Haploid Techniques

Abstract

During a systematic screening of reactions of various parts of flowers of Datura innoxia cultured in vitro, Guha and Maheshwari (1964) observed the development of haploid plants from anthers containing immature microspores. Later, especially tobacco anthers were extensively investigated by various research groups, and mostly microspores of this species were used to test the suitability of this technique for hybrid breeding programs. Meanwhile, the production of haploids of several hundreds of plant species has been reported in the literature. Of these, only a few have been used, with limited success, in breeding programs; some reasons for this will be discussed later.

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During a systematic screening of reactions of various parts of flowers of Datura innoxia cultured in vitro, Guha and Maheshwari (1964) observed the development of haploid plants from anthers containing immature microspores. Later, especially tobacco anthers were extensively investigated by various research groups, and mostly microspores of this species were used to test the suitability of this technique for hybrid breeding programs. Meanwhile, the production of haploids of several hundreds of plant species has been reported in the literature. Of these, only a few have been used, with limited success, in breeding programs; some reasons for this will be discussed later.

Application Possibilities

A prerequisite to use the heterosis effect reproducible in hybrid breeding is the availability of homozygous parent lines. The production of such inbred lines requires many back-crossings of heterozygotic parent material. Inbred lines with desired properties are also required for outbreeding plant species. A considerable reduction of the time required to produce such plant material can be achieved by the use of haploids. Haploid higher plants are infertile, and therefore before haploids can be used in breeding programs, a diploidization is required (e.g., using colchicines). With the methods described later, such dihaploid plants can be ideally produced within 1 year. Considering the time necessary for the selection of haploid plants for further use in hybrid breeding, and the propagation of the selected plants (usually by rooting), one needs about 5 years to produce the first hybrid seeds. A time schedule to produce a tobacco hybrid, out of the pioneer days of the technique, can be seen in the following summary:

• 1976: anther culture and raising of haploid plants

• 1977: propagation by cuttings, and selection of diploid twigs for rooting

• 1978: propagation of dihaploid plants by rooting

• 1979: crosspollination of selected dihaploid parents

• 1980: planting of F1-hybrids.

Various methods have been used for the diploidization of haploids, of which only two will be mentioned here. The method of Jensen (1974, 1986), originally described for barley, uses young plants (five-leaf stage) submersed in a solution of 0.1% colchicines (without chloroform) containing 2% DMSO, and 0.2–0.5 ml Tween for 5 h in the light at 20–22°C. For dicots, the method of Ockendon (1986) will be described. Here, a colchicine solution of 0.05% is applied directly to the shoot apex of a young plant, with a microsyringe (10 µl). An alternative is the application with cotton wool soaked with the colchicine solution. For both methods, a success of more than 70% has been reported.

Actually, three experimental approaches are available to produce haploid plants: (1) the anther culture method, or that of microspores derived thereof, (2) the embryogenesis of isolated unfertilized egg cells, and (3) production from hybrids of species from which the set of chromosomes of one parent has been eliminated during development. Before a detailed description of the first method will be given, the other two will be briefly summarized.

The now classical example for the utilization of interspecies hybridization is a crosspollination of Triticum aestivum and Hordeum bulbosum (the bulbosum method). Here, one set of chromosomes (Triticum) is organized at metaphase, whereas that of the other parent remains unorganized in the cell, and is lost during the following cell divisions. For the bulbosum method, also Hordeum vulgare can be used as a suitable parent (Fig. 6.1 ), and haploids, i.e., dihaploids, obtained by this method were soon used in breeding programs (Kasha and Rheinsberg 1980). Five years after initiation of this breeding program, the first new variety (Mingo) was available. During the last decades, many more examples of interspecies hybrids have been reported, and a summary can be obtained from Gernand et al. (2005). In this paper, experiments are described to also follow the fate of the chromosomes of the “loosing” partner of the hybridization of wheat × pearl millet, by elimination. All pearl millet chromosomes were eliminated between 1 and 3 weeks after pollination. Chromosome elimination involves the formation of nuclear extrusions, and the post-mitotic formation of micronuclei the chromatin of which is fragmented later.

