Review Article 
EVOLUTION OF CHILLI PEPPER BREEDING: FROM CONVENTIONAL METHODS TO CUTTING-EDGE GENETIC IMPROVEMENTS	

ABSTRACT
Chilli pepper is an important spice crop, with high medicinal and culinary properties along with high levels of vitamins and antioxidants. Both conventional and non-conventional breeding methods have been adopted for the development of promising cultivars and varieties of chilli worldwide. Varieties like CO 3 and CO 4 were introduced from Sri Lanka to India. Indian Institute of Horticultural Research, Bengaluru has adopted mass selection to develop varieties like Arka Basant and Arka Mohini. Arka Abhir and Arka Lohit are pureline selections from Dyavanur Dubba and IIHR 324 respectively. Jwalamukhi and Jwalasakhi are the popular hybrids in Kerala, developed through hybridisation and selection. Marker assisted backcrossing helped in introgressing a heat tolerant Hsp gene, from AVPP0702 (donor) into chilli variety Kulai, making it heat tolerant.  
Bhut Jolokia, once considered to be the world’s hottest chilli, is an interspecific hybrid between Capsicum chinense and Capsicum frutescens. Mutation breeding in K-1 chilli variety lead to the development of variety MDU-1. A widely cultivated variety in West Bengal,  Dalle Khursani was characterised and identified to be a naturally occurring polyploid in 2017.  Adoption of tissue culture techniques, especially anther culture, has helped in the development of purelines in chilli. The transcription factor dreb1A was transformed into a local chilli cultivar, G4 to impart drought tolerance in the cultivar. wAnthracnose resistance in chilli was mediated in chilli by editing NAC 72 gene locus through CRISPR/Cas9 mediated cytidine base editing system.
An integrated breeding strategy that combines the broad variability and farmer acceptance of conventional methods with the precision and speed of modern molecular tools is considered the most effective pathway for future chilli improvement programs. Such a combined approach can accelerate the development of high-yielding, disease-resistant, and climate-resilient chilli varieties suitable for diverse agro-ecological conditions.
KEY WORDS: Capsicum spp., Chilli breeding, Capsicum improvement, conventional breeding, modern breeding approaches

INTRODUCTION
Chilli, chilli pepper or pepper (Capsicum spp.), belongs to the Solanaceae family. It is a spice cum vegetable crop that is cultivated throughout the world and comes second in ranking after tomato among solanaceous crops. It has a chromosome number of 2n=2x=24. This crop in Mexico is considered to be the origin of chilli and South America is considered as the centre of diversity (Rao and Anilkumar, 2020). Fuchs in 1543 gave chilli its botanical name, ie., Capsicum which is derived from a greek word ‘capsa’, owing to the peculiar shape of the chilli fruits. Generally utilized for its inclusive flavor thus, called as 'Wonder Spice' varying in size, shape, color, flavor and degree of hotness, from mild to very hot.
	The genus Capsicum consists of around 30 species. Among these, only five are cultivated ones: “Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum pubescens and Capsicum frutescens. Five domesticated species are differentiated by morphological characters based on colour and morphology of flowers and seeds. Capsicum annuum is the widely and popularly cultivated Capsicum spp. across the world. The diploid Capsicum species are of chromosome number 2n=2x=24. The genomic size is 2700 Mb and around 30,000 genes” (Moscone, 2016, Pattika, 2024).
	Chilli has been in use from time immemorial by humans in several ways, as a spice, vegetable or condiment, due to its colour, flavour and pungency. In India, it is an important ingredient in curry blends. It is also used in Thai, Indonesian, and Malay cuisines. Rouille, a chilli-based sauce from France is quite famous (Charles, 2013). Among the major chilli producing countries, the greatest share is taken by India and hence, India is the major producer, consumer and exporter contributing one fourth of world production.
The medicinal properties of chilli have been under use from the time of our ancestors (Fattori et al., 2016). “The vitamin C content in Capsicum is reported to be twice as that found in citrus fruits. While, dry chillies are reported to have high content of β-carotene (a precursor of vitamin A). Chillies have been reported to contain bioflavonoids and antioxidants and to possess antibacterial and anticancer properties” (Pramanick and Srivastava, 2013). 
Nowadays the uses of chilli have been extended to the production of food dye, used as ornamental plants and for the extraction of various components that are utilised in pharmaceutical and cosmetic industries (Saleh, 2018).
	The great diversity among the chilli with respect to fruit and flower morphology, colour, capsaicin content and other qualitative and quantitative characters have built interest in researchers. 
Around the world, the focus has been to enhance the yield along with its nutritional and medicinal properties (Herath, 2021). Traits such as pungeny are more difficult to be manipulated and breeders have adopted various strategies and breeding methods to study the genetics, combining ability and heterosis in chilli. 
	The aim of this literature is to compile the breeding objectives and breeding methods adopted for genetic improvements in chilli and to further assess the scope of the breeding methods adopted, in developing high yielding quality chilli crop. 

