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**115**

unchanged.

**Chapter 8**

**Abstract**

boundaries.

**1. Introduction**

from Thailand

Polyploidy in the Ginger Family

*Kesara Anamthawat-Jónsson and Puangpaka Umpunjun*

Polyploidy is common in the ginger family Zingiberaceae. The aims of the present paper are (1) to provide a general introduction on species diversity with emphasis on conservation; (2) to highlight the human-use significance of this family, focusing on the two major genera, *Zingiber* (ginger) and *Curcuma* (turmeric); (3) to present chromosome number data from 45 natural and cultivated *Curcuma* taxa from Thailand, of which polyploids are predominant; and (4) to describe our own work on cytotaxonomy of selected Thai *Curcuma* species. We obtained somatic chromosome numbers from root tips and analysed meiotic chromosome behaviour from flowers. We also used the molecular cytogenetic method of ribosomal gene mapping on chromosomes to infer mechanism of polyploidization and reveal genomic relationships among closely related species. The main results of our cytogenetic studies include the following. The most sought-after medicinal *Curcuma* cultivars growing on a large-scale basis are secondary triploids, so as taxa in natural habitats that are harvested for local utilisation. These triploids are sexually deficient, due to meiotic pairing abnormalities, but they are propagated asexually via rhizomes. The ribosomal mapping results indicate natural triploidization process via hybridisation, either within populations or across the species

**Keywords:** *Curcuma*, cytogenetics, cytotaxonomy, ethnobotany, ginger,

Taxonomic classification of the ginger family (Zingiberaceae) is still under revision for many floras, as more than 3000 species names have been used worldwide, but only half of these are accepted. These aromatic herbs grow in moist areas of the tropics and subtropics, including some regions that are seasonably dry. The ginger family comprises about 50 genera and more than 1300 species worldwide, and in Thailand 21 genera with about 200 species have been described. Numerous species are endemic to Thailand, but the majority has a wider distribution, especially over Southeast and South Asia. A few species of this family are commercially cultivated, such as ginger (*Zingiber officinale* Rosc.), turmeric (*Curcuma longa* L.) and aromatic ginger (*Kaempferia galanga* L.). Interestingly, these widely cultivated species are sexually deficient triploid or pentaploid plants—the elite cultivars are therefore propagated by rhizomes. These polyploid species are superior to their diploid relatives in terms of growth and yield, while the sought-after quality characters remain

medicinal plants, polyploidy, triploidy, turmeric, *Zingiber*

#### **Chapter 8**

## Polyploidy in the Ginger Family from Thailand

*Kesara Anamthawat-Jónsson and Puangpaka Umpunjun*

#### **Abstract**

Polyploidy is common in the ginger family Zingiberaceae. The aims of the present paper are (1) to provide a general introduction on species diversity with emphasis on conservation; (2) to highlight the human-use significance of this family, focusing on the two major genera, *Zingiber* (ginger) and *Curcuma* (turmeric); (3) to present chromosome number data from 45 natural and cultivated *Curcuma* taxa from Thailand, of which polyploids are predominant; and (4) to describe our own work on cytotaxonomy of selected Thai *Curcuma* species. We obtained somatic chromosome numbers from root tips and analysed meiotic chromosome behaviour from flowers. We also used the molecular cytogenetic method of ribosomal gene mapping on chromosomes to infer mechanism of polyploidization and reveal genomic relationships among closely related species. The main results of our cytogenetic studies include the following. The most sought-after medicinal *Curcuma* cultivars growing on a large-scale basis are secondary triploids, so as taxa in natural habitats that are harvested for local utilisation. These triploids are sexually deficient, due to meiotic pairing abnormalities, but they are propagated asexually via rhizomes. The ribosomal mapping results indicate natural triploidization process via hybridisation, either within populations or across the species boundaries.

**Keywords:** *Curcuma*, cytogenetics, cytotaxonomy, ethnobotany, ginger, medicinal plants, polyploidy, triploidy, turmeric, *Zingiber*

#### **1. Introduction**

Taxonomic classification of the ginger family (Zingiberaceae) is still under revision for many floras, as more than 3000 species names have been used worldwide, but only half of these are accepted. These aromatic herbs grow in moist areas of the tropics and subtropics, including some regions that are seasonably dry. The ginger family comprises about 50 genera and more than 1300 species worldwide, and in Thailand 21 genera with about 200 species have been described. Numerous species are endemic to Thailand, but the majority has a wider distribution, especially over Southeast and South Asia. A few species of this family are commercially cultivated, such as ginger (*Zingiber officinale* Rosc.), turmeric (*Curcuma longa* L.) and aromatic ginger (*Kaempferia galanga* L.). Interestingly, these widely cultivated species are sexually deficient triploid or pentaploid plants—the elite cultivars are therefore propagated by rhizomes. These polyploid species are superior to their diploid relatives in terms of growth and yield, while the sought-after quality characters remain unchanged.

#### **2. The ginger family (Zingiberaceae), with emphasis on** *Curcuma*

The ginger family or Zingiberaceae comprises about 50 genera and more than 1300 species worldwide [1, 2]. The family distribution is pantropical, with centre of species diversity in South and Southeast Asia. Some species are found in America and subtropical and warm-temperate Asia. In China, 20 genera and 216 species (141 endemic, four introduced) have been recorded [1]. Geographically, Thailand is part of the Indochinese region that harbours the highest ginger genetic resources [3, 4]. Several of these species are rare and endemic to Thailand [5]. A large number of Thai taxa of Zingiberaceae are known as edible, ornamental or medicinal plants, from which commercial products beneficial to human can be developed.

Two best known genera in the context of cultivation and human uses worldwide are *Zingiber* Miller (ginger) and *Curcuma* Linnaeus (turmeric). The largest genus *Zingiber*, which comprises 100–200 species, is native to Southeast Asia especially in Thailand [6], China [7], the Indian subcontinent and New Guinea [8]. It contains the true gingers, plants grown for their medicinal and culinary value. The best known is *Z. officinale*, the garden ginger.

*Curcuma* is a genus of about 120 accepted species in the family Zingiberaceae that contains such species as turmeric (*C. longa*) and Siam tulip (*C. alismatifolia* Gagnep.). They are native to Southeast Asia, southern China, the Indian subcontinent, New Guinea and northern Australia [6, 8–11]. Tropical Asia and South Asia are the diversity hotspots of the genus. Although the species diversity is very high and new species are being discovered regularly, other species are disappearing. Habitat loss, due to global warming, deforestation, agricultural expansion and anthropogenic activities, is one of the main causes of biodiversity loss worldwide [12–14]. In addition, overharvesting for use in traditional medicine has raised a serious concern that wild plants will be disappearing from nature.

According to the IUCN Red List of Threatened Species, seven *Curcuma* species have been declared endangered to extinction (EN) and six additionally critically endangered (CR). The endemic EN species are *C. caulina* J. Graham, India [15]; *C. colorata* Valeton, Indonesia [16]; *C. coriacea* Mangaly & M. Sabu, India [17]; *C. corniculata* Skornick., Lao [18]; *C. prasina* Skornick., Thailand [19]; *C. sahuynhensis* Skornick. & N.S. Lý, Vietnam [20]; and *C. vitellina* Skornick. & H.D.Tran, Vietnam [21]. The endemic CR species are *C. bhatii* (R.M.Sm.) Skornick. & M. Sabu, India [22]; *C. leonidii* Skornick. & Luu, Vietnam [23]; *C. newmanii* Skornick., Vietnam [24]; *C. pygmaea* Skornick. & Sida f., Vietnam [25]; *C. supraneeana* (W.J. Kress & K. Larsen) Skornick., Thailand [26]; and *C. vamana* M. Sabu and Mangaly, India [27]. There clearly is an urgent need to protect these *Curcuma* species in their natural habitats while at the same time encouraging ex situ conservation and supporting researches aiming to find viable methods for sustainable cultivation of species of economic potential.

#### **3. Recent publications on the ginger family**

The survey of recent (2019) publications on Zingiberaceae in the Web-of-Science database, using "ginger" as keyword in titles (**Figure 1**), shows that the genus *Zingiber* (ginger) is by far the most investigated worldwide. The most researched topics concern medicinal properties and health benefits of ginger (1); pharmaceutical, biochemical and molecular characterisation (2); applications in food science and chemistry (3); other technologies and industrial applications (4); as well as some effort in improving cultivation (5). On the other hand, research on *Zingiber* diversity, taxonomy, ecology and genetics (6) is limited.

**117**

**Figure 1.**

*Polyploidy in the Ginger Family from Thailand DOI: http://dx.doi.org/10.5772/intechopen.92859*

Ginger (*Z. officinale*) is a very popular spice used worldwide, whether it be used to spice up meals, or as a medicine [28]. Ginger can be used for a variety of food or medicine items, as vegetables, candy, soda, pickles and alcoholic beverages. It is one of the most versatile, ancient, significant, medicinal, nutritional herbs with several ethnomedical values. This plant is recognised due to its therapeutic properties, including antibiotic, antimicrobial, antioxidant and anti-inflammatory effects [29]. Phenolic acids, diarylheptanoids, terpenoids and flavonoids are reported to exist in ginger rhizomes [30]. A list of 72 gingerols and diarylheptanoids derivatives from ginger rhizomes is presented in Asamenew et al. [31], and among these compounds, gingerol- and shogaol-related derivatives are the principal medicinally active components contributing to the characteristic pungent flavour of ginger together with essential oil major component, zingerone. These bioactive compounds have been shown in experiments to be effective for inflammatory diseases [32] and osteoarthritis [29], to help induce apoptosis in cancer cells [33] and to show anti-leukaemic

*Distribution of recent publications by research topics in 2019, obtained from web-of-science database (webofknowledge.com/WOS\_), using the single keyword "ginger" in title. Research topics (x-axis): 1, medicinal properties and health benefits; 2, pharmaceutical, biochemical and molecular research; 3, food science and chemistry; 4, other technologies and industrial applications; 5, cultivation and agriculture; and 6, biodiversity, taxonomy, ecology and genetics. Y-axis: Percentages of the number of publications in 166 totals. Blue columns include papers on* Zingiber*, ginger, gingerols and ginger-related topics. Red columns include papers on* Curcuma*, turmeric, curcumins and related topics. Grey columns include other species in the ginger family Zingiberaceae.*

Our survey of recent (2019) publications on Zingiberaceae in the Web-of-Science database, using "*Curcuma*" as keyword in titles (**Figure 2**), shows that *C. longa* (turmeric, saffron turmeric) is the single most researched *Curcuma* species. The results show that this species has received much attention in the area of pharmaceutical research and medicinal applications. Turmeric is commonly used as spice, dye, drug and cosmetics [35], but recent research efforts have further characterised its medicinal properties and have identified its biochemical components in high resolution and specificity. The genus *Curcuma* is rich in flavonoids, tannins, anthocyanin, phenolic compounds, oil, organic acids and inorganic compounds [36]. The biological activities of *Curcuma* have been attributed to the non-volatile ingredients of the rhizome, cucurminoids (e.g. curcumin), as well as to the volatile terpenoids [37]. Curcumin has been shown in experiments to have strong anti-inflammatory and antioxidant effects [29, 36]. The European Union has

effect [34]. Ginger has a great pharmaceutical potential.

*Polyploidy in the Ginger Family from Thailand DOI: http://dx.doi.org/10.5772/intechopen.92859*

#### **Figure 1.**

*Chromosomal Abnormalities*

known is *Z. officinale*, the garden ginger.

**2. The ginger family (Zingiberaceae), with emphasis on** *Curcuma*

from which commercial products beneficial to human can be developed.

serious concern that wild plants will be disappearing from nature.

methods for sustainable cultivation of species of economic potential.

*Zingiber* diversity, taxonomy, ecology and genetics (6) is limited.

**3. Recent publications on the ginger family**

The ginger family or Zingiberaceae comprises about 50 genera and more than 1300 species worldwide [1, 2]. The family distribution is pantropical, with centre of species diversity in South and Southeast Asia. Some species are found in America and subtropical and warm-temperate Asia. In China, 20 genera and 216 species (141 endemic, four introduced) have been recorded [1]. Geographically, Thailand is part of the Indochinese region that harbours the highest ginger genetic resources [3, 4]. Several of these species are rare and endemic to Thailand [5]. A large number of Thai taxa of Zingiberaceae are known as edible, ornamental or medicinal plants,

Two best known genera in the context of cultivation and human uses worldwide are *Zingiber* Miller (ginger) and *Curcuma* Linnaeus (turmeric). The largest genus *Zingiber*, which comprises 100–200 species, is native to Southeast Asia especially in Thailand [6], China [7], the Indian subcontinent and New Guinea [8]. It contains the true gingers, plants grown for their medicinal and culinary value. The best

*Curcuma* is a genus of about 120 accepted species in the family Zingiberaceae that contains such species as turmeric (*C. longa*) and Siam tulip (*C. alismatifolia* Gagnep.). They are native to Southeast Asia, southern China, the Indian subcontinent, New Guinea and northern Australia [6, 8–11]. Tropical Asia and South Asia are the diversity hotspots of the genus. Although the species diversity is very high and new species are being discovered regularly, other species are disappearing. Habitat loss, due to global warming, deforestation, agricultural expansion and anthropogenic activities, is one of the main causes of biodiversity loss worldwide [12–14]. In addition, overharvesting for use in traditional medicine has raised a

According to the IUCN Red List of Threatened Species, seven *Curcuma* species have

been declared endangered to extinction (EN) and six additionally critically endangered (CR). The endemic EN species are *C. caulina* J. Graham, India [15]; *C. colorata* Valeton, Indonesia [16]; *C. coriacea* Mangaly & M. Sabu, India [17]; *C. corniculata* Skornick., Lao [18]; *C. prasina* Skornick., Thailand [19]; *C. sahuynhensis* Skornick. & N.S. Lý, Vietnam [20]; and *C. vitellina* Skornick. & H.D.Tran, Vietnam [21]. The endemic CR species are *C. bhatii* (R.M.Sm.) Skornick. & M. Sabu, India [22]; *C. leonidii* Skornick. & Luu, Vietnam [23]; *C. newmanii* Skornick., Vietnam [24]; *C. pygmaea* Skornick. & Sida f., Vietnam [25]; *C. supraneeana* (W.J. Kress & K. Larsen) Skornick., Thailand [26]; and *C. vamana* M. Sabu and Mangaly, India [27]. There clearly is an urgent need to protect these *Curcuma* species in their natural habitats while at the same time encouraging ex situ conservation and supporting researches aiming to find viable

The survey of recent (2019) publications on Zingiberaceae in the Web-of-Science database, using "ginger" as keyword in titles (**Figure 1**), shows that the genus *Zingiber* (ginger) is by far the most investigated worldwide. The most researched topics concern medicinal properties and health benefits of ginger (1); pharmaceutical, biochemical and molecular characterisation (2); applications in food science and chemistry (3); other technologies and industrial applications (4); as well as some effort in improving cultivation (5). On the other hand, research on

**116**

*Distribution of recent publications by research topics in 2019, obtained from web-of-science database (webofknowledge.com/WOS\_), using the single keyword "ginger" in title. Research topics (x-axis): 1, medicinal properties and health benefits; 2, pharmaceutical, biochemical and molecular research; 3, food science and chemistry; 4, other technologies and industrial applications; 5, cultivation and agriculture; and 6, biodiversity, taxonomy, ecology and genetics. Y-axis: Percentages of the number of publications in 166 totals. Blue columns include papers on* Zingiber*, ginger, gingerols and ginger-related topics. Red columns include papers on* Curcuma*, turmeric, curcumins and related topics. Grey columns include other species in the ginger family Zingiberaceae.*

Ginger (*Z. officinale*) is a very popular spice used worldwide, whether it be used to spice up meals, or as a medicine [28]. Ginger can be used for a variety of food or medicine items, as vegetables, candy, soda, pickles and alcoholic beverages. It is one of the most versatile, ancient, significant, medicinal, nutritional herbs with several ethnomedical values. This plant is recognised due to its therapeutic properties, including antibiotic, antimicrobial, antioxidant and anti-inflammatory effects [29]. Phenolic acids, diarylheptanoids, terpenoids and flavonoids are reported to exist in ginger rhizomes [30]. A list of 72 gingerols and diarylheptanoids derivatives from ginger rhizomes is presented in Asamenew et al. [31], and among these compounds, gingerol- and shogaol-related derivatives are the principal medicinally active components contributing to the characteristic pungent flavour of ginger together with essential oil major component, zingerone. These bioactive compounds have been shown in experiments to be effective for inflammatory diseases [32] and osteoarthritis [29], to help induce apoptosis in cancer cells [33] and to show anti-leukaemic effect [34]. Ginger has a great pharmaceutical potential.