Fig. 6.1

Results of crossbreeding of Hordeum vulgare (V) and Hordeum bulbosum (B). Note the high percentage of fruit set (50–90%) with 20–100% of embryo development (after Jensen 1986)

As will be discussed later, plants produced by such methods exhibit a higher degree of cytogenetic stability, compared to those derived by the anther culture method. Whereas in principle the anther or microspore method is applicable to all plants, the bulbosum method depends on the availability of suitable parent species.

Although basically there exists the possibility of obtaining haploid plant material by culture of immature egg cells, due to the easier handling of anthers their microspores are preferred. A summary on gynogenesis was published some years ago by Keller et al. (1987), in which nine successful example are listed: Hordeum, Triticum, Oryza, Beta vulgaris, Gossypium, Ephedra, Nicotiana, Crepis, and Lolium. During recent years, there has been intensified research to exploit the possibility of gynogenesis—let’s wait and see!

A comparison of androgenetic and gynogenetic derived plants will be given below. An example is the use of protoplasts of dissected ovules. Whereas unfertilized protoplasts of barley did not divide, those fertilized developed into microcalli, and if co-cultivated with microspores undergoing embryogenesis, these developed embryonic structures and eventually fertile plants. If cultured alone, these microcalli degenerated (Holm et al. 1994). Another way is to use a floral-dip method, as for Arabidopsis for genetic transformation with Agrobacterium tumefaciens carrying the gus gene and the 35S promoter. Five days or more before anthesis gus activity was detected only in developing ovules, and not in pollen or pollen tubes. This selectivity could be due to the special developmental path of Arabidopsis flowers. Here, the gynoecium develops as an open structure to form closed locules about 3 days before anthesis (Desfeux et al. 2000).

Reports can be found describing superiority for androgenic plants, others for gynogenic, often only in one or the other trait. Androgenesis, however, was generally considered as more efficient than gynogenesis (Foroughi-Wehr and Wenzel 1993); the success of both techniques seems genetically controlled, and broad variations of genotypes can be observed. In a tobacco system, doubled haploids of either origin and their self progeny were about equal, but the androgenic material exhibited more vigor and was highly variable (Kumashiro and Oinuma 1985). A more recent paper compares androgenic and gynogenic monoploid plants of Solanum phurea (Lough et al. 2001). In contrast to gynogenic plants, androgenic plants had an increase in leaf size of 15–20%, and total tuber yield was about doubled to tripled. Plant height, however, was significantly reduced in androgenic lines. Gynogenesis is often employed to surpass the high percentage of albino plants often observed in androgenic systems.

Another possibility to obtain gynogenic plants could be the use of X-ray irradiated pollen for fertilization. This should make pollen inactive as gene donor, but still capable of inducing cellular division of the ovule. Plant material obtained without fertilization, and only with the maternal set of genetic material could be produced.

Physiological and Histological Background

In many publications, a stress requirement is described as a prerequisite to induce androgenic development. The requirement of a stress treatment depends on the plant species, as well as the species genotype. This can be starvation and osmotic stress induced by a mannitol supplement to the culture medium, as for barley, or a combination of starvation and heat shock for tobacco and wheat, or heat shock alone for rapeseed and pepper. Other stress factors can be colchicines, nitrogen starvation (Heberle-Bors 1983), gamma irradiation, or cold shock; a summary is given by Maraschin et al. (2005). In other reports, androgenesis can be induced without an obvious shock treatment. Considering the high concentration of sucrose (2% and more) in most media, already the transfer of isolated tissue invokes some osmotic stress. Furthermore, the confrontation of cells in vitro with often rather high concentrations of phytohormones in embryogenic systems has to be considered as stress factor. Here, positive influences of ABA on embryogenesis match those of osmotic stresses. Also for the induction of somatic embryogenesis, a number of stress factors are under discussion as being necessary, such as wounding, osmotic stress, starvation, and heavy metal ions. Often a separation of explants from their origin in the intact plant, as well as setting a wound at explantation are considered as stress factors, and as prerequisites to induce somatic embryogenesis. The relevance of these factors is discussed elsewhere. Actually, compared to the cells in the original mother plant, all in vitro culture systems incur stress conditions for the cells of cultured explants.

