Protocol for effective doubled haploids production by androgenesis for polish breeding materials of wheat (Triticum aestivum L.)
Summary: In the process of androgenesis, genetic information necessary for the regeneration of the whole parental organism, is expressed in a microspore, a haploid (n) cell of male gametophyte, which naturally (in planta) is determined to develop into pollen grain. Mature pollen grains contains male gametes which take part in the process of double fertilization and formation of a diploid zygotic embryo possessing both parental genomes. However, under a very specific conditions, microspores can induce an alternative pathway of development. Omitting fertilization, stress treated and transferred to in vitro culture microspores can developed into androgenic embryo-like structures (ELSs) which possess only haploid genome (n). These haploid ELSs can regenerate into fully functional haploid plants. The final effect of androgenesis induction are so called doubled haploids (DHs), received by spontaneous or induced chromosome doubling. As totally homozygous organisms, DHs are highly advantageous in many research areas, biotechnology and bioengineering. The most beneficial is the deployment of DHs into breeding practice, as total homozygosity is highly desirable feature. Especially that, its gaining by classical breeding methods is practically impossible. High level of homozygosity is a prerequisite for a new variety registration, and is usually received by series of subsequent inbreedings and selections, repeated for several generations. Shortening a breeding-cycle for a few years could have a great impact on competitiveness of breeding companies.
DH production in wheat, the object of proposed study, is possible at present, however, with the use of the another method, by so called ‘distant crosses’. These procedures are complex, highly time-, labour- and cost-consuming. Foreign breeding companies, which offer commercial production of wheat DHs probably possess efficient procedures based on androgenesis however none of them have ever been made public, probably because of some confidentiality agreements. Standard methods that have evolved for particular, usually responsive genotypes, are used repeatedly despite the fact of their low effectiveness in broad spectrum of breeding materials. Even effective androgenesis induction (high number of ELS) observed in numerous genotypes, does not mean high final efficiency of the process (high number of green doubled haploid plants). It is the effect of low regeneration potential of produced ELS and high frequency of albinotic, chlorophyll-lacking plants, not able to function independently, outside in vitro cultures. Up to now, only one- or two-factor(s) modifications with small final effects on the process or of high genotype-selectivity have been reported. Proposed project is aimed on comprehensive work out of each phase of the procedure, what should result in significant improvement of DH technology effectiveness and its optimization for wide spectrum of breeding materials.
The main factor responsible for triggering an alternative microspore developmental pathway is a stress treatment. Next phases of microspore development require a transfer to in vitro culture and a supply of all substances and factors important for proper functioning, differentiation and regeneration. Both, the type of a stress, its intensity and duration as well as in vitro culture conditions determine the effectiveness of androgenesis (the number of produced DH lines) and should be precisely optimized. Various stress factors (high or low temperature, osmotic stress, starvation) and parameters of stress treatment (intensity and duration) will be analysed enabling the selection of the best androgenesis-initiating pre-treatment procedure. Several substances, which reduce oxidative stress and stimulate antioxidative system activity, modify endogenous phytohormones level, stimulate chlorophyll biosynthesis and control the process of programmed cell death (apoptosis) will be used. Some reports, including papers published by scientific team of IPP PAS, confirm the importance of cell redox potential and beneficial effect of antioxidants. However, up to now all studies and all modifications concerned the stage of in vitro culture. For the first time, the preliminary phase of androgenesis induction will be optimized, what should result in increase of microspore viability and its stress tolerance just before isolation and transfer to in vitro conditions. Next, conditions of in vitro androgenesis-induction, regeneration and rooting will be optimized enabling more efficient green plant regeneration and higher genome doubling frequency. For these purposes, for the first time, some substances known as biostimulators will be selected on the base of newest literature data and results received by IPP PAS scientific team.
Newest reports and conclusions from our own studies suggest that significant improvement of androgenesis effectiveness is possible. What’s more, it is expected that the results that will have been produced in the frame of the project should have universal character and will find application in other, still recalcitrant, plant species.