Our survey of recent (2019) publications on Zingiberaceae in the Web-of-Science database, using "*Curcuma*" as keyword in titles (**Figure 2**), shows that *C. longa* (turmeric, saffron turmeric) is the single most researched *Curcuma* species. The results show that this species has received much attention in the area of pharmaceutical research and medicinal applications. Turmeric is commonly used as spice, dye, drug and cosmetics [35], but recent research efforts have further characterised its medicinal properties and have identified its biochemical components in high resolution and specificity. The genus *Curcuma* is rich in flavonoids, tannins, anthocyanin, phenolic compounds, oil, organic acids and inorganic compounds [36]. The biological activities of *Curcuma* have been attributed to the non-volatile ingredients of the rhizome, cucurminoids (e.g. curcumin), as well as to the volatile terpenoids [37]. Curcumin has been shown in experiments to have strong anti-inflammatory and antioxidant effects [29, 36]. The European Union has

#### **Figure 2.**

*Distribution of recent publications by research topics in 2019, obtained from web-of-science database (webofknowledge.com/WOS\_), using the single keyword "Curcuma" in title. Research topics (x-axis): 1, medicinal properties and health benefits; 2, pharmaceutical, biochemical and molecular research; 3, food science and chemistry; 4, other technologies and industrial applications; 5, cultivation and agriculture; and 6, biodiversity, taxonomy, ecology and genetics. Y-axis: Percentages of the number of publications in 200 totals. Red columns include papers on* Curcuma longa *(turmeric). Yellow columns include papers about all other*  Curcuma *species, e.g.* C. zedoaria *(white turmeric, 7%),* C. caesia *(black turmeric, 5%),* C. xanthorrhiza *(Javanese ginger/turmeric, 5%),* C. amada *(mango ginger, 5%),* C. aromatica *(fragrant turmeric, 3%) and 17 other* Curcuma *species with less than 3% each.*

recommended the use of numerous medicinal plants for the treatment of gastrointestinal disorders, and among them are ginger root (*Z. officinale*) and turmeric root (*C. longa*) [38].

Some 23 other *Curcuma* species have recently been explored in search for new medicinal applications (**Figure 2**). The top five *Curcuma* species investigated are *C. zedoaria* (Christm.) Rosc. (white turmeric, native to South and Southeast Asia, cultivated in Thailand), *C. caesia* Roxb. (black turmeric, native to Northeast India, natural species of Thailand), *C. xanthorrhiza* Roxb. (Javanese ginger/turmeric, originated from Java island, cultivated in Thailand), *C. amada* Roxb. (mango ginger, originated from East India, natural species of Thailand) and *C. aromatica* Roxb. (fragrant turmeric, natural species of South Asia, cultivated in Thailand). Bioactive ingredients, including terpenes (more than 40 monoterpenes and sesquiterpenes), antioxidants flavonoids and phenolic compounds are present in all these species [39, 40]. Curcumin from *C. zedoaria*, as from *C. longa*, shows good anti-inflammatory effects [36]. Zederone and zedoarondiol, from rhizomes of En-Lueang (*Curcuma cf. amada*), show strong cytotoxicity in a leukaemic cell line and in peripheral blood mononuclear cells, as well as having antioxidant and haemolysis properties [41]. Dry extracts from rhizomes of *C. xanthorrhiza* and *C. zedoaria* have been shown to have anticancer and antiviral properties [42–44]. Furthermore, volatile oils extracted from leaves of *C. caesia* have broad antioxidant, antiinflammatory and antimicrobial effects in vitro [45, 46]. In contrast to *C. longa*, many of the medicinal *Curcuma* species are not in large-scale cultivation, and this increases the risk of overharvesting of rhizomes from wild plants. The good news is that researchers are beginning to improve local cultivars and finding suitable methods of micropropagation of these *Curcuma* species, for example, *C. angustifolia* Roxb. [47].

**119**

*Polyploidy in the Ginger Family from Thailand DOI: http://dx.doi.org/10.5772/intechopen.92859*

**4. The genus** *Curcuma* **in Thailand**

ovarian hormone deficit [53, 54].

*C. alismatifolia* Gagnep.

*C. bella* Maknoi\*, K. Larsen & Sirirugsa**a** *C. bicolor* J.Mood & K. Larsen

*C. candida* (Wall.) Techapr.\***c**

*C. cochinchinensis* Gagnep.

*C. glans* K. Larsen &

J. Mood

**Species of** *Curcuma* **L. 2***n* **chromosome** 

**number**

*C. aromatica* Roxb. 42, 63, 86 Di-, tri-,

*C. ecomata* Craib x

*C. glacillima* Gagnep. ca. 32 x

*C. latifolia* Roxb. 63, 84 Tri-,

*C. aeruginosa* Roxb. 63 Triploid x 4, 6, 9

*C. amada* Roxb. 42 Diploid x 1, 2, 4, 9, 10 *C. angustifolia* Roxb. 42 (64) Diploid x 4, 6, 9, 10

tetraploid

*C. aurantiaca* van Zijp 42 Diploid x x 4, 6, 9, 10

*C. caesia* Roxb.\***b** 63 Triploid x 9, 10

*C. comosa* Roxb. 42, 63 Di-, triploid x x 5, 9, 11

*C. elata* Roxb.*\****d** 63 Triploid x 4, 5, 7, 9, 11 *C. flaviflora* S.Q.Tong 42 Diploid x 7

*C. hermandii* Gagnep. 20 x x 9

*C. leucorhiza* Roxb. Triploid x 6,

tetraploid

Forty-five species are found in Thailand or almost 50% of the total species diversity of *Curcuma* worldwide (**Table 1**). At least 12 of these species are endemic to Thailand. New species have recently been described. For example, *C. saraburiensis* Boonma & Saensouk from Saraburi province, Central Thailand [48] and *C. putii* Maknoi & Jenjitt [49]. Several species of *Curcuma* are cultivated throughout Thailand for commercial purposes. The whole plant has economic values: the above-ground part of the plant bears attractive flowers that have been exported worldwide as cut flowers, such as Siam tulip (*C. alismatifolia*) and *C. parviflora* Wall. [50], whereas the below-ground rhizomes are harvested and sold in local markets for use as crude extracts in the traditional medicine or for the production of certified pharmaceutical products. Medicinal species, such as *C. comosa* Roxb., has received much attention in recent years for being a phytoestrogen-producing plant (e.g. [51, 52]). Products from rhizomes of *C. comosa* have been developed for use as an anti-inflammation remedy and for treatment of uterine abnormalities and

**Ploidy Natural Cultivated References**

x x

x 1, 2, 3, 4, 6,

7, 9, 10

32 x x 9

42 Diploid x 12

x

x

x x 4, 5, 9, 11

*Chromosomal Abnormalities*

recommended the use of numerous medicinal plants for the treatment of gastrointestinal disorders, and among them are ginger root (*Z. officinale*) and turmeric root

*Distribution of recent publications by research topics in 2019, obtained from web-of-science database (webofknowledge.com/WOS\_), using the single keyword "Curcuma" in title. Research topics (x-axis): 1, medicinal properties and health benefits; 2, pharmaceutical, biochemical and molecular research; 3, food science and chemistry; 4, other technologies and industrial applications; 5, cultivation and agriculture; and 6, biodiversity, taxonomy, ecology and genetics. Y-axis: Percentages of the number of publications in 200 totals. Red columns include papers on* Curcuma longa *(turmeric). Yellow columns include papers about all other*  Curcuma *species, e.g.* C. zedoaria *(white turmeric, 7%),* C. caesia *(black turmeric, 5%),* C. xanthorrhiza *(Javanese ginger/turmeric, 5%),* C. amada *(mango ginger, 5%),* C. aromatica *(fragrant turmeric, 3%) and 17* 

Some 23 other *Curcuma* species have recently been explored in search for new medicinal applications (**Figure 2**). The top five *Curcuma* species investigated are *C. zedoaria* (Christm.) Rosc. (white turmeric, native to South and Southeast Asia, cultivated in Thailand), *C. caesia* Roxb. (black turmeric, native to Northeast India, natural species of Thailand), *C. xanthorrhiza* Roxb. (Javanese ginger/turmeric, originated from Java island, cultivated in Thailand), *C. amada* Roxb. (mango ginger, originated from East India, natural species of Thailand) and *C. aromatica* Roxb. (fragrant turmeric, natural species of South Asia, cultivated in Thailand). Bioactive ingredients, including terpenes (more than 40 monoterpenes and sesquiterpenes), antioxidants flavonoids and phenolic compounds are present in all these species [39, 40]. Curcumin from *C. zedoaria*, as from *C. longa*, shows good anti-inflammatory effects [36]. Zederone and zedoarondiol, from rhizomes of En-Lueang (*Curcuma cf. amada*), show strong cytotoxicity in a leukaemic cell line and in peripheral blood mononuclear cells, as well as having antioxidant and haemolysis properties [41]. Dry extracts from rhizomes of *C. xanthorrhiza* and *C. zedoaria* have been shown to have anticancer and antiviral properties [42–44]. Furthermore, volatile oils extracted from leaves of *C. caesia* have broad antioxidant, antiinflammatory and antimicrobial effects in vitro [45, 46]. In contrast to *C. longa*, many of the medicinal *Curcuma* species are not in large-scale cultivation, and this increases the risk of overharvesting of rhizomes from wild plants. The good news is that researchers are beginning to improve local cultivars and finding suitable methods of micropropagation of these *Curcuma* species, for example,

**118**

*C. angustifolia* Roxb. [47].

(*C. longa*) [38].

*other* Curcuma *species with less than 3% each.*

**Figure 2.**

### **4. The genus** *Curcuma* **in Thailand**

Forty-five species are found in Thailand or almost 50% of the total species diversity of *Curcuma* worldwide (**Table 1**). At least 12 of these species are endemic to Thailand. New species have recently been described. For example, *C. saraburiensis* Boonma & Saensouk from Saraburi province, Central Thailand [48] and *C. putii* Maknoi & Jenjitt [49]. Several species of *Curcuma* are cultivated throughout Thailand for commercial purposes. The whole plant has economic values: the above-ground part of the plant bears attractive flowers that have been exported worldwide as cut flowers, such as Siam tulip (*C. alismatifolia*) and *C. parviflora* Wall. [50], whereas the below-ground rhizomes are harvested and sold in local markets for use as crude extracts in the traditional medicine or for the production of certified pharmaceutical products. Medicinal species, such as *C. comosa* Roxb., has received much attention in recent years for being a phytoestrogen-producing plant (e.g. [51, 52]). Products from rhizomes of *C. comosa* have been developed for use as an anti-inflammation remedy and for treatment of uterine abnormalities and ovarian hormone deficit [53, 54].



*\*Species references: a, Maknoi et al. [55]; b, Puangpairote [56]; c, Jenjittikul and Larsen [57]; d, Larsen [6]; e, Maknoi et al. [49]; f, Boonma and Saensouk [48]; g, Chen et al. [58]; and h, Puangpairote et al. [59]. Chromosome/ploidy references: 1. Ramachandran [60]; 2. Ramachandran [61]; 3. eFlora [9]; 4. Leong-Skornikova et al. [10]; 5. Soontornchainaksaeng and Jenjitikul [51]; 6. Zaveska et al. [62]; 7. Chen et al. [63]; 8. Chen et al. [58]; 9. Puangpairote [56]; 10. Rice et al. [64]; 11. Puangpairote et al. [59]; 12. Nopporncharoenkul et al. [65].*

#### **Table 1.**

*List of* Curcuma *species found in Thailand, based on Maknoi [11] (except \*), with 2*n *somatic chromosome number, ploidy level and distribution.*

**121**

*(h, j–k) [66]; and (i) [56].*

**Figure 3.**

*Our own research work on selected* Curcuma *species from Thailand. (a) Triploid* Curcuma comosa *plants, showing the above-ground part of the plant with 60–150-cm-tall leafy shoots and 15–32-cm-long inflorescences with short peduncle and dark pink flowers. (b) Rhizome of the triploid* Curcuma *sp. "*elata-latifolia*". Typical rhizome of this species is ovoid-ellipsoid in shape and about (7–15) × (6–10) cm in size, with 2–7 lateral rhizomes, 2–12 cm long and up to 5 secondary lateral rhizomes. (c) Mitotic metaphase cell of* C. candida *showing diploid chromosome number 2n = 42. (d) Mitotic metaphase cell of* C. comosa *showing diploid chromosome number 2n = 42. (e) Male meiotic cell of the diploid* C. candida *showing normal chromosome pairing with 21 bivalents at metaphase-I. (f) Male meiotic cell of the triploid cytotype of* C. comosa *showing chromosome pairing at metaphase-I with 21 trivalents, indicating autotriploidy. (g) Male meiotic cell of the triploid* C. latifolia *showing irregular synapsis at metaphase-I chromosome pairing with 21 trivalents, indicating allotriploidy. (h) A mitotic interphase cell of the triploid cytotype of* C. comosa *showing three major sites of the 45S ribosomal genes, confirming triploidy in this species. (i) A mitotic interphase cell of the triploid cytotype of* C. comosa *showing three major sites of the 45S ribosomal genes, one large* C. comosa *marker site Cc1 and two smaller sites. (j) A mitotic interphase cell of the triploid cytotype of* C. elata *showing three major sites of the 45S ribosomal genes and three minor sites, confirming triploidy in this species. (k) A mitotic interphase cell of the triploid cytotype of* C. elata *showing three major sites of the 45S ribosomal genes, again confirming triploidy in this species. Scale bars represent 5 μm. References: (a–b) [51]; (c, e) [65]; (d, f–g) [59];* 

*Polyploidy in the Ginger Family from Thailand DOI: http://dx.doi.org/10.5772/intechopen.92859* *Chromosomal Abnormalities*

*C. maehongson* C. Maknoi

*C. nakornsawan* C. Maknoi

*C. putii* Maknoi & Jenjitt.\***e**

*C. rhabdota* Sirirugsa & M. Newman

*C. rubrobracteata* Skornickova, Sabu & Prasanth k.

*C. saraburiensis* Boonma & Saensouk\***f**

*C. sparganiifolia* Gagnep.

*C. ubonratchani* C.Maknoi

J. Chen\***g**

Rosc.

*C. woodii* N.H.Xia &

*C. zedoaria* (Christm.)