Breeding objectives in chilli
	“Breeding of pepper, has four main macro-objectives to achieve and related to:
(a) main agronomic traits such as yield, fruit features such as color and shape, plant habit, and fruit set; (b) resistances to abiotic stresses such as drought and salinity which limit the cultivation in certain areas; (c) resistances to a plethora of bacterial, fungal, and viral disease causing severe damage to cultivations and loss of quality of the production; (d) quality, for which breeding objectives are mainly related to the improvement of various bioactive compounds such as capsaicinoids, isoprenoids, flavonoids, and vitamin C” (Tripodi and S. Kumar,2019).
  	The breeding objectives in chilli mainly depend upon the end users’ demand. It also differs for both hot and bell pepper based on country of cultivation, cultivation conditions, consumption preference of customers etc.
         The main aim of a chilli breeder is to develop a variety with high yield potential, improved quality, high pungency, protection against biotic and abiotic stresses and other quality traits.  
         The foremost breeding objective in Capsicum is breeding for increased yield as it is with any other crop. This is to meet the increasing demand due to an increasing population. Complex traits such as number of fruits per plant, fruit length, fruit diameter, total fruit weight etc. affects the yield in chilli. In previous studies, selection for fruit number, fruit weight and early flowering were found to enhance the yield to a greater extent.  Genetic information along with efficient breeding methods are important factors in development of a high yielding chilli variety (Hasanuzzaman, and Golam, 2011).  Heterosis breeding by the utilisation of male sterility system is focused upon for this objective. Both genetic male sterility (GMS) and cytoplasmic male sterility (CMS) is utilised but CMS system is the most widely exploited in chilli. 
          “Another objective of breeding is against diseases in Capsicum.  Disease resistance breeding starts with identification and collection of resistant sources, understanding the genetic background of the resistant source and then utilisation of this resistant source to introgress the same into desirable genotypes” (Srivastava and Mangal, 2019). Important diseases in chilli at present include diseases caused by viruses (Potyvirus, Topoviruses, Begomoviruses, Cucumoviruses etc.), fungus (anthracnose, phytophthora root rot etc.), bacteria (bacterial spot, bacterial wilt) and pests such as nematodes, whiteflies, thrips etc. New resistance sources are to be identified in chilli with the evolution of new strains of pathogen. 
	Increased tolerance to high temperature, drought and wide adaptability and breeding for horticultural and biochemical traits are also focused upon. Genes/QTLs that contribute to various economic traits as well as to disease and insect resistance are listed in table 1.
Table 1. Genes/QTLs contributing to various economic traits, disease and insect resistance
Character	Genes/QTLs	Source	
Fruit shape	fs3.1,  fs10.1, fs2.1, fs4.1, fs1.1, fs8	Dwivedi et al., 2013, Rao et al, 2003	
Fruit Length	Qfl.iivr.3.2, Qfl.iivr.3.4, fl1.1 and fl8.2, fl2.1, fl1.2 and fl7.1, FL3.1, FL3.2, FL3.3, FL3.4, FL3.5	Dwivedi et al., 2013, Rao et al, 2003, Han et al, 2016	
Fruit width	Qfw.iivr-2.1, FD-1, FD-2, FD-3.1, FD-3.2, FD-11	Dwivedi et al., 2013, Han et al, 2016	
Plant height	Qpht.iivr.5.1, PH-2, PH-4, PH-6, PH-7, PH-8.1, PH-8.2, dw1-dw8	Dwivedi et al., 2013, Han et al., 2016,  Wang and Paul,2006	
Total Fruit Weight	Qtofw.iivr-1.1, fw2.1, fw4.1, fw4.2, fw11.1, fw11.2, fw1.1, FW-6.1, FW-6.2	Dwivedi et al., 2013, Zygier et al, 2005, Rao et al, 2003	
Yield	yld1.1 and yld8.1,	Rao et al. (2003)	
Number of fruits per plant	Qnfp.iivr.2.1, fno2.1	Dwivedi et al., 2013, Rao et al, 2003	
Pericarp Thickness	Qpt.iivr-2.1, perwd3.1, perwd8.1	Dwivedi et al., 2013	
Locule number	Nlo2.1, Nlo8.1, Nlo12.1
and Nlo12.2, CaBRX	Palloix et al., 2009, Ma et al., 2021	
Flowering	flw1.1, flw2.1, flw2.2, flw3.1, flw4.1, flw4.2, flw6.1, flw8.1, flw10.1	Rao et al, 2003	
Maturity	mat1.1, mat4.1, mat5.1, mat9.1	Rao et al, 2003	
Seed weight	swt2.1, swt8.1, swt12.1	Rao et al, 2003	
Anthracnose	Anr-5, co1, co2, co4,Co5, 	Lin et al. 2002, Mahasuk et al. 2009, G arg et al, 2014	
Leaf curl virus	peplcv-1, peplcv-7, and peplcv-12	Siddique et al., 2022	
phytophthora capsici	Pc5.1, Pc8.1, Pc9	Mallard et al, 2013, Lozada et al., 2021	
Chili veinal mottle virus	Cvr1, cvr4	Lee et al., 2017	
Cucumber mosaic virus	qcmv.hb-8.2, qcmv.hb-4.1, qcmv.hb-7.1, qcmv.hb-
8.1, qcmv.hb-8.3	Yao et al., 2013	
Bacterial spot	Bs-1,Bs-2, Bs-3,Bs-4	Sahin and Miller, 1997	
Male sterility	Ms8, Ms10	Bartoszewski  et al., 2012, Aulakh et al., 2016	

