Pollen of two I. cylindrica genotypes, one having spike with brown anthers (Ic-Pbr) and other with yellow anthers (Ic-Pye) was collected from three collection environments (early, mid and late flowering) and preserved in sealed Petri dishes under three preservation regimes, P1 (4 °C temperature and 60% RH), P2 (–20 °C and 65% RH) and P3 (–80 °C temperature and 45% RH). The pollen viability and germination tests of the preserved pollen were conducted after every 15 days till complete loss of viability. The study revealed that Ic-Pbr was superior to Ic-Pye in terms of the mean absolute pollen viability (APV) under preservation conditions. Among the collection environments, pollen collected just after first anthesis, that is, early flowering was superior in terms of the preservation potential. The preservation regime, P2 was found to be best among the three for better preservation of I. cylindrica pollen. By utilizing the one month old preserved pollen for pollination of wheat spikes, pollen from P2 regime induced haploid embryos to the tune of 25.53%. The study concluded that by preserving I. cylindrica pollen at –20 °C, the period of DH production in wheat can be extended by at least one month. The preserved pollen will also open avenues for exploring the possibilities of inducing haploids in other cereals like rice and maize whose flowering does not coincide with I. cylindrica naturally.
Aegiolops kotschyi cytoplasmic male sterile system often results in part of haploid plants in wheat (Triticum aestivum L.). To elucidate the origin of haploid, 235 wheat microsatellite (SSR) primers were randomly selected and screened for polymorphism between haploid (2n = 3x = 21 ABD) and its parents, male-sterile line YM21 (2n = 6x = 42 AABBDD) and male fertile restorer YM2 (2n = 6x = 42 AABBDD). About 200 SSR markers yielded clear bands from denatured PAGE, of which 180 markers have identifiable amplification patterns, and 20 markers (around 8%) resulted in different amplification products between the haploid and the restorer, YM2. There were no SSR markers that were found to be distinguishable between the haploid and the male sterile line YM21. In addition, different distribution of HMW-GS between endosperm and seedlings from the same seeds further confirmed that the haploid genomes were inherited from the maternal parent. After haploidization, 1.7% and 0.91% of total sites were up- and down-regulated exceeding twofold in the shoot and the root of haploid, respectively, and most of the differentially expressed loci were up/down-regulated about twofold. Out of the sensitive loci in haploid, 94 loci in the shoot, 72 loci in the root can be classified into three functional subdivisions: biological process, cellular component and molecular function, respectively.
Large numbers of genetically stable, homozygous plants are needed for classical and molecular breeding programmes. In vitro anther culture has proved to be a useful tool for haploid/doubled haploid (DH) induction in pepper (Capsicum annuum L.) for more than twenty years. The present paper reports on a great improvement in the in vitro haploid induction and genome duplication methods routinely used for resistance breeding in sweet and spice peppers by two Hungarian research institutions, the Agricultural Biotechnology Center in Gödöllő and the Budapest Research Unit of the Vegetable Crops Research Institute. As a result of the colchicine-stimulated early genome induction method, the critically low (<0.1%) regeneration frequency of spice pepper types became ten times greater, reaching a value of around 1.0%, though this was still considerably lower than that achieved in pepper varieties for fresh consumption (5-10%). Moreover, the ratio of useful doubled haploids was far higher (H:DH = 1:2 or 1:4) in some cases after colchicine treatment than that of untreated control plants (H:DH = 2:1 or 3:1, depending on the genotype). An efficient method with good reproducibility, requiring less manual work, was elaborated for the in vitro genome duplication of pepper haploid regenerants using colchicine. When the haploid induction ability of plants conventionally cultured in the greenhouse was compared to that of plants raised under artificial conditions in phytotron chambers (satisfactory day and night temperatures, illumination, humidity), the responsiveness of the latter microspores (ratio of plant regeneration) was found to be almost twice as high. The application of 3% maltose for six days at 35°C resulted in a 1.45% increase in the ratio of responding anthers and a 0.34% increase in plant regeneration, averaged over all the variety types. Phenosafranin staining was used for the analysis of microspore viability. The reduction in viability during the induction period proved to be less pronounced in lines with better androgenetic responses than in those with poorer responsiveness.