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Haploids and Haploid Production in Citrus






In Citrus natsudaidai, haploid seedlings were fi rst obtained by the application of gamma rays (Karasawa, 1971).

One haploid embryo was obtained in an immature seed from a diploid (Clementine mandarin) ´ diploid (Pearl tan- gelo) cross (Esen and Soost, 1972).


 

Haploids can be induced mainly through two strategies: from the female gamete, and from the male gamete. Although a lot of research has been carried out on gametic embryogenesis in Citrus spp. and their relatives (Germanà, 1997), not much of it has been successful (Tables 7.1 and 7.2).

Anther culture technique has been reg- ularly employed to recover haploids in Citrus. Nevertheless, since many Citrus species proved to be recalcitrant to this method, other methods have been and could be attempted to produce haploids.

Moreover, a comparison of doubled haploid plants coming from male or female gametes could be interesting because the method of producing doubled haploids seems to produce differences in doubled haploid fi eld performance. The maternally derived homozygous regenerants are gener- ally more vigorous after in vivo transfer and have less gametoclonal variation than dou- bled haploids obtained from male gametes (Snape et al., 1988; Wernsman et al., 1989).


 

 

Table 7.2. Haploids and doubled haploids (DH) obtained in Citrus and its relatives by anther culture.

 

Genotypes Response References
Poncirus trifoliata L. Raf. (´) plantlets Hidaka et al., 1979
C. madurensis Lour. (´) (2´) plantlets Chen et al., 1980
Hybrid No. 14 of C. ichangensis ´ C. reticulata (´) plantlet Deng et al., 1992
Citrus clementina Hort. ex Tan. (´) DH (3´) plantlets Germanà et al., 1994,
    2000a, 2005, Germanà and
    Chiancone, 2003
C. limon L. Burm. f. (´) calli Germanà et al., 1991
Mapo tangelo (C. deliciosa ´ C. paradisi) (´) embryoids Germanà and Reforgiato, 1997
Clausena excavata (´) embryoids Froelicher and Ollitrault, 2000

 


 

Gynogenesis

Selection of seedlings

Parthenogenesis (the production of an embryo from an egg cell without the partic- ipation of the male gamete) and apogamy (the production of an embryo from a game- tophytic cell other than the ovum) are the causes of spontaneously occurring hap- loids.

Spontaneously developed haploids were reported in over 100 angiosperm species (Kasha, 1974). Most of the haploids in fruit trees (especially pome and stone fruits) are of spontaneous origin (Zhang et al., 1990). Spontaneous haploid plants were obtained in apple, pear, peach, plum, apri- cot, etc., but in very low number and they were not very viable (Zhang et al., 1990).

 

In situ parthenogenesis induced by irradiated pollen followed by in vitro culture of embryos

Parthenogenesis induced in vivo by irradi- ated pollen, followed by in vitro culture of embryos, can be an alternative method of obtaining haploids. Gynogenesis by in situ pollination with irradiated pollen has been successfully used for Petunia (Raquin, 1985), Cucumis melo (Sauton and Dumas de Vaulx, 1987), Daucus carota (Rode and Dumas de Vaulx, 1987), Malus domestica (Zhang and Lespinasse, 1991; Hofer and Lespinasse, 1996), Pyrus communis (Bouvier et al., 1993), Actinidia deliciosa (Pandey et al., 1990; Chalak and Legave, 1997) and Citrullus lanatus (Sari et al., 1994).

The production of nine haploid plantlets, which did not survive, and two embryogenic callus lines was achieved in clementine (Citrus clementina Hort. ex Tan.), cv. SRA 63 after in situ parthenogen- esis induced by pollen of Meyer lemon (Citrus meyeri Y. Tan.) irradiated at 300, 600 and 900 Gy from a 60Co source (Ollitrault et al., 1996). Flowers of clemen- tine SRA 63 were pollinated in the fi eld with the irradiated pollen; fruits were picked at maturity and embryos were culti- vated in vitro.


 

The method is based on the in vitro cul- ture of immature seeds or embryos obtained as a result of pollination with pollen irradi- ated by g-rays from 60Co, and it should be tested in those species in which in vitro anther culture and gynogenesis have not been successfully applied. Irradiation does not hinder pollen germination, but prevents pollen fertilization, stimulating the devel- opment of haploid embryoids from ovules. The success of this technique is dependent on the choice of radiation dose, the devel- opmental stage of the embryos at the time of culture, the culture conditions and the media requirements.

 

In situ or in vitro parthenogenesis induced by triploid pollen followed by in vitro culture of embryos

Three haploid plants were obtained from two monoembryonic diploid (clementine and ‘Lee’) ´ triploid hybrid of ‘Kawano nat- sudaidai’ (C. natsudaidai) in vivo crosses (Oiyama and Kobayashi, 1993). Triploidy of pollen, like irradiation, does not hinder pollen germination, but prevents pollen fer- tilization and stimulates the development of haploid embryoids from ovules. Haploid and diploid embryoids did not show any difference in their size; however, haploid seedlings grew very slowly in the soil. Restriction endonuclease analyses of nuclear rDNA and of chloroplast DNA determined the maternal origin of these haploids.

Haploid plantlet regeneration through gynogenesis in C. clementina Hort. ex Tan., cv. Nules, has been induced by in vitro pol- lination with triploid pollen (Germanà and Chiancone, 2001). The pollen source chosen was ‘Oroblanco’, a triploid grape- fruit-type citrus obtained in 1958 through a cross between an acidless pummelo (Citrus grandis Osbeck) and a seedy, tetraploid grapefruit (C. paradisi Macf.) (Soost and Cameron, 1980).

The in vitro stigmatic pollination tech- nique consists of applying pollen to the apical part of the stigma of an excised gynoecium implanted in solid culture


 

 

 

Fig. 7.1. (a) An excised gynoecium of Citrus clementina, cv. Nules implanted in solid medium and polli- nated with triploid pollen grains. (b) Stigma exudate and ‘Oroblanco’ pollen grains on the stigma of a pistil taken from a mature bud fl ower. (c) Gynogenic embryoids breaking through the ‘Nules’ ovary four months after in vitro pollination with triploid ‘Oroblanco’ pollen grains.