 Breeding approaches adopted for Capsicum Improvement
1.	Classsical breeding methods 
	Conventional plant breeding is the process of combining beneficial traits through selective crossbreeding and choosing favorable recombinants from the resulting progeny. This approach is used to create improved plant varieties that can adapt to diverse climates, enhance flavor or nutrition, resist diseases and pests, and make more efficient use of water or nutrients.

1. Non-conventional breeding approaches - The integration of plant tissue culture and recombinant DNA technology has enabled the transfer of genes across different species. Conventional plant breeding can also involve wider crosses, where unrelated species or even genera are hybridized using advanced techniques like tissue culture, marker-assisted breeding, and genetic engineering. While modern methods such as transgenic breeding, genetic manipulation, and marker-assisted selection provide valuable tools, they serve as complementary techniques rather than replacements for traditional breeding practices.
 List 1 :    Conventional and Non-Conventional Breeding Approaches
Conventional	Non-conventional	
Introduction	Tissue culture 	
Selection	Genetic transformation	
a) Mass selection	Marker assisted selection	
b) Pureline selection		
Hybridisation and selection		
a) Pedigree selection		
b) Single-seed descent (SSD) method		
c) Backcross method		
Heterosis breeding		
Wide hybridisation		
Mutation breeding		
Polyploidy Breeding		
Resistance Breeding		

1.	Introduction
              Plant introduction is a practice of introducing plants, genotypes or a group of genotypes from their place of adaptation to a new environment. The chilli crop is under cultivation and use since 7500 BC. It was first introduced from South America into Central America from where it was introduced into Europe by Colombus when he discovered America (Srivastava and Mangal, 2019). From the European continent, it further spread to the Asian countries and was introduced in India through the Portuguese travellers (Singh et al., 2004). Thus, introduction may involve new varieties of a crop already grown in that area, a wild relative of the crop species or totally a new crop species for that area. It is important that the material which is being introduced should not carry any pest and diseases.
            CO 3 and CO 4 are improved varieties of India, developed from introduced Sri Lankan varieties. California Wonder, Yolo Wonder, World Beater, Chinese Giant, Golden Wonder, Bull Nose, Sweet Banana etc are other varieties which have been introduced in India from USA through Indian Agricultural Research Institute (Roy et al., 2019). 

