BIOTECHNOLOGICAL APPROACH FOR CONSERVATION

The biotechnological methods are used to select genotypes with desirable traits, to identify and insert the important desirable genes that induce resistance and tolerance to biotic and abiotic stress. Plant tissue culture method particularly micropropagation techniques are now used to multiply and preserve plant genetic biodiversity outside its natural habitat. Furthermore, plant tissue culture methods offer a safe means for the international exchange of germplasm, which allow the establishment of extensive collections with minimal space requirements.

Biotechnological approaches also allow a valuable supply of in vitro regenerated plant materials for recovery of wild population, they promise the repository of pathogen-free material and elite plants and assist the progress of the performance of molecular investigations and ecological studies (Tandon and Kumaria, 2005). Some biotechnological techniques, such as in vitro culture, are very helpful in keeping up ex situ gennplasm collections of plant species that have asexual mode of propagation than the species that are impossible to maintain as seeds or in field gene banks (Chatteijee and Ghosh, 2015, 2016; Haque and Ghosh, 2013a, 2013b, 2016, 2017; Kundu et al., 2015).

Totipotency is a unique inherent character of all plant cells that are now been exploited through in vitro teclmiques, this concept was basically proposed by Haberlandt (1902) and initially, it was clearly demonstrated by Steward et al. (1958). In vitro plant regeneration via callus culture, multiplication, and clonal propagation of the plant are essential for improvement of disease-free plant and it is a compulsory application of biotechnology branch to plant genetic improvement. It is also important for the conservation of genetically pure planting materials.

Micropropagation is now widely used for large scale propagation of novel plants as an alternative of conventional propagation methods, even genetically modified as well as hybrid cultivars are also multiplied in this biotechnological technique. This method is also used for mass-propagation of new genotypes. Meristem culture also adopted for generation of large-scale virus-free and other pathogen-fiee plant material that trigger the impressive improvement the yield of established cultivars.

17.5.1 IN VITRO CULTURE AND MICROPROPAGATION

Plant tissue culture is an ideal procedure that is now widely used in academic as well as commercial puiposes. Several aseptic in vitro culture procedures are now adopted especially in as in vitro seed germination, micropropagation, callus and cell suspension culture (Wilson and Roberts, 2012).

In contrast to any other plant propagation system, the in vitro aseptic micropropagation method gain highest appreciation for large-scale multiplication of any elite clone without natural climatic influences like temperature, light, humidity, any seasonal variation, etc. (Costa et al., 2013). Even micropropagation method generated genetically stable can also be used for the purpose of conservation of recalcitrant and endanger species. In vitro culture and micropropagation form the base for establishing tissue cultures and developing in vitro and cryo-conservation technology.

Plant tissue-cultured should be maintained for their genetic stability as well as morphological nature. Micropropagation provides continuous stable supply of any plant genetic resources that can serve in two way firstly it involved in different crop improvement program and secondly, for germplasm conservation purpose. A huge number of plant species are now enlisted in successfully in vitro micropropagation system including horticultural species. Among the most important in vitro technique applications, is the micropropagation of vegetable plants, medicinal and aromatic threatened plant species (Matkowski, 2008; Purohit, 2013).

In current scenario, conservation of a particular plant species and plant biodiversity of a population conservation is an important issue and this problem can be satisfied by in vitro micropropagation that can provide huge number of propagules from a cross-section of the genetic diversity of a region (Rogers, 2003) it also provides additional backup collections and provides alternative propagation and conservation of Capsicum species (Fig. 17.4) (Haque et al., 2016; Haque and Ghosh, 2017). Recent reports on in vitro multiplication of Capsicum (Fig. 17.5) and conservation (Haque et al., 2016, Haque and Ghosh, 2017).

Flow diagram of plant tissue culture for mass propagation and conservation

FIGURE 17.4 Flow diagram of plant tissue culture for mass propagation and conservation.

Tissue culture of Capsicum, (a) Shoot multiplication and (b) complete regenerated plant with shoot and roots, (c) In vitro flowering and (d) ex vitro condition after hardening

FIGURE 17.5 Tissue culture of Capsicum, (a) Shoot multiplication and (b) complete regenerated plant with shoot and roots, (c) In vitro flowering and (d) ex vitro condition after hardening.

The in vitro plant cell culture of Capsicum is an important system for understanding the mechanism of synthesis of capsaicin and other commercially viable secondary metabolites both at cellular and molecular level (Kumari et al., 2015). In spite of this, a collection of research work has been directed to explore the morphogenic potential and to attain the successful repeatable protocol for regeneration of whole plantlets of chili and constant efforts are still in progress to achieve more improvement in the arena of pepper biotechnology.