*C.* cf*. Zedoaroides* Chaveer. & Tanee\***h**

**Total number of taxa = 45**

*number, ploidy level and distribution.*

**Species of** *Curcuma* **L. 2***n* **chromosome** 

**number**

*C. pierreana* Gagnep. x

*C. ranong* C.Maknoi x

*C. saraburi* C.Maknoi x

*C. stenochila* Gagnep. x *C. tak* C.Maknoi x

*C. viridiflora* Roxb. x

*C. longa* L. 63 (32, 48, 62-64) Triploid x 1, 2, 4, 6,

*C. mangga* Val. 42 (63) Diploid x 4, 9

*C. parviflora* Wall. 28, 30, 32, 36, 42 x 9 *C. petiolata* Roxb. 42, 64 Di-, triploid x x 9, 10

*C. roscoceana* Wall. 42 Diploid x x 4, 9 *C. rubescens* Roxb. 63 Triploid x 9

*C. singularis* Gagnep. 42 x 9

*C. xanthorrhiza* Roxb. 63 Triploid x 3, 6, 7, 9, 10

pentaploid

63, 64, 84, 105 Tri-, tetra-

*\*Species references: a, Maknoi et al. [55]; b, Puangpairote [56]; c, Jenjittikul and Larsen [57]; d, Larsen [6]; e, Maknoi et al. [49]; f, Boonma and Saensouk [48]; g, Chen et al. [58]; and h, Puangpairote et al. [59].*

*Chromosome/ploidy references: 1. Ramachandran [60]; 2. Ramachandran [61]; 3. eFlora [9]; 4. Leong-Skornikova et al. [10]; 5. Soontornchainaksaeng and Jenjitikul [51]; 6. Zaveska et al. [62]; 7. Chen et al. [63]; 8. Chen et al. [58]; 9. Puangpairote [56]; 10. Rice et al. [64]; 11. Puangpairote et al. [59]; 12. Nopporncharoenkul et al. [65].*

*List of* Curcuma *species found in Thailand, based on Maknoi [11] (except \*), with 2*n *somatic chromosome* 

**Ploidy Natural Cultivated References**

x

x

24 x x 9

63 Triploid x 7

x

x

42 Diploid 8

63 Triploid x 11

**33 19**

x x

x 2, 4, 6, 9,

10, 11

9, 10

**120**

**Table 1.**

#### **Figure 3.**

*Our own research work on selected* Curcuma *species from Thailand. (a) Triploid* Curcuma comosa *plants, showing the above-ground part of the plant with 60–150-cm-tall leafy shoots and 15–32-cm-long inflorescences with short peduncle and dark pink flowers. (b) Rhizome of the triploid* Curcuma *sp. "*elata-latifolia*". Typical rhizome of this species is ovoid-ellipsoid in shape and about (7–15) × (6–10) cm in size, with 2–7 lateral rhizomes, 2–12 cm long and up to 5 secondary lateral rhizomes. (c) Mitotic metaphase cell of* C. candida *showing diploid chromosome number 2n = 42. (d) Mitotic metaphase cell of* C. comosa *showing diploid chromosome number 2n = 42. (e) Male meiotic cell of the diploid* C. candida *showing normal chromosome pairing with 21 bivalents at metaphase-I. (f) Male meiotic cell of the triploid cytotype of* C. comosa *showing chromosome pairing at metaphase-I with 21 trivalents, indicating autotriploidy. (g) Male meiotic cell of the triploid* C. latifolia *showing irregular synapsis at metaphase-I chromosome pairing with 21 trivalents, indicating allotriploidy. (h) A mitotic interphase cell of the triploid cytotype of* C. comosa *showing three major sites of the 45S ribosomal genes, confirming triploidy in this species. (i) A mitotic interphase cell of the triploid cytotype of* C. comosa *showing three major sites of the 45S ribosomal genes, one large* C. comosa *marker site Cc1 and two smaller sites. (j) A mitotic interphase cell of the triploid cytotype of* C. elata *showing three major sites of the 45S ribosomal genes and three minor sites, confirming triploidy in this species. (k) A mitotic interphase cell of the triploid cytotype of* C. elata *showing three major sites of the 45S ribosomal genes, again confirming triploidy in this species. Scale bars represent 5 μm. References: (a–b) [51]; (c, e) [65]; (d, f–g) [59]; (h, j–k) [66]; and (i) [56].*

We have studied *C. comosa* and its related species, collectively called wan-chakmotluk in Thai language for its phytoestrogen properties. The plant produces bright colourful flowers in the form of inflorescences (**Figure 3a**). Its rhizomes are ovoid to ovate spheroidal in shape and about 8–15 cm in diameter (**Figure 3b**). Wan-chakmotluk belongs to three *Curcuma* species: *C. comosa*, *C. elata* Roxb. and *C. latifolia* Rosc. [51]. *Curcuma comosa* is recognised by its inflorescences with short peduncles (**Figure 3a**), whereas the other two species have long peduncles more suitable for flower arrangements. *Curcuma elata* and *C. latifolia* produce large and branchy rhizomes (**Figure 3b**).

Our chromosome number investigations have shown that the three wan-chakmotluk species can be further separated into five cultivars or cytotypes ([51]; see also **Table 1**): *C. comosa* has two cytotypes, diploid with 2*n* = 2*x* = 42 (**Figure 3d**) and triploid with 2*n* = 3*x* = 63 (**Figure 3f**); *C. elata* (and *C*. cf. "*elata-latifolia*") is triploid with 2*n* = 3*x* = 63; but *C. latifolia* has two cytotypes, triploid with 2*n* = 3*x* = 63 (**Figure 3g**) and tetraploid with 2*n* = 4*x* = 84. The group of wanchak-motluk has been extended to cover more Thai taxa [59], including triploid *C. caesia*, triploid *C*. cf. *zedoaroides* Chaveer. & Tanee and tetraploid *C*. cf. *zedoaria* (Christm.) Rosc.

We have also recently described cytotaxonomy of the white flowering *C. candida* (Wall.) Techapr., to be diploid with 2*n* = 2*x* = 42 ([65]; **Figure 3c**, **e**). *C. candida* is a conservation-vulnerable species, rare and endemic to the Tenasserim Range bordering Thailand and Myanmar. As this species has the potential to be developed as an ornamental or medicinal plant [67], efforts are being made to promote cultivation rather than harvesting it from the wild.

#### **5. Polyploidy in** *Curcuma*

Our studies and those of others have shown that while most Thai *Curcuma* species are diploid (2*n* = 42), other species are polyploid (**Table 1**). This ploidy level determination is based on the meiotic chromosome pairing in pollen mother cells, i.e. a diploid plant shows 21 bivalents, resulting from a complete synapsis of homologous chromosomes at metaphase-I of the meiotic cell division (e.g. **Figure 3e**). Therefore, we have concluded that the base chromosome number for *Curcuma*, at least the Thai species investigated, to be 21 (*x* = 21), but we have also identified this as "secondary" base chromosome number, possibly deriving from three times primary base number *x* = 7 [59, 66]. Leong-Skornickova et al. [10] measured genome size of 51 Indian *Curcuma* taxa using flow cytometry and obtained chromosome counts from about one-third of the plants. They established that the base number was *x* = 7 for Indian *Curcuma* because all the 2*n* numbers in their study were multiples of seven, from hexaploids (2*n* = 42) up to 15-ploids. This *x* = 7 is most likely an ancestral base number of *Curcuma*. Most angiosperms, woody and herbaceous, are considered being ancient polyploids with the original base numbers *x* = 6 and *x* = 7 [68]. The major crop plants of the world are polyploid, for example, wheat, maize, potatoes, banana, cotton, oilseed rape and coffee beans, and most of these highly productive plants are ancient polyploids [69]. Therefore, in this context, all Thai *Curcuma* species (**Table 1**) are basically (ancient) polyploids, ranging from 2*n* = 42 (primary hexaploid, secondary diploid) to 2*n* = 63 (primary 9-ploid, secondary triploid) and 2*n* = 84 (primary 12-ploid, secondary tetraploid). However, for the matter of consistency among our studies, we treat all Thai *Curcuma* taxa based on the secondary base number *x* = 21. This is in line with most other chromosome studies, whereby the meiotic

**123**

such gains in polyploidy.

*Polyploidy in the Ginger Family from Thailand DOI: http://dx.doi.org/10.5772/intechopen.92859*

*C. zedoaria* [71].

tion and polyploidization.

analysis is used to determine ploidy levels, for example, the most cultivated

Polyploidy is indeed very common in the ginger family Zingiberaceae.

The genus *Curcuma* contains chromosome numbers spanning the full range of the family Zingiberaceae, from 2*n* = 20 to 105 [10, 51, 59, 61, 63, 64, 70], but is characterised by chromosomes of particularly small sizes, usually less than 2 μm. A large number of *Curcuma* species (at least 25 species) have the diploid chromosome number 2*n* = 42 (base number *x* = 21), several (ca. 12) species have 2*n* = 63, and other numbers such as 20, 24, 32, 34, 84 and 105 have also been reported.

Fluorescent in situ hybridization (FISH) mapping of the tandemly repeated 45S (18S–25S) ribosomal DNA on chromosomes of wan-chak-motluk supports the occurrence of triploidy among the species and cytotypes with 2*n* = 63 [66]. Sets of three ribosomal FISH signals (loci) are apparent in the triploid *C. comosa* (**Figure 3h**, **i**) and the triploid *C. elata* (**Figure 3j**, **k**). In addition, the meiotic figure obtained from the triploid cytotype of *C. comosa* comprises of 21 trivalents; each is a pairing of three homologous chromosomes (**Figure 3f**). Cytogenetic characteristics of triploidy have been observed in other *Curcuma* species, such as *C. longa* [60] and

This triploidization is likely to be the outcome of hybridization between unreduced (2*n*) and normal (1*n*) gametes within or between the diploid populations. Such mechanism has been well documented [72, 73]. In Zingiberaceae, multiple occurrences of triploid formation have been shown in the ornamental ginger genus *Globba* L. from Southeast Asia, based on molecular phylogenetic analysis of both chloroplast and nuclear genes [74]. The situation with *Curcuma*—wan-chakmotluk—is similar to that of *Globba* in that tetraploids (2*n* = 84) are extremely rare (**Table 1**) and the triploids are variable both morphologically and cytogeographically [51]. The molecular study by Zaveska et al. [75] has shown that in *Curcuma*, the genus of palaeopolyploid origin, its evolution is most likely driven by hybridiza-

Once a triploid has arisen, it could easily survive because *Curcuma*, like other genera in Zingiberaceae, reproduces predominantly by vegetative means, i.e. the plants often propagate by rhizomes and numerous bulbils produced on the inflorescence. In the context of cultivation and utilisation of wan-chak-motluk, triploid cultivars (with 2n = 63) are indeed very popular among the growers, for example, for having larger rhizomes. We have also found that triploid and tetraploid plants do have proportionally larger genome sizes compared with the diploid plants [59]. Polyploidization in plants often increases cell size as well as growth rates, giving rise to plant phenotypes having higher physiological capacity and productivity [76]. Increasing the ploidy level is known to be positively correlated with plant production, both biomass and yield [69]. Furthermore, polyploids are often said to have a broader ecological tolerance than their diploid progenitors [77]. This is thought to be due to the effects of increased heterozygosity providing metabolic flexibility to cope with wider arrays of conditions [76]. In addition, the advantages of having more copies of the genes should allow polyploids to thrive in environments that pose challenges to their diploid progenitors [78]. In Zingiberaceae, triploids are highly successful in cultivation, mainly due to their productive rhizomes. In natural environments, triploids may be superior as a likely result of the plant's fitness as described above. *Curcuma* triploids are indeed common and widespread over a vast geographical range throughout Asia [9, 10, 62, 63, 70, 71]. Future studies combining cytogenomics, genetics, physiology and ecology should shed light onto the underlying physiological mechanism and its genetic basis of

turmeric species *C. longa* is triploid with 2*n* = 63 [60, 64].

#### *Polyploidy in the Ginger Family from Thailand DOI: http://dx.doi.org/10.5772/intechopen.92859*

*Chromosomal Abnormalities*

rhizomes (**Figure 3b**).

(Christm.) Rosc.

rather than harvesting it from the wild.

**5. Polyploidy in** *Curcuma*

We have studied *C. comosa* and its related species, collectively called wan-chakmotluk in Thai language for its phytoestrogen properties. The plant produces bright colourful flowers in the form of inflorescences (**Figure 3a**). Its rhizomes are ovoid to ovate spheroidal in shape and about 8–15 cm in diameter (**Figure 3b**). Wan-chakmotluk belongs to three *Curcuma* species: *C. comosa*, *C. elata* Roxb. and *C. latifolia* Rosc. [51]. *Curcuma comosa* is recognised by its inflorescences with short peduncles (**Figure 3a**), whereas the other two species have long peduncles more suitable for flower arrangements. *Curcuma elata* and *C. latifolia* produce large and branchy

Our chromosome number investigations have shown that the three wan-chakmotluk species can be further separated into five cultivars or cytotypes ([51]; see also **Table 1**): *C. comosa* has two cytotypes, diploid with 2*n* = 2*x* = 42 (**Figure 3d**) and triploid with 2*n* = 3*x* = 63 (**Figure 3f**); *C. elata* (and *C*. cf. "*elata-latifolia*") is triploid with 2*n* = 3*x* = 63; but *C. latifolia* has two cytotypes, triploid with 2*n* = 3*x* = 63 (**Figure 3g**) and tetraploid with 2*n* = 4*x* = 84. The group of wanchak-motluk has been extended to cover more Thai taxa [59], including triploid *C. caesia*, triploid *C*. cf. *zedoaroides* Chaveer. & Tanee and tetraploid *C*. cf. *zedoaria*

We have also recently described cytotaxonomy of the white flowering *C. candida* (Wall.) Techapr., to be diploid with 2*n* = 2*x* = 42 ([65]; **Figure 3c**, **e**). *C. candida* is a conservation-vulnerable species, rare and endemic to the Tenasserim Range bordering Thailand and Myanmar. As this species has the potential to be developed as an ornamental or medicinal plant [67], efforts are being made to promote cultivation

Our studies and those of others have shown that while most Thai *Curcuma* species are diploid (2*n* = 42), other species are polyploid (**Table 1**). This ploidy level determination is based on the meiotic chromosome pairing in pollen mother cells, i.e. a diploid plant shows 21 bivalents, resulting from a complete synapsis of homologous chromosomes at metaphase-I of the meiotic cell division (e.g. **Figure 3e**). Therefore, we have concluded that the base chromosome number for *Curcuma*, at least the Thai species investigated, to be 21 (*x* = 21), but we have also identified this as "secondary" base chromosome number, possibly deriving from three times primary base number *x* = 7 [59, 66]. Leong-Skornickova et al. [10] measured genome size of 51 Indian *Curcuma* taxa using flow cytometry and obtained chromosome counts from about one-third of the plants. They established that the base number was *x* = 7 for Indian *Curcuma* because all the 2*n* numbers in their study were multiples of seven, from hexaploids (2*n* = 42) up to 15-ploids. This *x* = 7 is most likely an ancestral base number of *Curcuma*. Most angiosperms, woody and herbaceous, are considered being ancient polyploids with the original base numbers *x* = 6 and *x* = 7 [68]. The major crop plants of the world are polyploid, for example, wheat, maize, potatoes, banana, cotton, oilseed rape and coffee beans, and most of these highly productive plants are ancient polyploids [69]. Therefore, in this context, all Thai *Curcuma* species (**Table 1**) are basically (ancient) polyploids, ranging from 2*n* = 42 (primary hexaploid, secondary diploid) to 2*n* = 63 (primary 9-ploid, secondary triploid) and 2*n* = 84 (primary 12-ploid, secondary tetraploid). However, for the matter of consistency among our studies, we treat all Thai *Curcuma* taxa based on the secondary base number *x* = 21. This is in line with most other chromosome studies, whereby the meiotic

**122**

analysis is used to determine ploidy levels, for example, the most cultivated turmeric species *C. longa* is triploid with 2*n* = 63 [60, 64].