2.Selection
           Selection refers to the process of favoring certain genotypes with desirable traits, leading to their increased reproduction while eliminating less favorable ones. It is a crucial component of all plant breeding programs, helping to enhance yield, improve key traits, and meet specific breeding goals. Selection is typically based on the plant's phenotype. For instance, efforts to develop high-yielding chilli varieties began at Lam, Guntur in 1928, resulting in the creation of improved cultivars.
2. 1. Mass selection
            Mass selection is one of the simplest and most widely used method for genetic improvement of Capsicum in the past especially by the indigenous people of South America (Padilha  and Barbieri, 2016). Using this method, multiple traits with simple inheritance are improved simultaneously without utilising pedigree information. Characters with high heritability are fixed and a considerable amount of variability is also preserved. 
            During mass selection, individual plants are selected from a phenotypically diverse population. The seeds from the selected individual plants are bulked and are used to grow next generation. This method is usually used to improve open pollinated varieties or land races in chilli.
            Arka Basant, a mass selected variety from a Hungarian cultivar ‘Soroksari’ is suitable for both kharif and rabi season in India. It has an average fruit weight of 50-80g and a yield of 15 t ha-1 is a mass selected variety released by the Indian Institute of Horticultural research (IIHR), Bengaluru (Ashok et al., 2020). 
            Arka Mohini, developed through mass selection from the variety ‘Golden Calwonder’ is also released by the IIHR. It has blocky fruits and yield upto 16 t ha-1 in 150 days.
            Arka Gaurav, released in 1984, was an improvement over ‘Golden Calwonder’ followed by mass selection. It yields about 16t/ha in 150 days with an average fruit weight of 130-150g.
 2.2 Pureline selection
            During pureline selection, a large number of plants are selected from a self-pollinated crop. Seeds are harvested individually from the selected plants and sown in the next season to grow individual plant progenies. The individual plant progenies are evaluated and the best progeny is released as a pureline variety. 
            Pureline selection conducted on a local variety IIHR 324 at Indian Institute of Horticultural Research led to the development of Arka Lohit, a highly pungent chilli variety. The fruits are straight with pointed tips and yield about 25t/ha. Arka Abhir was a pureline selection from DyavanurDubba. The fruits are light green and wrinkled with lesser pungency. It yields about 2t/ha in 160-180 days. 
          Surakta is a pureline selection developed from PDKV, Akola. It has a yield of 6.8 t/ha for red fruits. Other pureline varieties released in India are Bhagyalaxmi, Musalwadi, Sindhur, Patna Red, Pant C 1, PLR1, KI etc (Gopalakrishnan (2007), Ramachandran (2013))
3. Hybridisation and selection
          This involves crossing between two superior parents and further selection of superior progenies from F1 or subsequent generations.
3.1. Pedigree selection
          Pedigree selection is a method for handling segregating generations in which individual plants are selected from F2 and subsequent generations and their progenies are grown. A record of all parent-offspring relationship is maintained during the study.
          Arka Suphal is a variety developed using pedigree selection at IIHR, Bengaluru. It is an F9 derivative of the cross, Pant C1 X IHR 517 A. It is tolerant to powdery mildew and viruses and has a yielding capacity of 25 t ha-1 in case of green chilli and 3 tonnes per ha for dry chilli. 
          Some other examples of varieties developed by pedigree selection are: Andhra Jyoti, Pusa Jwala, Pusa Sadabahar, K2, Punjab Lal and Jawahar 218.
          Jwala Mukhi and Jwala Sakhi are the varieties developed by pedigree selection from Kerala Agricultural University.  
3.2. Single-seed descent (SSD) method
          Single seed descent method is a modification of bulk method where, a single seed is selected randomly from each selected plant to make a bulk, instead of bulking the whole seed lot. 