 

 


medium (Fig. 7.1a and b). Some ovaries were transformed into brownish and friable callus, sometimes breaking to reveal ovules. From this kind of ovary, the gynogenic embryoids emerged (Fig. 1c) 4–5 months after in vitro pollination, which is practi- cally the same time required for androgene- sis. The pollination and mature stage of pistils were necessary for gynogenic embry- oid regeneration. Although unlike clemen- tine anther culture (Germanà et al., 1994, 2000a), embryogenic calluses were not obtained, parthenogenesis induced in vitro by triploid pollen can be usefully employed in attempting to obtain haploids in monoembryonic genotypes of Citrus for which androgenesis has not yet been suc- cessful in haploid production.

 

 

Anther and isolated microspore technique

The in vitro anther or isolated microspore culture technique is usually the most effec- tive and widely used method of producing haploids and doubled haploids.

Regeneration from male gametes has


been reported in about 200 species belong- ing to some families, such as Solanaceae, Cruciferae and Gramineae; many other fam- ilies (Leguminosae and woody plants) appear, instead, rather recalcitrant (Dunwell, 1986; Hu and Yang, 1986;

Sangwan-Norrel et al., 1986; Bajaj, 1990; Raghavan, 1990; Wenzel et al., 1995). Since 1970, extensive research has been carried out to obtain haploids by anther culture or gynogenesis for perennial species breeding, with not always satisfactory results.

The cellular, biochemical and molecu- lar bases for the transformation of microspores into pollen embryoids have not yet been completely understood. However, it is already possible to indicate some fi ndings. For example, it is known that the capacity to regenerate from a male gamete is genetic and inheritable, and that the stage of microspore development is crit- ical for induction. Usually in the period around the fi rst haploid mitosis (late unin- ucleate or early bicellular pollen stage), male gametes become competent to differ- entiate in a different way from the gameto- phytic pathway with continued growth and


 


 

division. Moreover, external stresses are necessary to enable competent microspores to undergo embryogenic development. The stress can be physical (also wounding con- nected to the anther excision and culture), thermal (heat, cold) or chemical (water stress, starvation). The induced micro- spores are characterized by an altered syn- thesis and an accumulation of RNA and proteins, and it seems that the genes involved in this reprogramming are stress- related and/or associated with the zygotic embryogenesis.

 

Anther culture

Anther culture is an easier and more com- monly used method than isolated microspore culture, because the isolation of microspores requires a higher degree of skill and better equipment than anther cul- ture (Heberle-Bors, 1989).

Research on haploidization by anther culture has been carried out on several fruit trees, especially in pome and stone fruits, and sometimes produced embryoids that rarely germinated (Zhang et al., 1990). The induction of embryogenesis from cultured apple anthers is still low and highly geno- type dependent (Hofer, 1995, 1997).

As regards Citrus and their relatives, by anther culture, haploid plantlets have been recovered from Poncirus trifoliata L. Raf. (Hidaka et al., 1979) and C. madurensis Lour. (Chen et al., 1980), and one doubled haploid plantlet has been obtained from the hybrid No. 14 of C. ichangensis ´ C. reticu- lata (X.X. Deng et al., 1992); haploid plantlets and highly embryogenic haploid calli of C. clementina Hort. ex Tan. (Germanà et al., 1994, 2000a; Germanà, 2003a; Germanà and Chiancone, 2003); haploid, but albino embryoids of Mapo tan- gelo (C. deliciosa ´ C. paradisi) (Germanà and Reforgiato, 1997) were produced; hap- loid and diploid calli, embryoids and leafy structures but no green plants of C. limon L. Burm. f. (Germanà et al., 1991); and haploid embryoids of Clausena excavata (Froelicher and Ollitrault, 2000) have also been achieved (Table 7.2).


 

In Citrus and its relatives, Hidaka et al. (1979) fi rst reported anther culture in P. tri- foliata and later in C. aurantium (1981) and

C. sinensis (1984a, b); Chen et al. (1980) reported anther culture in C. madurensis; and Chaturvedi and Sharma (1985) worked on C. aurantifolia anther culture. Ling et al. (1988) studied C. madurensis anther cul- ture. Further studies on anther culture of several Citrus genotypes were carried out by Starrantino (1986) and Geraci and Starrantino (1990). X.X. Deng et al. (1992) reported on P. trifoliata and a hybrid of C. ichangensis ´ C. reticulata anther culture. Germanà reported research on C. limon (Germanà et al., 1991) and C. clementina (Germanà et al., 1994, 2000a; Germanà, 2003a), C. reticulata (Germanà et al., 1994) and tangelo Mapo (Germanà and Reforgiato, 1997) anther culture.

Only heterozygous plantlets have been obtained by anther culture in C. aurantium (Hidaka et al., 1981; Germanà et al., 1992; Germanà, 2003b), C. sinensis (Hidaka, 1984b), C. aurantifolia (Chaturvedi and Sharma, 1985), C. madurensis (Ling et al., 1988), C. reticulata (Germanà et al., 1994; Germanà, 2003b) (Fig. 7.2) and P. trifoliata (Deng et al., 1992). Anther culture produced embryonal structures in C. sinensis and C. paradisi (Starrantino, 1986), and mixoploid calli were obtained from C. reticulata, C. deli- ciosa, C. sinensis, C. limon, C. paradisi and Mapo tangelo (Geraci and Starrantino, 1990).

 

 
 

Fig. 7.2. A cluster of somatic embryoids from Citrus reticulata (cv. Avana) anther culture.


 

 

 

Fig. 7.3. (a) Anther of C. clementina Hort. ex Tan. with microspores at the uninucleate stage before culture.

(b) A microspore at the uninucleate stage.

 

 


In these cases, anther culture could be regarded as a method of obtaining somatic embryogenesis, which is a very effi cient method of regeneration. Embryogenic callus is valuable for propagation or genetic improvement and can be used for somatic hybridization by protoplast fusion, genetic transformation, synthetic seed production and germplasm storage.

 

Anther culture technique in Citrus

Floral buds, with the pollen grains at a spe- cifi c stage of development, are collected from the donor plant. After pre-treatment, the buds are surface sterilized by immer- sion for 3 min in 70% (v/v) ethyl alcohol, followed by immersion in sodium hypochlorite solution (~1.5% active chlo- rine in water) containing a few drops of Tween-20 for 15–20 min, and fi nally rinsed three times for 5 min with sterile distilled water. Petals are aseptically removed with small forceps, and anthers (Fig. 7.3a) are carefully dissected and placed into the medium.

