Anther and Microspore Culture and Production of Haploid Plants

Anther and Microspore Culture and Production of Haploid Plants

Haploid and double haploid plants attract the interest of geneticists, plant embryologists, physiologists, and breeders. Their genetic characteristics make them an elegant experimental system for genetic studies as well as an integral part of breeding programs, especially in generating pure lines. Haploid plants can be induced by the male as well as female gametophytes. This article focuses on haploid plants derived from the male gametophyte, i.e., anther and microspore cultures, concisely discussing the origin of haploid plants and techniques applied to their production, after a historical overview.

The development and viability of pollen play a key role in the fertility of plants. Besides its importance in sexual reproduction, pollen can be used for haploid plant production. Haploid plants are genetically characterized as plants containing only one set of chromosomes. The haploid state occurs due to the reduction of zygotic (diploid) chromosome number to the gametic (haploid) number during meiosis. In nature, haploid plants appear via abnormal fertilization, i.e., chromosome elimination or mispairing during the crossing-over. Haploid plants are sterile and therefore doubling of the chromosome set is required to produce fertile plants, which are called double haploids (DHs) or homozygous diploids. Two basic genetic features make DHs distinct for genetic studies and breeding.

The best-known application of haploids is the F1 hybrid system for the production of homogeneous hybrid varieties. The DH lines are also used for targeted genetic manipulation, mutant breeding, and selection, which considerably reduces the time required for the production of new cultivars.

Historical Overview
In 1922, Blakeslee and co-workers first discovered the appearance of natural haploid embryos and plants, which were derived from gametophytic cells of Datura stramonium.  To date, naturally occurring haploid plants are described in about 100 species of angiosperms. In 1954, Tucker, for the first time, observed that mature pollen grains of a gymnosperm Ginkgo biloba can be induced to proliferate in culture to form a haploid callus, but the direct formation of embryolike haploid structures from another culture of Datura innoxia was first reported by Guha and Maheswari in 1964.

Their experiments clearly demonstrated the feasibility of induction of haploid structures from anther tissues. In 1967, Bourgin and Nitsch succeeded in producing the first haploid plants from cultured anthers of Nicotiana sylvestris and Nicotiana tabacum. Later in 1974, the first description of microspore culture was also made by Nitsch. Since then, the techniques of microspore and anther cultures were optimized for a wide range of economically important dicotyledonous and monocotyledonous plants.

Origin of Haploids
Androgenesis Two basic strategies are applied to the induction of haploids from higher plants: in vivo and in vitro induction by various physical, chemical, or biological stimulants. The first method for haploid production, developed by Kasha and Kao in 1970, is based on chromosome elimination in hybrid embryos. This methodology exploits the fact that when two unrelated plant species are crossed, the chromosome sets of both parents fail to pair during the crossover stage of meiosis. For example, with crosses between common barley (Hordeum vulgare) and its wild ancestor (Hordeum bulbosum), the chromosomes of H.

Anther and pollen culture represent the major techniques for in vitro induction of haploid plants. The development of haploid plants can be induced from pollen via embryogenesis. The formation of embryos from the androgenic (male) tissues is called androgenesis. In this case, the microspores are switched from their normal gametophytic fate to sporophytic development.

Different physical and chemical stimuli have been studied for the induction of androgenesis. The most efficient and widely applied techniques include
1) cold pretreatment of spikes;
2) starvation—the cultivation of dissected anthers in media without carbon source; and 
3) incubation under higher temperatures. 

Genotype, physiological state of donor plants, stage of microspore development, culture media, and culture conditions are also important determinants of androgenesis. The anthers containing microspores in the mid- or late-uninucleate stage are most suitable for the induction of androgenesis. 

Of the different media components, the carbon source, its concentration, and the ratio of nitrate and ammonium ions (NH4 +) are important in achieving embryogenesis and the development of green plants from microspores.[7,8] Based on these findings several basal media were developed for androgenic cultures. Phytohormones, especially the content of cytokinins and auxin; aeration and permanent supply of fresh, well-buffered medium; increased osmotic pressure; and temperature are all critical for successful establishment of androgenic cultures. Regeneration of haploid plantlets from androgenic cultures can be achieved by direct embryogenesis from microspores or via organogenesis.

The technique of another culture is relatively simple and efficient and requires minimal facilities. The androgenesis can be induced by pretreating whole inflorescences (cold pretreatment) or by pretreating dissected anthers (high temperature, starvation). Anthers are cultured on a solid or in a liquid medium on a rotary shaker at 50–60 rpm. The cultures are kept at 24–27C.

First, anthers are cultured on the callus induction medium for about 2 weeks in darkness and subsequently transferred to a regeneration medium containing phytohormones and organic substances at 16-hour photoperiods (2000–8000 lux) for shoot regeneration. Developing plantlets are then transferred to a rooting medium containing a lower concentration of carbohydrates and other nutrients. The regeneration frequency of androgenic cultures is usually very high. In barley, it ranges from 4.8 to 50 green plants per single anther.

Plant breeding companies routinely use another culture for the production of haploid plants. The only disadvantage of this technique is the regeneration of plants with different ploidy due to the presence of both gametophytic and sporophytic cells in the culture.

The technique of microspore (pollen) culture was developed more recently than anther culture. In this technique, pollen grains are separated from the anther tissues and cultured in a liquid medium. Microspores provide haploid single cells that can be utilized for various biological studies. Different techniques are applied for the isolation of microspores.

 The most efficient is technique of microlending, in which small pieces of the inflorescence are put in a blender and quickly cut to release microspores into the isolation solution. The crude preparation is filtered through a sieve and the microspore suspension is centrifuged to separate microspores. The plating density (the number of viable microspores per volume of medium) is an important factor in the induction of androgenesis. The optimal population density depends on the genotype, the quality of donor material, and the isolation technique. The induction of androgenesis occurs either while they are still inside the spikes (cold pretreatment) or directly after the isolation (high temperatures, starvation). Microspores are cultured in a liquid induction medium on a rotary shaker.

They are kept in the dark at 24–27C for 3–4 weeks. The emerging calli of visible size are transferred to a solidified medium. Further cultivation of microspore-derived cultures is similar to those derived from the dissected anthers. The isolated microspore culture offers the possibility of combining selection procedures with the advantages of a haploid system. The nutritional requirements of the isolated microspores are much more complex than those of dissected anthers. The use of isolated microspore culture finds wide application in different fundamental studies and provides greater opportunities for cells.

Pollen (microspore) and another culture can be used for the production of haploid and diploid plants. Haploid plants are valuable material for breeding new varieties and biotechnological applications. Fertile diploid plants represent an essential source for producing pure inbred lines, which are homogeneous and show no segregation. Haploid and diploid plants considerably accelerate and simplify breeding and selection processes. 

Using the method of haploidization it is possible to obtain homozygosity for genes in cases where this is normally difficult to achieve, for example for self-incompatible alleles. Haploid cell cultures are also useful material for mutation analysis and cell modification. Microspores as single cells represent an ideal system for in vitro cell selection and genetic manipulation. Microspores can be used for microinjection, electroporation, particle bombardment and cocultivation with Agrobacterium tumefaciens resulting in transgenic haploid and homozygous plants.
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