The maximum success of in vitro regeneration of chili has been attained through organogenesis process, but the other methods of regeneration through tissue culture, like somatic embiyogenesis and anther of pollen culture for androgenic haploid production, have also been explored. A summary list of in vitro morphogenic response of different explants, composition of culture media in approach toward the micropropagation of different species of Capsicum is given below (Table 17.2).

17.5.1.1 EFFECT OF DIFFERENT CULTIVARS ON IN VITRO RESPONSE

The genotype is one of the main factors that influence the organogenic response of cultures in different plant species. Cultivar specific regeneration protocol is now essential because cultivar, as well as specific genotype or chemotype, also plays an important differential role for regeneration purposes due to cultivars specific differential gene expression and culture media response. So, in this context, recommendation of single standard protocol not acceptable it demands cultivar or genotype-specific protocol.

Various factors like explant type, composition of nutrient media, different physical conditions (light, temperature, humidity, volume of the culture vessels, etc.) highly influenced the regeneration efficacy of the plant species Capsicum. It is reported by Ochoa-Alejo and Ireta-Moreno (1990) that the same explant type ware used from different sources of cultivars showed clear differential response when cultured in same PGRs supplemented MS media. The differential shoot bud morphogenic response was observed in different cultivars when culture was maintained with same condition.

Differential response in organogenic potentiality of chili have also been observed in different genotypes (Rodeva et al., 2006; Valadez-Bustos et al., 2009; Manzur et al., 2013; Mythili et al., 2017), different cultivars (Ezura et al., 1993; Szasz et al., 1995; Sanatombi and Sharma, 2008; Orlinska and Nowaczyk, 2015; Hegde et al., 2017), various species (Christopher and Rajam, 1996; Rodeva et al., 2006; Joshi and Kothari, 2007; Kehie et al., 2013; Maligeppagol et al., 2016), and different culture media (Ochoa-Alejo and Ireta-Moreno, 1990; Ezura et al., 1993; Rodeva et al., 2006; Bonilla and Chen, 2015; Ashwani et al., 2017; Mythili et al., 2017).

17.5.1.2 CHOICE OF THE EXPLANTS

The selection of right explant is veiy important aspect in plant tissue culture practices, particularly growth and morphogenesis, including the different genotypes of chili. The regeneration process of chili is also controlled by the selection of appropriate explant. Various types of explants including cotyledons,

s.

No.

Species

Explants/tissues

PGR's+medium

References

1

Capsicum annuum

Zygotic embryo

BAP (5.0 rngL"1)

Agrawal and Chandra (1983)

2

C. annuum

Hypocotyl. cotyledon, stem, leaf, root, shoot-tip. embryo

BAP (5.0 mgL'1)

Agrawal etal. (1989)

3

C. annuum

shoot-tip and hypocotyl

BAP (5.0 mgL-‘)+NAA(0.1 mgL-1)

Ebida and Hu (1993)

4

C. annuum

Shoot tip, seedlings

BAP (2.0 mgL'1)

Madhuri and Rajam (1993)

5

C. praetermissum

Shoot tip

BAP (2.0 mgL"1)

Christopher and Rajam (1994)

6

C. annuum

Cotyledon

BAP (8.88 pM)+IAA (2.85 pM)+AgNOs (5.85 pM)

Hyde and Phillips (1996)

7

C. annuum

Hypocotyl. cotyledon, leaf

BAP (13.3 pM)+IAA (5.71 pM), BAP (44.4 pM), BAP (22.2 pM)

Christopher and Rajam (1996)

8

C. praetermissum

Hypocotyl. cotyledon, leaf

BAP (13.3 pM)+IAA (5.71 pM), BAP (44.4 pM), BAP (22.2 pM)

Christopher and Rajam (1996)

9

C. baccatum

Hypocotyl. cotyledon, leaf

BAP (13.3 pM)+IAA (5.71 pM), BAP (44.4 pM), BAP (22.2 pM)

Christopher and Rajam (1996)

10

C. annuum

Cotyledon

BAP (13.35 pM)+IAA (3.4-5.9 pM)+EBR (0.1 pM)

Franck-Duchenne et al. (1998)

11

C. annuum

Cotyledon

BAP (5.0 mgL-‘)+PAA (2.0 mgl1)

Husain et al. (1999)

12

C. annuum

Zygotic embryos

BAP (5.0 mg L4)+NAA (1.0 rngL"1)

Arous et al. (2001)

13

C. annuum

Seedling explants, embryonal explants

TDZ (1.0-2.0 rngL"1)

Dabauza and Pena (2001)

14

C. annuum

Leaf, cotyledon

TDZ (1.0-3.0 mgL"1)

Venkataiali et al. (2003)

15

C. annuum

Cotyledon

TDZ (1.5 pM) + IAA (0.5 pM)

Siddique and Anis. (2006)

s.