To induce the potential for somatic embryogenesis, the dedifferentiation, or rather transdifferentiation (or more modern, reprogramming) of microspores, egg cells, or somatic cells is a prerequisite that is brought about by the environment under in vitro conditions—e.g., nutrient media, and temperature.

Following Maraschin et al. (2005), androgenic development, and probably any other somatic embryogenesis, consists of three major phases: acquisition of embryogenic potential, initiation of cell division, and pattern formation of the dividing cells. Initially, a stress (or phytohormones) induces a reprogramming of cellular metabolism, including a repression of gene expression related to starch biosynthesis, and the induction of proteolytic genes and stress-related genes. This is followed by the activation of key regulators of embryogenesis—e.g., the so-called BABY BOOM transcription factor. After induction of cell division during pattern formation of embryos, programmed cell death seems also to play an essential role. This is apparently related to the loss of extracellular ATP (Chivasa et al. 2005), to date of unclear function.

Metabolic rearrangement can be brought about by the ubiquitin-26S proteosomal system, which degrades molecules; autophagy for degrading and recycling organelles, in animals via lysosomes, and in plants via vacuoles is involved. The breakdown products are eventually metabolized.

Ideally, the induction of androgenesis basically consists of an induction of somatic embryogenesis in microspores after isolation from the mother plant. In many cases, however, first the formation of a callus can be observed, in which later embryos develop. Even more often, root and shoot formation occurs first, and both join later to produce an intact plant.

At the suitable developmental stage (see, e.g., Fig. 6.2 ), surface-sterilized flower buds are opened under sterile conditions, and the anthers are severed by means of a forceps, and placed onto an agar medium (see 6.3 ). A suitable medium is given in Table 3.3 (see also NN medium). Two or 3 weeks later, the appearance of the first embryos can be observed (Fig. 6.3 ). Highly significant for success is the proper developmental stage of the anthers, i.e., their microspores. The optimal stage for microspores of tobacco is the first mitosis that produces the first vegetative and the first generative nuclei. As far as is known, the haploid embryo develops from the vegetative nucleus. Some rough correlation exists between the development of the flower bud, and that of the pollen, providing guidelines for the proper developmental stage to obtain the anthers for the experiment. For tobacco, this stage is reached as soon as the corolla can be seen emerging from the calyx (Fig. 6.2 ). By putting the severed buds overnight into the refrigerator prior to taking the anthers, a 10–15% increase in the generally low number of haploid plants (usually below 0.1%) can be reached.

Fig. 6.2

Isolation of anthers (after Reinert and Yeoman 1982)

Fig. 6.3

Various stages of anther culture. Top Anthers of Datura on agar, middle initiation of embryo development in a ruptured anther, bottom embryonic structures isolated from an androgenic anther (photographs by E. Forche)

Heberle-Bors (1983) reports results on some factors that are significant in determining the potential to perform androgenesis, and consequently the success per experimental setup. Tobacco was used as model. For a start, a dimorphism of pollen was observed in mature anthers. Unfertile pollen, called P-pollen, are embryogenic, and can be separated by density gradient centrifugation from fertile pollen. P-pollen occurs mainly under stress situations promoting male sterility. Examples for this are short day conditions, low temperature, or nitrogen deficiency. Under these conditions, the efficiency of the nutritive tissue of the anther, i.e., the tapetum, is restricted or disturbed, and the pollen can not develop to maturity. The amount of P-pollen can also be increased by an application of growth substances that promote male sterility (auxins, antigibberellins), and therefore its increase is not solely due to disturbance of the nutritional system of the anther. Furthermore, also plants with a genetically based male sterility usually exhibit a high potential of androgenesis. Beside influences of variations in the growth conditions of the parent plant on the success to produce haploid plants from its microspores, an experimental system to increase the production of haploid plants from normal and healthy mother plants is described by Moreno et al. (1989). Here, microspores at an early developmental stage (early two-nuclei stage) are cultured in a sugar-free nutrient medium (topreserve isoosmotic conditions, sucrose is replaced by mannose) for 1 week, resulting in a dominance of P-pollen. The abundance of haploid plants seems to be limited only by the survival rate of the pollen following this treatment.