The stage of pollen development is


commonly determined by staining one or more anthers per bud with acetocarmine, Schiff’s reagent or 4¢, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining (Fig. 7.3b).

Usually, tubes are employed for Citrus anther culture (Hidaka et al., 1979, 1981; Hidaka, 1984a; Chatuvedi and Sharma, 1985; Starrantino, 1986; Hidaka and Omura 1989b; Geraci and Starrantino, 1990; X.X. Deng et al., 1992), but Petri dishes (Starrantino, 1986; Germanà et al., 1991,

1992, 1994, 1997, 2000a; Germanà and Chiancone, 2003) and bottles or fl asks have also been used (Chen, 1985; Ling et al., 1988).

 

Isolated microspore culture

Pollen culture is performed by removing somatic anther tissue. This technique, although more diffi cult and laborious, is ideal for studying the mechanism of pollen embryogenesis, because it eliminates the unknown effects of the sporophytic anther tissue, thereby allowing a greater control over the culture process.


 


 

Isolated microspore culture technique in

Citrus

 

Investigation of isolated microspore culture of several Citrus species (lemon, orange, clementine, sour orange, grapefruit) and a related genus (Poncirus) has been carried out (Germanà et al., 1996). Anthers at the uninucleated stage from cold-pre-treated (4˚ C for 10 days) and surface-sterilized fl ower buds are excised and pre-cultured on either liquid or solid medium in Petri dishes. After 5–15 days at 27 ± 1˚ C in the dark, anthers are gently squeezed with a glass pestle in 2 ml of liquid medium. The anther mixture is fi ltered through a sterile nylon sieve (40 mm) and microspores are centrifuged (1000 r.p.m. for 10 min) and washed twice with fresh medium. Finally, the clean isolated microspores are resus- pended in fresh liquid medium at a density of 103–104 grains/ml, placed in a thin layer (3 ml) in Petri dishes (6 cm in diameter), sealed with parafi lm and incubated at 27 ± 1˚ C under cool white fl uorescent lamps with a photosynthetic photon fl ux density of 35 mmol/m2/s for a photoperiod of 16 h of light per day. After various periods of time (1–4 months), the isolated microspores of almost all investigated Citrus species pro- duced multinucleated structures and devel- oped into small proembryos, which failed

 

 
 

Fig. 7.4. Pseudobulbil produced after about eight months in a Citrus limon (L CNR 26) isolated microspore culture.


 

to develop any further, although several media and different methods (double layer, liquid medium, soft agar, solid agar, etc.) were employed. Formation of ‘pseudobul- bils’, white or green spherical bodies, described in Citrus by Button and Kochba (1977), has been obtained only in those genotypes (clementine and lemon) that had also produced haploids by anther culture (Fig. 7.4).

Medium for isolated microspore cul- ture is more complex than that for anther culture. The NTH (Nitsch and Nitsch, 1969) or N6 (Chu, 1978) liquid fi lter sterilized medium was supplemented with casein, glutamine, malt extract, biotin, myo-inosi- tol, glycine, pyridoxine, thiamine, serine, coconut water and ascorbic acid, with galactose and sorbitol as carbon source and a complex combination of growth regula- tors (2, 4-dichlorophenoxyacetic acid (2, 4-

D) + kinetin + zeatin + giberellic acid).

 

 

Factors affecting in vitro pollen embryogenesis

Although progress in pollen embryogenesis has taken many steps forward in recent years, several aspects of this phenomenon remain unclear, particularly the induction process and the factors that control it. The identifi cation of the inhibitory and stimula- tory factors is of fundamental importance, especially in recalcitrant species such as Citrus.

In vitro pollen embryogenesis is affected by numerous factors: genotype; the pre-treatment applied to anthers or to fl oral buds; pollen developmental stage; donor plant growth conditions; culture media (macro- and microelements, carbon source and plant growth regulators); and incuba- tion conditions.

Experience in several genotypes has shown that often a particular modifi cation in the procedures (such as the use of malt- ose in barley, the stress in wheat, and so on) can result in breakthroughs in making the process work. Research in recent years regarding factors affecting gametic embryo-


 


genesis in Citrus (Germanà et al., 2000a; Germanà and Chiancone, 2003) has resulted in an increase in the number of the cultivars responding to the phenomenon, an improvement in the induction rate of haploid production and an understanding of the ways in which the diffi culties of in vivo acclimatization can be overcome. As a result, to date, more than 100 homozygous embryogenic callus lines, each coming from single anthers of the different clementine cultivars Nules, Sra 63 and Monreal, have become available.

 

Genotype

The induction rate of pollen embryos is mainly infl uenced by the genotype and dif- ferent culture factors (Heberle-Bors, 1980). ‘In most species only a few genotypes can be induced to undergo androgenetic devel- opment’ (Vasil, 1980).

Because the number of microspores competent to undergo embryogenesis (‘E- grains’, Sunderland, 1978; or ‘P-grains’, Heberle-Bors, 1982) depends on the geno- type (Heberle-Bors, 1985), it is necessary to know the optimal conditions to turn the development of pollen towards a sporo- phytic pathway and to avoid embryo abor- tion.

Almost all research carried out on pollen embryogenesis in Citrus recognizes the preponderant infl uence of the genotype (Khuroshvili et al., 1982; Hidaka, 1984a; Chen, 1985; Germanà et al., 1991, 1994), even though the frequency of P-grains in Citrus has not been the object of any research.

The proof that the genotype has a fun- damental infl uence on the success of the gametic embryogenic process is evident from Table 7.2; to date, in the genus Citrus, almost all haploids and doubled haploids have been regenerated in different cultivars of the one species C. clementina, although research on anther culture of numerous genotypes has been reported (Germanà, 1992, 1997).

Research (Germanà et al., 1994; and unpublished results) carried out simultane-


ously on several Citrus cultivars (four culti- vars of clementine, two of mandarin, four of sweet orange, four of sour orange, fi ve of lemons and four of grapefruits) resulted in plantlets being obtained from haploid embryogenic callus only in C. clementina cv. Nules, haploid callus in one cultivar of

C. limon, and diploid and highly embryo- genic callus from two cultivars of C. reticu- lata (Avana and Tardivo di Ciaculli) and two cultivars of C. aurantium (A.A. CNR 10 and A.A. CNR 23). All experiments were carried out under the same culture condi- tions and pre-treatments (4˚ C for 4 days), testing 11 different media. Further research improved the plantlet induction rate of clementine and confi rmed its androgenetic response (Germanà et al., 2000a; Germanà and Chiancone, 2003).