The genus *Curcuma* contains chromosome numbers spanning the full range of the family Zingiberaceae, from 2*n* = 20 to 105 [10, 51, 59, 61, 63, 64, 70], but is characterised by chromosomes of particularly small sizes, usually less than 2 μm. A large number of *Curcuma* species (at least 25 species) have the diploid chromosome number 2*n* = 42 (base number *x* = 21), several (ca. 12) species have 2*n* = 63, and other numbers such as 20, 24, 32, 34, 84 and 105 have also been reported. Polyploidy is indeed very common in the ginger family Zingiberaceae.

Fluorescent in situ hybridization (FISH) mapping of the tandemly repeated 45S (18S–25S) ribosomal DNA on chromosomes of wan-chak-motluk supports the occurrence of triploidy among the species and cytotypes with 2*n* = 63 [66]. Sets of three ribosomal FISH signals (loci) are apparent in the triploid *C. comosa* (**Figure 3h**, **i**) and the triploid *C. elata* (**Figure 3j**, **k**). In addition, the meiotic figure obtained from the triploid cytotype of *C. comosa* comprises of 21 trivalents; each is a pairing of three homologous chromosomes (**Figure 3f**). Cytogenetic characteristics of triploidy have been observed in other *Curcuma* species, such as *C. longa* [60] and *C. zedoaria* [71].

This triploidization is likely to be the outcome of hybridization between unreduced (2*n*) and normal (1*n*) gametes within or between the diploid populations. Such mechanism has been well documented [72, 73]. In Zingiberaceae, multiple occurrences of triploid formation have been shown in the ornamental ginger genus *Globba* L. from Southeast Asia, based on molecular phylogenetic analysis of both chloroplast and nuclear genes [74]. The situation with *Curcuma*—wan-chakmotluk—is similar to that of *Globba* in that tetraploids (2*n* = 84) are extremely rare (**Table 1**) and the triploids are variable both morphologically and cytogeographically [51]. The molecular study by Zaveska et al. [75] has shown that in *Curcuma*, the genus of palaeopolyploid origin, its evolution is most likely driven by hybridization and polyploidization.

Once a triploid has arisen, it could easily survive because *Curcuma*, like other genera in Zingiberaceae, reproduces predominantly by vegetative means, i.e. the plants often propagate by rhizomes and numerous bulbils produced on the inflorescence. In the context of cultivation and utilisation of wan-chak-motluk, triploid cultivars (with 2n = 63) are indeed very popular among the growers, for example, for having larger rhizomes. We have also found that triploid and tetraploid plants do have proportionally larger genome sizes compared with the diploid plants [59]. Polyploidization in plants often increases cell size as well as growth rates, giving rise to plant phenotypes having higher physiological capacity and productivity [76]. Increasing the ploidy level is known to be positively correlated with plant production, both biomass and yield [69]. Furthermore, polyploids are often said to have a broader ecological tolerance than their diploid progenitors [77]. This is thought to be due to the effects of increased heterozygosity providing metabolic flexibility to cope with wider arrays of conditions [76]. In addition, the advantages of having more copies of the genes should allow polyploids to thrive in environments that pose challenges to their diploid progenitors [78]. In Zingiberaceae, triploids are highly successful in cultivation, mainly due to their productive rhizomes. In natural environments, triploids may be superior as a likely result of the plant's fitness as described above. *Curcuma* triploids are indeed common and widespread over a vast geographical range throughout Asia [9, 10, 62, 63, 70, 71]. Future studies combining cytogenomics, genetics, physiology and ecology should shed light onto the underlying physiological mechanism and its genetic basis of such gains in polyploidy.

### **6. Conclusion**

The most widely cultivated plants belong to the two largest genera of this family, the ginger genus (*Zingiber*) and the turmeric genus (*Curcuma*). They are also the best researched plants from this family, and the most researched topics concern medicinal properties and health benefits, pharmaceutical, biochemical and molecular characterisation, as well as applications in food science and technology. The present study identifies numerous polyploid species in the turmeric genus (*Curcuma*) from Thailand. In particular, triploid species and/or cultivars are popular for a large-scale cultivation. The plants are easily propagated via underground rhizomes, which are also the part of the plant that contains bioactive compounds with medicinal properties. Rhizomes of triploid cultivars are bigger than those of the diploid, wild relatives and thus are more economically valuable. Triploids are also the most adaptable plants in diverse environments. On the other hand, the overharvesting of wild plants, in search for novel or better bioactive compounds, poses a serious risk of species extinction. Cytogenetic research, such as that presented here, can provide useful information for both types of activities, i.e. in the plant improvement for cultivation and in the conservation of natural biodiversity.

### **Acknowledgements**

This work was supported by Mahidol University and University of Iceland. We appreciate the contribution in taxonomic identification of plant materials from Dr. Thaya Jenjittikul of Mahidol University. We thank both Dr. Tidarat Puangpairote from Prince of Songkla University and PhD student of Mahidol University, Nattapon Nopporncharoenkul, for their accurate cytogenetic work on Zingiberaceae of Thailand.

### **Author details**

Kesara Anamthawat-Jónsson1 and Puangpaka Umpunjun2 \*

1 Institute of Life and Environmental Sciences, University of Iceland, Reykjavík, Iceland

2 Department of Plant Science, Faculty of Science, Mahidol University, Bangkok, Thailand

\*Address all correspondence to: puangpaka.ump@mahidol.ac.th

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**125**

*Polyploidy in the Ginger Family from Thailand DOI: http://dx.doi.org/10.5772/intechopen.92859*

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#### **References**

*Chromosomal Abnormalities*

**6. Conclusion**

**124**

**Author details**

**Acknowledgements**

Zingiberaceae of Thailand.

Iceland

Thailand

Kesara Anamthawat-Jónsson1

provided the original work is properly cited.

and Puangpaka Umpunjun2

1 Institute of Life and Environmental Sciences, University of Iceland, Reykjavík,

This work was supported by Mahidol University and University of Iceland. We appreciate the contribution in taxonomic identification of plant materials from Dr. Thaya Jenjittikul of Mahidol University. We thank both Dr. Tidarat Puangpairote from Prince of Songkla University and PhD student of Mahidol University, Nattapon Nopporncharoenkul, for their accurate cytogenetic work on

The most widely cultivated plants belong to the two largest genera of this family, the ginger genus (*Zingiber*) and the turmeric genus (*Curcuma*). They are also the best researched plants from this family, and the most researched topics concern medicinal properties and health benefits, pharmaceutical, biochemical and molecular characterisation, as well as applications in food science and technology. The present study identifies numerous polyploid species in the turmeric genus (*Curcuma*) from Thailand. In particular, triploid species and/or cultivars are popular for a large-scale cultivation. The plants are easily propagated via underground rhizomes, which are also the part of the plant that contains bioactive compounds with medicinal properties. Rhizomes of triploid cultivars are bigger than those of the diploid, wild relatives and thus are more economically valuable. Triploids are also the most adaptable plants in diverse environments. On the other hand, the overharvesting of wild plants, in search for novel or better bioactive compounds, poses a serious risk of species extinction. Cytogenetic research, such as that presented here, can provide useful information for both types of activities, i.e. in the plant improvement for cultivation and in the conservation of natural biodiversity.

2 Department of Plant Science, Faculty of Science, Mahidol University, Bangkok,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: puangpaka.ump@mahidol.ac.th

\*

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[69] Leitch AR, Leitch IJ. Perspective— Genomic plasticity and the diversity of polyploidy plants. Science.

Medicine. 2008;**6**:27-51

[67] Picheansoonthon C, Koonterm S. Notes on the genus *Kaempferia L.* (Zingiberaceae) in Thailand. Journal of Thai Traditional and Alternative

[65] Nopporncharoenkul N, Jenjittikul T, Chuenboonngarm N, Anamthawat-Jónsson K, Umpunjun P. Cytogenetic verification of *Curcuma candida* (Zingiberaceae) from Thailand and Myanmar. Thai Forest Bulletin

2013;**48**:525-530

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*Chromosomal Abnormalities*

An endangered medicinal plant of north East India. Industrial Crops and

[46] Deeki LT, Manivannan SJ, Sujata U, Biswajit RG. Targeting metabolic profiling of black turmeric (*Curcuma caesia* Roxb.) accessions for industrially important compounds. Research Journal [53] Jaipakdee N, Limpongsa E, Sripanidkulchai B, Piyachaturawat P. Preparation of *Curcuma comosa* tablets using liquisolid techniques: In vitro and in vivo evaluation. International Journal of Pharmaceutics. 2018;**553**:157-168

[54] Tabboon P, Tantiyasawasdikul S, Sripanidkulchai B. Quality and stability assessment of commercial products containing phytoestrogen diarylheptanoids from *Curcuma comosa*. Industrial Crops and Products.

[55] Maknoi C, Sirirugsa P, Larsen K. *Curcuma bella* (Zingiberaceae), a new species from Thailand. Thai Journal of

[57] Jenjittikul T, Larsen K. *Kaempferia candida* wall. (Zingiberaceae), a new record for Thailand. Thai Forest Bulletin

[58] Chen J, Lindstrom AJ, Xia NH. *Curcuma woodii* (Zingiberaceae), a new species from Thailand. Phytotaxa.

[59] Puangpairote T, Maknoi C, Jenjittikul T, Anamthawat-Jónsson K, Soontornchainaksaeng P. Natural triploidy in phyto-oestrogen producing *Curcuma* species and cultivars from Thailand. Euphytica. 2016;**208**:47-61

[60] Ramachandran K. Chromosome numbers in the genus *Curcuma* Linn. Current Science India. 1961;**30**:194-196

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Botany. 2011;**3**:121-124

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clonal propagation of *Curcuma angustifolia* ensuring genetic fidelity of micropropagated plants. Plant Cell, Tissue and Organ Culture.

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[51] Soontornchainaksaeng P, Jenjittikul T. Chromosome number variation of phytoestrogen-producing *Curcuma* (Zingiberaceae) from

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**131**

**1. Introduction**

**Chapter 9**

**Abstract**

Maize Chromosome Abnormalities

and Breakage-Fusion-Bridge

*Margarida L.R. Aguiar-Perecin, Janay A. Santos-Serejo,* 

The maize karyotype was first characterized by the observation of pachytene chromosomes. The somatic chromosomes were identified by C-banding and FISH with repetitive DNA sequences. C-banding was useful for the identification of chromosome abnormalities in callus cultures. In the present review, we focus on the involvement of heterochromatic knobs on the occurrence of chromosome abnormalities in callus cultures. In a previous work we detected anaphase bridges resulting from delayed chromatid separation at knob regions and typical bridges derived from dicentric chromatids in cultures. The analysis of altered chromosomes showed they were derived from a chromatid-type breakage-fusion-bridge (BFB) cycle. Fluorescent in situ hybridization (FISH) showed signals of telomere sequences in the broken chromosome arm, thus giving evidence of de novo telomere formation on the broken chromosome end. Further observations of long- and short-term cultures have shown the presence of chromosome alterations derived from BFB cycles followed by chromosome healing. Additionally, the occurrence of unequal crossing over in a knob region was observed in callus culture. These results are of interest for

studies on the mechanisms of chromosome alterations during evolution.

**Keywords:** maize, heterochromatic knobs, chromosomal rearrangements,

Maize is an important crop plant and model organism. The maize karyotype was first characterized by the observation of pachytene chromosomes obtained from pollen mother cells, since the pioneering work by McClintock [1]. The early cytological maps were constructed based on the identification of chromosome relative lengths, arm ratios, heterochromatin patterns, prominent chromomeres, and nuclear organizer region [2–5]. Structures containing heterochromatin were described: heterochromatic knobs, centromeric heterochromatin, B chromosomes, abnormal chromosome 10, and nucleolus organizer region localized on chromosome 6 [6]. Chromosome abnormalities were detected in several investigations, and collections were organized containing reciprocal translocations (A-A translocations), B-A translocations (interchanges between B chromosome and arms of the A set), inversions, and trisomics, available at the Maize Genetics Cooperation

callus culture, breakage-fusion-bridge cycle, unequal crossing over

Cycles in Callus Cultures

*José R. Gardingo and Mateus Mondin*

#### **Chapter 9**

## Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures

*Margarida L.R. Aguiar-Perecin, Janay A. Santos-Serejo, José R. Gardingo and Mateus Mondin*

#### **Abstract**

The maize karyotype was first characterized by the observation of pachytene chromosomes. The somatic chromosomes were identified by C-banding and FISH with repetitive DNA sequences. C-banding was useful for the identification of chromosome abnormalities in callus cultures. In the present review, we focus on the involvement of heterochromatic knobs on the occurrence of chromosome abnormalities in callus cultures. In a previous work we detected anaphase bridges resulting from delayed chromatid separation at knob regions and typical bridges derived from dicentric chromatids in cultures. The analysis of altered chromosomes showed they were derived from a chromatid-type breakage-fusion-bridge (BFB) cycle. Fluorescent in situ hybridization (FISH) showed signals of telomere sequences in the broken chromosome arm, thus giving evidence of de novo telomere formation on the broken chromosome end. Further observations of long- and short-term cultures have shown the presence of chromosome alterations derived from BFB cycles followed by chromosome healing. Additionally, the occurrence of unequal crossing over in a knob region was observed in callus culture. These results are of interest for studies on the mechanisms of chromosome alterations during evolution.

**Keywords:** maize, heterochromatic knobs, chromosomal rearrangements, callus culture, breakage-fusion-bridge cycle, unequal crossing over

#### **1. Introduction**

Maize is an important crop plant and model organism. The maize karyotype was first characterized by the observation of pachytene chromosomes obtained from pollen mother cells, since the pioneering work by McClintock [1]. The early cytological maps were constructed based on the identification of chromosome relative lengths, arm ratios, heterochromatin patterns, prominent chromomeres, and nuclear organizer region [2–5]. Structures containing heterochromatin were described: heterochromatic knobs, centromeric heterochromatin, B chromosomes, abnormal chromosome 10, and nucleolus organizer region localized on chromosome 6 [6]. Chromosome abnormalities were detected in several investigations, and collections were organized containing reciprocal translocations (A-A translocations), B-A translocations (interchanges between B chromosome and arms of the A set), inversions, and trisomics, available at the Maize Genetics Cooperation

Stock Center [7] (www.maizegdb.org). These materials have been important tools for gene mapping.

The somatic chromosomes were identified by the C-banding procedure which was useful for the identification of chromosomal abnormalities in callus cultures [8–10]. The unequivocal identification of the somatic chromosomes is difficult due to their degree of condensation, and the use of the C-banding procedure was supplemented by an analysis of pachytene chromosomes of the lines from which callus cultures were derived. C-bands correspond to knobs visualized on meiotic chromosomes [11].

The characterization of meiotic and somatic chromosomes was improved by fluorescence in situ hybridization (FISH) using as probes repeated DNA sequences and genes, thus allowing the study of the molecular structure of chromosome components, such as centromere, neocentromere, B chromosome heterochromatic knobs, and gene mapping [12–20].

