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Biotechnological strategies for development of line
- 2. Introduction One of the goals in plant breeding program is the production of F1 hybrids. The main limitation of this scope is the long period required to produce inbred lines. The most time-consuming and labor-intensive aspect of developing these hybrids is the traditional inbreeding process that requires manual self pollination necessary to generate homozygous parental lines. This process requires six or more generations of inbreeding to establish adequately stable lines that can be used in hybrid combinations. In many species in vitro methods have allowed to speed up the production of homozygous lines, as alternative to the slower inbreeding process. Haploid plants can be obtained by anther, non-fertilized ovule, ovaries or whole flowers buds culture.
- 3. The haploid chromosome number means that meiotic recombinations and recessive gene effects are manifested at plant level. Then, spontaneous or induced chromosome doubling consent the regeneration of doubled haploids (DHs) homozygous materials, with restored fertility, that can be used in different breeding strategies. Because all the alleles of DHs lines are fixed, selection e.g. for quantitative characters is often more reliable than in conventional populations.
- 4. Haploids Haploids are plants (sporophytes) that contain a gametic chromosome number (n). Blakeslee first described this phenomenon in Datura stramonium in 1922. This was subsequently followed by similar reports in tobacco (Nicotiana tabacum), wheat (Triticum aestivum) and several other species (Forster et al., 2007). The potential of haploidy for plant breeding arose in 1964 with the achievement of haploid embryo formation from in vitro culture of Datura anthers ( Guha and Maheshwari, 1964, 1966)
- 5. Production of haploids and doubled haploids Haploids produced from diploid species (2n=2x), known as monoploids, contain only one set of chromosomes in the sporophytic phase (2n=x). They are smaller and exhibit a lower plant vigour compared to donor plants and are sterile due to the inability of their chromosomes to pair during meiosis. To propagate them through seed and to include them in breeding programs, their fertility has to be restored with spontaneous or induced chromosome doubling. The obtained DHs are homozygous at all loci and can represent a new variety (self- pollinated crops) or parental inbred line for the production of hybrid varieties (cross- pollinated crops).
- 6. The main factors affecting haploid induction and subsequent regeneration of embryos are: genotype of the donor plants, physiological condition of donor plants (i. e. growth at lower temperature and high illumination), developmental stage of gametes, microspores and ovules, pre-treatment (i.e. cold treatment of inflorescences prior to culture, hot treatment of cultured microspores), composition of the culture medium (including culture on “starvation” medium low with carbohydrates and/or macro elements followed by transfer to normal regeneration medium specific to the species), physical factors during tissue culture (light, temperature).
- 7. Haploid Techniques Induction of maternal haploids In situ induction of maternal haploids:-In situ induction of maternal haploids can be initiated by pollination with pollen of the same species (e.g., maize), pollination with irradiated pollen, pollination with pollen of a wild relative (e.g., barley, potato) or unrelated species (e.g., wheat). Wide hybridization:- Wide crossing between species has been shown to be a very effective method for haploid induction and has been used successfully in several cultivated species. In vitro induction of maternal haploids – gynogenesis:- In vitro induction of maternal haploids, so-called gynogenesis, is another pathway to the production of haploid embryos exclusively from a female gametophyte. It can be achieved with the in vitro culture of various un-pollinated flower parts, such as ovules, placenta attached ovules, ovaries or whole flower buds. Androgenesis can be induced with in vitro culture of immature anthers, a technically simple method consisting of surface sterilization of pre-treated flower buds and subsequent excision of anthers under aseptic conditions.
- 8. a) Induction of maternal haploids In situ induction of maternal haploids:- In situ induction of maternal haploids can be initiated by pollination with pollen of the same species (e.g., maize), pollination with irradiated pollen, pollination with pollen of a wild relative (e.g., barley, potato) or unrelated species (e.g., wheat). Pollination can be followed by fertilization of the egg cell and development of a hybrid embryo, in which paternal chromosome elimination occurs in early embryogenesis or fertilization of the egg cell does not occur, and the development of the haploid embryo is triggered by pollination of polar nuclei and the development of endosperm.
- 9. 2. Wide hybridization:- Wide crossing between species has been shown to be a very effective method for haploid induction and has been used successfully in several cultivated species. It exploits haploidy from the female gametic line and involves both inter- specific and inter- generic pollinations. The fertilization of polar nuclei and production of functional endosperm can trigger the parthenogenetic development of haploid embryos, which mature normally and are propagated through seeds (e.g., potato). In other cases, fertilization of ovules is followed by paternal chromosome elimination in hybrid embryos. The endosperms are absent or poorly developed, so embryo rescue and further in vitro culture of embryos are needed (e.g., barley). The ‘bulbosum’ method was the first haploid induction method to produce large numbers of haploids across most genotypes and quickly entered into breeding programs.
- 11. 3. In vitro induction of maternal haploids – gynogenesis:- In vitro induction of maternal haploids, so-called gynogenesis, is another pathway to the production of haploid embryos exclusively from a female gametophyte. It can be achieved with the in vitro culture of various un- pollinated flower parts, such as ovules, placenta attached ovules, ovaries or whole flower buds. Although gynogenetic regenerants show higher genetic stability and a lower rate of albino plants compared to androgenetic ones, gynogenesis is used mainly in plants in which other induction techniques, such as androgenesis and the pollination methods above described, have failed. Gynogenic induction using un-pollinated flower parts has been successful in several species, such as onion, sugar beet, cucumber, squash, gerbera, sunflower, wheat, barley etc. but its application in breeding is mainly restricted to onion and sugar beet.
