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Advances from Conventional to Modern Plant Breeding Methodologies
Plant Breed. Biotech. 2022;10:1-14
Published online March 1, 2022
© 2022 Korean Society of Breeding Science.

Sashi Lamichhane1*, Sapana Thapa2

1Department of Agronomy, Plant Breeding and Agri-statistics, Institute of Agriculture and Animal Sciences, Lamjung 33603, Nepal
2Department of Agronomy, Plant Breeding and Agri-statistics, Institute of Agriculture and Animal Sciences, Paklihawa 33007, Nepal
Corresponding author: Sashi Lamichhane,, Tel: +977-9849299533, Fax: +977-4315252
Received December 16, 2021; Revised February 10, 2022; Accepted February 10, 2022.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
First initiation of breeding started thousands of years ago when human practiced selection based in visually appealing traits. Further, domestication of wild plants eased adaptation of plant breeding. With increase in population the demand for food also increased which resulted in development of various breeding methodologies. Conventional breeding is a selective breeding methodology where crops are selected based on superior performances. Pure-line selections, mass selection, back cross breeding, recurrent selection, hybridization were most famous traditional breeding methods. It is a longer breeding method and is over-dependent on phenotype of plants. However, phenotypes of a plant are affected by various externalities. So, selection based on phenotypic expression is not accurate. As a result, breeder started integrating various branches of biology in plant breeding and developed modern breeding practices. After Mendelian theory and identification of DNA and RNA, plant breeding diverted to molecular era. People started breeding based on less environmentally susceptible parameters like genotypes, visual and genetic markers, image analysis and loci mapping. Some of the most common modern breeding practices include genomic selection, markers assisted breeding, high throughput phenotyping and CRISPR-Cas9. Despite these, plant breeding has fired up the problems of gene erosion due to loss of local landraces and wild-type plants.
Keywords : Plant breeding, Conventional breeding, Modern breeding, Plant domestication, Genetic erosion

The initiation of modern human civilization dates back to 10,000 BC when people were still dependent on hunting and gathering. Then, primordial humans started domesti-cation of sheep and other animals after 2300 years. Gradually, wheat, maize, potatoes were cultivated in sepa-rated plots. It was during 2900 BC when plough and irrigation system was introduced. After that domestication of various crops caught a speed (Agronomy and Agriculture 2021). The first existence of domesticated crops was found in South-West Asia during Neolithic age, with Einkorn and Emmer wheat being the first to be domesticated (Zohary et al. 2021). Just before 20 or 30 centuries ago, Neolithic man domesticated all of staple cereals and crops consumed today (Borlaug 1983). At present 2500 species of plants have undergone through the process of domestication (Dirzo et al. 2003) with 250 species being completely domesticated (Gepts et al. 2012).

Domestication of plants can be categorized into three stages: “gathering”, “cultivation” and “domestication”. Initially, people started collecting plants from wild types. Then, they started cultivating them on their field and finally practiced visual selection of those plants with advan-tageous characters. People initiated selection of crops to obtain vital beneficial characteristics from their wild types during domestication. Domestication of seed crops were based on certain traits like larger size of seed, thinner seed coats, lack of dormancy, increased yield, determinate growth pattern, etc. Within a plant only those species with these desirable traits were selected for domestication (Meyer and Purugganan 2013). Domestication of plants did not start in a single area but were spread across multiple areas (Meyer et al. 2012). As selection is the first step in plant breeding, it is believed that primitive human practiced plant breeding sub-consciously since initial stages of crop domestication.

Plant breeding is simply the integration of crop genetics in plants to produce plants according to plant ideotype. Plant breeding is a repetitive process of selection in parents as well as their progenies for desirable traits. Only after Mendel’s law of inheritance in 1865, concrete concept of plant breeding started. As Mendel studied sweet-pea to formulate his theories, it encouraged people to apply his theory in applied plant genetics. Prior to 19th century plant improvement was done by large scale farmers who selected seeds from plants with desirable visual characters and cultivated them. After restoration of Mendel’s law in 1900s, people started crossing of superior varieties and creating hybrids of required characters (Hickey et al. 2017). The importance of plant breeding is immense in present world. During 1965, Norman E. Borlaug pioneered “Green Revolution” through use of Plant genetics to breed semi-dwarf, fertilizer responsive and disease resistant wheat varieties. International Rice Research Institute also released rice variety resistant to lodging and photo-period insensitive (Lee et al. 2015). These were arguably the most important achievements in agriculture, attained with the aid of plant breeding and genetics. Some developed countries have minority of people involved in agriculture. These minority needs to handle tedious responsibility of feeding majority of people in the country (Breseghello and Coelho 2013). It is possible because plant breeding has been successful to improve crop yields even without increasing cropping area or people involved in it.