          Not many varieties have been developed using the SSD method in chilli but, chilli lines that were resistant to bacterial spots were developed using this method (Moreira et al., 2009)
3.3. Backcross breeding
          Backcross breeding method is adopted by plant breeders to incorporate simply inherited traits from unadapted donor parents into recipient lines through repeated cycles of crossing of the recurrent parent to the F1 line, followed by selection for the trait that is being transferred. Backcross breeding can be adopted to transfer both dominant and recessive traits. 
Aulakh et al in 2017 confirmed the efficiency of AVRDC-PP12 marker linked to male sterility gene ms10 for utilising in marker assisted backcrossing of three out of four crosses. Inheritance and pattern of segregation of fruiting habit traits such as fruits number node-1 and their orientation in chilli were studied by Anilkumar and Rao in 2018 by adopting back cross method. Biallelic monogenic inheritance of number of fruits per node and orientation of fruits was observed.   Usman et al. (2020) introgressed heat tolerant Hsp gene (that impart heat tolerance) from AVPP0702 (donor) to Kulai using marker assisted backcrossing. A successful transfer of a genetic male sterility ms10 gene from Capsicum annuum var. annuum (hot pepper) to C. annuum var. grossum (heat tolerant bell pepper) via marker-assisted backcrossing, thus pyramiding ms10 with non-pungent (pun1) gene (Rani et al., 2021).                  
4. Heterosis breeding
	Development of hybrid varieties is one of the quickest methods to increase productivity in crops including chilli. Qualitative and quantitative characters in chilli exhibit high variability and hence, there is great hope in improving yield in chilli using diverse inbred lines (Singh et al., 2014). The first and foremost important step in chilli heterosis breeding, as in any other crop, is the selection of diverse inbred lines as parents. This is achieved by studying the combining ability (General combining ability (GCA) and specific combining ability (SCA)) of the parents. 
	Hybrid chillies are produced by hand emasculation or by exploiting male sterile system. Presently in chilli, GMS and CMS are under use commercially for the development of hybrid varieties.
	Heterosis in chilli was reported by Doshi and Shukla (2000) who found out that characters such as number of fruits per plants, fruit length, fruit girth and shape and fruit volume showed high heterosis. Meena et al. (2018) identified promising hybrids for high yield production along with special focus on seed and bioactive compound under temperature stress regimes, using CMS lines.
	Arka tejaswi is a high yielding F1 hybrid that is tolerant to chilli leaf curl virus and with yield potential of 3-3.5 t acre-1, developed by IIHR, Bengaluru. Some hybrids that were developed based on CMS system are: Kashi Surkh, Kashi Early, Arka Meghna, Arka Sweta, Arka Khyati , Arka Harita. Examples of hybrids that were developed based on GMS system are: CH-1 ( MS-12 x LLS)  , CH-3 (MS-12 x S-2530) and CH-27 . 
	CH-27 is a multiple disease resistant chilli hybrid that was developed by Punjab Agricultural University. It was developed by crossing MS-12 with S-343, and is highly resistant to chilli leaf curl virus, fruit rot disease, resistant to root knot nematode and tolerant to insect pests such as thrips and mites.
5. Wide hybridisation
             Wild relatives and different species of the same genera of crops act as reservoirs of many useful genes such as genes for disease and insect resistance etc. that can be used as donors in hybridisation programmes. Crossability studies are to be undertaken beforehand and necessary methods to by-pass the fertilisation barriers is an important step during wide hybridisation (Table 2). 		
Table 2. Wild and cultivated species as source of disease resistance
Capsicum species	Resistant source	Diseases and resistant genes	
C. annuum	CM334	Phytophthora capsici
Pfo (Phytophthora foliar rot)
Pfr(Phytophthora fruit rot)
Psr (Phytophthora stem rot)
Pvr4 (PVY pathotypes 0, 1 and 2 and PepMoV)	
	PM217 (PI201234)	Meloidogyne spp.	
	PM687 (inbred
PI322719-a local Indian population)	Bacterial wilt (Ralstonia solanacearum)
Meloidogyne spp.	
C. chinense 	7204 
CNPH725 
ECU-973
PBC932	Tsw (tomato spotted wilt tospovirus)
Tsw (tomato spotted wilt tospovirus)
Tsw (tomato spotted wilt tospovirus)
co1, co2, co3 (Colletotrichum capsici)	