The maize chromosome structure has been extensively reviewed [21–25]. In the present review, we focus on the involvement of heterochromatic knobs on the occurrence of chromosome abnormalities in maize callus cultures. The size and number of knobs are variable, and they may be present in each of the 10 chromosomes of the complement at fixed positions in modern maize and its relatives, including species of *Zea* (teosintes) and *Tripsacum* [26, 27]. Knobs were mapped on the meiotic chromosomes [2, 3, 5], and recently they were mapped relative to the maize reference genome assembly [19]. Knobs are composed primarily of two tandemly repeated sequences, the 180-bp knob repeat and the 350-bp TR-1 element or a mixture of both [13, 17, 19]. Knobs also contain retrotransposons [28, 29].

One genetic effect attributed to knobs is their influence on recombination [6, 19], and it was revealed that knobs in heterozygous condition can reduce local recombination [19]. Another interesting genetic effect of knobs is their activity as neocentromeres resulting in meiotic drive. This meiotic event is a mechanism by which regions of the genome are preferentially transmitted to the progeny. In maize, meiotic drive is due to an uncommon form of chromosome 10, the abnormal chromosome 10 (Ab 10). In the presence of this chromosome, the knobs of other chromosomes are converted into motile neocentromeres. Thus the knobbed chromosomes preferentially segregate during female meiosis [30, 31]. The origin of maize polymorphism, including size and number, has been discussed in several reports, and it was proposed that meiotic drive was responsible for the evolution of knobs [32]. Recently, a cluster of eight genes on Ab10 was identified, called *Kinesin driver* (*Kindr*) complex, which are required for both neocentromere motility and preferential transmission. It was revealed that Kindr is a strong minus-end-directed motor that interacts specifically with neocentromeres containing 180-bp knob repeats [33].

The effect of knobs on chromosome break and origin of abnormalities in maize callus culture is presented in this review.

#### **2. Chromosome abnormalities in maize callus culture**

#### **2.1 Heterochromatin involvement in chromosome breakage**

Callus culture is an important step for genetic transformation in plants. The identification of maize genotypes showing high ability to form embryogenic callus type II (friable) and regenerate plants has progressed since the report by Green and Phillips [34]. The genotypes identified since then were adapted to temperate regions [35], and maize genotypes of tropical and subtropical origin have also been shown to produce friable type II calli capable to develop somatic embryogenesis [36–38].

**133**

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures*

breakage associated with heterochromatin was shown in several plant callus

**2.2 Breakage-fusion-bridge cycle in callus culture and de novo telomere** 

The first reports on breakage-fusion-bridge (BFB) cycles were made by McClintock [49, 50]. In investigations on the behavior in successive nuclear divisions of a chromosome broken at meiosis, it was shown that the chromatid type of BFB cycle initiated by broken chromosome ends occurs in gametophyte mitoses and in the endosperm. In the zygote the broken chromosome ends heal. BFB cycles have also been observed in other species including wheat (*Triticum aestivum*), in which reverse tandem duplications were observed [51]. The chromatid-type BFB cycle initiated in meiosis continued through pollen mitoses and in early endosperm

Investigations of mitotic cells in maize callus cultures detected anaphase bridges resulting in delayed separation at knob regions and typical bridges originated from dicentric chromatids. The observation of C-banded anaphases showed that the chromatids were held together at C-band sites (corresponding to knob) [8]. Typical bridges with and without C-bands were observed. These events were interpreted as derived from a chromatid type of BFB cycle initiated by chromatids that were

The analysis of abnormalities in chromosomes 7 and 9 of maize callus cultures gave evidence of their origin from BFB cycles [8–10]. As illustration, we show here the mechanism that would have originated these abnormalities. The callus culture was induced from a hybrid between two sister inbred lines derived from a tropical maize variety (Jac Duro [JD]). These JD lines possessed the same knob composition: K6 L2, K6 L3, K7S, K7L, K8 L1, K8 L2, and K9S [8, 20]. K refers to knob, the number identifies the chromosome, and S refers to short arm and L to long arm. Numbers 1, 2, and 3 refers to knob positions, according to the literature [27]. Thus, chromosome 7 possessed large knobs on both arms, and chromosome 9 had a very large knob on the short arm. Therefore, these chromosomes were more prone to suffer alterations. An abnormal chromosome 7 carrying two knobs on the short arm was observed in metaphases of a callus culture designed 3–57. This abnormality was interpreted as being a deficiency-duplication (Df-Dp) derived from a BFB cycle and healing of the broken arm, for it was observed in various cells of the culture [8]. **Figure 1** [9, 25] shows the mechanism that would have originated this aberration. The two knobs on the short arm would bear a deficiency in the terminal region (RTD). Therefore this

Various studies have shown the occurrence of cytogenetic and genetic variability in plants regenerated from maize callus cultures [39, 40]. This so-called somaclonal variation [41] is undesirable when genetic stability is required, but interesting for the study of mechanisms that give rise to chromosome abnormalities. Chromosome

Breakpoints involved in chromosome abnormalities associated with heterochromatin were previously detected in maize regenerated plants. The analysis of pachytene chromosomes of these plants revealed that most breakpoints were localized in chromosomes bearing a knob. The authors hypothesized that late-replicating heterochromatin would replicate later in tissue culture, giving origin to bridges in anaphases and occurrence of breakage between the knob and centromere [47]. This would explain the presence of knobs in chromosomes involved in abnormalities observed in regenerated plants. The authors identified in meiotic cells alterations in chromosome structure, such as translocations, intercalary deficiencies, and heteromorphic pairs in 91 of 189 plants regenerated from callus cultures originated from

*DOI: http://dx.doi.org/10.5772/intechopen.88876*

an Oh43-A188 genetic background [47].

divisions, but did not continue in embryo mitoses.

broken during the primary event.

cultures [8–10, 42–48].

**formation**

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures DOI: http://dx.doi.org/10.5772/intechopen.88876*

Various studies have shown the occurrence of cytogenetic and genetic variability in plants regenerated from maize callus cultures [39, 40]. This so-called somaclonal variation [41] is undesirable when genetic stability is required, but interesting for the study of mechanisms that give rise to chromosome abnormalities. Chromosome breakage associated with heterochromatin was shown in several plant callus cultures [8–10, 42–48].

Breakpoints involved in chromosome abnormalities associated with heterochromatin were previously detected in maize regenerated plants. The analysis of pachytene chromosomes of these plants revealed that most breakpoints were localized in chromosomes bearing a knob. The authors hypothesized that late-replicating heterochromatin would replicate later in tissue culture, giving origin to bridges in anaphases and occurrence of breakage between the knob and centromere [47]. This would explain the presence of knobs in chromosomes involved in abnormalities observed in regenerated plants. The authors identified in meiotic cells alterations in chromosome structure, such as translocations, intercalary deficiencies, and heteromorphic pairs in 91 of 189 plants regenerated from callus cultures originated from an Oh43-A188 genetic background [47].

#### **2.2 Breakage-fusion-bridge cycle in callus culture and de novo telomere formation**

The first reports on breakage-fusion-bridge (BFB) cycles were made by McClintock [49, 50]. In investigations on the behavior in successive nuclear divisions of a chromosome broken at meiosis, it was shown that the chromatid type of BFB cycle initiated by broken chromosome ends occurs in gametophyte mitoses and in the endosperm. In the zygote the broken chromosome ends heal. BFB cycles have also been observed in other species including wheat (*Triticum aestivum*), in which reverse tandem duplications were observed [51]. The chromatid-type BFB cycle initiated in meiosis continued through pollen mitoses and in early endosperm divisions, but did not continue in embryo mitoses.

Investigations of mitotic cells in maize callus cultures detected anaphase bridges resulting in delayed separation at knob regions and typical bridges originated from dicentric chromatids. The observation of C-banded anaphases showed that the chromatids were held together at C-band sites (corresponding to knob) [8]. Typical bridges with and without C-bands were observed. These events were interpreted as derived from a chromatid type of BFB cycle initiated by chromatids that were broken during the primary event.

The analysis of abnormalities in chromosomes 7 and 9 of maize callus cultures gave evidence of their origin from BFB cycles [8–10]. As illustration, we show here the mechanism that would have originated these abnormalities. The callus culture was induced from a hybrid between two sister inbred lines derived from a tropical maize variety (Jac Duro [JD]). These JD lines possessed the same knob composition: K6 L2, K6 L3, K7S, K7L, K8 L1, K8 L2, and K9S [8, 20]. K refers to knob, the number identifies the chromosome, and S refers to short arm and L to long arm. Numbers 1, 2, and 3 refers to knob positions, according to the literature [27]. Thus, chromosome 7 possessed large knobs on both arms, and chromosome 9 had a very large knob on the short arm. Therefore, these chromosomes were more prone to suffer alterations.

An abnormal chromosome 7 carrying two knobs on the short arm was observed in metaphases of a callus culture designed 3–57. This abnormality was interpreted as being a deficiency-duplication (Df-Dp) derived from a BFB cycle and healing of the broken arm, for it was observed in various cells of the culture [8]. **Figure 1** [9, 25] shows the mechanism that would have originated this aberration. The two knobs on the short arm would bear a deficiency in the terminal region (RTD). Therefore this

*Chromosomal Abnormalities*

for gene mapping.

chromosomes [11].

knobs, and gene mapping [12–20].

Stock Center [7] (www.maizegdb.org). These materials have been important tools

The somatic chromosomes were identified by the C-banding procedure which was useful for the identification of chromosomal abnormalities in callus cultures [8–10]. The unequivocal identification of the somatic chromosomes is difficult due to their degree of condensation, and the use of the C-banding procedure was supplemented by an analysis of pachytene chromosomes of the lines from which callus cultures were derived. C-bands correspond to knobs visualized on meiotic

The characterization of meiotic and somatic chromosomes was improved by fluorescence in situ hybridization (FISH) using as probes repeated DNA sequences and genes, thus allowing the study of the molecular structure of chromosome components, such as centromere, neocentromere, B chromosome heterochromatic

The maize chromosome structure has been extensively reviewed [21–25]. In the present review, we focus on the involvement of heterochromatic knobs on the occurrence of chromosome abnormalities in maize callus cultures. The size and number of knobs are variable, and they may be present in each of the 10 chromosomes of the complement at fixed positions in modern maize and its relatives, including species of *Zea* (teosintes) and *Tripsacum* [26, 27]. Knobs were mapped on the meiotic chromosomes [2, 3, 5], and recently they were mapped relative to the maize reference genome assembly [19]. Knobs are composed primarily of two tandemly repeated sequences, the 180-bp knob repeat and the 350-bp TR-1 element or a mixture of both [13, 17, 19]. Knobs also contain retrotransposons [28, 29].

One genetic effect attributed to knobs is their influence on recombination [6, 19], and it was revealed that knobs in heterozygous condition can reduce local recombination [19]. Another interesting genetic effect of knobs is their activity as neocentromeres resulting in meiotic drive. This meiotic event is a mechanism by which regions of the genome are preferentially transmitted to the progeny. In maize, meiotic drive is due to an uncommon form of chromosome 10, the abnormal chromosome 10 (Ab 10). In the presence of this chromosome, the knobs of other chromosomes are converted into motile neocentromeres. Thus the knobbed chromosomes preferentially segregate during female meiosis [30, 31]. The origin of maize polymorphism, including size and number, has been discussed in several reports, and it was proposed that meiotic drive was responsible for the evolution of knobs [32]. Recently, a cluster of eight genes on Ab10 was identified, called *Kinesin driver* (*Kindr*) complex, which are required for both neocentromere motility and preferential transmission. It was revealed that Kindr is a strong minus-end-directed motor that interacts specifi-

The effect of knobs on chromosome break and origin of abnormalities in maize

Callus culture is an important step for genetic transformation in plants. The identification of maize genotypes showing high ability to form embryogenic callus type II (friable) and regenerate plants has progressed since the report by Green and Phillips [34]. The genotypes identified since then were adapted to temperate regions [35], and maize genotypes of tropical and subtropical origin have also been shown to produce friable type II calli capable to develop somatic embryogenesis [36–38].

cally with neocentromeres containing 180-bp knob repeats [33].

**2. Chromosome abnormalities in maize callus culture**

**2.1 Heterochromatin involvement in chromosome breakage**

callus culture is presented in this review.

**132**

#### *Chromosomal Abnormalities*

abnormal chromosome 7 possessed reverse tandem duplications of these knobs and of a segment designated "b."

This chromosome 7 carrying a deficiency on K7S and duplications of the knob and of a "b" segment (Df-Dp7) was stable in culture and was transmitted to regenerated plants. Thus, R0 plants regenerated from the 3–57 culture were heterozygous for this chromosome alteration. R1 and R2 plants were recovered and analyzed. Homozygotes for normal chromosome 7 and heterozygotes for the Df-Dp7 were detected. Plants homozygous for the Df-Dp7 were not recovered. Presumably, seeds carrying homozygotes were not viable. **Figure 2A** [9, 25] shows a metaphase of a

**Figure 1.**

*BFB cycle that would give rise to chromosome 7 bearing a deficiency and duplication (Df-Dp) showing normal chromosome (A); anaphase with delayed separating chromatids and breakage at K7S (B); chromatid with a deficient K7S (C), fused after replication (D); breakage at anaphase (E); chromatid with duplication of the "b" segment (F), fused at broken ends (G); anaphase bridge and breakage (H); resulting chromatid with two knobs and reverse tandem duplications (RTD) of the "b" segment. Arrows at anaphases indicate breakpoints [9, 25].*

#### **Figure 2.**

*Somatic chromosomes of R1 plants derived from the 3–57 culture. C-banded mitotic metaphases homozygous for normal chromosome 7 showing knobs (C-bands) on the long and short arms (A); heterozygote for the Df-Dp chromosome 7 with two knobs; telomeric FISH signals on early and full metaphases (C, D); homologous pairs of chromosome 7 [9, 25].*

**135**

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures*

regenerated plant homozygous for normal chromosome 7, and **Figure 2B** shows a metaphase of a plant heterozygous for the aberration. The distal knob (K7S) is subterminal, for there is a tiny terminal euchromatic segment on the short arm. Fluorescent in situ hybridization (FISH) using the telomeric sequence (TTTAGGG)6 showed signals in all the somatic chromosomes of the regenerated plants, including the Df-Dp7 chromosome (**Figure 2C**–**E**) [9, 25]. This result gives evidence of telomere healing at the end of the broken short arm. In these DAPIstained metaphases, bands corresponding to the knobs could be clearly visualized. In the less condensed metaphases, the telomeric signals could be detected at the end

The healing of chromosome ends, i.e., the addition of telomere sequences to the broken chromosome ends, has been observed in diverse plant species. In wheat, FISH telomeric signals were detected at the broken ends of deleted chromosomes and at the centromeric regions of telocentric chromosomes [52, 53]. The expression of telomerase has been reported for diverse plant tissues, such as the meristematic tissue and suspension cell cultures [54]. In barley, there was a decrease in the number of telomeric sequences in differentiated cells, and the number of telomeric sequences increased in callus cultures [55]. High telomerase activity was observed in calli derived from tobacco plants, while in leaves the activity was very low [56]. In wheat, during the divisions of the gametophyte, dicentric chromosomes undergo BFB cycles. De novo addition of telomere sequences occurs gradually during the

The present study showing the telomere healing of the broken short arm of chromosome 7 gives evidence of telomerase expression in maize callus culture. The addition of telomeric repeats occurred on a euchromatic region, which was

The meiosis of the regenerated plants heterozygous for the Df-Dp chromosome 7 was normal. The terminal euchromatic segment was clearly observed at pachytene stage on the duplicated short arm. In the diplotene and diakinesis stages, a heteromorphic pair corresponding to chromosome 7 was observed, as expected for

C-banded metaphases of subcultures prepared after 18 months of the initiation of the 3–57 callus culture were analyzed during a cultivation period from the 18-month-old original culture to 42-month-old cell lines. The subcultures were designated as cell lines 1-MS2, 2-MS-2, 1-MS1, 2-MS1, 1 N6, and 2 N6. Feulgen-

The investigation of mitotic instability by the analysis of Feulgen-stained anaphases showed abnormalities similar to those previously described [8] and shown in **Figure 3**: (i) bridges resulting from delayed chromatids held together at knob sites (**Figure 3A**), (ii) broken bridges (**Figure 3B**), (iii) typical bridges (**Figure 3C**, **D**), and (iv) fragments (not shown) [9]. The analysis of the frequency of these abnormalities showed a tendency of decreasing frequency with time in culture. Three samples of each cell line were harvested in different periods of cultivation, except 1-MS1 from which seven samples were analyzed. The frequency of anaphase abnormalities observed varied from 4 to 10% in the first sample and from 0.67 to 5.33% in the last sample. This tendency of decreasing frequency was a consequence of the healing of the broken chromosomes, therefore, avoiding an accumulation of BFB cycles [9], as discussed below. Interestingly, the total frequency of abnormalities varied from 0.67 to 10%, and this result was quite similar to the ones observed in a previous study of

**2.3 Chromosome 7 and 9 abnormalities in long-term subcultures**

5-month-old cultures derived from related inbred lines [38].