World population at present is anticipated to increase by a billion every fourteen years. By 2040 A.D., it is expected to reach 10 billion at this rate. However to cope with this increment in population, food supply needs to be increased by 70-100% (Jonathan et al. 2011). This goal can be easily achieved by crop improvement through plant breeding. Plant breeding uses different strategies like chromosome alteration, artificial crossing and mutagenesis for creation of variation (Lee et al. 2015). Creation of variation aids in selection of desirable characters from a plant. For last 150 years, plant breeding has been successful to cut-short the life cycle of plant (Ahmar et al. 2020). Shorter lifecycle has enabled other major crops to be integrated in yearly cropping calendar.

The primary intent of this paper is to discuss about various breeding methodologies. The paper also tries to differentiate between conventional breeding and modern plant breeding. We hope the paper would act as a perfect knowledge source for scientists interested in Plant breeding.


Conventional Breeding is the process of developing new plant cultivars without the use of futuristic molecular breeding technologies. Conventional breeding does not violate the natural law of inheritance. Conventional breeding is a selective breeding methodology where pants are selected based on superior performance on selected characters. The required characters are attained from closely related individuals and incorporated into new cultivars using hybridization (Al-Khayri et al. 2016). Variability in conventional breeding is created through hybridization. Selection based on phenotype is done to identify the most important genotype. Then, the selected cultivars are evaluated and then released as varieties. In conventional breeding, identical parents do not produce variation as there would be no segregation of gametes (Acquaah 2009). Conventional breeding is a longer process and usually takes more than 10 years to release a new cultivar (Bharti and Chimata 2019). Conventional breeding is over-dependent on phenotypic expressions of cultivars for identifying superior ones. Thus selected cultivars are not free from errors as phenotypes are highly influenced by genotype by environment interactions (Lema 2018). Conventional breeding is applied science and it leans towards experiences, observations and skills of breeders for judgment (Allard 1961). For instance, a most extensively grown potato in America, Burbank, was discovered through abrupt observations of growers by conventional breeding methodologies (Fehr 1987). The inconsistent result of conventional breeding is mainly due to over-dependence on subjective analysis. However, scientific and modern plant breeding is less subjective and more science. Thus, modern breeding practices is more effective and efficient (Jiang 2013).


For last 20 years modern technologies are being amalgamated with conventional breeding practices. With time the breeding objectives of plants are moving beyond the limits to improve crop yield only. More unique traits like weed resistant, improved nutrition and responsiveness to soil and microbial community are studied robustly. To overcome these unusual goals, conventional breeding are used in combination with other branches of science for thorough study of genomics (Fu 2015). Newly used technologies like Genomic selection, enviromics and High Throughput Phenotyping (HTP) are practiced to improve genetic gain of cultivars. These new technologies gave rise to modern breeding. Genomics, Enviromics and Phe-nomics are considered to be parts of modern plant breeding triangle (Crossa et al. 2017). Conventional breeding has been using many genetic and molecular techniques that have accelerated the speed of crop enhancement. These modern techniques has been widely used in staple crops like rice, wheat, sorghum, maize, etc. (Ricroch et al. 2014). Various supplementary approaches are used to enhance global food production. Modern plant breeding tries to explore characters that help a variety to perform superiorly throughout distant locations (Ewing et al. 2019). These breeding hypotheses use parents with consistent phe-notypes which show less effect through genotype by environment interactions (Finlay and Wilkinson 1963). Despite its effectiveness, modern breeding programs are alleged to cause genetic erosion. However, these alle-gations are deemed as false (Huang et al. 2007) (Table 1).

