Androgenesis—Technology for Obtaining Genetically Stable Breeding Material of Capsicum annuum L.

Androgenesis in vitro is a basic method of obtaining haploid plants and DH (doubled haploid) lines of major crops such as potato, rapeseed, tomato, pepper, wheat, maize, and barley, and also many different minor crops and species with lower agricultural impact. Diploid plants appearing among androgenic regenerants are the effect of spontaneous doubling of the chromosome number in haploid cells during an embryo’s early developmental stages and are valuable fully homozygous breeding material. The subject of the presented research is spontaneous diploidization occurring in the development of androgenic, haploid pepper regenerants. In the presented experiment, the formation of diploid seeds was observed in the progeny of an androgenic, haploid plant derived in an anther culture of a hybrid (Capsicum annuum L. ATZ × Capsicum annuum L. `Corno di toro`)F2. Agromorphological and molecular analyses concerned eight diploid plants being progeny of the anther-derived haploid regenerant. Five of the plants constituted a phenotypically balanced group with valuable agromorphological features. Their genetic homogeneity was confirmed using 10 RAPD markers and 16 ISSR markers. Based on the results, it was concluded that anther-derived haploid plants of Capsicum can be the source of diploid, apomictic seeds, and the obtained offspring may constitute genetically stable, valuable breeding material.

Androgenic Haploid Plant Development Via Embryogenesis With Simultaneous Determination of Bioactive Metabolites in Cambod Tea (Camellia Assamica ssp. Lasiocalyx)

This pioneering work reports successful androgenic plant development via embryogenesis from microspore calluses in anther cultures and estimation of bioactive metabolites in in vitro regenerants and parent plant (control) of Cambod tea, Camellia assamica ssp. lasiocalyx (Planch MS) cultivar TV19. Anthers bearing microspores at early-to-late uni-nucleate stage were selected to initiate androgenesis. A pre-treatment of 50 C for five days in the dark was most effective to initiate profusely growing white callusing from microspores within 10 weeks of culture on MS medium (6% sucrose) supplemented with high cytokinins/ auxin ratio maintained by benzyl adenine (BAP) and 2,4-dichlorophenxyacetic acid (2, 4-D). Nodular structures on the callus surface differentiated into embryos. Further developement of the embryos occurred on embryogenesis medium but, with ten times reduced concentration of growth regulators and additives. Germination of embryos into complete plantlets was achieved when major salts in medium were reduced to half MS (½ MS) and augmented with BAP, GA3 and IBA along with glutamine and serine. Cytological examination of the root-tip cells revealed that regenerated plantlets were haploids (2n=x=15), which was further confirmed through flow cytometry. The hot-water extracts from in vitro haploid calluses, embryos and field-grown donor plant were utilized for quantification of (+)-catechin, (-)-epicatechin, (-)-epigallocatechin gallate, caffeine and theophylline. Our findings revealed that the metabolite profile of in vitro regenerated haploid cultures is comparable to that of the mother plant, thereby presenting them as potential source for genome duplication and development of genetically stable homozygous pure breeding lines.

Modern issues of sugar beet (Beta vulgaris L.) hybrid breeding

High efficiency of the cultivation of unfertilized sugar beet ovules and preparation of haploid regenerants (microclones) of pollinators – maintainers of О-type sterility and MS forms of the RMS 120 hybrid components has been shown. A technological method that accelerates the creation of new uniform starting material is proposed. It speeds up the breeding process two to threefold. The identification of haploid regenerants with sterile cytoplasm in initial populations is of great theoretical and practical importance for breeding, as it facilitates the production of homozygous lines with cytoplasmic male sterility and high-performance hybrids on sterile basis. As shown by molecular analysis, a single-nucleotide polymorphism never reported hitherto is present in the mitochondrial genome of the haploid plant regenerants. It allows identification of microclones as fertile and sterile forms. It has been found that DNA markers of the sugar beet mitochondrial genome belonging to the TR minisatellite family (TR1 and TR3) enable reliable enough identification of haploid microclonal plants as MSor O-type forms. Fragments of 1000 bp in length have been detected in monogenic forms in the analysis of 11 sugar beet plants cultured in vitro by PCR with the OP-S4 random RAPD primer. Testing of the OP-S4 marker’s being in the same linkage group as the genes responsible for expression of the economically valuable trait monogermity demonstrates its relative reliability. By the proposed method, dihaploid lines (DH) of the male-sterile form and the О-type sterility maintainer of the RMS 120 sugar beet hybrid have been obtained in in vitro culture. These lines are highly uniform in biomorphological traits, as proven under field conditions.

Production of haploid embryos and plants in Iranian melon (Cucumis melo L.) through irradiated pollen-induced parthenogenesis.

The irradiated pollen technique (IPT) is the most successful haploidization technique within Cucurbitaceae. The influence of gamma-ray doses (250, 350, 450 and 550 Gy), genotypes and stage of development of embryos obtained by IPT on the induction of haploid embryos were studied in several Iranian melon cultivars as well as their hybrids with alien cultivars. Female flowers were pollinated using pollen that had been irradiated with gamma rays. Different shapes and stages of embryos were excised 21-25 days after pollination and cultured on E20A medium. Direct culture, liquid culture and integrated culture methods were used; integrated culture and liquid culture methods showed advantages in increasing the efficiency of haploid plant production in melon breeding programmes. Results revealed that 550 Gy of gamma irradiation was successful in inducing parthenogenesis and fruit development, whereas lower irradiation doses were not effective in inducing haploid embryos. The percentages of embryos per seed were the highest in 'Samsoori' (1.2%) and 'Saveh' (1.1%) cultivars. Some of the heart-shaped and cotyledon-shaped embryos developed into haploid plants. In total, 52 parthenogenic melon plantlets were recovered from 274 embryos via IPT. Production of haploid embryos and haploid plants was strongly influenced by gamma-ray dose, embryo stage and genotype. Indirect methods and chromosome counting performed on the root cells of regenerated plants showed that these plants were haploid (n = x = 12).