*DOI: http://dx.doi.org/10.5772/intechopen.88876*

of the euchromatic segment of the duplicated short arm.

early mitotic divisions in the sporophyte [57].

heterozygotes bearing a duplication [9].

stained anaphases were also observed.

certainly non-telomeric.

#### *Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures DOI: http://dx.doi.org/10.5772/intechopen.88876*

regenerated plant homozygous for normal chromosome 7, and **Figure 2B** shows a metaphase of a plant heterozygous for the aberration. The distal knob (K7S) is subterminal, for there is a tiny terminal euchromatic segment on the short arm.

Fluorescent in situ hybridization (FISH) using the telomeric sequence (TTTAGGG)6 showed signals in all the somatic chromosomes of the regenerated plants, including the Df-Dp7 chromosome (**Figure 2C**–**E**) [9, 25]. This result gives evidence of telomere healing at the end of the broken short arm. In these DAPIstained metaphases, bands corresponding to the knobs could be clearly visualized. In the less condensed metaphases, the telomeric signals could be detected at the end of the euchromatic segment of the duplicated short arm.

The healing of chromosome ends, i.e., the addition of telomere sequences to the broken chromosome ends, has been observed in diverse plant species. In wheat, FISH telomeric signals were detected at the broken ends of deleted chromosomes and at the centromeric regions of telocentric chromosomes [52, 53]. The expression of telomerase has been reported for diverse plant tissues, such as the meristematic tissue and suspension cell cultures [54]. In barley, there was a decrease in the number of telomeric sequences in differentiated cells, and the number of telomeric sequences increased in callus cultures [55]. High telomerase activity was observed in calli derived from tobacco plants, while in leaves the activity was very low [56]. In wheat, during the divisions of the gametophyte, dicentric chromosomes undergo BFB cycles. De novo addition of telomere sequences occurs gradually during the early mitotic divisions in the sporophyte [57].

The present study showing the telomere healing of the broken short arm of chromosome 7 gives evidence of telomerase expression in maize callus culture. The addition of telomeric repeats occurred on a euchromatic region, which was certainly non-telomeric.

The meiosis of the regenerated plants heterozygous for the Df-Dp chromosome 7 was normal. The terminal euchromatic segment was clearly observed at pachytene stage on the duplicated short arm. In the diplotene and diakinesis stages, a heteromorphic pair corresponding to chromosome 7 was observed, as expected for heterozygotes bearing a duplication [9].

#### **2.3 Chromosome 7 and 9 abnormalities in long-term subcultures**

C-banded metaphases of subcultures prepared after 18 months of the initiation of the 3–57 callus culture were analyzed during a cultivation period from the 18-month-old original culture to 42-month-old cell lines. The subcultures were designated as cell lines 1-MS2, 2-MS-2, 1-MS1, 2-MS1, 1 N6, and 2 N6. Feulgenstained anaphases were also observed.

The investigation of mitotic instability by the analysis of Feulgen-stained anaphases showed abnormalities similar to those previously described [8] and shown in **Figure 3**: (i) bridges resulting from delayed chromatids held together at knob sites (**Figure 3A**), (ii) broken bridges (**Figure 3B**), (iii) typical bridges (**Figure 3C**, **D**), and (iv) fragments (not shown) [9]. The analysis of the frequency of these abnormalities showed a tendency of decreasing frequency with time in culture. Three samples of each cell line were harvested in different periods of cultivation, except 1-MS1 from which seven samples were analyzed. The frequency of anaphase abnormalities observed varied from 4 to 10% in the first sample and from 0.67 to 5.33% in the last sample. This tendency of decreasing frequency was a consequence of the healing of the broken chromosomes, therefore, avoiding an accumulation of BFB cycles [9], as discussed below. Interestingly, the total frequency of abnormalities varied from 0.67 to 10%, and this result was quite similar to the ones observed in a previous study of 5-month-old cultures derived from related inbred lines [38].

*Chromosomal Abnormalities*

of a segment designated "b."

abnormal chromosome 7 possessed reverse tandem duplications of these knobs and

This chromosome 7 carrying a deficiency on K7S and duplications of the knob and of a "b" segment (Df-Dp7) was stable in culture and was transmitted to regenerated plants. Thus, R0 plants regenerated from the 3–57 culture were heterozygous for this chromosome alteration. R1 and R2 plants were recovered and analyzed. Homozygotes for normal chromosome 7 and heterozygotes for the Df-Dp7 were detected. Plants homozygous for the Df-Dp7 were not recovered. Presumably, seeds carrying homozygotes were not viable. **Figure 2A** [9, 25] shows a metaphase of a

*BFB cycle that would give rise to chromosome 7 bearing a deficiency and duplication (Df-Dp) showing normal chromosome (A); anaphase with delayed separating chromatids and breakage at K7S (B); chromatid with a deficient K7S (C), fused after replication (D); breakage at anaphase (E); chromatid with duplication of the "b" segment (F), fused at broken ends (G); anaphase bridge and breakage (H); resulting chromatid with two knobs and reverse tandem duplications (RTD) of the "b" segment. Arrows at anaphases indicate breakpoints* 

*Somatic chromosomes of R1 plants derived from the 3–57 culture. C-banded mitotic metaphases homozygous for normal chromosome 7 showing knobs (C-bands) on the long and short arms (A); heterozygote for the Df-Dp chromosome 7 with two knobs; telomeric FISH signals on early and full metaphases (C, D); homologous pairs* 

**134**

**Figure 2.**

**Figure 1.**

*[9, 25].*

*of chromosome 7 [9, 25].*

#### **Figure 3.**

*Feulgen-stained anaphase cells of the cell lines derived from the culture 3–57, with a laggard chromosome showing delayed separating chromatids (A), broken bridge (after the primary event) (B), typical bridge (C), and double bridge (D). Scale bar = 10 μm [9].*

The analysis of the cell line pedigree showing the types of chromosomes 7 and 9 observed in C-banded metaphases in different subcultures of the six cell lines is displayed in **Figure 4** [9, 25]. A karyotype diversity among cell lines was detected in this analysis, but homogeneity within some of them was observed in samples harvested at different age transfers. Then, new abnormal chromosomes were stable in different subcultures. Gross aberrations were not observed in chromosomes 6 and 8 that possess knobs smaller than those found in chromosomes 7 and 9.

Different types of abnormal chromosomes 7 and 9 were observed in the cell lines. In the original 18-month-old callus culture 3–57, two types of chromosome 7 were detected. One of the chromosomes possessed two knobs on the short arm (K7S) corresponding to the Df-Dp chromosome 7 described above, and the other type possessed K7S on an interstitial position of a duplicated short arm (**Figure 4**). This chromosome would have originated from a mechanism similar to that shown in

**137**

**Figure 4.**

(42-month-old culture, **Figure 6**) [9].

*subcultures of the six cell lines derived from culture 3–57 [9, 25].*

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures*

**Figure 1** [9, 25]. The following types of chromosome 7 were distinguished in the cell lines (**Figure 5A**): 7A, normal type, with a terminal K7S and a subterminal K7L; 7B, with a duplicated short arm and a subterminal K7S; 7C, with two knobs on the short arm and a terminal euchromatic segment (similar to **Figure 2B**); 7D, similar to 7C, but without the terminal euchromatic segment; 7E, similar to 7D, with a smaller deficient and terminal K7S; 7F, with a larger short arm, a very large interstitial K7S and without the K7L on the long arm. The 7A, 7B, and 7C types were found in the original 18-month-old culture (**Figure 4**). **Figure 5B**, **C** illustrates the 7D and 7B types, respectively, and the 7E type can be seen in **Figure 5D**. **Figure 5E** illustrates the 7C chromosome. The 7F type can be seen in the pedigree of the 1-MS1 cell line

*Cell line pedigree showing the types of chromosomes 7 and 9 observed in C-banded metaphases in different* 

Different types of altered chromosome 9 were also observed in the samples of cell lines (**Figure 5A**) [9]: the normal type corresponds to 9A; a smaller terminal K9S corresponds to 9B; a smaller subterminal K9S corresponds to 9C; 9D is a chromosome without the knob; and 9E is a minichromosome derived from chromosome 9.

*DOI: http://dx.doi.org/10.5772/intechopen.88876*

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures DOI: http://dx.doi.org/10.5772/intechopen.88876*

**Figure 4.**

*Chromosomal Abnormalities*

**136**

somes 7 and 9.

**Figure 3.**

*and double bridge (D). Scale bar = 10 μm [9].*

The analysis of the cell line pedigree showing the types of chromosomes 7 and 9 observed in C-banded metaphases in different subcultures of the six cell lines is displayed in **Figure 4** [9, 25]. A karyotype diversity among cell lines was detected in this analysis, but homogeneity within some of them was observed in samples harvested at different age transfers. Then, new abnormal chromosomes were stable in different subcultures. Gross aberrations were not observed in chromosomes 6 and 8 that possess knobs smaller than those found in chromo-

*Feulgen-stained anaphase cells of the cell lines derived from the culture 3–57, with a laggard chromosome showing delayed separating chromatids (A), broken bridge (after the primary event) (B), typical bridge (C),* 

Different types of abnormal chromosomes 7 and 9 were observed in the cell lines. In the original 18-month-old callus culture 3–57, two types of chromosome 7 were detected. One of the chromosomes possessed two knobs on the short arm (K7S) corresponding to the Df-Dp chromosome 7 described above, and the other type possessed K7S on an interstitial position of a duplicated short arm (**Figure 4**). This chromosome would have originated from a mechanism similar to that shown in

*Cell line pedigree showing the types of chromosomes 7 and 9 observed in C-banded metaphases in different subcultures of the six cell lines derived from culture 3–57 [9, 25].*

**Figure 1** [9, 25]. The following types of chromosome 7 were distinguished in the cell lines (**Figure 5A**): 7A, normal type, with a terminal K7S and a subterminal K7L; 7B, with a duplicated short arm and a subterminal K7S; 7C, with two knobs on the short arm and a terminal euchromatic segment (similar to **Figure 2B**); 7D, similar to 7C, but without the terminal euchromatic segment; 7E, similar to 7D, with a smaller deficient and terminal K7S; 7F, with a larger short arm, a very large interstitial K7S and without the K7L on the long arm. The 7A, 7B, and 7C types were found in the original 18-month-old culture (**Figure 4**). **Figure 5B**, **C** illustrates the 7D and 7B types, respectively, and the 7E type can be seen in **Figure 5D**. **Figure 5E** illustrates the 7C chromosome. The 7F type can be seen in the pedigree of the 1-MS1 cell line (42-month-old culture, **Figure 6**) [9].

Different types of altered chromosome 9 were also observed in the samples of cell lines (**Figure 5A**) [9]: the normal type corresponds to 9A; a smaller terminal K9S corresponds to 9B; a smaller subterminal K9S corresponds to 9C; 9D is a chromosome without the knob; and 9E is a minichromosome derived from chromosome 9.

#### **Figure 5.**

*Types of chromosomes 7 and 9 observed in C-banded metaphase of the cell lines from culture 3–57 (A); metaphase cells of the 1-MS1 cell line (B) and 1MS2 (C); chromosomes 7 and 9 of the N6 cell line(D, E). Scale bar = 10 μm [9, 25].*

The 9A, 9C, and 9E types can be seen in **Figure 5B**, and the 9B type is shown in **Figure 5D**, **E**. **Figure 6** shows the 9D type, which appears in the 31-month-old subculture of the 1-MS1 cell line. This figure illustrates the different types of chromosomes 7 and 9 detected in 1-MS1 cell line [9, 25].

Therefore, the analysis of metaphases of the cell lines showed new abnormalities in chromosomes 7 and 9. The occurrence of delayed chromatid separation and bridges in anaphases provided evidence of BFB cycle events, and healing of the broken chromosomes could be inferred by the stability of the same abnormal chromosome in different subcultures of the same cell line [9, 25].

In most cell lines, the original abnormal chromosomes 7 (7B and 9C types) were maintained. The 7E type (with a smaller distal K7S) was found in the 42-month-old subculture of the 2-N6 cell line (see **Figure 4**) [9, 25]. The 7D type (chromosome 7 without the terminal euchromatic segment) was observed in the 1-MS1 and 2-MS1. These data suggest that cells bearing the original Df-Dp chromosome 7 (7B or 7C types) were highly adapted in culture and that the new types (7D and 7E) found in

**139**

**Figure 6.**

*bar = 10 μm [9].*

this knob in the 7F type.

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures*

some subcultures were derived from the original altered chromosome 7 (7C type) through new events of delayed chromatid separation at the knob region and breakage. The 7F type would be a new alteration of the normal chromosome and was detected in the 42-month-old subculture of the 1-MS1 cell line (**Figures 4, 6**) [9, 25]. Its origin would be through a delay in sister chromatids on K7S at anaphase, and an amplified subterminal knob would appear if the duplicate knob did not separate and a breakage occurred at an adjacent euchromatic region. A delayed separation of chromatids on K7L at anaphase, followed by breakage, would explain the absence of

*Types of chromosomes 7 and 9 observed in C-banded metaphases of the 1-MS1 and 2-MS1 cell lines. Scale* 

Chromosome 9 suffered alterations in most cell lines, except for the 1-MS2 and 1-N6 cell lines. The 9D type (K9S deleted) was detected in the 2-MS2 and 2-MS1 cell lines, and the 9B type (partial deletion of the knob) was observed in the 2-N6 cell line (see **Figure 4** [9, 25]). A total or partial deletion of K9S would have occurred after a delay of separation of the chromatids on this knob region and breakage totally or partially eliminating the knob or a segment of it. Interestingly, in the cell line 1-MS1, two types of chromosome 9 appeared, the 9C displaying a subterminal smaller K9S (9C type) and a chromosome without the knob (9D type).

