**5. Ploidy detection and chromosome doubling**

The use of haploid plants, which are very valuable for breeding, depends on chromosome doubling of these plants, thus bringing their chromosome number to the number before haploidization and making them 100% homozygous. Following haploidization procedures, chromosomal set numbers of the regenerated plants are determined by using different ploidy analysis techniques. Although various methods have been used to determine the ploidy in eggplant, flow cytometry analysis is becoming more widespread, since it is more practical and faster.

For chromosome doubling, haploid plants are exposed especially to colchicine or oryzalin, trifluralin and other chemicals with antimitotic effect in either in vitro or in vivo conditions. It has been reported that the lanolin treatment with 0.5%

**79**

mL<sup>−</sup><sup>1</sup>

*Development of Androgenesis Studies on Eggplant (*Solanum melongena *L.) in Turkey…*

colchicine for 48 h in darkness to the buds starting from the secondary axillary buds

In Turkey, Ellialtioglu et al. [40] compared in vitro and in vivo colchicine treatments. In in vitro treatment, micro shoots were incubated in the colchicine solution containing 0.5 or 1%, for 1 or 2 h. In in vivo treatment, the acclimatized haploid plants transferred to greenhouse were pruned, and then the cotton pieces absorbed with same concentrated colchicine (0.5 or 1%) were placed in their axillary buds for 1 or 2 h. One hundred percent chromosome doubling and dihaploid shoots can be achieved in in vivo by using both 0.5% colchicine treatment for 2 h and 1% colchicine for 1 h from the well-grown haploid plants, of which leaves were pruned after the development of four to five nodes. However, this way takes longer time than in vitro method. Each of the waiting steps in in vivo method consisting of the plant growth in greenhouse, shooting of the buds, formation of the first flower and finally selfing requires some time. In addition, all the procedures may need to be repeated if the doubling did not happen. The in vitro method has been identified as a way that can be an alternative to in vivo and may even be seen as advantageous for saving time. Although there have been a few losses in in vitro method during the applications and acclimatization, these losses will remain unimportant when working with a large number of materials. It has been shown by Ellialtioglu et al. [40] that especially the application of 0.5% colchicine for 2 h in in vitro can be used for dihaploidization of haploid shoots in the eggplant. The most important advantage of this method is that the regenerated plants developed from this application thrive as the complete diploid plants, which offers earlier flowering and thus the possibil-

In the study conducted by Özdemir Çelik [47], the in vitro method described by Ellialtioglu et al. [40] was used to double the chromosome numbers of haploid plants obtained from anther culture, and chromosome doubling was successfully performed.

Among the eggplant androgenesis studies conducted in Turkey so far, anther culture technique has been used predominantly, and feasible androgenetic results are generally derived from these cultures. The highest microspore embryogenesis rate in all the anther culture studies conducted in Turkey was recorded as 320 embryos/100 anthers from A117 F1 [46] cultivar. This was followed by 59.6% from Mabel cultivar [76], 38.4% from Anamur F1 [77] and 36.4% [56] from open pol-

As for the efficiency of microspore embryogenesis in eggplant androgenesis studies in the world, as far as we know, the highest embryogenesis rates obtained from anther culture studies were 237.5% (237.5 embryos/100 anthers) from DH36 line developed from Bandera F1, 146.5% from Bandera F1, the parent of DH36 line

In the studies of isolated microspore culture, the first remarkable improvement was achieved by Corral-Martinez et al. [27]. After the refinement studies, the highest success was reported by Rivas-Sendra et al. [81] from the first generation of DH population lines (DHS1 lines) developed from Bandera F1. The

DH15, DH41, DH40 and DH36 lines, respectively. According to the literature, in order to obtain DH eggplant plants via isolated microspore culture nowadays, it is attempted to get callogenesis at first and then to develop regenerated plants

and 267.36 callus mL<sup>−</sup><sup>1</sup>

, 76.86 callus mL<sup>−</sup><sup>1</sup>

from Bandera F1 (control) and

, 92 callus

[79], 60% from Ecavi F1 [33] and 53% from Cristal F1 [80].

callus yields were obtained to be 65.08 callus mL<sup>−</sup><sup>1</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.88299*

ity of earlier selfing.

linated cultivar Aydın Siyahı.

, 149.11 callus mL<sup>−</sup><sup>1</sup>

**6. Microspore embryogenesis efficiency**

in in vivo conditions resulted in 50–70% doubling [21].

*Development of Androgenesis Studies on Eggplant (*Solanum melongena *L.) in Turkey… DOI: http://dx.doi.org/10.5772/intechopen.88299*

colchicine for 48 h in darkness to the buds starting from the secondary axillary buds in in vivo conditions resulted in 50–70% doubling [21].

In Turkey, Ellialtioglu et al. [40] compared in vitro and in vivo colchicine treatments. In in vitro treatment, micro shoots were incubated in the colchicine solution containing 0.5 or 1%, for 1 or 2 h. In in vivo treatment, the acclimatized haploid plants transferred to greenhouse were pruned, and then the cotton pieces absorbed with same concentrated colchicine (0.5 or 1%) were placed in their axillary buds for 1 or 2 h. One hundred percent chromosome doubling and dihaploid shoots can be achieved in in vivo by using both 0.5% colchicine treatment for 2 h and 1% colchicine for 1 h from the well-grown haploid plants, of which leaves were pruned after the development of four to five nodes. However, this way takes longer time than in vitro method. Each of the waiting steps in in vivo method consisting of the plant growth in greenhouse, shooting of the buds, formation of the first flower and finally selfing requires some time. In addition, all the procedures may need to be repeated if the doubling did not happen. The in vitro method has been identified as a way that can be an alternative to in vivo and may even be seen as advantageous for saving time. Although there have been a few losses in in vitro method during the applications and acclimatization, these losses will remain unimportant when working with a large number of materials. It has been shown by Ellialtioglu et al. [40] that especially the application of 0.5% colchicine for 2 h in in vitro can be used for dihaploidization of haploid shoots in the eggplant. The most important advantage of this method is that the regenerated plants developed from this application thrive as the complete diploid plants, which offers earlier flowering and thus the possibility of earlier selfing.