Methods for Practical Application

Basically, two methods are available, which are practiced with many variations. In the first method, which seems to be the more original, whole anthers are placed on an agar medium, and in the second the anthers are cultured in a liquid medium in which the pollen is liberated by agitation. Using anthers of tobacco, an example for each method will be given.

Agar culture of anthers

• Severing the flower buds with a corolla length of 15–25 mm

• For surface sterilization, the buds are transferred into a hypochlorite solution (0.1% active chlorine) supplemented with some drops of Tween for 10–15 min

• Washing the buds at the sterile working bench with sterilized water, and transfer onto a sterile Petri dish

• Removal of the calyx and corolla with a flamed forceps

• Severing the anthers, and transfer into a sterile Petri dish. Gentle removal of the filaments.

• Transfer of the anthers onto an agar nutrient medium (Table 6.1, and see below; 5 ml medium per 50 × 18 mm Petri dish). The two pollen sacs should touch the surface of the agar, with the furrow oriented toward the air. The dishes are sealed with Parafilm.

• The cultures are transferred to a dark growth cabinet at 28°C

• At the appearance of the first embryonic stages after about 2–4 weeks, the cultures are illuminated (16/8 h, 20–25°C).

Table 1

Table 6.1 Agar medium for anther cultures (mg/l)

Additionally, a freshly prepared EDTA-Fe solution: 27.9 mg FeSO₄ × H₂O and 37.2 mg EDTA-Na

Of this culture medium, 5 ml is transferred into Petri dishes (50 × 18 mm) on which, after cooling, the anthers are placed.

This medium was suggested by Sunderland (1984), and originally contained neither phytohormones nor activated charcoal, as used in many systems later. A supplement of 0.5% charcoal, or of 1% naphthylacetic acid (NAA), however, often clearly increases the number of haploid plants obtained.

Liquid culture of anthers

This method includes a pre-treatment of the anthers before culture to stimulate the development of the microspores into plantlets. This pre-treatment consists of a “cold stress”:

• Transfer of the freshly isolated flower buds into a sealable container (Petri dish, polyethylene bag)

• Placement of the container for 3 weeks in the dark into the refrigerator (7–9°C). Following Sunderland, this “cold” treatment increases the number of haploid plants 10- to 15-fold.

• To obtain the anthers, the method described for agar cultures is followed

• Transfer of up to 50 anthers into a Petri dish (50 × 18 mm) containing 5 ml of the nutrient medium given above without agar. The anthers float on the surface of the liquid medium.

After a few days, a sufficient number of pollen falls out of the anthers, which can then be used again to start a new culture by transfer to another Petri dish.

After about 2 weeks, the first embryonic structures can be observed; until transplantable young plants are available, the nutrient medium has to be renewed several times.

Still not completely understood is the significance of activated charcoal. This supplement is somehow associated with the inactivation (via absorption) of some agar components that can inhibit androgenesis, and the release of other, ­water-soluble substances that can promote it (Forche et al. 1981). As mentioned before, another key factor is temperature. A number of plant species are reported to require a storage of the anthers at low temperature (2–4°C) for at least 24–48 h prior to cultivation. Temperature is important also during culture, and its requirement seems to depend on genetic factors. For example, whereas the tobacco variety “Wisconsin” requires 22°C to initiate androgenesis, this can be achieved for “Xanthi” only at 28°C. The requirements discussed here can be regarded only as tendencies, and the exact conditions to induce androgenesis have to be determined for each species or variety. Many examples can be found in the literature on the internet.