*DOI: http://dx.doi.org/10.5772/intechopen.88876*

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures DOI: http://dx.doi.org/10.5772/intechopen.88876*

#### **Figure 6.**

*Chromosomal Abnormalities*

**138**

**Figure 5.**

*bar = 10 μm [9, 25].*

The 9A, 9C, and 9E types can be seen in **Figure 5B**, and the 9B type is shown in **Figure 5D**, **E**. **Figure 6** shows the 9D type, which appears in the 31-month-old subculture of the 1-MS1 cell line. This figure illustrates the different types of

*Types of chromosomes 7 and 9 observed in C-banded metaphase of the cell lines from culture 3–57 (A); metaphase cells of the 1-MS1 cell line (B) and 1MS2 (C); chromosomes 7 and 9 of the N6 cell line(D, E). Scale* 

Therefore, the analysis of metaphases of the cell lines showed new abnormalities in chromosomes 7 and 9. The occurrence of delayed chromatid separation and bridges in anaphases provided evidence of BFB cycle events, and healing of the broken chromosomes could be inferred by the stability of the same abnormal

In most cell lines, the original abnormal chromosomes 7 (7B and 9C types) were maintained. The 7E type (with a smaller distal K7S) was found in the 42-month-old subculture of the 2-N6 cell line (see **Figure 4**) [9, 25]. The 7D type (chromosome 7 without the terminal euchromatic segment) was observed in the 1-MS1 and 2-MS1. These data suggest that cells bearing the original Df-Dp chromosome 7 (7B or 7C types) were highly adapted in culture and that the new types (7D and 7E) found in

chromosomes 7 and 9 detected in 1-MS1 cell line [9, 25].

chromosome in different subcultures of the same cell line [9, 25].

*Types of chromosomes 7 and 9 observed in C-banded metaphases of the 1-MS1 and 2-MS1 cell lines. Scale bar = 10 μm [9].*

some subcultures were derived from the original altered chromosome 7 (7C type) through new events of delayed chromatid separation at the knob region and breakage. The 7F type would be a new alteration of the normal chromosome and was detected in the 42-month-old subculture of the 1-MS1 cell line (**Figures 4, 6**) [9, 25]. Its origin would be through a delay in sister chromatids on K7S at anaphase, and an amplified subterminal knob would appear if the duplicate knob did not separate and a breakage occurred at an adjacent euchromatic region. A delayed separation of chromatids on K7L at anaphase, followed by breakage, would explain the absence of this knob in the 7F type.

Chromosome 9 suffered alterations in most cell lines, except for the 1-MS2 and 1-N6 cell lines. The 9D type (K9S deleted) was detected in the 2-MS2 and 2-MS1 cell lines, and the 9B type (partial deletion of the knob) was observed in the 2-N6 cell line (see **Figure 4** [9, 25]). A total or partial deletion of K9S would have occurred after a delay of separation of the chromatids on this knob region and breakage totally or partially eliminating the knob or a segment of it. Interestingly, in the cell line 1-MS1, two types of chromosome 9 appeared, the 9C displaying a subterminal smaller K9S (9C type) and a chromosome without the knob (9D type).

#### *Chromosomal Abnormalities*

In addition, a minichromosome (9E type) appeared in the subcultures possessing one of these abnormal types. These abnormalities could have resulted from the mechanism suggested in **Figure 7** [9, 25]. The primary event would be a delay in the separation of chromatids at K9S region followed by breakage originating a deficient knob. Then, two types of BFB cycles, the chromosome [58] and the chromatid types, would have originated the 9C, 9D, and 9E chromosome types. The 9C and 9E chromosome types were observed in several subcultures, thus providing evidence of healing of the broken chromosome ends. In the cell lines analyzed, abnormalities were detected only in chromosomes 7 and 9. These alterations were derived from a primary event of chromatid delayed separation at knob sites in anaphases, followed by breakage and BFB cycle. The presence of large knobs in these chromosomes would lead to this kind of primary event. A case of elimination of chromosome segments from knobbed chromosomes was reported by Rhoades and Dempsey [59]. In the presence of B chromosomes, a bridge formation would occur due to delayed replication of the knob at the second microspore division. Chromosomes containing large knobs would be involved more frequently in this kind of event.

#### **Figure 7.**

*Mechanism that would originate different types of chromosome 9 in the 1-MS1 cell line: Normal chromosome (A); anaphase with delayed separating chromatids and breakage (B); the resulting chromatids, one normal and the other with a deficient knob (C) fused after replication (D); nondisjunction of sister chromatids (E) resulting in a dicentric chromosome (F, G); double bridge (H) giving rise to a chromatid with a deficiency at K9S and another chromatid without knob (I), which after duplication and fusion(J) suffered breakage in the next anaphases (K); resultant chromosomes: One with a deficient K9S, another without the knob, and a minichromosome originated after deletions whose mechanism is unclear (L). Arrows at anaphases indicate breakpoints [9, 25].*

**141**

**Figure 8.**

*amplified K7L (C). Scale bar = 10 μm [10].*

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures*

**2.4 Heterochromatic knob amplification resulting from unequal crossing over** 

*Somatic chromosomes of regenerated plants (R1) derived from the 12-F culture: C-banded prometaphase of the homozygote for normal chromosome 7 (A); homozygote for the amplified K7L (B); heterozygote for the* 

The observation of mitotic and meiotic aspects of an amplification of the knob localized on the long arm of chromosome 7 (K7L), in plants regenerated from a long-term callus culture designated 12-F, was carried out aiming to investigate the origin of this amplification. The 12-F original culture was 28 months old when the R1 plants were obtained [10]. The original callus 12-F was heterozygous for the amplified K7L. Therefore, segregation was expected in R1 progenies derived by selfing R0 plants. Plants homozygous for the normal and amplified K7L, and plants heterozygous for the amplified K7L, were recovered (**Figure 8**) [10]. The frequency of plants homozygous for the amplification was lower than expected according to Mendelian segregation, while the frequency of plants homozygous for the normal K7L was higher than expected. The frequency of heterozygotes was according to the

*DOI: http://dx.doi.org/10.5772/intechopen.88876*

**and BFB cycle**

expected value.

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures DOI: http://dx.doi.org/10.5772/intechopen.88876*

#### **2.4 Heterochromatic knob amplification resulting from unequal crossing over and BFB cycle**

The observation of mitotic and meiotic aspects of an amplification of the knob localized on the long arm of chromosome 7 (K7L), in plants regenerated from a long-term callus culture designated 12-F, was carried out aiming to investigate the origin of this amplification. The 12-F original culture was 28 months old when the R1 plants were obtained [10]. The original callus 12-F was heterozygous for the amplified K7L. Therefore, segregation was expected in R1 progenies derived by selfing R0 plants. Plants homozygous for the normal and amplified K7L, and plants heterozygous for the amplified K7L, were recovered (**Figure 8**) [10]. The frequency of plants homozygous for the amplification was lower than expected according to Mendelian segregation, while the frequency of plants homozygous for the normal K7L was higher than expected. The frequency of heterozygotes was according to the expected value.

#### **Figure 8.**

*Chromosomal Abnormalities*

kind of event.

In addition, a minichromosome (9E type) appeared in the subcultures possessing one of these abnormal types. These abnormalities could have resulted from the mechanism suggested in **Figure 7** [9, 25]. The primary event would be a delay in the separation of chromatids at K9S region followed by breakage originating a deficient knob. Then, two types of BFB cycles, the chromosome [58] and the chromatid types, would have originated the 9C, 9D, and 9E chromosome types. The 9C and 9E chromosome types were observed in several subcultures, thus providing evidence of healing of the broken chromosome ends. In the cell lines analyzed, abnormalities were detected only in chromosomes 7 and 9. These alterations were derived from a primary event of chromatid delayed separation at knob sites in anaphases, followed by breakage and BFB cycle. The presence of large knobs in these chromosomes would lead to this kind of primary event. A case of elimination of chromosome segments from knobbed chromosomes was reported by Rhoades and Dempsey [59]. In the presence of B chromosomes, a bridge formation would occur due to delayed replication of the knob at the second microspore division. Chromosomes containing large knobs would be involved more frequently in this

*Mechanism that would originate different types of chromosome 9 in the 1-MS1 cell line: Normal chromosome (A); anaphase with delayed separating chromatids and breakage (B); the resulting chromatids, one normal and the other with a deficient knob (C) fused after replication (D); nondisjunction of sister chromatids (E) resulting in a dicentric chromosome (F, G); double bridge (H) giving rise to a chromatid with a deficiency at K9S and another chromatid without knob (I), which after duplication and fusion(J) suffered breakage in the next anaphases (K); resultant chromosomes: One with a deficient K9S, another without the knob, and a minichromosome originated after deletions whose mechanism is unclear (L). Arrows at anaphases indicate* 

**140**

**Figure 7.**

*breakpoints [9, 25].*

*Somatic chromosomes of regenerated plants (R1) derived from the 12-F culture: C-banded prometaphase of the homozygote for normal chromosome 7 (A); homozygote for the amplified K7L (B); heterozygote for the amplified K7L (C). Scale bar = 10 μm [10].*

Some plants whose karyotype was investigated were selected for meiotic analysis. The homologous chromosomes were completely synapsed on knobs and terminal euchromatic segment on the long arm at pachytene in plants homologous for normal and amplified K7L (**Figure 9A**, **B**) [10]. In plants heterozygous for the amplification, the knobs and the terminal euchromatic segments were completely synapsed in some cells (**Figure 9C**) [10], but synapsis failure was also detected in these regions (**Figure 9D**) [10]. In chromosomes bearing the K7L amplification and in normal chromosomes, the size of the distal euchromatic segment was similar, but the size of the amplified knob was significantly larger than the normal knob. In a possible case of delay of chromatid separation at this knob site followed by breakage and a BFB cycle, the distal euchromatic segment would be lost as discussed below [10].

Other abnormalities such as translocations, inversions, duplications, and deletions were not found in the chromosomes of these plants derived from a long-term callus culture [10].

The analysis of microsporocytes at the diakinesis stage showed the presence of two types of univalents: one larger with two C-bands, thus corresponding to chromosome 7, and a small one. The frequency of univalents was low for both types. The frequency of the large univalents in heterozygous plants was higher (2.88%) than control plants (0.55%). Differences were not observed in the frequency of small univalents in the heterozygotes (1.14%) in comparison with control plants (1.92%) [10]. Therefore, the meiosis was normal in most microsporocytes, and R2 progenies were also obtained.

The investigation of short-term cultures derived from inbred lines and hybrids related to the inbred line donor of culture 12-F showed interesting alterations on the

#### **Figure 9.**

*Carmine-stained meiotic chromosomes of R1 plants derived from the 12-F culture. Pachytene of a homozygote for the normal K7L (A); homozygote for the amplified K7L (B); heterozygote for the amplified K7L, showing completely synapsed chromosomes (C); pairing failure in the knob region of the heterozygote. Scale bar = 10 μm [10].*

**143**

**Figure 10.**

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures*

occurrence of unequal sister chromatid recombination [10].

long arm of chromosome 7 bearing K7L. The cytogenetic analysis of these cultures detected abnormalities in chromosomes 7 and 9 and other chromosomes with and without knobs. Here we focus only on alterations in the long arm of chromosome 7 to infer the origin of these abnormalities, aiming to understand the origin of the K7L amplification observed in plants derived from culture 12-F. A total of 5223 cells of the callus cultures from 6 genotypes were examined. In three cells from different cultures, chromosome 7 bearing with asymmetric C-bands (corresponding to K7L) was observed: one band was amplified, and the other was reduced in sister chromatids (**Figure 10A, B**). These band alterations would have appeared due to the

Unequal crossing over in regions containing duplicate genes or repetitive DNA has been demonstrated in several organisms, such as yeast [60], apes [61], and humans [62]. Two reciprocal products, a directly amplified tandem duplication and a deletion, can result from unequal crossing over between homologous chromosomes. In patients with chromosome duplications involving some types of Charcot–Marie–Tooth disease, the occurrence of unequal recombination between homologous segments (interchromosomal) and sister chromatids (intrachromosomal) has been shown [62]. Thus, the generation of deletions and duplications by unequal recombination can affect the copy number of repeated genes and noncod-

*Types of alterations in the long arm of chromosome 7 observed in C-banded metaphases of the short-term callus cultures: K7L with different sizes in the sister chromatids (A, B); K7L duplication (C); K7L amplification (D); K7L reduction (E); K7L in the telomeric position (F); absence of K7L (G); diagrammatic representation of the abnormal chromosomes (H). Arrows point to the altered chromosomes. N, normal; Dp, duplication; amp,* 

*amplification; Rd., reduction; T, terminal; Del, deletion. Scale bar = 10 μm [10].*

*DOI: http://dx.doi.org/10.5772/intechopen.88876*

ing repeated DNA sequences.

#### *Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures DOI: http://dx.doi.org/10.5772/intechopen.88876*

long arm of chromosome 7 bearing K7L. The cytogenetic analysis of these cultures detected abnormalities in chromosomes 7 and 9 and other chromosomes with and without knobs. Here we focus only on alterations in the long arm of chromosome 7 to infer the origin of these abnormalities, aiming to understand the origin of the K7L amplification observed in plants derived from culture 12-F. A total of 5223 cells of the callus cultures from 6 genotypes were examined. In three cells from different cultures, chromosome 7 bearing with asymmetric C-bands (corresponding to K7L) was observed: one band was amplified, and the other was reduced in sister chromatids (**Figure 10A, B**). These band alterations would have appeared due to the occurrence of unequal sister chromatid recombination [10].

Unequal crossing over in regions containing duplicate genes or repetitive DNA has been demonstrated in several organisms, such as yeast [60], apes [61], and humans [62]. Two reciprocal products, a directly amplified tandem duplication and a deletion, can result from unequal crossing over between homologous chromosomes. In patients with chromosome duplications involving some types of Charcot–Marie–Tooth disease, the occurrence of unequal recombination between homologous segments (interchromosomal) and sister chromatids (intrachromosomal) has been shown [62]. Thus, the generation of deletions and duplications by unequal recombination can affect the copy number of repeated genes and noncoding repeated DNA sequences.

#### **Figure 10.**

*Chromosomal Abnormalities*

callus culture [10].

were also obtained.

Some plants whose karyotype was investigated were selected for meiotic analysis. The homologous chromosomes were completely synapsed on knobs and terminal euchromatic segment on the long arm at pachytene in plants homologous for normal and amplified K7L (**Figure 9A**, **B**) [10]. In plants heterozygous for the amplification, the knobs and the terminal euchromatic segments were completely synapsed in some cells (**Figure 9C**) [10], but synapsis failure was also detected in these regions (**Figure 9D**) [10]. In chromosomes bearing the K7L amplification and in normal chromosomes, the size of the distal euchromatic segment was similar, but the size of the amplified knob was significantly larger than the normal knob. In a possible case of delay of chromatid separation at this knob site followed by breakage and a BFB cycle, the distal euchromatic segment would be lost as discussed below [10].

Other abnormalities such as translocations, inversions, duplications, and deletions were not found in the chromosomes of these plants derived from a long-term

The analysis of microsporocytes at the diakinesis stage showed the presence of two types of univalents: one larger with two C-bands, thus corresponding to chromosome 7, and a small one. The frequency of univalents was low for both types. The frequency of the large univalents in heterozygous plants was higher (2.88%) than control plants (0.55%). Differences were not observed in the frequency of small univalents in the heterozygotes (1.14%) in comparison with control plants (1.92%) [10]. Therefore, the meiosis was normal in most microsporocytes, and R2 progenies

The investigation of short-term cultures derived from inbred lines and hybrids related to the inbred line donor of culture 12-F showed interesting alterations on the

*Carmine-stained meiotic chromosomes of R1 plants derived from the 12-F culture. Pachytene of a homozygote for the normal K7L (A); homozygote for the amplified K7L (B); heterozygote for the amplified K7L, showing completely synapsed chromosomes (C); pairing failure in the knob region of the heterozygote. Scale* 

**142**

**Figure 9.**

*bar = 10 μm [10].*

*Types of alterations in the long arm of chromosome 7 observed in C-banded metaphases of the short-term callus cultures: K7L with different sizes in the sister chromatids (A, B); K7L duplication (C); K7L amplification (D); K7L reduction (E); K7L in the telomeric position (F); absence of K7L (G); diagrammatic representation of the abnormal chromosomes (H). Arrows point to the altered chromosomes. N, normal; Dp, duplication; amp, amplification; Rd., reduction; T, terminal; Del, deletion. Scale bar = 10 μm [10].*

#### *Chromosomal Abnormalities*

From this scenario, we can assume that the asymmetric chromosome 7 observed in callus cultures resulted from unequal chromatid recombination at the K7L site. Therefore, the amplification of K7L detected in R1 plants derived from the 12F culture would have originated from an unequal recombination at K7L. This event would not alter the size of the distal euchromatic segment, as observed here [10].