			
C. pubescens	PI235047	Bacterial spot (Xanthomonas campestris pv. vesicatoria)	
C. chacoense	PI260435	Bs2 (Xanathomonas campestris pv. vesicatoria), Tswv	

           In a study conducted by Srivastava et al. (2021), Solanum pseudocapsicum, a member of solanaceae family and considered as the cousin of chilli, was screened for chilli leaf curl virus, ChiLCV. They observed that S. pseudocapsicum was free of symptoms under both conditions even after continuous exposure to the virus for 4 years.  The results showed the presence of virus in all the materials, proving that S. pseudocapsicum is a carrier of the disease but is immune to show the symptoms. Hence, S.pseudocapsicum is a good source of resistance to ChiLCV but crossability studies have to be conducted to transfer the tolerance gene to the Capsicum  genera.
           Bhut Jolokia is an interspecific hybrid between Capsicum chinense and Capsicum frutescens. It is also known as Ghost Pepper (Bhutan pepper in Assamese) due to its very high Scoville rating of 1,041,427 SHUs. 
6.Mutation breeding 
           Utilisation of physical and chemical mutagens to create variability in a population, followed by the development of new variety using this population is known as mutation breeding (Srivastava and Mangal, 2019). In chilli, seeds are the most desirable part used for mutation treatment. Some of the achievements in mutation breeding in chilli are mentioned in table 3.
Table 3. Achievements in mutation breeding in chilli
Mutation technique	Achievements	
X-rays	Male-sterile lines 
resistance to cucumber mosaic virus (CMV), improved flavor, higher yield, and compact plant stature	
Ultraviolet irradiation	Increased level of vitamin C and E 	
Gamma irradiation	Male-sterile lines, 
Resistance to cucumber mosaic virus, Improved flavor, 
Higher yield,
Compact plant stature	
	Mutants with changed shoot architecture in hot pepper	
	Increased β-carotene and orange color on maturity	
TILLING 
(‘Yuwol-cho’ cultivar)	Resistance to the tobacco etch virus	

	MDU 1 is a gamma ray mutant of K-1 developed by Tamil Nadu Agricultural University. This mutant has a compact plant type with higher yield and capsaicin content.
7.  Polyploidy Breeding
	Polyploidisation events are associated with an increase in vigour followed by its adaptation to new environmental conditions. The superiority of the polyploids over diploids is often considered to be due to transgressive segregation. 
	Polyploid capsicum is reported to express stunted growth and presence of larger and greener leaves. Increased dry weight in leaf, stem and root, leaf area and thickness were exhibited by tetraploid Capsicum  when compared to their diploid counterparts (Sjahril et al., 2020).
	A polyploid Capsicum species, Dalle Khursani was morphologically and cytogenetically characterised by Jha et al. (2017). Dalle Khursani is a widely cultivated variety in West Bengal with high pungency. In their study, they analysed seven populations of Dalle Khursani, namely Jalpaiguri, Kalimpog, Mungpoo, Darjeeling, Kolakham, Takdah and Ramdhura, for leaf, flower and fruit traits along with the diploid species of Capsicum. 
           Giemsa staining of the chromosomes of Dalle Khursani showed 2n= 4x= 48 chromosomes in all the seven populations. 
            Even though it is widely cultivated in West Bengal, its use is reduced in Capsicum breeding. This is due to the confusion in the identification and its position in the taxonomy. Thus, the analysis of morphological and cytologenetical data will play a major role in species identification within the genome. 
            They also concluded that Dalle Khursani is a natural allopolyploid and in order to identify its parents, further studies are to be conducted. 
8. Tissue culture
             In plant breeding, plant tissue culture is utilised to develop disease free plants and to produce transgenic plants, for somatic embryogenesis, anther and ovule culture for post fertilisation barrier, embryo rescue as well as for inducing polyploidy (Tefera, 2019).
             In chilli, either somatic embryogenesis or organogenesis can be utilised to develop plants with the desired morphology (Table 4). 
             Heidari et al. (2017) investigated embryo induction through anther culture in several F1 chilli hybrids viz, Cadia, Magno, Plato, King Arthur and Maratus and evaluated the effects of genotype, heat shock and cold treatment (4o C, 24 h) on the efficiency of anther culture. It was found that both cold pretreatment and heat shock improved embryogenesis efficiency when compared to the control. It was also found that genotypes Cadia, Magno and Maratus showed more response than King Arthur and Plato. They also developed an efficient standard regeneration protocol for purelines from anther culture. The doubled haploids obtained can be assessed under green house conditions to isolate appropriate lines that can be utilised in further breeding programmes.
Table 4. In vitro plant regeneration in chilli (Kothari et al., 2010)
Species	Tissue	Mode of 
regeneration	PGR’s + medium	
Capsicum annuum
C. frutescens	Cotyledon, 
hypocotyl	Organogenesis	BA (8.88 μM) + IAA 
(2.85–5.71 μM	
C. annuum	Zygotic 
embryo	Organogenesis	BA (22.2 μM)	
C. annuum 	Wounded 
hypocotyls	Organogenesis	MS without PGRs	
C. annuum	Cotyledon 	Organogenesis	BA (22.2–31 μM) + PAA 
(14.7 μM)	
C. frutescens	Shoot tip 	Axillary 
proliferation	BA (22.2 μM) + Kin 
(4.6 μM)	
C. annuum
C. frutescens
C. chinense	Leaf, 
cotyledon, 
hypocotyl	Organogenesis 	BA (8.8 μM) + IAA 
(11.4 μM)	