Table 1 . Differences between conventional and modern plant breeding methods.

Conventional plant breedingModern plant breeding
∙ The breeding method is based more on phenotype which creates unreliability in determining pure line of genes.∙ It is genotype based method of breeding which is more accurate in determining the pure gene line.
∙ It is more time consuming to release a new variety.∙ It is comparatively less time consuming to release a new variety.
∙ Variability in conventional breeding is created through hybridization.∙ Newly used technologies like Genomic selection, enviromics and High Throughput Phenotyping (HTP) are practiced to improve genetic gain of cultivars.
∙ Generally, dominant genes are only selected. The selection of recessive alleles needs to undergo a long procedure.∙ The recessive alleles can be selected through the use of markers and identification of specific sites of gene which is quite shorter procedure than of the conventional.
∙ It is less effective since it depends more on skills, arts and subjective analysis of breeder. This makes the result less reliable.∙ Modern Plant Breeding is more effective and trusted since it is based on scientific research and findings.
∙ Conventional plant breeding demands less technical skills and scientific knowledge of genetics∙ In the same place, modern plant breeders are required with more technical skills and genetic science knowledge.
∙ It is less expensive as it can be carried with local techniques and tools.∙ It is highly expensive as it requires high tech machineries and methodologies.


From Neolithic age, first initiation of domestication of crop, to twenty-first century, the golden age of plant breeding, people have applied various strategies for crop improvement. The extensive use of conventional breeding in correspondence with other disciplines of science has certainly made modern plant breeding more reliable and effective. In comparison with traditional breeding, modern plant breeding has following advantages:

a. By using some techniques like Marker Assisted Breeding and Quantitative Trait Loci Mapping, selections of superior plant can be done even at seedling stage. The later occurring unwanted genes can be removed by using different markers.

b. Since conventional selection is dependent on phenotypic expression, only dominant genes are expressed on. Thus, case of conventional breeding only dominant genes can be selected. In order to select recessive alleles, test cross and selfing are done which is lengthy procedure. However with modern breeding several recessive alleles can also be selected by use of markers and identification of specific sites of the gene.

c. In traditional breeding, phenotypic selection is difficult because of presence of masking effects. Some of the quantitative traits which are governed by multiple alleles are difficult to select because their phenotypic expression varies. However, with new breeding technologies like gene editing and genomic selection it is easier.

d. Although constant breeding are conducted through conventional breeding, the results has not been very fruitful. Genetic gains of quantitative traits have not been effective as they are largely affected by genotype by environment (G × E) interaction. But, modern breeding is based on stable genotypes which are less affected by G × E interaction. This makes the breeding program effective and efficient.

e. Conventional breeding is more dependent on skills, art and subjective analysis of breeder (Anderson 2013). However, modern breeding is more of a science than art. It is based on scientific research and findings. Thus, it is more trustworthy and effective.


Although modern plant breeding can address major problems of conventional breeding, it is difficult to execute these programs due to some setbacks. Modern breeding requires skilled, expert and effective manpower for execution. Breeder needs to have knowledge about not only plant breeding, but also other branches of biology. Modern breeding methodologies are difficult to handle because of economic, institutional and technical challenges (Morris and Bellon 2004). The use of modern breeding programs like speed breeding is difficult to adopt in developing countries due to unavailability of trained individuals and technicians (Wanga et al. 2021). Some of the major challenges of modern plant breeding are discussed below:

a. Global weather and climatic patterns are changing due to global warming. As a result the extent of effect of abiotic and biotic stress factors are also changing with uncertainty. Some crops like legumes have started showing double stress symptoms. It has been quite challenging for modern breeding programs to develop varieties resistant to more than one stress. Sometimes there is presence of negative correlation between stress resisting genes and quantitative traits which reduces productivity of crops (Kumar et al. 2011).

b. There have been problems related to genetic erosion with development of plant breeding. In Canada over last 100 years, genetic diversity has rapidly declined because of breeding efforts (Fu and Dong 2015). Because of low variability, experimental errors might be high. Thus, it might be the condition that replications might be increased. It is challenging for modern breeding programs to establish same level of variation in offspring.

c. The technical knowhow required for modern breeding is high. Traditionally, plant breeding could be done through experiences, skills and little help. However, modern plant breeding requires scientific knowledge of biotechnology, plant breeding and agri-statistics. Thus, skilled manpower is required for operation of modern breeding.

d. Modern plant breeding uses modern high-tech machi-neries and methodologies. These machineries and methodologies are expensive and difficult to operate. As of a study conducted by Brennan and Martin (2006), the total cost estimation of conducting a Wheat breeding program is approximately equal to 1.1 million USD. It seems these programs are very difficult to be conducted by local farmers or in developing nations.