Other alterations were observed on the long arm of chromosome 7 in the callus cultures analyzed: duplication of K7L (**Figure 10C**), amplification of K7L (**Figure 10D**), reduction of K7L (**Figure 10E**), K7L localized on telomeric position (**Figure 10F**), and the absence of K7L (**Figure 10G**). **Figure 10H** shows a diagrammatic representation of these abnormalities. The frequency of these aberrations varied from 0.98 to 4.82% in the six genotypes evaluated [10].

These abnormalities could have originated from a delay of sister chromatid separation at the K7L region, followed by breakage and BFB cycles as suggested in **Figure 11** [10]. After the primary event of delayed chromatid separation, breakage could occur in three different positions at the knob region, terminal (1), proximal (2), and middle (3), giving rise to the different types of abnormalities shown in **Figure 10C**–**G**. Note that in all possible events, the terminal euchromatic "b" segment would be lost. Regenerated plants homozygous for these aberrations probably would not survive with the deletion of the terminal segment. The recovery of regenerated plants homozygous for the K7L amplification gives support to

#### **Figure 11.**

*Diagrammatic representation of the BFB cycles that would result in abnormalities in the long arm of chromosome 7 observed in the short-term callus cultures. The normal chromosome (A) duplicates after DNA replication (B) and delayed separation of the chromatids is observed (C). Breakage occurs at three possible positions: Terminal (1), proximal (2), and middle (3). The resulting chromatids are the following: Breakage at position 1 originates a chromatid with a terminal knob or an amplified knob after a new mitotic cycle (D); breakage at position 2 results in chromatids with the absence of the knob (E); breakage at position 3 results in a reduced terminal knob after the primary event, in a reduced knob and a terminal euchromatic segment after a new mitotic cycle or a duplicated knob after a third mitotic cycle (F). "A" and "b" represent segments near the knobs at a proximal and at a distal position, respectively. Note that in all events, the "b" segment is lost [10].*

**145**

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures*

cycles, they would not survive in homozygous regenerated plants.

the hypothesis that this amplification was originated from an unequal chromatid

Therefore, the results show that knob amplification or reduction can appear as a result of BFB cycles or unequal crossing over, but if they are originated from BFB

The presence of some chromosome abnormalities in maize callus cultures can be explained by the occurrence of delay of chromatid separation in mitotic anaphases. This primary event gives origin to a bridge followed by a breakage-fusion-bridge cycle and chromosome healing. FISH using telomere sequences as probes gave evidence of de novo telomere formation at broken chromosome ends. Amplifications and deficiencies in the knobs may also occur via unequal chromosome crossing over evidenced in culture by the presence of chromosome 7 showing differences in the

The data suggest interesting questions for further investigations such as the mechanism underlying the delay in chromatid separation at knob sites and that of de novo telomere formation at the broken chromosome ends in callus culture. Changes in DNA methylation could be the cause of unusual later replication of

The observations on the chromosome healing of chromosomes 7 and 9 showed that this event occurred in euchromatic and heterochromatic regions, certainly non-telomeric sites. Mechanistic information on telomere formation is available through studies on *Saccharomyces cerevisiae*. In this species, telomere sequences were added to non-telomeric broken chromosome ends, but a strong preference for telomerase action was observed at GT, TG, or GG nucleotides [63]. In wheat, de novo telomere formation was initiated by 2-to-4 nucleotide target motifs in an rDNA sequence localized in terminal position [53]. Homologous recombination, nonhomologous end joining, and de novo telomere formation are different mechanisms that repair DNA double strand. De novo telomere formation would be a rare event [64]. In the present report, the healing of chromosomes 7 and 9 possibly occurred in the presence of internal sequences to which telomerase was

In conclusion, mechanisms of chromosomal evolution like the related here might occur in plants. It has been suggested that structural chromosomal rearrangements

The authors acknowledge: Canadian Science Publishing for the permission to use figures and data of the article [9]. S. Karger AG for the permission to use figures and data of the article [10]. Nova Science Publishers Inc. for the permission to use figures and data of the review [25]. Fundação de Amparo à Pesquisa do Estado de

frequently appear in euchromatin-heterochromatin borders [65].

*DOI: http://dx.doi.org/10.5772/intechopen.88876*

size of K7L (C-band) in sister chromatids.

amplification.

**3. Conclusions**

knobs (see [40]).

recruited.

**Acknowledgements**

**Conflict of interest**

São Paulo (FAPESP) for research support.

The authors declare no conflict of interest.

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures DOI: http://dx.doi.org/10.5772/intechopen.88876*

the hypothesis that this amplification was originated from an unequal chromatid amplification.

Therefore, the results show that knob amplification or reduction can appear as a result of BFB cycles or unequal crossing over, but if they are originated from BFB cycles, they would not survive in homozygous regenerated plants.

#### **3. Conclusions**

*Chromosomal Abnormalities*

observed here [10].

From this scenario, we can assume that the asymmetric chromosome 7 observed in callus cultures resulted from unequal chromatid recombination at the K7L site. Therefore, the amplification of K7L detected in R1 plants derived from the 12F culture would have originated from an unequal recombination at K7L. This event would not alter the size of the distal euchromatic segment, as

Other alterations were observed on the long arm of chromosome 7 in the callus cultures analyzed: duplication of K7L (**Figure 10C**), amplification of K7L (**Figure 10D**), reduction of K7L (**Figure 10E**), K7L localized on telomeric position (**Figure 10F**), and the absence of K7L (**Figure 10G**). **Figure 10H** shows a diagrammatic representation of these abnormalities. The frequency of these aberrations

These abnormalities could have originated from a delay of sister chromatid separation at the K7L region, followed by breakage and BFB cycles as suggested in **Figure 11** [10]. After the primary event of delayed chromatid separation, breakage could occur in three different positions at the knob region, terminal (1), proximal (2), and middle (3), giving rise to the different types of abnormalities shown in **Figure 10C**–**G**. Note that in all possible events, the terminal euchromatic "b" segment would be lost. Regenerated plants homozygous for these aberrations probably would not survive with the deletion of the terminal segment. The recovery of regenerated plants homozygous for the K7L amplification gives support to

*Diagrammatic representation of the BFB cycles that would result in abnormalities in the long arm of chromosome 7 observed in the short-term callus cultures. The normal chromosome (A) duplicates after DNA replication (B) and delayed separation of the chromatids is observed (C). Breakage occurs at three possible positions: Terminal (1), proximal (2), and middle (3). The resulting chromatids are the following: Breakage at position 1 originates a chromatid with a terminal knob or an amplified knob after a new mitotic cycle (D); breakage at position 2 results in chromatids with the absence of the knob (E); breakage at position 3 results in a reduced terminal knob after the primary event, in a reduced knob and a terminal euchromatic segment after a new mitotic cycle or a duplicated knob after a third mitotic cycle (F). "A" and "b" represent segments near the knobs at a proximal and at a distal position, respectively. Note that in all events, the "b" segment is lost [10].*

varied from 0.98 to 4.82% in the six genotypes evaluated [10].

**144**

**Figure 11.**

The presence of some chromosome abnormalities in maize callus cultures can be explained by the occurrence of delay of chromatid separation in mitotic anaphases. This primary event gives origin to a bridge followed by a breakage-fusion-bridge cycle and chromosome healing. FISH using telomere sequences as probes gave evidence of de novo telomere formation at broken chromosome ends. Amplifications and deficiencies in the knobs may also occur via unequal chromosome crossing over evidenced in culture by the presence of chromosome 7 showing differences in the size of K7L (C-band) in sister chromatids.

The data suggest interesting questions for further investigations such as the mechanism underlying the delay in chromatid separation at knob sites and that of de novo telomere formation at the broken chromosome ends in callus culture. Changes in DNA methylation could be the cause of unusual later replication of knobs (see [40]).

The observations on the chromosome healing of chromosomes 7 and 9 showed that this event occurred in euchromatic and heterochromatic regions, certainly non-telomeric sites. Mechanistic information on telomere formation is available through studies on *Saccharomyces cerevisiae*. In this species, telomere sequences were added to non-telomeric broken chromosome ends, but a strong preference for telomerase action was observed at GT, TG, or GG nucleotides [63]. In wheat, de novo telomere formation was initiated by 2-to-4 nucleotide target motifs in an rDNA sequence localized in terminal position [53]. Homologous recombination, nonhomologous end joining, and de novo telomere formation are different mechanisms that repair DNA double strand. De novo telomere formation would be a rare event [64]. In the present report, the healing of chromosomes 7 and 9 possibly occurred in the presence of internal sequences to which telomerase was recruited.

In conclusion, mechanisms of chromosomal evolution like the related here might occur in plants. It has been suggested that structural chromosomal rearrangements frequently appear in euchromatin-heterochromatin borders [65].

#### **Acknowledgements**

The authors acknowledge: Canadian Science Publishing for the permission to use figures and data of the article [9]. S. Karger AG for the permission to use figures and data of the article [10]. Nova Science Publishers Inc. for the permission to use figures and data of the review [25]. Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for research support.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Chromosomal Abnormalities*

#### **Author details**

Margarida L.R. Aguiar-Perecin1 \*, Janay A. Santos-Serejo2 , José R. Gardingo3 and Mateus Mondin1

1 Department of Genetics, Luiz de Queiroz Agriculture College, University of São Paulo, Piracicaba, SP, Brazil

2 Embrapa Cassava and Fruits, Brazilian Agricultural Research Corporation, Cruz das Almas, BA, Brazil

3 University of Ponta Grossa, Ponta Grossa, PR, Brazil

\*Address all correspondence to: mlrapere@usp.br

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**147**

*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures*

crossing over. Cytogenetic and Genome Research. 2018;**154**:107-118.

[11] Aguiar-Perecin MLR, Vosa CG. C-banding in maize II. Identification of somatic chromosomes. Heredity. 1985;**54**:37-42. DOI: 10.1038/hyd.1985.6

[12] Ananiev EV, Phillips RL, Rines HW. A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: Are chromosome knobs megatransposons? Proceedings of the National Academy of Sciences of the United States of America. 1998;**95**:10785- 10790. DOI: 10.1073/pnas.95.18.10785

DOI: 10.1159/000488067

[13] Chen CC, Chen CM,

[14] Page BT, Wanous MK, Birchler JA. Characterization of maize chromosome 4 centromeric sequence: Evidence for an evolutionary relationship with B chromosome centromere. Genetics. 2001;**159**:291-302

[15] Zhong CX, Marshall JB, Topp C, Mroczek R, Nagaki K, et al. Centromeric retroelements and satellites interact with maize kinetochore protein CENH3. Plant Cell. 2002;**14**:2825-2836. DOI:

[16] Nagaki K, Song J, Stupar SM, Parokonny AS, Yuan Q, Ouyang S, et al. Molecular and cytological analyses of large tracks of centromeric DNA reveal the structure and evolutionary dynamics of maize centromeres. Genetics. 2003;**163**:759-770

[17] Kato A, Lamb JC, Birchler JA. Chromosome painting using repetitive

s001220051445

10.1105/tpc.006106

Hsu FC, Wang CJ, Yang JT, Kao YY. The pachytene chromosomes of maize as revealed by fluorescence *in situ* hybridization with repetitive DNA sequences. Theoretical and Applied Genetics. 2000;**101**:30-36. DOI: 10.1007/

*DOI: http://dx.doi.org/10.5772/intechopen.88876*

[1] McClintock B. Chromosome morphology in Zea mays. Science.

Research. 1939;**59**:475-490

Verlag; 1994. pp. 432-441

p. 468

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[7] Maize Genetics Cooperation Stock Center. Available from: www.maizegdb.

[8] Fluminhan A, Aguiar-Perecin MLR, Santos JA. Evidence for heterochromatin involvement in chromosome breakage in maize callus culture. Annals of Botany. 1996;**78**:73-81. DOI: 10.1006/

[9] Santos-Serejo JA, Aguiar-Perecin MLR. Breakage-fusion-bridge cycles and de novo telomere formation on broken chromosomes in maize callus cultures. Genome. 2016;**59**:367-378.

[10] Santos-Serejo JA, Gardingo JR, Mondin M, Aguiar-Perecin MLRA. Alterations in heterochromatic knobs in maize callus culture by breakagefusion-bridge cycle and unequal

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*Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures DOI: http://dx.doi.org/10.5772/intechopen.88876*

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**Author details**

Mateus Mondin1

Margarida L.R. Aguiar-Perecin1

Paulo, Piracicaba, SP, Brazil

das Almas, BA, Brazil

\*, Janay A. Santos-Serejo2

1 Department of Genetics, Luiz de Queiroz Agriculture College, University of São

2 Embrapa Cassava and Fruits, Brazilian Agricultural Research Corporation, Cruz

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

3 University of Ponta Grossa, Ponta Grossa, PR, Brazil

\*Address all correspondence to: mlrapere@usp.br

provided the original work is properly cited.

, José R. Gardingo3

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Madison: American Society of Agronomy; 1988. pp. 259-343

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DOI: 10.1073/pnas.28.11.458

1973;**64**:12-18

1980;**284**:426-430

pnas.77.4.2158

2002;**18**:74-82

s00412-013-0404-2

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Beverley SM, Kan Y, Wilson AC. Rapid duplication and loss of genes coding for α chains of hemoglobin. Proceedings of the National Academy of Sciences of the United States of America. 1980;**77**:2158-2162. DOI: 10.1073/

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[63] Ribeyre C, Shore D. Regulation of telomere addition at DNA double –strand breaks. Chromosoma. 2013;**122**:159-173. DOI: 10.1007/

Chromatin elimination induced by the B chromosome of maize: I mechanism of loss and the pattern of endosperm variegation. Journal of Heredity.

s004380050918

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Genetics. 1995;**140**:1069-1085

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10.1007/soo4380051150

pnas.93.25.14422

pnas.92.21.9555

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10.1139/g92-128

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**150**

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### *Edited by Tülay Aşkın Çelik and Subrata Dey*

Chromosomes are vital components of genetic material, and, as such, distruption or changes to the structure of chromosomes can result in different health problems and deficits. This book explains chromosomal abnomalities and their effects on living organisms, including humans and plants. Classical and molecular cytogenetics techniques have a considerable number of potential applications, especially in clinical trials and biomedical diagnosis, making them a strong and insightful complement to other molecular and genomic approaches. Chapters cover topics including Down syndrome, fetal ultrasounds, acute myeloid leukemia, and Phelan-McDermid syndrome, among others.

Published in London, UK © 2020 IntechOpen © koya79 / iStock

Chromosomal Abnormalities

Chromosomal Abnormalities

*Edited by Tülay Aşkın Çelik and Subrata Dey*