9. Genetic Transformation
               Combined with plant tissue culture, genetic transformation for the development of transgenic crops is an efficient tool in plant breeding to develop plants with desired traits in a less span of time. Agrobacterium mediated genetic transformation is carried out in dicotyledonous plants while biolistics is useful in both dicots and monocots. 
               In chilli, Agrobacterium tumefaciens is used as the transformation tool to transfer desirable genes. But, due to the recalcitrant nature of chilli, transformation studies in chilli is limited. 
               Maligeppagol et al. (2015) transformed local chilli cultivar, G4 with a transcription factor dreb1A that is known to impart drought tolerance in chilli. They also optimised in vitro regeneration of the two local cultivars: G4 and LCA334. The explants taken were cotyledonary leaf and hypocotyls and among them, cotyledonary leaves were found to show a higher (51%) regeneration compared to hypocotyls. They confirmed the presence of transgene using PCR and southern blotting. Among the transformants, some of them showed better drought tolerance when compared to the control plants. They concluded that the regeneration protocol that they developed along with the transformation may help in optimisation of transformation in chilli for other agronomic traits of interest. 
                  Only reports of cotyledons, leaf tissues and hypocotyls successful in developing callus are available (Rao and Anilkumar, 2020) (Table 5). 

Table 5. Genetic transformations in chilli.
Genotype	Explant	Agrobacterium strain	Selection marker	Gene of
interest/   Reporter
gene	Vector	
C. frutescens	Pollen transformation	EHA 101	Hygromycin	GUS	pCAMBIA 1301	
‘Arka Gaurav’, ‘Arka Mohini’	Hypocotyl	EHA 105	Kanamycin	GUS	pBinARbC1	
‘K1’,‘K2’, ‘PLR1’	Shoot tips	EHA 105	Cefotaxime and
Kanamycin	GUS	pIG121 Hm	
Pusa Sadabahar, Pusa Jwala	Cotyledons,
Hyotcotyls	LBA 4404	Kanamycin	GUS	pCAMBIA2301	
California Wonder		LBA 4404	Kanamycin and rifampicin	GUS	pBI121	
P915, P4090, P410, P101	Hypocotyls,
Cotyledon	EHA 105, LBA
4404	Kanamycin	TMV-CP, PPII	pCAMBIA	

