Wheat is one of the most important food crops in the world, and its importance will increase with increasing global population. Common wheat originated from the spontaneous amphiploidization of tetraploid T. turgidum (2n=28, AABB) with the diploid Aegilops tauschii (2n=14, DD).
However, it is believed that only a few accessions of the donor species contributed to the evolution of common wheat, thereby excluding the larger genetic diversity of its parental species.
Plant breeding using the doubled-haploid (DH) system is a key technology used to speed up the breeding process. In traditional plant breeding, it generally takes at least five generations after crossing before a sufficiently homozygyous population is obtained, while DH produces a homozygous plant in one generation. Therefore, DH technology dramatically increases the speed of the inbred developmental processes by reducing several time-intensive generations of inbreeding and by making phenotyping and genotyping more predictive. DHs have thus become a key component to the product development process1.
Haploid technology is also an important biotechnological tool in breeding programs, as well as in genetic and developmental studies. It is significant that the totipotent nature of a haploid cell is exploited in several facets of biological research. In those genotypes where these technologies have been successfully applied, improvement programs receive significant advantages. Worldwide, DH technology has become routine in a wide range of crops, with the largest number of varieties derived from DH from barley, followed by rapeseed2. Haploid techniques can be a valuable tool for the rapid production of homozygous transgenic plants, thus assisting in the establishment of transformation techniques. Additionally, DHs are indispensable in species with inbreeding depression, in perennial woody species with long juvenile periods, and in plants with barriers to self incompatibility and dioecism3.
Use of Doubled Haploids in Plant Breeding
Among the various techniques in crop improvement, the use of haploids is considered to be the most significant. To produce DHs, various approaches have been followed with varying degrees of success; namely, in vivo modified pollination methods (chromosome elimination, in situ pollination with irradiated pollen, pollen from a triploid, etc.), or in vitro culture of gametophytes (gynogenesis, pollen embryogenesis through anther and microspore culture). DH systems are also the method of choice for mutant selection, due to the ease of selection and fixation of mutations and the desired recombinants, especially when quantitative traits are concerned.
Haploid technology has tremendous use for accelerating breeding technologies when combined with Marker Assisted Selection (MAS). MAS, when combined with double haploidy, is a timesaving method of performing backcross conversion to select an elite line with a particular trait. By combining molecular markers and DHs, it is possible to stack resistance genes. DHs are also useful for Bulked Segregant Analysis (BSA), which compares individuals from two extreme populations for a given trait. DH populations can be used as permanent mapping populations because they are stable and constant. DHs offer a unique opportunity to improve selection efficiency because selection is based on gametophytic instead of sporophytic cells.
When combined with somatic hybridization, DHs can also lead to selection of desired genotypes. Alternatively, protoplast fusion techniques can be utilized to combine the genomes (nuclear or cytoplasmic) of different lines. DHs are also indispensible for reverse breeding (RB), a novel technique designed to directly produce parental lines for any heterozygous plants. DH reduces genetic recombination in the selected heterozygote by eliminating meiotic crossing over and generating in vitro homozygous doubled haploids. Reverse breeding can also be used to generate chromosome substitution lines, and it allows the reshuffling of chromosomes between two homozygous plants in all possible ways4. Reverse breeding also aids in understanding the genetic control of chromosome pairing inherently present in allopolyploids, such as bread wheat, durum wheat, and oats2.
DHs provide a link between conventional breeding and genomics. DHs are also a key feature in genomic programs for integrating genetic and physical maps. They have widespread application in quantitative genetics and SNP discovery and in establishing chromosome maps, resulting in reliable information on the location of major genes and QTLs for important traits. DHs have also played an important role in the development and exploitation of structured mutant populations for forward and reverse genetics approaches, especially TILLING. The haploid chromosome set, added to embryogenic competence, makes microspores one of the most attractive cellular targets for transfer and stable integration of recombinant DNA into plant genomes by both direct gene transfer and Agrobacterium-based methods.
Doubled Haploids and Wheat Transgenic
In addition to its role in plant breeding, chromosome doubling may also be used in genetic transformation studies, and DHs are prime targets for transformation and genetic manipulation. Transgenes can be applied to both haploid and DH plants, resulting in stable transgenics, which has led to a resurgence of interest in haploid research, not only for their application and utility for crop improvement programs, but also as an important tool to study gamete and embryo biology and genetics involving gene mapping, gene discovery, and identification5. Although discovered in 1922, this technique in now yielding real and tangible results in both basic and applied biology. Wheat cultivars developed from DHs have been released for cultivation in Canada, China, Europe, Brazil, etc2.
Wheat transformation is a lengthy process, and for most introduced genes it is essential to produce homozygous lines of transgenic plants to characterize the effects of a phenotype. The identification process requires the screening of numerous plants to separate homozygous from heterozygous transgenics in the T1 and T2 generations, and it is therefore labor-intensive6. Anther culture is not only useful for the rapid generation of haploids, it allows genetic and functional analysis when coupled with transgenic technology. Anther culture has been used successfully to regenerate fertile, non-transgenic wheat. Success of the procedure is dependent on various factors, including the culture medium and growth conditions during anther pre-treatment, callus formation, and plantlet regeneration.
Colchicine treatment during isolated microspore and anther culture enables chromosomal doubling, resulting in the production of homozygous dihaploids. During transformation, haploid embryos may be produced and used as targets for bombardment procedures or co-cultivated with Agrobacterium to obtain transgenic double haploids that are homozygous for the transgene(s). However, only a few studies have been undertaken towards producing transgenic haploid plants in wheat, and none of these studies reports the production of transgenic doubled haploid plants.
