Microspore embryogenesis is the most commonly used method to produce doubled haploids. It is based on the ability of a single haploid cell, the microspore, to de-differentiate and regenerate into a whole plant after being exposed to stresses, such as low or high temperatures, carbon starvation and colchicine. Some stresses such as temperature treatments and carbon starvation have been used with success in many plant species, whereas others such as colchicine had limited application in a few species. Reports on the application of whole plant treatments with feminizing agents on inflorescences and buds are scarce. Furthermore, the technical means to apply some stresses such as γ-irradiation are not readily available. Recently, novel stresses such as pH, inducer chemicals, carrageenan oligosaccharides and heavy metals were reported to induce microspore embryogenesis. It remains to be seen, however, whether these stresses are effective in a wider range of species. Finally, pretreatment of cultured cells with high concentrations of 2,4-D efficiently induces somatic embryogenesis in several species (carrot, alfalfa). However, reports on the use of this particular chemical stress are not available in microspore embryogenesis. The paper presented here gives an overview of various stresses and mechanisms of action of these stresses in inducing microspore embryogenesis.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1399-3054.2006.00675.x

Stresses applied for the re‐programming of plant microspores towards in vitro embryogenesis

In the early 1990s, many basic protocols were developed for haploidy and doubled haploidy, but most were inefficient. During the last decade, progress in technology has been achieved mainly by empirical, time and cost consuming testing of protocols; as a consequence success was proportional to the number of laboratories involved. In the most frequently studied crops (barley, wheat, triticale, maize, rice and rapeseed) improved protocols are now used routinely in breeding and although several problems remain the benefits make doubled haploidy well worthwhile. Significant advances have also been achieved in vegetable, fruit, ornamental, woody and medicinal species, though responses in many remain low with legumes being particularly recalcitrant. There has been resurgence in doubled haploids over the last few years with protocols published for almost 200 plant species. The present review aims to show the recent progress in haploid and doubled haploid technology of higher plants.

Progress in Doubled Haploid Technology in Higher Plants

Albinism is a common problem encountered in interspecific crosses and tissue culture experiments including anther culture and generation of doubled haploids. It is characterized by partial or complete loss of chlorophyll pigments and incomplete differentiation of chloroplast membranes. This in turn impairs photosynthesis and the plants eventually die at a young stage without reaching maturity. Environmental conditions such as light, temperature, media composition and culture conditions play some role in determining the frequency of albino plant formation. Genetic factors are even more important, and are major determinants in albinism. Genetic studies in different crops show that it is a recessive trait governed by many loci. Both the nuclear and chloroplast genomes affect albinism and incompatibilities between the two are a probable cause of many pigment defects in hybrid progenies. Such incompatibility has been reported in a large number of angiosperms. The mechanisms behind these incompatibilities are p...

Albinism in plants: a major bottleneck in wide hybridization, androgenesis and doubled haploid culture.

Haploids in the Improvement of Poaceae

Isolated microspore cultures of two spring triticale (x Triticosecale Wittm.) cultivars were used to examine the effect of various stress treatments (either high—32°C or low—5°C temperature with or without nitrogen/carbohydrate starvation) applied to excised anthers on the effectiveness of microspore embryogenesis induction. To quantify the effects of pretreatment conditions, the activity of antioxidative enzymes (catalase, peroxidase and superoxide dismutase) together with respiration rate and heat emission were measured. It was observed that heat shock treatment applied as the only one stress factor increased the activity of antioxidative enzymes which suggests intensive generation of reactive oxygen species. Such pretreatment effectively triggered microspore reprogramming but drastically decreased microspore viability. After low temperature treatment, the activity of antioxidative enzymes was similar to the control subjected only with the stress originated from the transfer to in vitro culture conditions. This pretreatment decreased the number of microspores entering embryogenesis but sustained cell viability and this effect prevailed in the final estimation of microspore embryogenesis effectiveness. For both, low- and high-temperature treatments, interaction with starvation stress was beneficial increasing microspore viability (at 5°C) or efficiency of embryogenesis induction (at 32°C). The latter treatment significantly reduced cell metabolic activity. Physiological background of these effects seems to be different and some hypothetical explanations have been discussed. Received data indicate that in triticale, anther preculture conditions could generate oxidative stress and change the cell metabolic activity which could next be reflected in the cell viability and the efficiency of microspore embryogenesis.

Stress-related variation in antioxidative enzymes activity and cell metabolism efficiency associated with embryogenesis induction in isolated microspore culture of triticale (x Triticosecale Wittm.)

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium Adventitious embryogenesis was observed during in vitro development of triticale microspores Albino and green plantlets were regenerated from embryo-like structures More than 50% of regenerants were albino In total, 126 green plantlets were produced, transplanted and established in soil Cytological evidence revealed that 90% of the transplanted regenerants were haploid

/pdf/in-vitro-androgenesis-of-triticale-in-isolated-microspore-gs2ygdf12q.pdf

In vitro androgenesis of triticale in isolated microspore culture

To determine the value of phenotypic variability of populations of different origins, two DH varieties (GK Delibab, GK Szindbad), a DH variety candidate (Ambitus) and two conventional winter wheat varieties (GK Istvan, GK Kincső) were compared for seven agronomic traits. The aim of the study was to find out if values of phenotypic variability of theoretically homozygous DH varieties are lower than variability of not homozygous traditional varieties, which have some genetic variation. On the basis of our analysis it can be established that there is no obvious difference in the variability of examined characters (plant height, spike length, spike width, spikelet length, spiklet width, number of node/spike and number of seeds/spike) of populations of different origins. The value of coefficients of variations (CV) ranged between 5-10%, which is characteristic for the balanced population. Only the value CV of seed-number/spike was higher. In conclusion, it appears that the examined populations of different origins cannot be separated by the value of variability.

K. Lökös Tóth

Papers

In vitro androgenesis of triticale in isolated microspore culture

Agronomic performance of doubled haploid wheat varieties