 

Stage of pollen development

The pollen developmental stage is a com- plex factor that affects the success of anther culture. The suitable stage differs depend- ing on the crop species tested. Generally, pollen grains between the uninucleate and early bicellular stage are cultured. After the pollen grains begin to accumulate storage reserves, they usually lose their embryo- genic capacity and follow the gametophytic developmental pathway (Heberle-Bors, 1989; Raghavan, 1990). Usually, the stage of pollen development is tested in one anther per fl oral bud size by the acetic–carmine method (Sharma and Sharma, 1972). The anthers are collected from fl ower buds at different stages of development and squashed in 1% acetocarmine in 45% acetic acid for observation under an optical microscope to determine the stage of pollen development. DAPI fl uorescent staining has also been used. However, different develop- mental stages have been observed within a single anther, and between different anthers of the same fl ower bud in Citrus and in Poncirus as well as in many other genera (Vasil, 1967; Shull and Menzel, 1977; Hidaka et al., 1979, 1981; Chen,

1985).

Hidaka et al. (1979), studying the effect


 


 

of different developmental stages of P. trifo- liata pollen grains on the formation of embryoids, pseudobulbils and calli, indi- cated the early uninucleate stage as most suitable for embryoid production. Anthers at other developmental stages (from pollen mother cell (PMC) to bicellular stage) pro- duced only calli. Pseudobulbils could not be obtained at PMC, tetrad or bicellular stages. In C. aurantium, Hidaka et al. (1981) obtained embryoids from anthers only at the late uninucleate stage and callus pro- duction from anthers at all other develop- mental stages except for the PMC in the meiosis and the tetrad stage. Various authors have used different developmental stages in Citrus anther culture including: the uninucleate stage (Hidaka, 1984a, b; Hidaka and Omura, 1989b; Germanà et al., 1990, 1991, 1994; X.X. Deng et al., 1992;

Germanà and Chiancone, 2003); tetrads at the uninucleate stage (Chaturvedi and Sharma, 1985); the middle anaphase period (Chen, 1985); and the stage of just released spores to the first haploid mitosis (Starrantino, 1986; Geraci and Starrantino, 1990).

 

Physiological condition of the donor plant

The number of P-grains also depends on the growth conditions of the donor plants (Heberle-Bors, 1985), because their forma- tion in vivo and/or in vitro seems to be con- nected with a nitrogen starvation phenomenon (Heberle-Bors, 1983).

Although the physiological condition of the donor plant can dramatically affect the androgenic process, this parameter has been investigated only in herbaceous plants because of the diffi culties of determining it in open-air cultivated, perennial woody plants. Signifi cant seasonal variations in anther response have been observed in many genotypes, and it has been noticed that anthers removed from field-grown plants give a better response than those picked from greenhouse-grown plants (Vasil, 1980). In fact, the physiological and growth conditions of the donor plant, which affect the endogenous levels of hor-


 

mones and the nutritional status of the tis- sues of the anther (Sunderland and Dunwell, 1977), are important for the suc- cess of the embryogenetic process. This parameter has not been considered in the studies regarding Citrus anther culture.

The infl uence of the physiological con- dition of the donor plant, affected by cli- matic (temperature, photoperiod and light intensity) and pedological conditions, should be investigated, because this could help to explain the reasons why the response to anther culture is so season- dependent, although the same conditions (pollen development stage, fl oral bud pre- treatments, medium, light and temperature conditions of culture) are employed.

 

Pre-treatment

It has been observed that stress treatment (such as chilling, high temperature, high humidity, water stress, anaerobic treatment, centrifugation, sucrose and nitrogen starva- tion) applied to excised fl oral buds or to anthers before culture acts as a trigger for inducing the sporophytic pathway, pre- venting the development of fertile pollen (gametophytic pathway) (Sangwan-Norrel et al., 1986; Touraev et al., 1997), although sometimes different results have been obtained (Sunderland, 1974).

The stress, triggering the microspore development from gametophytic to sporo- phytic, seems to act by altering the polarity of the division at the fi rst haploid mitosis involving reorganization of the cytoskele- ton (Nitsch and Norreel, 1973; Reynolds, 1997), delaying and modifying pollen mito- sis (two equal-size vegetative-type nuclei instead of one vegetative and one genera- tive), blocking starch production or dissolv- ing microtubules (Nitsch, 1977), or maintaining the viability of the cultured P- grains (Heberle-Bors, 1985).

Cold pre-treatment is routinely employed in anther culture of many crops, and its effect is genotype-dependent (Powell, 1988; Osolnik et al., 1993).

Cold temperature is the most common physiological stress applied in anther cul-


 


ture of Citrus. The different cold pre-treat- ments employed in Citrus anther culture are: 5˚ C for 2 h (Starrantino, 1986), overnight (Geraci and Starrantino, 1990) and 3–5 days (Ling et al., 1988); and 4˚ C for 4 days (Germanà et al., 1991), for 6 days (Germanà et al., 1994), for 2–6 days (X.X. Deng et al., 1992) and for 10 days (Germanà et al., 1997, 2000a).

In a study carried out by Chen (1985) on the effects of 0–25 days of 3˚ C cold pre- treatment on C. madurensis Lour., a dura- tion of 5–10 days was favourable for inducing callus and embryoids.

A study carried out on factors affecting anther culture in C. clementina showed a negative effect on callus production of cen- trifugation (4000 r.p.m. for 5 min) and high temperature (40˚ C for 24 h) when compared with chilling (4˚ C for 10 days) (Germanà et al., 2000a). These and other results (Germanà and Chiancone, 2003) indicated that high temperature applied to the fl oral buds before culture is not recommended for androgenesis in Citrus cv. Nules, contrast- ing with what has been reported in Solanum melongena L. (Rotino, 1996), in Brassica oleracea (Keller et al., 1983) and in Solanum chacoense (Cappadocia et al., 1984).

 

Culture medium

The diverse genotypes show very different basal medium requirements to induce pollen-derived plant formation.

