The influence of cultivar, donor plant and culture procedure on the efficiency of androgenesis was studied in carrot anther culture. Experiments were carried out on five carrot cultivars: CxC 9900 F1, Lucky B F1, HCM, Beta III and Perfekcja, which were chosen because of their high carotene contents. Two procedures of anther culture were compared: (1) incubation in darkness for two weeks, followed by exposure to continuous light and transfer onto a fresh medium of the same composition; and (2) incubation in darkness until embryos appeared, without transfer onto a fresh medium. Temperature was +27oC all the time. Genotype played an important role in the process of androgenesis in carrot anther culture.The efficiency was the highest in cv. HCM ? 5.6 embryos per 100 anthers. Considerable differences in the capacity for androgenesis were observed between individual donor plants. The ratio of embryos obtained per 100 anthers for cv. HCM varied from 0.0 to 48.9. The second procedure of anther culture proved to be more efficient, cheaper and less complicated.
This paper provides an overview of the development of methods of cereals doubled haploid production over the last decade. The influence of genotype and albinism remain the main problem of androgenesis. Localisation of major genes influencing androgenic potential and manipulation with temperature during induction and regeneration offer possibilities of efficiency improvement. Isolated microspore culture in cereals is effective when microspores are co-cultured with sporophytic tissue. Gynogenic methods may be developed for some barley and apomictic wheat forms. Distant crosses that are followed by the elimination of chromosomes of the male parent from hybrid embryos are broadly applied. Maize and its relatives are used as effective pollinators of a wide range of cereal species since prezygotic barriers have not been found so far. Brief description of utilisation of doubled haploid lines in breeding programs, research and genetic transformation of cereals closes the overview.
The change in the developmental pathway of microspores from gametophytic to sporophytic is induced by stress during pretreatment of spikes and anthers. In our experiments, anther culture of three barley cultivars was tested with regard to the effect of chilling at 4?C for 28 days, starvation in 0.3 M mannitol solution for 4 days, and a combination of both methods. Chilling was shown to increase embryo/callus formation, while mannitol treatment favoured plant development, including development of green plants; simultaneous application of the two stress factors for 4 days proved to be ineffective. The tested cultivars exhibited a similar ability (calculated per 100 transferred embryos/calli) to develop plants without pretreatment; however, their responses to stress varied greatly. The collected data indicate that mannitol pretreatment, as compared to chilling, is more efficient in responsive cultivars.
In the study on double haploid (DH) lines of tobacco, two cultivars ? MN944 and BY103 transformed with a construct carrying the coat protein gene of Lettuce Mosaic Virus (LMV) CP were used. The hybrids F1 from crossing transgenic line BY103 with its non-transformed equivalent as well as transgenic MN944 line with cv. Wi?lica were used for producing haploids via inducted androgenesis. Double haploids were received by regeneration from stem fragments. The course of meiosis in pollen mother cells was analysed and pollen viability was estimated.
Two soft-flesh Capsicum frutescens L. lines were used in the study. The androgenesis process was induced according to the procedure described for C. annuum L. In the cultures established in summer, not many anthers forming the callus tissue were observed. Cytometric analysis showed the presence of cells with different content of DNA. Among 1.000 anthers displayed of one line, seven embryos were observed. Four embryos continued the development resulting in obtaining plants. Three of them had the DNA content on 1C level, the fourth one was diploid. The test of an androhaploid induction in autumn cycle, with the genotype of positive response in summer, was not successful.
The purpose of this work was to quantify the impact of spontaneous and X-radiation-induced chromosome rearrangements on survival rate of androgenetic rainbow trout (Oncorhynchus mykiss). Various doses of X irradiation (50, 150, 250, 350 Gy) were used for inactivation of nuclear DNA in oocytes. After the irradiation, eggs were inseminated with normal sperm from 4 males derived from a strain characterized by Robertsonian rearrangements and length polymorphism of the Y chromosome. The haploid zygotes were exposed to a high hydrostatic pressure (7000 psi) to duplicate the paternal DNA. Neither Robertsonian chromosome polymorphism nor the Y chromosome morphology impaired the viability of the androgenetic embryos and alevins. Moreover, survival of eyed embryos of the androgenetic rainbow trout increased significantly with increasing doses of oocyte X irradiation. After 6 months of rearing, only specimens from the 250 and 350 Gy variants survived. The number of fingerlings with remnants of the maternal genome in the forms of chromosome fragments was higher in the 250 Gy group. Intraindividual variation of chromosome fragment number was observed, and some individuals exhibited haploid/diploid mosaicism and body malformations. Individuals irradiated with less than 250 Gy died, presumably because of the conflict between intact paternally derived chromosomes and the residues of maternal genome in the form of chromosome fragments.