The success of microspore cultures depends also on the growing conditions of the donor plant. For example, temperature again seems to be of significance here. Higher yields of embryos were obtained if microspores originated from plants of rapeseed (Brassica napus) grown under a light/dark cycle of 16/8 h at 15/12°C, compared to 23/18°C (Lo and Pauls 1992). The authors relate this to a reduction in cytoplasmic granularity and/or exine density.

Finally, a description of haploid plants will be given. The tissue surrounding the haploid microspores in the anther (tapetum connective) is diploid. Thus, reliable methods are needed to distinguish between tapetum, haploid plants derived from the microspores, and diploids derived from the adjacent diploid tissue. Most reliable, of course, is to count the chromosomes in the dividing cells, e.g., in the cells of the root tip. However, usually only one root tip is available per plant, which one does not wish to “sacrifice”. As an alternative, the method described now has been used successfully in our investigations for many years, and it requires only a small piece of a leaf or callus. If the plant originates from microspores, then cells with 1C nuclei can be observed. An absence of such cells indicates that the plants are derived from diploid material of the anther. The 1C-value of the species can be determined using its microspores. Problems related to ploidy stability will be dealt with later (Chap. 13).

Microfluorometric determination of the ploidy level (Blaschke 1977; Blaschke et al. 1978): For the standardization of the method, the DNA was measured in the early tetrad stage of the nuclei in the developing microspore. The intensity of fluorescence of these structures is set equal to the DNA content of the haploid nucleus (1n). This old method is particularly suitable for using tissue with no, or only very few cell divisions available for chromosome counts. The plant material to be used for the investigation is first fixed in ethanol/glacial acetic acid (3:1) for 12 h, followed by an ascending alcohol series for dehydration. Then, the cells are embedded in paraffin, and with the help of a microtome sections of 12–15 µm are cut. The sections are fixed on a slide with a gelatine/glycerine solution (1 g pure gelatine dissolved in 100 ml aqua dest., supplemented with 15 ml glycerine and few crystals of thymol), and dried for 12 h at 40°C.

After removal of the paraffin on a heated plate, a descending alcohol series is applied, similar to other common histological methods. This is followed by staining with a 0.005% bisbenzimide solution (Dye Höchst 33258) for 10 min (pH 4.4–4.6), then rinsing for 5 min in running tap water, and differentiation for 15 min in 70% ethanol. After embedding the sections in “glycerine for fluorescence microscopy” (Merck), a cover glass is emplaced, and the next day the measurements can be made.

Measurements are made at a wavelength of >490 nm using a Xenon high-pressure lamp for illumination at 500-fold magnification. To determine ­fluorescence intensity, the lens of the microscope is adapted to the size and shape of the nuclei. For the measurements, one has to be careful to select nuclei that do not touch others nearby. The intensity of fluorescence is stable for at least 6 months. The reading can be compared to values obtained from tetrad-state microspores. After enzymatic maceration of cell suspensions, an automation of C-value determination can be performed by flow cytophotometry. Here, protoplasts are more suitable than intact cells.

Other methods

Recently, a method was published for DNA determination using genetically transformed Arabidopsis material (Zhang et al. 2005), by coupling GFP with the histone 2A. This construct (HTA6) complexed within chromatin, and it is therefore linearly related to the DNA content of the nucleus. This material could also be used for flow cytometry.

Sari et al. (1999) compared four different methods to determine the ploidy level, i.e., chromosome counting, flow cytometry, size and chloroplast number of the guard cells, and some morphological observations, using two cultivars of watermelon. All four were successful, and equally reliable. The most easy to perform was the method based on stomata measurements. The length of stomata of haploids was 17–18 µm, the diameter 10–12 µm, and the number of chloroplasts in the guard cells was 6–7. For diploids, the corresponding values were 23–24 µm, 18 µm, and 11–12. Chloroplasts with suitable equipment, a rough determination of the ploidy level could also be carried out on intact plants, in the greenhouse or in the field. Here, questions concerning relations of the ploidy level and the plastome should be followed in detail.