- 12. b) Induction of paternal haploids – Androgenesis:- Androgenesis is the process of induction and regeneration of haploids and double haploids originating from male gametic cells. Due to its high effectiveness and applicability in numerous plant species, it has outstanding potential for plant breeding and commercial exploitation of double haploids. Its major drawbacks are high genotype dependency within species and the recalcitrance of some important agricultural species, such as woody plants, leguminous plants and the model plant Arabidopsis thaliana.
- 13. Androgenesis can be induced with in vitro culture of immature anthers, a technically simple method consisting of surface sterilization of pre-treated flower buds and subsequent excision of anthers under aseptic conditions. The anthers are inoculated and cultured in vitro on solid, semi-solid or liquid mediums or two- phase systems (liquid medium overlaying an agar-solidified medium). The method relies on the ability of microspores and immature pollen grains to convert their developmental pathway from gametophytic (leading to mature pollen grain) to sporophytic, resulting in cell division at a haploid level followed by formation of calluses or embryos.
- 15. Applications of gynogenesis In practice, production of haploid plants by gynogenesis is not used as frequently as androgenesis in crop improvement programs because: The dissection of unfertilized ovaries and ovules is quite difficult; and The presence of small numbers of ovaries per flower compared to the large number of pollen grains in an anther. Now, if there is androgenesis which is highly successful, why should you use gynogenesis? Gynogenesis is mainly used to produce haploids of male-sterile plants or of dioecious plant species (each individual has only female or male flowers), such as mulberry and cannabis.
- 16. The advantage this method offers you over androgenesis is the low occurrence of albinos (white plants without chlorophyll) in cereals like wheat, maize and rice. In crop plants like onion, sugar beet, and melon, gynogenesis is the only way to produce haploid plants. Also, in some plant species such as rice, gynogenesis is more efficient than androgenesis.
- 17. Haploid Plants from Tissue Culture Specialized plant tissue culture methods have enabled the production of completely homozygous breeding lines from gametic cells in a shortened time frame compared to conventional plant breeding. Plants from gametic cells of an F1 hybrid represent a gametic array each having a different genetic contribution from the parents. Lines exhibiting the desired characteristics are chosen for large- scale field trials as a prelude to commercialization. Although the number of new plant varieties developed via this method has been limited, refinement of tissue culture techniques has extended the range of crop species from which haploid plants have been produced as well as the efficiency resulting in large-scale haploid plant production. Several varieties developed via this method are grown on considerable acreage while others are being tested as candidates to replace varieties developed by conventional methods.
- 18. Haploid Plants from Tissue Culture Application in Crop Improvement Haploid plants are of great interest to geneticists and plant breeders. Haploids offer geneticists the opportunity to examine genes in the hemizygous condition and facilitate identification of new mutations. Plant breeders value haploids as a source of homozygosity following chromosome doubling from which efficient selection of both quantitative and qualitative traits is accomplished (Griffing, 1975). As a tool in crop improvement strategies, it is imperative that haploids are produced from individual heterozygous genotypes in sufficiently large numbers to compensate for undesirable gene combinations that result from linkage and random assortment via meiosis. Just as plant breeders advance large populations through several generations (to maximize variability in later generations), application of haploidy in plant breeding is dependent on the ability to produce a haploid population of sufficient size to accommodate selection of desired gene combinations.
- 19. What is a doubled haploid (DH) plant? In a DH plant, the chromosome set of a haploid plant has been doubled spontaneously or artifi cially. Chromosome doubling is necessary since haploid plants are generally frail, have reduced organ size and are not fertile. The most commonly used chemical agent to render haploid plantlets diploid is colchicine, which blocks cell division without blocking chromosome . Quick guide duplication. This treatment acts like a ‘copy–paste’ of the haploid genome into a diploid genome. Consequently, in DH plants all loci are homozygous. Chromosome doubling creates ‘pure’ homozygotes or fully inbred lines.
- 20. Why is doubled haploid technology impactful for agriculture? Doubled haploid technology comprises both the production of haploid plants and the chromosome doubling process. It has become an important tool in plant breeding, since it shortens the time needed to create pure homozygous lines, which can either be released directly to farmers as cultivars or used as genitors (inbred lines) for the production of hybrid seeds. The primary advantage of DH plants is to possess a phenotypic stability due to the fact that all alleles are in a homozygous state. In short, DH technology increases the efficiency of plant breeding.
- 22. What future improvements are needed for DH technology? In plant breeding, and apart from maize, the production of DH plants requires at least an in vitro-based process, the success of which remins highly species- and genotype-dependent, as well as labor-intensive and time- consuming. Moreover, the DH technology has not yet been applied to all breeding programs, and there are still some major crop species (e.g., soybean, tomato, sunflower) that are recalcitrant the currently available procedures, or for which present protocols are not efficient enough. The understanding of the maize in situ system should help to extend the DH technology to more species or breeding programs by either directly knocking out the functional ortholog of the maize gene, or if needed by transferring the cellular and molecular knowledge acquired on maize.