Embryogenesis of European Radish (Raphanus sativus L. subsp. sativus convar. radicula) in Culture of Isolated Microspores In Vitro

The European radish is one of the most unresponsive crops in the Brassicaceae family to embryogenesis in in vitro microspore culture. The aim of this work was to study the process of embryogenesis of European radish and its biological features. In this study, the embryogenesis of European radish is described in detail with illustrative data for the first time. For the first time for the entire family Brassicaceae, the following were found: microspores with intact exines with ordered-like divisions; microspores completely free of exines; and a new scheme of suspensors attachment to the apical parts of embryoids. The morphology of double and triple twin embryoids was described, and new patterns of their attachment to each other were discovered. Uneven maturation of European radish embryoids at all stages of embryogenesis was noted. The period of embryoid maturation to the globular stage of development corresponded, in terms of time, to the culture of B. napus, and into the cotyledonary stage of development, maturation was faster and amounted to 17–23 days. The rate of embryoid development with and without suspensors was the same.

Plant Regeneration from Unpollinated Ovules of Sugar Beet (Beta vulgaris L.) on Growing Media with Different Carbohydrates

Sugar beet (Beta vulgaris L.) is referred to as a strategic species due to its exceptional economic and functional importance. Sugar beet is cultivated in order to provide material for sugar production as it is the world’s second source after sugar cane. However, in this species, the regeneration of haploid shoots is difficult in comparison to other cultures or isolated microspores. Haploid plants of sugar beet can be derived from in vitro culture mostly via gynogenesis. Therefore, the aim of this research has been to increase the effectiveness of shoot formation from unpollinated sugar beet ovules by optimising the regeneration technique via induced gynogenesis. Various types and concentrations of chosen carbohydrates in media were evaluated. The Murashige and Skoog medium containing 4.4 μmol/L of 6-benzylaminopurine was solidified by 0.7% of agar and enriched with either sucrose (0.06 mol/L or 0.09 mol/L), glucose (0.09 mol/L), fructose (0.09 mol/L), maltose (0.09 mol/L) or with a combination of sucrose (0.04 mol/L) and mannitol (0.04 mol/L) or with sucrose (0.04 mol/L) and fructose (0.04 mol/L). The control medium contained 0.09 mol/L sucrose without any cytokinins. Of all the analysed media, the best for shoot regeneration turned out to be the media with 4.4 µmol/L 6-benzylaminopurine, solidified with 0.7% agar, additionally containing 0.09 mol/L glucose or 0.06 mol/L sucrose. On those media, over three-fold more shoots compared with the control medium were produced.

Doubled Haploids in Eggplant

Eggplant is a solanaceous crop cultivated worldwide for its edible fruit. Eggplant breeding programs are mainly aimed to the generation of F1 hybrids by crossing two highly homozygous, pure lines, which are traditionally obtained upon several self crossing generations, which is an expensive and time consuming process. Alternatively, fully homozygous, doubled haploid (DH) individuals can be induced from haploid cells of the germ line in a single generation. Several attempts have been made to develop protocols to produce eggplant DHs principally using anther culture and isolated microspore culture. Eggplant could be considered a moderately recalcitrant species in terms of ability for DH production. Anther culture stands nowadays as the most valuable technology to obtain eggplant DHs. However, the theoretical possibility of having plants regenerated from somatic tissues of the anther walls cannot be ruled out. For this reason, the use of isolated microspores is recommended when possible. This approach still has room for improvement, but it is largely genotype-dependent. In this review, we compile the most relevant advances made in DH production in eggplant, their application to breeding programs, and the future perspectives for the development of other, less genotype-dependent, DH technologies.

Induction of Embryogenesis in the Culture of Isolated Microspores of Wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L.) haploids and doubled haploids are widely used in breeding, the investigations of a combinative variability and its stabilization in homozygotes. In four domestic varieties of winter wheats (Moskovskaya 56, Moskovskaya 39, Galina, Nemchinovskaya 24) and three domestic varieties of spring wheats (Ester, MIS, Amir). With spring wheat variety Falat as a control, the efficacy of embryogenesis in isolated microspores was tested using standard protocol for induction of direct embryo formation in the isolated microspore culture. In all winter varieties there was shown a low frequency of cytoplasmic strands, which are typical for the embryogenic microspores, whereas in the spring varieties it was high. After 4 days cultivation in the medium used for induction, the microspore viability decreased in winter varieties. and another 10 days later the Viable cells were not observed. The spring varieties developed the multicellular structures, which could produce embryos. The reference variety Falat produced 28 % of proembryoids, able mostly to further embryonic formation. Basing on these results, the protocol for inducing direct embryogenesis in wheat microspores was modified, including maltose concentration in medium, the conditions of spikelet heat treatment, the number of ovaries and time when they were added to the culture, the combination and concentration of hormones in the media for induction and cultivation.