The basal media most used in Citrus anther culture are: B5 medium (Gamborg et al., 1968; Khuroshvili et al., 1982); DB medium (Drira and Benbadis, 1975); MS medium (Murashige and Skoog, 1962; Hidaka et al., 1979, 1981; Khuroshvili et al., 1982; Hidaka, 1984a, 1984b; Starrantino, 1986; Ling et al., 1988; Hidaka and Omura, 1989b; Germanà, 1994); modified MS medium (Chaturvedi and Sharma, 1985; Chen, 1985; Germanà et al., 1994); Murashige and Tucker (1969) medium (Geraci and Starrantino, 1990; X.X. Deng et al., 1992; Froelicher and Ollitrault, 2000);

N6 medium (Chu, 1978; Chen, 1985;


Starrantino, 1986; Germanà et al., 1997, 2000a; Germanà and Chiancone, 2003); SH medium (Schenk and Hildebrandt, 1972; Chaturvedi and Sharma, 1985); Sj-1 medium (Starrantino, 1986); Chaturvedi and Mitra (1974) medium (Germanà et al., 1994); and Nitsch and Nitsch (1969) medium (NTH) (Khuroshvili et al., 1982; Germanà et al., 1997, 2000a).

A study comparing three basal media in the induction media: MS (Murashige and Skoog, 1962); NTH (Nitsch and Nitsch, 1969); and N6 (Chu, 1978) showed a higher

effi ciency of the last two media in produc-

ing callus (Germanà et al., 2000a).

 

Carbon source

The carbon source and its concentration is an essential component in the medium for embryo induction. The effect of its concen- tration is probably related to osmotic pres- sure regulation during the induction phase (Sunderland and Dunwell, 1977; Sangwan and Sangwan-Norrel 1990). Furthermore, high concentrations of carbohydrate seem to be deleterious (Keller et al., 1975).

Sucrose is the most common carbon source used in the anther culture of Citrus and their relatives, at 5% concentration (Hidaka et al., 1979, 1981; Hidaka, 1984b; Hidaka and Omura, 1989b; Geraci and Starrantino, 1990; Froelicher and Ollitrault, 2000), although other concentrations have also been reported: 8% (Starrantino, 1986),

2% (Ling et al., 1988) and 2.5–5% (Drira and Benbadis, 1975; X.X. Deng et al., 1992). Hidaka (1984) studied the effects of sucrose concentration (1, 3, 5, 7 and 9%) on embry- oid and callus formation and found that 3% sucrose was the ideal concentration to form embryoids in P. trifoliata, 7% in C. auran- tium and 1% in C. sinensis (Trovita orange); 10% was found to be ideal in C. maduren- sis (Chen, 1985); and 3 and 6% in C. limon androgenesis (Germanà et al., 1991).

The infl uence on anther culture of two carbon sources (sucrose and glucose) was tested in two C. clementina and two C. reticulata cultivars (Germanà et al., 1994). Sucrose (5%) was the best, although with a


 


 

variable response depending on the species or the variety tested.

Glycerol in combination with sucrose stimulated callus production in C. clementina (Germanà et al., 2000a), while galactose was found to be very effective for the production of embryoids from Citrus calli (Hidaka and Omura, 1989a). More recent research has shown a positive infl u- ence of the combination of lactose and galactose in inducing haploid production in clementine (M. A. Germanà et al., unpublished).

 

Plant growth regulators

The effects of plant growth regulators have been widely investigated in anther culture of Citrus. In C. limon and C. medica anther culture, Drira and Benbadis (1975) obtained callus on a medium containing 1.0 mg/l 2, 4- D, 1.0 mg/l a-naphthaleneacetic acid (NAA) and 0.1 mg/l 6-benzylaminopurine (BA). Hidaka et al. (1979) found that a medium containing 0.2 mg/l of both indole-3-acetic acid (IAA) and kinetin (Kin) was the most effi cient in embryoid formation, while the addition of 2, 4-D increased callus formation in P. trifoliata anther culture. Furthermore, Hidaka et al. (1981) obtained embryoid pro- duction from C. aurantium by anther cul- ture in medium supplemented with 0.02 mg/l Kin and 0.02 or 2.0 mg/l IAA.

In C. sinensis, Hidaka (1984b) found that the best composition of pollen embryo- genesis induction medium was similar to that used for sour orange, having lower con- centrations (0.002 or 0.02 mg/l) of IAA and Kin. Chaturvedi and Sharma (1985) supple- mented medium with 0.5 mg/l BA and 1.0 mg/l IAA while studying androgenesis in C. auratifolia.

Chen (1985) found that 1.0 mg/l BA and 0.1 mg/l 2, 4-D was the best hormone combination for embryoid production and that the 2, 4-D concentration in the medium is crucial for embryoid production in C. madurensis: an increase of its concentra- tion promotes callus formation and inhibits embryoid development.

Starrantino (1986), testing numerous


 

media containing an auxin (0.1, 0.5, 1.0, 5.0 and 10.0 mg/l of IAA or 2, 4-D) in combina- tion with a cytokinin (0.1, 0.5, 1.0, 5.0 and

10.0 mg/l of BA or Kin), obtained two embryonal structures from two C. sinensis anthers in medium containing 1.0 mg/l 2, 4- D and 10.0 mg/l Kin, and one embryonal structure from an anther of C. paradisi in medium supplemented with 10 mg/l 2, 4-D and 0.5 mg/l Kin.

Ling et al. (1988) obtained the highest frequency (0.92%) of embryoid formation in C. madurensis by supplementing medium with 2.0 mg/l of both IAA and Kin. Geraci and Starrantino (1990) suc- ceeded in obtaining the highest percentage of callus proliferation (25%) in the pres- ence of 1 mg/l BA and 0.5 mg/l 2, 4-D in C. reticulata, C. deliciosa, C. paradisi and Mapo tangelo (C. deliciosa ´ C. paradisi), while in C. sinensis and C. limon, the high- est callus proliferation rate (44.2%) was obtained with 1.0 mg/l of both NAA and

BA.

X.X. Deng et al. (1992) reported the best response in P. trifoliata and an ichang papeda hybrid on medium having 0.1 mg/l NAA.

The anther culture of C. limon was suc- cessful on medium containing 2.0 mg/l Kin

+ 1.0 mg/l zeatin (ZI) + 0.1 mg/l NAA (Germanà et al., 1991).