There are various methods being practiced in conven-tional breeding for both self and cross pollinated plants. Some of the methods are described below:

Conventional breeding for self-pollinated crops

Pure-line selection

Selection within a same population is one of the most primitive methods of plant breeding. In some self- pollinated plants like rice and wheat, pure line selection is quite common (Breseghello and Coelho 2013). Pure lines are progenies produced by self-pollination of single homozygous parent. Since genetic make-up of progenies are same to their parents, new genotypes are impossible to be created through pure line selection. Pure line selection came into practice after Johannsen gave pure-line theory in 1903 (Poehlman 2012). A research conducted on wheat revealed that heterosis in yield increased by 15-20% than the higher performing parental line through pure line selection (Lane 1981). Pure lines have evenness in genetic structure and are highly used in conditions where uniformity in product is given much priority in market (Al-Khayri et al. 2016). Phenotypic variances present in pure-lines are only due to environmental effects. There-fore, selection in pure-lines are ineffective due to low heritability (Begna 2021).

In this method, superior homozygous parental genotypes are selected and they are self-pollinated. Progenies obtain-ed from each parental genotype are grown and assessed individually. Plants are evaluated for desirable traits and uniformity. Second generation progenies with superior types go through preliminary yield trials. Then, multi- location trials are conducted in presence of check varieties from selected individuals. After few years of conducting multi-locational trials superior genotypes are released as a new variety. The whole procedure of variety release takes around six or seven years.

Mass selection

Mass selection is practiced for both self and cross pollinated crops (Allard 1961). Mass selection is practiced to enhance productivity of base population by increasing the frequency of desirable traits. Those landraces which has been transferred from generation to generations since ages are improved through mass selection. Off- type plants are excluded from the cultivated population and desired plants are selected for further breeding and testing programs (Acquaah 2012). Selections of those plants are based on phenotypic expression. This type of selection is more effective if the selected traits are highly heritable (Brown and Caligari 2011). The presence of variance due to additive genes makes mass selection highly efficient (Wolff 1972). It is performed either as single parental (one type gamete controlled) or bi-parental (both male and female gamete controlled) mass selection. However, when both male and female gamete are controlled mass selection is more effective because both the parents are selected (Chao-ying et al. 2010).

In this method, a landrace is selected based on performance of traits like height, disease resistance and early or late maturing. The selected individual is sown in the field and harvested at maturity. The harvested seeds are mixed and used for next generation. In second year, the crops are grown in a bulk from mixed seeds and compared with a check variety. Preliminary yield trial is performed. For next 3 to 4 years, the varieties are tested at multi- locations in presence of check variety. On seventh year of selection, the selected plant is released as variety and distributed.

Back cross breeding

Backcross is the process of crossing F1 hybrid with one of the homozygous parents to produce progenies identical to the parent (Aleksoski 2018). Backcross breeding is a method of incorporating a desirable trait from less established plant into a well-established one without affecting other traits in latter. It is a method of producing hybrids which are similar to parents with higher number of desirable traits. Backcross breeding is recurring selection methodology (Fujimaki 1978). After successive backcross for three to four generations, the progenies are alike recurring parents. A backcrossed progeny has to recover almost 98% of recurrent parent genome. It is obtained through repetitive backcross of hybrid progenies with recurrent parent for five to six generations (Vogel 2009). Selection of characters other than transferred one is ineffective in those progenies as the traits nearly matched with repeatedly backcrossed parent (Briggs 2016). The most common feature of backcross breeding is uneven gene contribution in newly formed variety from two parental lines. More genes are contributed from recurrent parent and very few from donor parent (Singh 1982).