10. Gene silencing
	A technique utilised to regulate the gene expression in a cell, gene silencing is also called the gene knockdown mechanism. Methods utilising antisense oligonucleotides, ribozymes, RNA interference, three prime untranslated regions and microRNAs etc. are adopted in silencing a gene in a cell.
	A study was conducted by Barragan and Ochoa-Alejo, 2014,  to understand the involvement of certain transcription factors (MYB, MYC, and WD40), in anthocyanin biosynthetic pathway in Capsicum eximum. Virus induced gene silencing was used, with a vector, that was derived from  Tobacco rattle virus and it was concluded that MYB and WD40 genes were directly involved in anthocyanin biosynthetic pathway regulation in Capsicum seeds. A significant reduction in anthocyanin content was observed due to down regulation of early and late anthocyanin structural genes.
Rodriguez and Ajeho (2015) silenced AT3 gene that further reduced the expression of pAmt, BCAT, Kas and Acl genes that are involved in capsaicinoid biosynthesis in fruits of chilli variety Tampiqueno 74. They obtained about 81.1% reduced AT3 gene expression in the variety along with reduced capsaicin (89.6%) and dihydrocapsaicin (87.7%). From the results they concluded that AT3 silencing has a negative effect on genes that control the capsaicinoid biosynthesis. Further investigation is needed into the results obtained to understand the mechanism of the negative regulation of gene expression due to the silencing of AT3 gene.
Similarly, disease resistance against anthracnose was conferred in both chilli and tomato fruits using Host- -Induced Gene Silencing (HIGS) approach. The Colletotrichum gloeosporioides COM1 (CgCOM1) developmental gene was targeted to restrict the fungal infection (Mahto et al., 2020). 
11. Gene editing- CRISPR/Cas9
CRISPR/Cas9 mediated gene editing has been attempted predominantly to confer anthracnose resistance in chilli. It is reported that the management of anthracnose in chilli through conventional approaches did not meet much success. Hence, the first CRISPR/Cas9 mediated cytidine base editing system for editing the NAC 72 gene locus in chilli was carried out by Joshi (2019) using Agrobacterium mediated transformation. The cytidine base editor was able to successfully edit the targeted cytidine base leading to anthracnose resistance. A base editing efficiency of 69% was attained.  
The Anthracnose susceptibility gene, CaERF28 in C. annuum variety Arka Lohit, was altered through a single transcript unit CRISPR/Cas9, in order to enhance anthracnose resistance by Mishra et al (2021). The outcomes from these studies can be used to establish the utilisation of base editing in precision molecular breeding for specific trait improvement in plants. CRISPR/Cas9 mediated gene editing in chilli is a fast-track, secure and multifaceted technique that can be extended to other solanaceous crops for their improvement.
However, CRISPR/Cas9 mediated gene editing possesses the risk of development of escape mutants, giving rise to new virus variants, as was reported by Mehta et al. in 2019 in cassava. Escape mutants arise when the gRNA used is designed from coding regions. Hence targeting multiple genomic regions of the virus by multiplexing the gRNA was suggested as an alternative by Rybicki (2019). This was experimentally proved by Roy et al (2019) in N. benthamiana against chilli leaf curl virus. The experiment also confirmed that the multiplexed editing led to reduced disease symptom formation. This technique can be carried out on similar solanaceous vegetables including chilli for a stable and enhanced disease resistance.

Conclusion and future prospects
Conventional and non-conventional breeding approaches play complementary roles in the genetic improvement of chilli (Capsicum spp.). Conventional methods such as introduction, selection, hybridization, mutation breeding and polyploidy breeding have historically been the foundation of varietal development. These approaches are relatively simple, cost-effective, and rely on phenotypic selection, making them well suited for low-resource breeding programs and regional research stations. However, conventional breeding is often time-consuming and less precise, as selection is influenced by environmental factors and it can be difficult to manipulate traits governed by multiple genes.
In contrast, non-conventional breeding methods—including tissue culture, marker-assisted selection (MAS), genetic transformation, gene silencing, and genome editing—enable precise and targeted improvement of specific traits. These tools reduce breeding time and allow introgression or modification of genes associated with disease resistance, abiotic stress tolerance, and quality traits with minimal linkage drag. Nonetheless, their application requires advanced infrastructure, technical expertise, and often faces regulatory and biosafety challenges, particularly in the case of transgenic crops.
An integrated breeding strategy that combines the broad variability and farmer acceptance of conventional methods with the precision and speed of modern molecular tools is considered the most effective pathway for future chilli improvement programs. Such a combined approach can accelerate the development of high-yielding, disease-resistant, and climate-resilient chilli varieties suitable for diverse agro-ecological conditions.

Author(s) hereby declare that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. 
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