Microspores were cultured on the modified B5 liquid medium containing 2.4D (0.1 mg L?1), NAA (0.1 mg L?1), L-glutamine (500 mg L?1), L-serine (100 mg L?1), and sucrose (100 g L?1). The developmental stages of microspores and divisions were observed. Initially, the formation of binuclear and multicellular structures was noticed. Plants regenerated in the cultures in which the tetrad stage of microsporogenesis had predominated. Embryoids were still forming 24 weeks after the cultures were set up. Six weeks after the transfer of androgenetic embryos onto the B5 regeneration medium, they were converted into complete plants. Out of 90 androgenetic plants planted in a growth chamber, 42 plants adapted to the new conditions. All of those plants proved to be diploids in cytometric analysis.
The primary aim of the study was to establish the effectiveness of induced androgenesis in in vitro anther culture of two pepper (Capsicum annuum L.) breeding lines ? ATZ1 and PO, and a hybrid between these two lines (ATZ1 ? PO)F1. Anther culture was maintained according to the method developed by Dumas de Vaulx et al. (1981) with some modifications. The experiment revealed that the effectiveness of androgenesis ranged from 4% for the ATZ1 line to 1.5% for the (ATZ1 ? PO)F1 and strongly depended on the developmental stage of flower buds, as well as the conditions for anther culture maintenance. The development of androgenic embryos was successfully induced only in anthers which originated from the flower buds with petals equal or slightly longer than sepals and there was a clear relationship between the length of the period of anther induction on CP medium and the level of kinetin in R1 regeneration medium.
This review paper covers the results of tissue culture and biotechnology of rye (Secale cereale L.) published between 1990 and 2000. The following subjects were raised: somatic embryogenesis, haploid production, intergenetic hybridisation and transformation. Upon conclusion of all the results published to date, we may say that the progress of biotechnology in the case of rye is very limited in comparison to other cereals. However, a lot of work and efforts were involved to obtain the aforementioned result. Taking into account the progress in plant genetics and molecular biology, the authors deeply believe that the New Millennium will bring a brake-through in rye biotechnology.
Recent advances at Institute of Grassland and Environmental Research (Aberystwyth, U.K.) in cytogenetics of the Lolium/Festuca complex places us in the advantageous position of being able to map genes of agronomic importance onto chromosome arms using fluorescence in situ hybridization (FISH). The ability to physically map genes leads to the capability for 'dissecting' quantitative traits into their different components and will lead to better understanding of the complex physiological processes involved and the identification of their genetic control. By tagging genes of interest, using molecular and morphological markers, it will be possible to select and combine suites of desirable genes in a single genotype and thus produce novel cultivars by conventional breeding procedures. Programmes for introgression depend on the relationships between species and on levels of chromosome pairing. Phylogenetic relationships within the Lolium/Festuca complex are being determined using both genomic in situ hybridization (GISH) and FISH. With recent advances in genetic manipulation within the Lolium/Festuca complex, opportunities now arise for gene transfer from Lolium and Festuca species into other important agricultural crops.
Microspores cultured in vitro can be reprogrammed to divide and produce a bipolar embryo. The reaction to stress treatment is a signal for inducing the sporophytic pathway, preventing the development of fertile pollen grain ? the gametophytic pathway. The ultimate goal is to convert each microspore from a heterozygous F1 plant to a doubled haploid plant so that a population of doubled haploids fully represents the genetic variability of the preceding meiosis.
Miscanthus sinensis is a promising species for biomass production. Influences of genetic and nongenetic factors on androgenesis induction efficiency were investigated. This is the first report on successful induction of pollen-derived callus in M. sinensis. The callus yield was strongly affected by genotype. A beneficial influence of cold pretreatment of spikes on androgenesis induction was observed. The highest yield of calli was obtained in cultures on a modified C17 medium. The results suggest that the high callus yield might be caused by the late culture initiation. The beginning of anther culture at the end of the flowering season caused a 17-fold increase in callus yield, in comparison to culture initiated at the height of the flowering season (August). It is likely, however, that the efficiency of androgenesis induction in the case of M. sinensis anther culture beginning in October could be related to a positive influence of growing donor plants in conditions of cooler and shorter day, i.e. 11-h day with temperature around 11oC and 13-h night with temperature around 5oC. Results of this study can significantly support the development of effective methods of M. sinensis haploidization, which could be used in crop improvement by breeding.
This review was prepared mainly on the basis of papers published after 1997. It describes the current knowledge on the use of gametic embryogenesis for haploid production in vegetable crops. Data on the results of research on androgenesis and gynogenesis are presented for different species, induction methods and factors affecting the efficiency of the processes.
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