No.

Species

Explants/tissues

PGR's+medium

References

16

C. fmtescens

Shoot tip

Zeatin (10.0 mgL'1), BAP (5.0 mgl_1)+IAA (1.0 mgl-1)

Sanatombi and Sharma (2007)

17

C. annuum

Cotyledon

BAP (22.2 pM)+PAA (14.7 pM)

Joslii and Kothari (2007)

18

C. annuum

Leaf, cotyledon, hypocotyl

BAP (8.8 pM)+IAA(11.4 pM)

Sanatombi and Sharma (2008)

19

C. fmtescens

Leaf, cotyledon, hypocotyl

BAP (8.8 pM)+IAA(11.4 pM)

Sanatombi and Sharma (2008)

20

C. chinense

Leaf, cotyledon, hypocotyl

BAP (8.8 pM)+IAA(11.4 pM)

Sanatombi and Sharma (2008)

21

C. annuum

Cotyledons

BAP (6.0 mgL-‘)+IBA (1.0 mgL1)

Otroshy et al. (2011)

22

C. chmense

Nodal segments and shoot tips

BAP (35.52 pM)+TDZ (18.16 pM)

Keheiet al. (2012)

23

C. annuum

Cotyledon

BAP (6.0 mgL-‘)+IAA (0.3 mgL1)

Verma et al., (2013)

24

C. annuum

Cotyledonary leaves and hypocotyls

BAP (10.0 rngL4)+IAA (1.0 mgL-1)

Rizvvan et al. (2013)

25

C. annuum

Shoot

BAP (2.0 mgL-‘)+IAA (0.5 rngL"1)

Swarny et al. (2014)

26

C. chmense

Leaf

Kin (45.0 pM)+2,4-D (3.5pM) + AgNO} (35.0 pM)

Bora et al. (2014)

27

C. annuum

Nodal segments and shoot tips

IB A (1.0 mgL-‘)+NAA(1.0 mgL'l)+Spermidm (1.5 uiM)

Haque and Ghosh (2017)

28

C. annuum

Cotyledons

BAP (44.44 pM)+IAA (5.71 pM) AgN03 (10.0 pM)+n-morpholine (1.98 mgL'1)

Ashwani et al. (2017)

29

C. annuum

Cotyledons

BAP (44.38 pM)+TDZ (9.0 pM) GA3 (5.77 pM)+PAA (14.7 pM)

Mythili et al. (2017)

30

C. annuum

Hypocotyl

Zeatin (7.55 mgL'1) + GA3 (2.0 mgL'1)

Hegdeetal. (2017)

hypocotyls, leaves, shoot tips, zygotic embryos, embryonic leaves, stems, intemodes, mature seeds, and roots have been employed for plant regeneration in capsicum (Agrawal, 1983; Agrawal et al, 1989; Ebida and Hu, 1993; Ezura et al., 1993; Gatz and Rogozinska, 1994; Ramirez-Malagon and Ochoa-Alejo, 1996; Berljak, 1999; do Rego et al., 2016; Ashwani et al., 2017; Mythili et al., 2017, Hedge et al., 2017).

17.5.2 CALLUS CULTURE

Developments in biotechnology, particularly methods for callus culture, should provide new means for the commercial handling of economically important plants for continuous production of different cell lines, regenerated plants for conservation, and the secondaiy metabolites they provide (Mula- bagal and Tsay, 2004). This method can be used for the quality improvement program of elite genotype of chili plants that will be utilized as renewable resources of capsaicin production. It can provide the system to produce and accumulate maximum amount of capsaicin by manipulating the parameters of the tissue culture environment and medium, selecting high-yielding cell clones, precursor feeding, and elicitation in different cultivars of chili (Table 17.3). The naturally grown medicinal plants are moreover different from before, they are facing more and more insecticide, herbicide, and heavy metals, which will cause contamination to the metabolites and finally generate side effects.