Homozygous Transgenic Barley (Hordeum vulgare L.) Plants by Anther Culture

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

CULTURE USE PROBLEMS IN SELECTION OF ISOLATED MICROSPORES IN GRAIN

Production of haploid plants by culture of isolated microspores is a quick way of obtaining homozygous crop lines. Recessive features of mutant homozygous plants are also possible to determine by this biotechnology. Contrary from anthers culture, in which the presence of anther walls can lead to the development of diploid somatic calli and plants, the microspore culture produces only haploid or dihaploid lines. Isolated microspores culture in addition represents and has a unique identification system for studying the mechanisms of embryogenesis in in vitro culture. The usage of haploid technology extends the genetic basis of wheat breeding, since it allows increasing the frequency of new gene combinations. This technology significantly increases the efficiency of breeding new highly productive varieties of crops. On this basis, it becomes possible to quickly assess the prospects of dihaploids, which significantly improves the efficiency of the selection process. DH plants are completely fertile and, if necessary, may be used as parents or processed as a cultivar. DHs have been widely used for cultivar development, genetic mapping, mutagenesis, and the study of gene functions.

Improvement of methods of creating hybrids of cabbage

Relevance One of the basic directions of the cabbage crop breeding is the creation of F1 hybrids with a complex of economically valuable traits. This process is difficult and time-consuming as to get pure lines must be within 6-12 years hold inbreeding. Herewith not every line gives the desired heterotic effect that also requires additional verification. Methods Biotechnological method culture of isolated microspores in vitro, which allows in the first generation to receive a line with 100% homozygosity, was used to speed up the breeding process. Combination ability were performed in complete diallel cross on the basic morphological signs. Results Culture medium for cultivation of isolated microspores in vitro was optimized for each genotype of cabbage for the best embryoids regeneration. Maximum amount of embryoids was received on medium with pH 6.2 using ampicillin 100 mg/l and zeatin 1 mg/l: 466.7 ± 153.2 PCs/100 buds. A new source material for breeding – doubled haploid lines of cabbage was received. Lines – the best parents for F1 hybrids with high yield, compact rosette of leaves, with optimum inside and short outside cabbage stump was created. Studies have shown that optimization of breeding process in case of creation of pure lines of cabbage in 3 years with microspore culture requires to reduce the breeding process in 2 times.

Rapid development of homozygous lines through culture of isolated microspores in leafy crops of Brassicaceae Burnett

Relevance Biotechnological methods are generally used to speed up breeding programs and to enhance genetic diversity, so the culture of isolated microspore in vitro can be regarded as one of very suitable methods. Nontraditional and uncommon vegetable crops belonging to Brassicaceae Burnett. are becoming more popular. Methods Accessions of sarepta mustard (Brassica juncea L. Czern.) and rocket salad (Eruca sativa Mill.) were taken for the study with the aim to optimize the basic protocol for these species. Results As a result of the study the optimum cultivation conditions have been determined for the species. Sizes of buds 2.5-3.5 mm long for sarepta mustard and 7.0-7.5 long for rocket salad which were used for cultivation had been experimentally defined. It was also shown that the cold pretreatment had improved the embryo yield. The nutritional NLN-13 medium with pH 6.1 and pretreatment at 32°C during a cultivation day had been shown to be more favourable for all accessions. All conditions that had been used were suitable for embryo formation. First divisions had been seen after 4 days of cultivation, while the embryos at primary cotyledonary stage only appeared after 2 weeks of cultivation. The embryo yield per 5 buds reached 25-30 and 5-7 in the sarepta mustard and the rocket salad, respectively. It is worth noticing that the root formation and plant adaptation had passed better and faster in sarepta mustard than in rocket salad. Thus, whole process of homozygous line developing can be completed for 4-5 months, making the breeding program 3 times shorter.

Initiation of microcalli in culture of pea (Pisum sativum L.) isolated microspores

Innovative biotechnologies based on use of double haploids enables developing new varieties considerably faster compared to conventional plant breeding approaches. In pea, reliable methods of haploid plants production are not fully elaborated yet. The current research aimed at testing different conditions (genotype, medium, and stress treatment) for initiation of sporophytic developmental shift in culture of pea isolated microspores. Reprogramming pea microspores towards a sporophytic development was stimulated with temperature stress. Cold (+4°С) and heat (+35°С) stress treatments were applied to pea isolated flower buds and microspores, respectively. Microspores were isolated from plants of 6 pea genotypes, treated at 18 temperature regimes and cultivated in 8 liquid nutrient media with various chemical compounds including growth regulators, vitamins, sugars, glutamine, casein hydrolysate, and osmotic agents. Microcalli were produced from isolated microspores of pea breeding line 109b and variety Stabil in conditions of nutrient media KM-ар1 and MSB-M3 after cold (+4°С) stress treatment for 16 and 10 days, respectively. The media KM-ар1 and MSB-M3 contained a relatively low concentration of sugar (10 and 6 g L-1, respectively), and were supplemented with polyethylene glycol 6000 or mannitol as osmotic agents.