In this method, donor parents are those plants which contain desired character and recurrent parents are those plants which receives the selected genes. A cross is made between two parents to produce hybrid progeny (F1). Selection is done among the F1 progenies for desired traits. The selected F1 progenies are grown and then back crossed with recurrent parent to produce back cross hybrid (BC1). Selection is done among the BC1 generations for desired character and then grown in separate fields. After it, BC1 individuals are again back crossed with recurrent parents. The process is continued until sixth back cross generation (BC6) is produced. Thus produced BC6 generation is grown for seed production. Multiple yield trials are conducted at different locations in presence of recurrent parent as check variety. The newly formed variety should be similar to recurrent parent.

Conventional breeding for cross-pollinated crops

Recurrent selection

Recurrent selection is a term coined by Hull (1945) which means process of reselecting desirable characters generations after generation to increase its frequency only from the crosses between high performing individuals (Bangarwa 2021). Crossing is done between heterozygous recurrent parent and inbred individual. It helps in maintaining variability as well as increasing gene fre-quency of desired traits. The process has been exclusively used for breeding of cross-pollinated crops (Khadr 1964). Recurrent selection was initially and widely practiced in maize, latter applied in rice, millet, wheat and soybeans (Ramya et al. 2016). There are three different types of recurrent selections (Luckett and Halloran 2017), which are:

a. Simple recurrent selection:

This method is carried out for characters with high heritability. In this method, selection is done phenotypically from open pollinated crops. After selection, the selected individuals are selfed. The progenies from selfed parents are cultivated in a crossing block and intercrossed. The progenies of intercrossed individuals are again selected and grown in separate crossing block. The process is repeated until no any further improvement is found between intercrossed parents and selected progenies.

b. Recurrent selection for combining ability:

In this method a phenotypically superior parents and tester plants are selected. These tester plants may be heterozygous or homozygous. Two things are simultaneously conducted in this method in first year. First, selfing of superior parents is done and progenies obtained from these selfings are grown in a crossing block. In parallel, the selected population is test crossed with tester plants and progenies obtained are subjected to replicated yield trials for evaluation. Progenies with higher mean performance in yield trials are expected to have good combining ability. Selfed parent of high performing tester progenies are selected and cultivated again in a crossing block and intercrossed. The harvested seeds from these plants are sown and the cycle of test crossing and selecting is continued as in first year. The cycle can be continued until desired character is not obtained. This method is effective for characters governed by incomplete dominance or over-dominance.

c. Reciprocal recurrent selection:

In this method two original open pollinated popul-ations of plants are selected. This original population of plants acts as tester plant for one another. Selfing is done for each of those populations and progenies obtained are grown in a separated crossing block. At the same time, test cross with one another is con-ducted and progenies obtained from each test cross are subjected to replicated yield trial separately. Parents of test progenies showing higher performance are selected and again selfing and test crossing are done. This cycle can be repeated until desired cha-racter is not obtained. This method is carried out for character governed by both additive and non-additive gene action.


Hybridization is the process of producing hybrids by mating genetically distant parents. It may be either natural or artificial and can involve different species, genetically different individuals or same species. It is the process of combining characters of different parents to produce genetically superior progenies. Joseph Koerauter, a German botanist, was the first to adopt hybridization technique for crop improvement in 1760 (Begna 2021). Hybridization does not alter the genetics of plant but produce recombinant genes which might contain desirable traits. This process is also used to overcome different reproductive barriers encountered in traditional sexual crossings like sexual incompatibility, male or female sterility, etc. (Mwangangi et al. 2019). The two most common hybridizations are:

a. Interspecific hybridization:

It includes crossing of individuals from two separate species which are sexually incompatible in general. This method is also known as wide crossing. This method is popular for transferring traits from wild relatives or mixing of favorable traits from two different species (Lidder and Sonnino 2012). This method is often used when a specified set of character is unavailable. Not all species can be hybridized as there is presence of pre-fertilization and post- fertilization barriers of interspecific hybridization. Pre-fertilization barriers prevent the formation of embryos and post- fertilization barriers reduce em-bryo vigor. Failure of pollen germination and pollen incompatibility are pre-fertilization barrier and post fertilization barriers include hybrid sterility, poor vigor, seed lethality and seed abortion (Murray 2016).