Germanà et al. (1994) found the best hormonal combination for callus produc- tion (6–28%, depending on the genotype) in anther culture of C. clementina and C. reticulata in: 0.02 mg/l NAA + 0.5 mg/l ZI +

0.5 mg/l Kin.

In Clausena excavata, Froelicher and Ollitrault (2000) obtained the best results with the following hormonal conbination: BA at low concentration (0.1 or 0.3 mg/l), alone or with 2, 4-D (0.1 mg/l).

Further research (Germanà and Chiancone, 2003) showed that thidiazuron (TDZ), one of the most active cytokinins in woody plant tissue culture (Huetteman and Preece, 1993), is effective in inducing hap- loid embryoids and plantlet regeneration in

C. clementina cv. Nules.


 


Activated charcoal

The addition of activated charcoal (0.5-2 g/l) to the medium increases the effi ciency of androgenesis in several genera. It seems to act by removing inhibitory substances from the medium, and presumably from the anther wall, and by regulating the level of endogenous and exogenous growth regula- tors (Reinert and Bajaj, 1977; Vasil, 1980; Heberle-Bors, 1985).

X.X. Deng et al. (1992) found that medium containing activated charcoal was effective in the medium having P. trifoliata embryoid induction. However, no positive effect of activated charcoal addition (0.5 g/l) has been observed in anther culture of several Citrus species (Germanà et al., 1994;

M.A. Germanà et al., unpublished).

 

Other substances

The addition of glutamine was necessary to produce callus in C. limon anther culture (Drira and Benbadis, 1975). In our laboratory, the best results were obtained by the addition to media of casein, biotin and sometimes coconut water, together with glutamine.

Although the way in which they act is not completely understood, various natural undefi ned extracts, e.g. coconut water, are used since they improve pollen response. They probably provide one or more sub- stances which stimulate pollen division.

A recent study (Chiancone et al., 2006) showed that the addition to medium of sper- midine increased the number of clementine anthers producing haploid embryoids. Polyamines are compounds found in all living organisms, classifi ed as growth regu- lators and involved in many biological processes, such as growth, development and stress response (Kumar et al., 1997). Moreover, their involvement in in vitro organogenesis and embryogenesis and their capacity to inhibit ethylene biosynthesis has been highlighted.

 

pH

pH is another factor which can infl uence the gametic embryogenic process (Stuart et


al., 1987). In Citrus anther culture, the pH of the medium is usually adjusted to 5.7–5.8 before autoclaving. The effect of the pH level (4, 5, 6, 7 and 8) of the medium on anther culture of P. trifoliata, C. aurantium and C. sinensis has been studied by Hidaka (1984a), who found that both pH 5 and 6 were effective in embryoid formation in all genotypes tested.

 

Solidifying agents

Usually, Citrus anther culture media are solidifi ed by adding agar. Chaturvedi and Sharma (1985) obtained diploid plantlet regeneration by floating C. aurantifolia anthers on a liquid medium, then embed- ding them in a semi-solid medium. Generally, we obtained better results in a solid medium rather than a liquid one, also when pre-culturing anthers for pollen isola- tion. In the liquid medium, anthers initially swell, later turning brown and sometimes shrivelling. Probably the two steps (fi rst liquid and later semi-solid) in the culture are essential for success.

Research on C. clementina, Mapo tan- gelo, Fortunella margarita and C. paradisi anther culture showed the benefi cial effect of potato starch as a gelling agent on callus production (Germanà et al., 1997, 2000a;

M.A. Germanà et al., unpublished).

Froelicher and Ollitrault (2000) in anther culture of Clausena excavata added gelrite to solidify the medium.

 

Incubation conditions

The incubation conditions have not received much attention in Citrus anther culture (especially light quality and pho- toperiod).

Hidaka (1984a) reported inducing embryoids at 24 and 28˚ C in trifoliate orange, sour orange and ‘Trovita’ orange.

Chen (1985) observed that temperature seems to be more important than light in Citrus androgenesis, and obtained embry- oids at 20–25˚ C, especially under dark con- ditions (2.21% induction rate).

Different temperatures were used in


 


 

Citrus: 25 ± 1°C (Ling et al., 1988) and 27–28 ± 1°C (Hidaka et al., 1979, 1981;

Hidaka, 1984b; Chaturvedi and Sharma, 1985; Starrantino, 1986; Hidaka and Omura, 1989a; Geraci and Starrantino, 1990; Germanà et al., 1990, 1991, 1994; Deng et al., 1992; Germanà and Reforgiato, 1997; Froelicher and Ollitrault, 2000).

The light intensities used are: 500 lux with a 16 h photoperiod (Hidaka et al., 1979, 1981; Hidaka 1984a, b); 500–800 lux

with a 12 h photoperiod (Chen, 1985); 3000 lux with a 15–16 h photoperiod (Chaturvedi and Sharma, 1985; Ling et al., 1988); 1000 lux with a 16 h photoperiod (Starrantino, 1986; Geraci and Starrantino, 1990; X.X. Deng et al., 1992); and 3500 lux with a 16 h photoperiod (Germanà et al., 1991). An inductive period in darkness (10–15 days; 3–4 months Froelicher and Ollitrault, 2000) is usually applied in androgenesis research. In our experiments, Petri dishes are usually incubated at 27 ± 1˚ C for 15 days in the dark, and then placed under cool white fl uorescent lamps (Philips TLM 30W/84) with a photosynthetic photon fl ux density of 35 mmol/m2/s and a 16 h photoperiod (Germanà et al., 1994, 2000; Germanà and Reforgiato, 1997; M.A. Germanà et al.,

unpublished).

Preliminary research on the effect of light quality on anther culture of C. clementina Hort. ex Tan., cultivar Nules, testing, after one month of darkness, four light qualities: white, red, far-red and blue, and using as control conditions continuous darkness and white light under a photope- riod of 16 h, showed that gametic embry- oids and embryogenic callus were obtained only under photoperiodic conditions of white light, suggesting that the alternation of light and dark is necessary for the gametic embryogenesis process in clemen- tine (Germanà et al., 2005a).

 

 

Embryo development from microspores and the origin of haploids

Gametic embryogenesis (from male or female gametes) can be considered to be


 

 

Fig. 7.5. Direct embryogenesis in Citrus anther culture.