Moreover, due to the complex structures of secondary metabolites, chemical synthesis is not achieving maximum productivity in most cases. The huge demand for raw materials for photochemical production from various medicinal plant sources in pharmaceutical industry of the whole world can be satisfied by in vitro production of cell and tissue. Scientists are developed a different way to produce the conservation strategies and corresponding secondaiy metabolites from this way. A lot of effort is involved for the continuous round the year supply of pharmaceutically important bioactive compounds by manipulating the plant tissue culture system in different way.

This in vitro tissue culture methods are now involved for predictable production of raw materials that are independent of weather and season. In vitro plant callus and cell culture now involved for the production of different types of economically viable secondary metabolites. Synthesis of bioactive natural products from callus culture and plant cell cultures has been presently used for pharmaceutical industiy. Production of flavor components and secondaiy metabolites in vitro using immobilized cells is a perfect method

s.

No.

Species

Explants/tissues

PGR's+MS medium

References

1

Capsicum annuum

Cotyledon. Hypocotyl

BAP (1.0-3.0 mgL-‘)+IAA(1.0 mgL_1)+GA3 (O.SingL4)

Rodeva et al. (2006)

2

C. annuum

Leaf. Shoot and nodal region

2,4-D (2.0 ingL-‘)+Kin (0.5 mgl1)

Umamaheswai and Lalitlia (2007)

3

C. annuum

Cotyledon

Kin (0.5 mgL-‘)+2,4-D (5.0 mgL-‘)+NAA(2.0 lngL'1)

Rakshit et al. (2008)

4

C. annuum

Internode

BAP (2.0 pM)+2,4-D (10.0 pM)

Khanetal. (2011)

5

C. annuum

Hypocotyls

BAP (1.0 mgL'‘)+IAA(0.8 mgL“1)-AgNOJ (4.0 mgL'1)

Xie et al. (2013)

6

C. chinense

Immature green pods

2,4-D (2.0 ingL“l)+Kin (0.5 mgl1)

Mangang (2014)

7

C. annuum

Seedlings

BAP (2.0 ingL_1)+NAA(0.1 mgL4)

Swamy et al. (2014)

8

C. chinense

Stem

BAP (3.0 mgL4)+NAA (1.0 mgL'1)

Raj et al. (2015)

9

C. annuum

Seedlings

BAP (0.1 pM)+2,4-D (0.1 pM)

Suthar and Shall (2015)

10

C. chinense

Leaf and internod

BAP (6.66 pM)+2,4-D (9.05 pM)

Gayathri et al. (2015)

11

C. annuum

Leaf and stem

BAP (1.0-5.0 mgL"l)+GAj (0.5-1.5 mgL4)

Ikliajiagbe et al. (2016)

12

C. annuum

leaf, cotyledon, epycotyl and hypocotyl

BAP (4.0 mgL“l) + IAA (0.5 rugL4)+L2 vitamin.

Renfiyeni and Trisno (2016)

13

C. annuum

Leaf and Internode

BAP (2.2 pM)+2,4-D (18.10 pM)

Santos et al. (2017)

for spices crops like chili. Production of capsaicin was reported using such system (Johnson et al., 1996; Prasad et al., 2006; Umamaheswai and Lalitha, 2007; Kehie et al., 2014, 2016; Giri and Zaheer, 2016; Ferri et al., 2017).

17.5.3 SOMATIC EMBRYOGENESIS

Somatic embryogenesis is an unique in vitro method that can help to justify the totipotency concept in all plant cells. This potential character is now widely used in different aspects of plant biotechnology including ex situ conservation. It is also used to study the development of the embiyo it can be used to produce plants commercially or to carry out basic studies including cell differentiation, gene expression, molecular genetics, and many others aspects also. The reports of somatic embryogenesis study are huge that cover different aspects of induction and subsequent development of various natures from the role of plant growth regulators, mainly auxins and cytokinin, to the function of the components of the media of culture (Loyola-Vargas, 2016).

Somatic embryogenesis can accelerate the steps of genetic improvement program of commercial crop species (Stasolla and Yeung, 2003). Somatic embryogenesis refers to the development of structures that resemble zygotic embryo from somatic cells through an orderly chain of characteristic morphological stages and is contemplated superior over other in vitro propagation systems as it reduces the propagation time and potentially offers an effective system for regenerating whole plants with high-genetic uniformity (Kothari et al., 2010).