b. Intraspecific hybridization:

It is one of the most successful method of hybridi-zation and widely used in commercial plant breeding. As mating population are from a same species, there is no barriers of hybridization. This method is widely practiced for combining desirable traits from two or more plants of a same species and creating variations for selection (Murray 2016). Based on number of parents used, intraspecific hybridization may be Simple or Complex Cross. Simple cross involves crossing of two parents to produce F1 hybrids which are then either selfed to produce F2 or back crossed. Complex cross involves crossing of more than two parents to produce F1 hybrids. Complex cross is used to concentrate desirable traits from more than two parents into one hybrid offspring (“Hybridization in Plants: Types, Procedure and Consequence of Hybri-dization” 2021).


In order to make breeding programs more effective, efficient and swift, other branches of biology are being integrated into it. As a result modern specific approaches are being developed for breeding of various plants. Some of these modern breeding methods include:

Genomic selection

It is a type of modern breeding program where best individuals are selected for breeding based on predicted breeding values. Breeding values are merit of a selected individual based on average performance of its offspring. Genomic selection (GS) is more fruitful in comparison to conventional breeding because it aids in improving grain yield in less time. It is superior version of Marker assisted selection because it uses genome wide markers to speculate effect of quantitative gene loci thereby calculating esti-mated breeding value (Wang et al. 2018). A research con-ducted by International Maize and Wheat Improvement Center (CIMMYT) showed that breeding interval could be as less as half of conventional breeding in plants like maize (Crossa et al. 2017). On contrary to Linkage analysis and Genome-wide association studies, the main objective of GS is not to locate Quantitative Trait Loci (QTL) but to make assumptions on performances of future offspring based on DNAs collected at present. Breeding Values can be predicted through methods like penalized regression method and Bayesian method. However, the quality of prediction can be gauged by the correlation between estimated and actual breeding values (Rabier et al. 2016). These estimated and actual breeding values are calculated through two types of data sets used in Genomic selections: training population and validation population. The already phenotyped and genotyped training population helps to predict breeding values in validation population (Dwivedi et al. 2015). Genomic selection has been able to quite successfully map small-effect genes which have multiple QTL. These small effect genes are important in plant breeding because they govern most of economically and agronomically essential traits (Robertsen et al. 2019). With increase in price of phenotyping of crops, Genomic selec-tion can act as an effective measure for improvement of quantitative traits which are difficult or expensive to be recognized through phenotypic expressions (Sweeney et al. 2019).

Marker assisted selection

Marker Assisted Selection (MAS) is a method which uses various DNA markers for determining the genomic regions responsible for expression of desired traits in plants (Das et al. 2017). The availability of various DNA markers has made it easier to apply MAS is breeding programs. DNA markers are small segment of DNA which helps to identify variegation in alleles of genes among individuals of same gene pool (Nogoy et al. 2016). The most regularly used markers in plant breeding are Simple sequence repeats (SSRs), otherwise known as microsatellites. SSRs are quite popular due to easiness and cheap to use them (Collard and Mackill 2008). The uses of DNA markers are quite popular because they do not alter the phenotypic expression of plant and are affected neither by growing environment nor by inheritance (Ashraf et al. 2012). MAS is useful in selecting traits like grain yields, flower colors, seed characters, etc. which are expressed only in late reproductive stage. Through use of DNA markers such traits can be identified in a genotype even at early stage of plant development (Madhusudhana 2019). MAS is more effective approach while selecting traits that are less heritable, expensive and tedious. However, the method is only effective in case of selecting few quantitative traits which controls notable amount of variation in a population (Shu and Wu 2016). Some of the most commonly used DNA markers in Markers Assisted Breeding are (Jiang et al. 2013):

a. Restriction Fragment Length Polymorphism (RFLP):

