenesis and gynogenesis, embryo rescue, and flow cytometry analysis were developed
3-	Hybridization	Interspecific homoploid crosses	Interspecific diploid crosses resulted in viable diploid hybrids
		First interspecific interploid crosses	Reciprocal interspecific interploid crosses resulted in triploid, pentaploid and hexaploid hybrids
		Second interspecific and back-cross crossing cycles	Interspecific interploid crosses and back-crosses resulted in viable allopolyploid hybrids, including improved self-compatible allotetraploids
4-	Intergeneric hybridization	Intergeneric homoploid and interploid crosses	Reciprocal Hylocereus-Selenicereus crosses yielded viable intergeneric diploid and triploid hybrids
5-	Autopolyploidization	Artificially induced in situ genome duplication	Autotetraploid, autohexaploid and autooctaploid lines were obtained
6-	Homozygous lines	Anther and ovule morphology and development	The timing of pollen and of ovule developmental stages was determined
		Androgenesis	Homozygous lines were created via androgenesis
		Gynogenesis	Homozygous lines were created via gynogenesis
7-	Embryo rescue	Embryo development	The timing of the different embryo developmental stages was determined
		Homoploid interspecific crosses	Hybrids were obtained from embryo rescue following homoploid interspecific crosses
		Interploid interspecific crosses	Hybrids were obtained from embryo rescue following interploid interspecific crosses
8-	Further breeding and development	Breeding and selection for tolerance to extremely high temperatures	Allotetraploid cultivars with improved tolerance to high temperature were produced
		Grafting elite cultivars	Grafting produced plants with enhanced tolerance to high temperatures
9-	Nutritional value	Pigment identification/ profiling in red pulp pitaya	A novel betacyanin named “hylocerenin” was identified
		Breeding and selection for improved nutritional values	To be done
10-	Genomics and molecular genetics studies	Betalain biosynthesis	Conserved and novel miRNAs were identified Advanced knowledge in transcriptional regulation of genes associated with betalain biosynthesis was acquired. Candidate genes of the betacyanin pathway were identified and localized in the genome
		Tolerance to abiotic stresses	Identification of proteins related to cold stress Identification of putative genes and metabolites involved in heat- stress response
		Flowering process and anthesis time	Platform for studying the molecular mechanism controlling floral induction and regulation Identification of 33 aquaporin genes
		Gene editing	The first high-quality draft genome of H. undatus
		Molecular breeding	To be done