Harini and Lakslnni Sita (1993) first time reported the somatic embryogenesis and regeneration in chili from immature zygotic embryos. Both direct or indirect mode of somatic embryogenesis and plant regeneration successfully achieved from various explants, like immature or mature zygotic embiyos, seedlings, leaves, stem segments, etc. (Binzel et al., 1996; Buyukalaca and Mavituna, 1996; Kintzios et al., 2000; Kintzios et al., 2001; Kaparakis and Alderson, 2002; Steinitz et al., 2003; Khan et al., 2006; Santana-Buzzy et al., 2009; Aviles-Vinas et al., 2013; Venkataiah et al., 2016). MS medium supplemented with 2,4-D and different percentage of sucrose and sometimes undefined media has been observed to promote somatic embiyos induction in chili explants (Binzel et al., 1996; Buyukalaca and Mavituna, 1996; Aviles-Vinas et al., 2013, Venkataiah et al., 2016), whereas cytokinins seem not to have significant role or even instead of induction they can inhibit somatic embryogenesis in chili pepper (Kaparakis and Alderson, 2008).

In few cases, cytokinin is involved in the regulation of plant cell cycling, division, and differentiation, playing key roles in somatic embryogenesis (Zapata-Castillo et al., 2007; Santana-Buzzy et ah, 2009). Conversion of mature somatic embryos to complete plantlets has been induced by GA3 or TDZ, alone or in combination (Binzel et ah, 1996; Venkataiah et ah, 2016). Role of cytokinin on somatic embryos maturation and germination also reported by several groups (Harini and Lakshmi Sita, 1993; Binzel et ah, 1996; Solis-Ramos et ah, 2009), whereas abscisic acid (ABA) has also been used to promote maturation of somatic embryos (Buyukalaca and Mavituna,

  • 1996). Plant regeneration and direct somatic embryogenesis from lrypocotyl, stem segments, and shoot tips of C. annuum on TDZ supplemented medium has been studied by (Khan et ah, 2006; Aboshama, 2011).
  • 17.5.4 HAPLOID CULTURE

Haploid culture first reported from India, anther culture for the androgenic haploid plant was earned out on Datura innoxia by Guha and Maheshwari (1964). Haploid culture is one of the useful methods in plant breeding which may be utilized to facilitate the detection of mutations and the recovery of unique recombinants (Kothari et ah, 2010; Roshany et ah, 2013). Haploids may occur spontaneously in nature in some specific plant species or they may be induced experimentally to occur in Capsicum species (Pochard and Dumas de Vaulx, 1979; Ahmad et ah, 2006). Several reports are available on anther culture and regeneration of haploid plants of Capsicum species and hybrids (Wang et ah, 1973; Kuo et ah, 1973; George and Narayanaswamy, 1973; Sibi et ah, 1979; Silva Monteiro et ah, 2011; Roshany et ah, 2013; Barroso et ah, 2015; Nowaczyk et ah, 2015; Ari et ah, 2016). Wang et ah (1973), Kuo et ah (1973), and others have discussed haploid production of chili through anther culture and haploid plant regeneration (Table 17.4).

Chili is now recognized to be the third Solanaceous crop that could be defined as recalcitrant in regard to the response to androgenesis induction (Segui-SimaiTO et ah, 2011). Anthers with microspore at the uninucleate stage were cultured on MS medium modified in some micronutrients and vitamins and supplemented with either Kinetin, NAA, or 2,4-D. Several factors influence the haploid plant regeneration from microspore or anther culture of the Capsicum species, hybrid, cultivar, or even genotype (Munyon et ah, 1989; Mityko et ah, 1995; Nowaczyk and Kisiala, 2006; Koleva- Godeva et ah, 2007; Lantos et ah, 2009; Alremi et ah, 2014; Barroso et ah, 2015). Pretreatments of anther donor plants (Kristiansen and Andersen,

TABLE 17.4 Androgenic Culture of Chili.

s.

No.