RFLP is first developed DNA marker and is based on Southern-Blotting. They are most effective in comparative mapping. They are locus-specific and are highly reproducible. They are co-dominant, thus can differentiate between homozygous and heterozygous DNA. They exploit differences observed in restriction sites of DNA. Variation in a DNA sequence may be brought upon by deletion or insertion of base pairs in restriction sites. These variations can be identified by electrophoresis or DNA probing. However, RFLP can give effective results only in case of availability of high-quality DNA. Thus, use of RFLP has been restricted to few in plant breeding due to its demand of pure DNA and being laborious.

b. Random Amplified Polymorphic DNA (RAPD):

RAPD is Polymerase Chain Reaction (PCR) based marker and is made by amplification of a random primer which binds at various loci of DNA template. Polymorphism is found at primer binding sites which are visible through electrophoresis. RAPD is dominant marker and has high level of polymor-phism. They are easier to use and are efficient because they are devoid of blotting or hybridization. In comparison to RFLP, they require very small amount of DNA and the primers are universal.

c. Amplified Fragment Length Polymorphism (AFLP):

APLPs are both restriction and PCR based markers and are visualized by selective PCR. It requires purified DNAs that are free from proteins and con-taminations to give effective results. In case of un-availability of pure DNA, restriction does not occur and gives false result. The first step of AFLP involves digestion of genomic DNA by using restriction enzymes. Those fragments cut by use of frequent cutters like TaqI, MseI, etc. can only be amplified. AFLPs are very reproductive and are resistant to changes during PCR.

d. Simple Sequence Repeats (SSR) or microsatellites:

SSRs are PCR based markers and are random tandem repeats of nucleotide bases. These nucleotide repeats may be di, tri or tetra. The major source of polymor-phism is due to variation in tandem repeats. SSR markers are highly variable and show co-dominance. These markers are easily inspected under PCR and tracked in high resolution electrophoresis. SSR markers are highly popular because they require very small sample of DNA and have low initial cost. They are most widely used in molecular breeding, QTL mapping, MAS and genomic analysis.

e. Single Nucleotide Polymorphism (SNP):

A SNP marker contains variation in only one nucleo-tide between two DNA sequences. They are the simp-lest form of markers as variation is seen in only one base pair. They are the most common form of markers present in organisms. They are present at intervals of every 100-300 base pairs in plants. They are co-domi-nant markers and their simplicity has made them the most attractive marker in genetics and breeding. Yet, its high cost of operation and requirement of high quality DNA have restricted the use of it in various fields.

High Throughput Phenotyping (HTP)

Although selection of plant based on phenotype is still limited by environmental factors, it can be improved though use of remote sensing, new sensors and software. Various phenotypic traits like stress tolerance, disease resistance, yield, etc. can be easily accessed through high throughput phenotyping. This method involves pheno-typing, computation of data, analyzing and selection of plants based on analysis (Jangra et al. 2021). It is a nonde-structive method of analyzing complex plant traits (Irish Lorraine B. et al. 2019). HTP keeps tabs on crop develop-ment and responses to growing environment throughout its growth cycle and analyze the obtained data. HTP is effective in keeping track with the relation between geno-type and phenotype of plants (Moreira et al. 2020). One of the major advantages of HTP over traditional phenotyping by eye is that HTP is capable of keeping records of shift in phenotypes of hundreds of thousands of crops in response to alteration of genotypic and environmental factors in a single day (Rebetzke et al. 2018). Thus, HTP has succeed in collecting data from larger population with rigor and accuracy and reducing human diligence using automated equipment (Mir et al. 2019). HTP is based on various imaging techniques that captures images of crops over different time frames (Pasala and Pandey 2020). Some of the major imaging technique includes:

a. Visible light imaging:

Visible light imaging technique is the most popular method because of its low cost and ease of operation. Images are taken with normal cameras from visible band of light i.e. 400-750nm. Thus obtained raw data are expressed in intensity values of Red, Green and Blue bands (Li et al. 2014). This technology is favored in measuring plant structures, yield charac-ters, biomass, leaf morphology, etc.

b. Fluorescence imaging:

Fluorescence imaging technique is used to obtain status of metabolic activities within plant. When plants are illuminated by blue or actinic lights, chloro-phyll re-emits some of the absorbed shorter wave- length lights which are called fluorescence. These re-emitted light acts as a good indicator of photo- assimilation in plants. Fluorescence imaging is done with charge-coupled device (CCD) cameras. The technique is popular for characterizing plant health, physiology and disease resistance traits (Zhang and Zhang 2018).