Agrobacterium-mediated transformation has several desirable features over direct DNA delivery, such as the introduction of only a few copies of the genes and greater chances of recovery of single copy transgene, high co-expression of introduced genes, easy manipulation of the transgene, and the ability to transfer large segments of DNA with minimal rearrangements and at a lower cost7,8. With this in mind, doubled haploid plants were produced in wheat through anther culture and Agrobacterium-mediated transformation in commercial Indian bread wheat varieties.
In work performed at the Department of Plant Molecular Biology, University of Delhi, India9, wheat anther culture was investigated in six commercial Indian genotypes: UP2338, PBW343, HD2428, HD2329, HD2687, CPAN1676, and Chinese Spring. However, only CPAN1676 responded well to haploid embryo induction. This variety has been shown to respond excellently towards in vitro regeneration in different tissues employed as explants10. Therefore this cultivar was used for induction of haploids and subsequent transformation studies.
The donor tillers containing microspores at the late uninucleate stage were cut between the 2nd and 3rd nodes. Selected tillers with spikes in the penultimate leaf sheath were kept in the dark in Erlenmeyer flasks with tap water for cold pretreatment at 4° C for two weeks. Embryo-like structures (ELS) were visible as white beads in the liquid medium 4 – 6 weeks after isolation. The axenically isolated haploid embryos of CPAN1676 and Chinese Spring varieties were inoculated with Agrobacterium [LBA 4404 (pCAMBIA2301:HVA1)] for three hours, after which the embryos were placed on Petri plates containing co-cultivation medium4.
The Agrobacterium co-cultivated haploid embryos were incubated on regeneration medium supplemented with kanamycin (50 mg/L) for 3 - 4 weeks in a culture room. After one month of incubation, both green and albino plantlets emerged. For chromosome doubling, plantlets were treated with colchicine for 5 h, followed by overnight washing. Regenerated plantlets were checked for transgene integration in T0 generation, and positive transgenic haploid plants were selected. Stable transgenic doubled haploid plants were obtained and transgene expression was monitored through the T4 generation. Doubled haploid transgenic plants grown under post-anthesis water limitation had faster seed germination and seedling establishment and showed better drought tolerance than to non-transgenic, doubled haploid plants, as measured by percent germination, biomass accumulation, seedling growth, nitrate reductase enzyme activity, and plant yield. The transgene (HVAI) was stably integrated and expressed over generations, and transgenic plants had higher tolerance to simulated water stress. HVA1 is a group 3 LEA gene used in transformation for enhancing drought tolerance in both monocots and dicot plants. Previous results indicate the potential of LEA genes as molecular tools for the genetic improvement of plants for water limiting environments11.
We also found that the transgenic wheat, doubled haploid plants overexpressing HVA1 gene had much better root and shoot growth, even under 400 mM of mannitol. Though the exact mode of action of LEA proteins is not clear, several possibilities exist to explain their function. Reports indicate a possible role of LEA proteins in dehydration tolerance through the maintenance of protein and/or membrane structure, the sequestration of ions, and the binding of water, and they function as a chemical chaperon. Thus overexpression of HVA 1 may improve protein and membrane integrity, leading to better growth under water limiting conditions.
Photosystem II plays a key role in response to environmental changes. The efficiency of PSII can be measured by chlorophyll fluorescence, thereby indirectly indicating the photosynthetic capacity of plants. Photoinhibition in flag leaf shows decreased Fv/Fm due to post anthesis water stress. At field capacity, the flag leaves of WT plants displayed a drastic decrease in PSII activity after 10 days of water limitation, as measured by Fv/Fm and electron transport rate, while transgenic DH plants fared better in both PSII activity and the relative water content of the flag leaf.
Post-anthesis drought stress reduces wheat yield and number and weight of kernels per spike. Those characteristics, along with 1000 grain weight, were suggested as important criteria for selecting drought tolerant cultivars. In the present study we found that transgenic double haploid plants perform much better with respect to these parameters, especially seed weight per spike, than the WT plants. Therefore, the present study indicates the utility of DHs in raising stable transgenic with the gene(s) of interest.
Perspectives
It takes several years from the time of the initial transformation event to the generation of a variety with superior agronomic traits for commercial use. The production of transgenics, screening for desirable phenotypes, followed by the generation of homozygous lines are labor and resource intensive. We thus show the practical feasibility of generating stable transgenics for a desirable trait via doubled haploids and homozygous line generation in a crop species, saving both time and resources. Plants are stable for the transgene and show a consistent expression of the introduced gene. This also happens to be the first report of the production of double-haploid transgenic wheat through Agrobacterium-mediated transformation in a commercial cultivar for a desirable trait. However, further work is needed to develop a comparatively genotype-independent, anther culture protocol in wheat. Combining haploidy with other technologies, such as MAS, induced mutagenesis, and transgenic technology, would accelerate crop improvement. The totipotent nature of the microspore can be exploited for basic research into several facets of genetics and genomics, and the incorporation of transgenes would be useful in functional genomics of wheat and other allopolyploids of agronomic importance.
References
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Paramjit Khurana and Harsh Chauhan
Department of Plant Molecular Biology, University of Delhi South Campus
New Delhi-110021, India
param@genomeindia.org