Species

Culture

Media supplement

References

1

Capsicum

annuum

Anther

Kin (0.1 mgL4)+2,4-D (0.001 mgL4)+Vitamin Bp(0.04mgL-‘)

Rodeva et al. (2006)

2

C. annuum

Microspore

Zeatin (2.5 pM)+IAA(5.0 pM)

Supena et al. (2006)

3

C. annuum

Flower bud. Anther

Kin (1.0 mgL4)+IAA (0.001 mgl-1)+2,4-D (0.01 mgL4)

Koleva-Godeva et al. (2007)

4

C. annuum

Anther

BAP (1.0 mgL4)+NAA (4.0 rngL4)+AgNOj (15.0 mgL4)

Та skin et al. (2011)

5

C. annuum

microspore culture

Kin (0.2 mgL4)+2,4-D (0.1 mgL4)

Lantos et al. (2012)

6

C. annuum

Anther

BAP (0.5 mgL4)+NAA (0.5 mgL-‘)+2,4-D (0.1 mgL4)

Roshany et al. (2013)

7

C. annuum

Anther

Kin (0.1 mgL4)+2,4-D (0.01 mgL4)

Nowaczyk et al. (2015)

8

C. annuum

Anther

BAP (6.0 mgL4)+NAA (0.5 mgL4)+AgN03 (6.0 mgL4)

Taskin et al. (2013); Ari et al. (2016)

9

C. annuum

Anther

Kin (0.1 mgL4)+2,4-D (0.1 mgL4)

Akyol et al. (2016)

10

C. annuum

Anther

Zeatin (1.0 mgL4)+2,4-D (0.2 mgL4)+AgNO, (15.0 mgL4)

Hegdeetal. (2017)

  • 1993) or floral buds before in vitro culture are very common for increasing the regeneration ability of liaploids in chili pepper (Sibi et ah, 1979; Barroso et ah, 2015).
  • 17.5.5 TRANSFORMED CULTURE OF CHILI

Orthodox plant breeding has paid meaningfully to crop upgrading over the past 50 years. However, there is intense pressure to produce further improvements in crop quality and quantity due to the result of population growth, social demands, health requirements, environmental stress, and ecological considerations. Traditional plant breeding is not able to withstand this increasing demand due to the limited gene pool, restricted range of organism between which genes can be moved in the species barriers. Genetic transformation holds countless ability for improving these major restrictions to crop productivity. Chili is second only to tomato in terms of vegetable production in advanced countries, and its breeding and production, as with other major crops, are constantly faced by plentiful pests, diseases, and abiotic stresses (Rizwan et ah, 2013).

Genetic manipulation is an attractive proposition where it involves recombination of an efficient cell or tissue culture regeneration system with recombinant DNA technology, which would transfer specific genes from other taxa, or the modified expression of specific native genes (Kothari et ah, 2010). It is very common, Agrobacterium tumefaciens and A. rhizogenes has been used as the vector for genetic transformation of diverse dicotyledonous species, In the case of chili pepper, genetic transformation via Agrobacterium is surely an important tool to help genetic improvement against many diseases caused by phytopathogenic fungi, bacteria, and viruses (Aarrouf et ah, 2012; Rizwan et ah, 2013). However, advances in this area have been limited because of low efficiency for in vitro plant regeneration of the reported systems. Liu et ah (1990) published the first report on chili pepper genetic transfonnation employing in vitro seedling explants (hypocotyls, cotyledons, and leaves) cocultured with the wild tumorigenic strains A281 and C58 of A. tumefaciens and with a disarmed strain bearing the plasmid pGV 3850.

Callus normally developed from cotyledon and leaf tissues leaf-like structures. and occasional shoot buds in the presence of kanamycin. Although a number of kanamycin-resistant shoot buds were obtained, no further elongation and plant formation occurred. Usually Agrobacterium has been the only used vector for chili pepper genetic transfonnation (Liu et ah, 1990; Wang et al., 1991; Dong et al., 1992; Lee et al., 1993; Ye et al., 1993; Zhu et al., 1996; Kim et al., 1997; Christopher and Rajam, 1997; Subhash and Christopher, 1997; Lim et al., 1997; Mihalka et al., 2000; Aarrouf et al., 2012; Rizwan et al., 2013). Plant regeneration and transformation studies have been mainly focused on C. annuum (Liu et al., 1990; Dong et al., 1992; Christopher and Rajam, 1997; Lee et al., 1993; Ye et al., 1993; Zhu et al., 1996; Harpster et al., 2002; Kim et al., 2002; Shin et al., 2002; Dabauza and Pena, 2003; Li et al., 2003). Most transformation studies in chili pepper refer to the use of marker (npt II) or reporter genes (gus) in order to establish adequate protocols; however, some genes have also been utilized to generate transgenic plants with tolerance to Cucumber mosaic virus (Dong et al., 1992; Lee et al., 1993; Zhu et al., 1996; Kim et al., 1997; Chen et al., 2003; Lee et al.,

2009) or tolerant to multiple pathogenic organisms (Shin et al., 2002), and drought-tolerant (Maligeppagol et al., 2016).

 
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