Haploid Plants

The most obvious morphological characteristics of haploid plants are a reduced height, smaller leaves with a reduced diameter of the leaf lamina, and an excessive number of small fruits (Table 6.2, Fig. 6.4 ). In Fig. 6.4 , it can be seen that also haploid plants produce fruits. Seed formation, however, is absent, and since fruit size often depends on the development of seeds, the fruits remain small.

Fig. 6.4

Leaves, flowers and fruits of diploid (left), and haploid (right) Datura innoxia plants (photographs by E. Forche)

Table 6.2 Some morphological characteristics of haploid, diploid, and dihaploid plants of Nicotiana tabacum, var. Xanthi (Zeppernick 1988)

Furthermore, deformations of leaves and flowers often occur in haploid plants. In Datura plants, flowers with three petals, rather than five, have been observed, and in tobacco flowers a fused gynoecium and 7–8 anthers could be seen. It seems that two flowers were fused into one; such fusions have been observed also for the leaves. Such deformations have not been observed in normal diploid plants growing under the same conditions. Possibly, the population of diploid plants observed was too small to give a definite result on this problem.

Beside a reduction of time required to establish inbred lines, another benefit of haploid plants is the possibility of bringing recessive genes to realization hidden in the parent generation. To consider the “gene dosage effect” in judging the genomes of plants derived by androgenesis, dihaploid plants are required. Such plants can be produced by applying, e.g., colchicines to the buds (see above). In some plant species (e.g., tobacco), dihaploids are spontaneously produced from leaf buds. If these dihaploids are propagated by cuttings, then the properties of these genomes can be preserved. With such material, China was the first to breed new varieties of tobacco, corn, wheat, and other plants, now since many years in use by farmers.

A clear characteristic of haploid plants is a reduced leaf diameter, at more or less normal leaf length (Table 6.2; cf. Figs. 6.5 and 6.6 ). Furthermore, haploids enter the flowering stage about 1 week earlier than dihaploids, consistent with the higher concentration of gibberellins in the leaves of the former (Table 6.3). Because spraying dihaploids with a gibberellin solution (GA₃) induces the same leaf shape, it is reasonable to predict a strong influence of the ploidy level on the native gibberellin system of plants. Nevertheless, it is difficult to see in which way the gibberellin system and the ploidy level are related. Although the synthesis and the general metabolism of this group of phytohormones is quite well understood, its action as a signal to promote morphogenic responses such as leaf shape still requires investigations (for a summary, see Thomas and Sun 2004).

Table 6.3 Gibberellin activity of haploid and dihaploid strains, and an F1 hybrid (8 ´ 4) determined by a dwarf rice method (Tanginbozu) after separation of the ethylacetate extract of leaves of a defined size by thin layer chromatography (expressed as gibberellin equivalents, ng/g fresh weight; average of four replicates per strain; harvest 1988)

It is difficult to decide to which extent the variations in leaf shape of the three dihaploid strains in Fig. 6.6 are due to the expression of recessive genes. It should be pointed out that these strains were derived from anthers of the same diploid mother plant. These dihaploid plants are fertile, and can be used for crossings. The figure shows clear differences that were preserved through several generations of propagation by seeds. As can be seen from the images of comparable leaves of the two parents and a hybrid, a clear heterosis can be observed for leaf size (Fig. 6.5 ). The plant height of the hybrid, however, is between those of the parents. Heterosis can be seen again for the concentration of nicotine in the leaves (Table 6.4).

Fig. 6.6

Shapes of leaves of two tobacco strains derived by androgenesis and an F1 hybrid

Fig. 6.5

Shape of leaves of three strains of dihaploid (2n) tobacco plants derived by androgenesis

Table 6.4 Nicotine concentrations of two dihaploid strains of Nicotiana tabacum, and a hybrid thereof (F1; pot experiments, average of 3 consecutive years; Zeppernick 1988)