In the study conducted by Özdemir Çelik [47], the in vitro method described by Ellialtioglu et al. [40] was used to double the chromosome numbers of haploid plants obtained from anther culture, and chromosome doubling was successfully performed.

#### **6. Microspore embryogenesis efficiency**

Among the eggplant androgenesis studies conducted in Turkey so far, anther culture technique has been used predominantly, and feasible androgenetic results are generally derived from these cultures. The highest microspore embryogenesis rate in all the anther culture studies conducted in Turkey was recorded as 320 embryos/100 anthers from A117 F1 [46] cultivar. This was followed by 59.6% from Mabel cultivar [76], 38.4% from Anamur F1 [77] and 36.4% [56] from open pollinated cultivar Aydın Siyahı.

As for the efficiency of microspore embryogenesis in eggplant androgenesis studies in the world, as far as we know, the highest embryogenesis rates obtained from anther culture studies were 237.5% (237.5 embryos/100 anthers) from DH36 line developed from Bandera F1, 146.5% from Bandera F1, the parent of DH36 line [79], 60% from Ecavi F1 [33] and 53% from Cristal F1 [80].

In the studies of isolated microspore culture, the first remarkable improvement was achieved by Corral-Martinez et al. [27]. After the refinement studies, the highest success was reported by Rivas-Sendra et al. [81] from the first generation of DH population lines (DHS1 lines) developed from Bandera F1. The callus yields were obtained to be 65.08 callus mL<sup>−</sup><sup>1</sup> , 76.86 callus mL<sup>−</sup><sup>1</sup> , 92 callus mL<sup>−</sup><sup>1</sup> , 149.11 callus mL<sup>−</sup><sup>1</sup> and 267.36 callus mL<sup>−</sup><sup>1</sup> from Bandera F1 (control) and DH15, DH41, DH40 and DH36 lines, respectively. According to the literature, in order to obtain DH eggplant plants via isolated microspore culture nowadays, it is attempted to get callogenesis at first and then to develop regenerated plants

*Sustainable Crop Production*

**4.6 Culture conditions**

source × PGR application'.

darkness at 25°C

ness at 25°C

are germinated at 2,000–3,000 lux [12].

regeneration.

According to the studies conducted in Turkey and the world, the cold shocks applied to the eggplant buds taken at the appropriate stage did not generally resulted in positive response for microspore embryogenesis. On the contrary, it is generally accepted that high-temperature shocks such as 35°C for 8 days at dark conditions have positive effects on induction of microspore embryogenesis and

Ellialtioglu et al. [77], of which details were mentioned above previously, compared the growth performance of haploid embryos cultured under fluorescent lamps or LED lighting conditions. The ratio of haploid embryo formation ranging between 0 and 38.4% was reported to differ under the interaction of 'cultivar × light

After the anthers or microspores are isolated and cultured in a nutrient medium and a heat shock treatment is applied, the culture conditions based on climate data of the growth environment greatly affect the androgenesis success. The temperature and light to be two important variables of environmental conditions should be optimized. In in vitro conditions, light intensity can be used between 300 and 10,000 lux depending on the plant species, explant type, nutrient medium [78] and culture stage. It is recommended that light intensity should be low for anther cultures or even in the dark during the early days of culture. Thus, anthers are usually cultivated in the dark during the first period of culture, and then the 300–1,500 lux light intensity is applied to the cultures for embryo development. The embryos

As a result of the large number of studies made in both the world and Turkey,

• Firstly, to exposure the cultures in DDV-C medium to a heat shock pretreat-

• As additional pretreatment, to keep the cultures in the same nutrient medium for 4 more days but under photoperiod conditions consisting of 16/8 h of light/

• At the end of 12 (8 + 4) days, to transfer the cultures to DDV-R medium and culture them under photoperiod conditions consisting of 16/8 h of light/dark-

The use of haploid plants, which are very valuable for breeding, depends on chromosome doubling of these plants, thus bringing their chromosome number to the number before haploidization and making them 100% homozygous. Following haploidization procedures, chromosomal set numbers of the regenerated plants are determined by using different ploidy analysis techniques. Although various methods have been used to determine the ploidy in eggplant, flow cytometry analysis is

For chromosome doubling, haploid plants are exposed especially to colchicine or oryzalin, trifluralin and other chemicals with antimitotic effect in either in vitro or in vivo conditions. It has been reported that the lanolin treatment with 0.5%

ment consisting of 35°C in the dark for 8 days temperature shock

the climatic conditions preferred for eggplant anther cultures are:

**5. Ploidy detection and chromosome doubling**

becoming more widespread, since it is more practical and faster.

**78**

through organogenesis [47, 81]. However, an efficient direct embryogenesis protocol have not yet been developed in the isolated microspore culture studies in eggplant so far.