 

 

one example of cellular totipotency, usu- ally defi ned as the capacity of the somatic cell to regenerate an entire new plant and, evolutionarily, an important survival adap- tation mechanism (Reynolds, 1997).

The developmental process of a plant from a single microspore is referred to as microspore embryogenesis, although the

 

 
 

Fig. 7.6. Secondary embryogenesis in Citrus anther culture.


 


 

Fig. 7.7. A non-morphogenic callus from anther culture.


genic calli differentiate into a clump of embryoids (Fig. 7.10a and b).

The well-structured embryoids develop normally like zygotic embryos, through the globular, the heart, the torpedo and the cotyledonary stages, and often pro- duce secondary embryoids. Often ter- atomatal structures, cotyledonary fused (Fig. 7.11a and b), pluricotyledonary (Fig. 7.12) and thickened embryoids are observed. Sometimes pseudobulbils, with or without callus, are produced in Citrus anther culture (Fig. 7.13a and b).


 


route of regeneration may be via direct embryogenesis (Fig. 7.5), secondary embryogenesis (Fig. 7.6) or, less frequently, organogenesis. In other cases, microspores in culture produce undifferentiated calli, instead of embryoids.

After one week of culture, most of the anthers are swollen and after 2–3 months they start to produce calli or embryoids. Most of the calli are non-morphogenic (Fig. 7.7), but many of them appear highly embryogenic and they maintain their potential for a long time. The morphogenic calli appear friable (Fig. 7.8) and white. Sometimes calli develop from two different lobes of an anther (Fig. 7.9). The embryo-

 

Fig. 7.8. An embryogenic, haploid, friable, white callus emerging after three months of culture from anther culture of the cultivar Nules of C. clementina Hort. ex Tan.


 

 

Fig. 7.9. Calli emerging from two different lobes of an anther of cv. Nules clementine.

 

Green, compact and non-morphogenic calli emerging from anthers were also observed in Poncirus, C. clementina and C. limon (Fig. 7.14).

Divisions in pollen grains of various species start at different intervals after the fi rst pollen mitosis, and after the trauma of wounding and culture, depending on the degree of repression of the sporophytic gene programme (Heberle-Bors, 1985).

Several studies have been carried out on early nuclear division events of the microspores of herbaceous species. Hidaka and Omura (1989b) described cytologically the development of embryoids from microspores in C. aurantium and P. trifoli- ata. They observed three main routes of development. In route A, the microspores


 

 

 

 

Fig. 7.10. (a) Two Petri dishes after four months of culture containing haploid embryogenic calli from clementine Nules anthers. (b) Haploid embryo- genic calli and embryoids in different stages devel- oping from a single anther of clementine SRA 63 in culture.

 


lose their contents. In route B, microspores develop as in vivo, normal nuclear division with one vegetative and one or two genera- tive nuclei; this route is rarely observed. Route C is divided into two routes. In route C1, two types of morphologically similar


nuclei are observed: vegetative-type nuclei (C1a) or generative-type nuclei (C1b). Route C2, the repeated division of only the vege- tative-type nucleus or the repeated division of both vegetative and generative-type nuclei independent of each other, seems to


 

 

Fig. 7.11. (a and b) A cotyledonary fused embryoid.


 

 

Fig. 7.12. A pluricotyledonary embryoid.

 

 

form the most embryoids in Citrus anther culture.

When the nucleus divides without cell division, a multinucleate pollen grain is initially formed which later gives rise to a multicellular structure, then developing into a proembryoid (Fig. 7.15) and fi nally into an embryoid, until the exine rupture. Moreover, nuclear fusion among vegetative and generative nuclei has been observed, and this can explain an increase in ploidy level.

A morphological and ultrastructural study, at the cellular and subcellular level, of early microspore embryogenesis in sev- eral embryogenic varieties of C. clementina is in progress in a joint project between the University of Palermo (Italy) and the Centro de Investigaciones Biologicas (Spain). Microscopic analysis has revealed very important aspects of this embryogenic


 

 
 

 

Fig. 7.14. A green and compact callus emerging from inside a Poncirus trifoliata anther.

 

 
 

 

Fig. 7.15. A proembryoid located inside the anther. Fragments of the broken exine are still at its periphery. (2 mm semi-thin section stained with toluidine blue.) This photo was taken at the labora- tory of Dr Maria Carmen Risueñ o, Centro de Investigaciones Biologicas, CSIS, Spain.


 

 

 

Fig. 7.13. (a and b) Pseudobulbils from Citrus anther culture.


 

 

 

Fig. 7.16. (a and b) Haploid embryoid germination.

 

 


process, indicating differences between Citrus microspore-derived embryos and those derived from other embryogenic species, such as starch accumulation during the fi rst embryonic stages (Ramirez et al., 2003). Moreover, different cellular types have been observed in these embryos after the exine breakdown.

 

 

Fig. 7.17. Haploid plantlet of Nules clementine obtained from embryoid germination.


Plant recovery, hardening and characterization

Plantlet formation from cultured anthers may occur either directly through embryo- genesis of microspores or indirectly through organogenesis or embryogenesis of microspore-derived callus.

The highly embryogenic haploid callus is multiplied in MS medium supplemented with 5% sucrose, 0.02 mg/l NAA and 0.8% agar, maintaining its embryogenic potential for several years. As the embryos appear, they are germinated in Petri dishes (Fig. 7.16a and b) with MS medium containing 3% (w/v) sucrose, 1 mg/l GA3, 0.01 mg/l

NAA and 0.75% (w/v) agar (germination

medium), and they are later transferred to Magenta boxes (Sigma V8505) or to test tubes (Fig. 7.17).

The embryoids develop normally through the globular, heart, torpedo and cotyledonary stages, and often produce sec- ondary embryoids. Haploid embryoids of clementine vigorously germinate in vitro; in contrast, haploid plantlets grow slowly in soil, presumably due to recessive harmful genes expressed in homozygosity. These plantlets, when transplanted in vivo, usu- ally die as a result of fungal contamination. Better results have been obtained by graft- ing in vitro homozygous small shoots (2–3 mm) on to etiolated 20-day-old Troyer cit-


 


 

 

Fig. 7.18. I n vitro grafting of homozygous Citrus clementina, cv. Nules on to etiolated ‘Troyer’ cit- range seedlings to improve the material survival.

 

 

range seedlings (Fig. 7.18). After 3–4 months, the grafted plantlets obtained were washed with sterile water to remove the medium from their roots and then trans- ferred to sterilized pots containing peat moss, sand and soil in the ratio 1: 1: 1 for the acclimation phase (Fig. 7.19). The new scions obtained were later grafted on to 2- year-old sour orange seedlings. They showed a more compact habitus (Fig. 7.20a and b) and a decrease in vigour, with sig- nifi cantly smaller leaves, shorter internodes and more thorns when compared with the heterozygous parent of the same age of grafting (Germanà et al., 2000b).

More vigorous growth has been observed by grafting the second time on to

C. macrophylla instead of on to C. auran- tium (unpublished).

Ploidy of androgenetic plants

Haploid, and especially triploid, diploid, aneuploid and mixoploid calli and plantlets have been produced from Citrus


and its relatives by anther culture. Non- haploids may arise from: (i) somatic tissue of anther walls; (ii) the fusion of nuclei; (iii) endomitosis within the pollen grain; or (iv) irregular microspores formed by meiotic irregularities (D’Amato, 1977; Sunderland and Dunwell, 1977; Narayanaswamy and George, 1982; Sangwan-Norrel, 1983). The developmental stage of the pollen at the time of the culture can cause ploidy varia- tion in regenerated plants: in particular, the older the stage, the higher the ploidy level of the embryoids obtained (Maheshwari et al., 1980).

Besides regeneration from somatic tissue of anther, heterozygosity can also be observed when the plants are regenerated from unreduced microspores or in the case of new variation induced at the diploid level (gametoclonal variation) (Wenzel et al., 1977; Morrison and Evans, 1987).

 

CYTOLOGICAL CHARACTERIZATION OF REGENERANTS.

Chromosome number has been counted in root tip cells from regenerated embryos and plantlets, using the standard Feulgen tech- nique (Lillie, 1951). The explants were pre- treated with 0.05% (w/v) aqueous solution of colchicine for 2 h at room temperature, fi xed overnight in 3: 1 (v/v) ethanol: glacial acetic acid, and stored in 70% ethanol until viewing.

Chromosome counts carried out on root apices of embryos and of plantlets obtained

 

 
 

 

Fig. 7.19. A haploid plantlet of Citrus clementina,

cv. Nules transferred to soil.


 

 

 

Fig. 7.20. Doubled haploid Nules grafted on to sour orange seedlings 1 year (a) and 5 years (b) after grafting.

 


from in vitro androgenesis of clementine showed the haploid set of chromosomes (n = x = 9) (Fig. 7.21) (Germanà et al., 1991, 1994, 2000a, b; Germanà, 1997). During culture, haploid calli spontaneously diploidize, pro- ducing doubled haploid embryoids and plantlets (Germanà, 1997), and sometimes the presence of a triploid number of chro- mosomes (Fig. 7.22) in homozygous calli cells has also been observed.

 

FLOW CYTOMETRY. Identifi cation of regener- ants from anther culture of clementine and Clausena excavata has also been performed by fl ow cytometry (Ollitrault et al., 1996; Froelicher and Ollitrault, 2000; Germanà et al., 2005b).


Isozyme analyses

Because of the spontaneous diploidization of the haploid calluses, cytological analysis cannot always identify androgenic plants, and isozyme analyses have been employed to decide the gametic origin of calluses and plantlets (Germanà et al., 1991, 1994, 2000a, b; Germanà and Reforgiato, 1997). Isozyme techniques allow the distinction to be made between androgenetic and somatic tissue when the enzyme is heterozygotic in the diploid condition of the donor plant and the regenerants show lack of an allele.

To identify the origin of calli, embry- oids and plantlets obtained, their crude extracts are analysed using two enzyme sys-


 


 

Fig. 7.21. A haploid set of chromosomes from a root tip cell of a regenerated plantlet (n = x = 9).

 

 

tems: phosphoglucoisomerase (PGI) and phosphoglucomutase (PGM), as reported by Grosser et al. (1988). Numbering for isozymes (PGI-1) and lettering for different allozymes are the same as used by Torres et al. (1978).

Citrus clementina is heterozygous for PGI-1 and PGM. According to Torres et al.

 
 

Fig. 7.22. A triploid set of chromosomes from a homozygous callus cell (n = 3 x = 27).


(1978), the heterozygous clementine parent is FI (F = allele which specifi es fast migration toward the anode enzyme; I = intermediate) in PGM, and WS (W = allele which specifi es an enzyme migrating faster than F; S = allele which specifi es a slowly migrating enzyme) in PGI. For analysis of calli and leaves obtained from anther culture, the presence of a single band was retained as the homozy- gous state (Fig. 7.23a and b). With one or two exceptions out of more than 100 samples analysed, both enzyme systems confi rmed the androgenic nature of regenerants because of the contemporary lack of an allele.

X.X. Deng et al. (1992) used GOT (glu- tamate oxaloacetate transminase) isozyme analysis to show that the diploid plantlet obtained from anther culture of hybrid No.

14 of C. ichangensis ´ C. reticulata was

homozygous and so pollen derived.

PGI, PGM2 and isocitrate dehydroge- nase (IDH) have been used to characterize regenerants in gynogenesis by Ollitrault et al. (1996).

PGI and PGM have been employed by Germanà and Chiancone (2001) for the characterization of gynogenetic haploids in clementine.

 

RAPD analyses

A preliminary characterization of several doubled haploids of C. clementina has been carried out during a collaboration between our laboratory and the Istituto Sperimentale per l’Agrumicoltura of Acireale, using isozyme and random amplifi ed polymophic DNA (RAPD) markers (Germanà et al., 2000b). The aberrant transmission of RAPD markers due to the presence of a band found only in doubled haploids has been observed in homozygous clementine as well as in other genotypes (Pooler and Scorza, 1995). Further studies are in progress to determine the nature of these fragments.

 

Microsatellites

Microsatellites have also been employed to assess homozygosity and to characterize


 

 

 

Figs. 7.23. Isozyme pattern of PGI (a) and PGM (b) of calli and leaves. The fi rst lane on the left and the last lane on the right are the zymogram of the heterozygous Nules parent, the others are those of doubled haploid cultures.

 


regenerants obtained from citrus anther cul- ture (Germanà and Chiancone, 2003; Germanà et al., 2005b).

 

 


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