c. Thermal imaging:

It works under the principle of visualization of infra-red light and provides idea on surface temper-ature of object. It is sensitive within spectral range of 3-14 mm (Li et al. 2014). A low or higher surface temperature reflects opening or closing of stomata. Images are taken through thermal cameras which instantly distinguishes temperature differences in plant canopy. This method is used in studying abiotic and biotic stress, water relations in plants and opening or closing of stomata.

d. Spectral imaging:

This method is used in remote sensing of vegetation. It records the solar radiation produced from plants and studies them. A single leaf shows low reflectance and when the vegetation increases the reflectance elevates. These reflectances are used in studying the vegetation indices of plants. Spectral imaging is used for studying chlorophyll senescence, plant water status and pathological conditions (Li et al. 2014).

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9)

CRISPR is originally a defensive immune system in bacteria against DNA and plasmids of phages. The system detects foreign particles in bacteria and with the use of restriction enzymes these nucleic acids are dissolved (Chen et al. 2019). Mechanism of CRISPE-Cas9 happens in three different steps. At first, the invading foreign DNA is recognized and a spacer sequence from foreign DNA is integrated into host CRISPR array for immunological memory. Then, Cas9 protein is formed and the CRISPR array is transcribed to precursor CRISPR RNA (pre- crRNA). The non-coding pre-crRNA is hybridized to form mature CRISPR RNA (crRNA). Finally, with the help of crRNA and Cas9 protein foreign nucleotides are identified and cleaved with the help of enzymes (Wang et al. 2019). Initiation of molecular breeding with CRISPR started in crops like rice and tobacco in 2013. However, insertion of CRISPR complex is not easy and can be done through three methods. (i) Agrobacterium mediated transfer: It is a biological method in which Agrobacterium transforms the T-DNA into plant genome. T-DNA ensures the expression of CRISPR complex in plants. (ii) PEG method: It uses plant protoplast for Cas9-sgRNA insertion. This method allows easier introduction of RNPs, proteins and plasmids. (iii) Particle gun method: This method can introduce wide range of molecules in plant so, it is widely accepted. Genome editing of maize, soybeans and wheat has been done through this method (Sugano et al. 2018). Crop improvement through CRISPR is done either by addition of important genes in plants or removal of harmful genes. Over the year about one-third of crops are improved by knocking-off of negatively affecting factors (Liu et al. 2021). For instance, in rice grain shape affects quality of grains. Gene regulators GW2, GW5 and GW6 have negative effect on grain size. These regulators are disrupted with the use of CRISPR to improve grain quality (Xu et al. 2016). Also, CRISPR introduces disease resistance in plants not only by integrating dominant genes but also by disrupting host susceptibility against biotic stresses. For example, Bacterial blight in rice is caused by Xanthomonas oryzae pv. Oryzae. The bacterium initiates the transcription of SWEET genes that increases disease proneness. However, through CRISPR technologies scientists have modified the promoter region of SWEET 11, SWEET 13 and SWEET 14 genes which develops broad-spectrum disease resistance in rice (Oliva et al. 2019). Besides, CRISPR is used in creating herbicide resistance, male- sterility, manipulating self-incompatibility, haploid intro-duction, easing hybrid vigor, etc. (Zhu et al. 2020).


With improvements in science and technologies, breed-ing programs are also evolving. Human started plant breed-ing through direct selection of visual characters. However, these selections were inaccurate because visual symptoms are affected by various environmental factors. With integration of other branches of science conventional breeding programs are made swift and effective. Since modern plant breeding methodologies take short time to complete and are less affected by environmental factors, they are considered as effective means of breeding plants. However, executing modern breeding techniques are not easier either. Some of the developing countries are unable to establish infrastructures needed for modern breeding techniques. Therefore, in order to make modern practices reachable, we need to find a way to reduce price of operation. Although breeding has helped us to incorporate desired traits in plants, it has also raised the risk of losing local landraces and wild- type plants. As per population genetic theory, genetic drift and inbreeding diminishes variation and even favorable alleles and ability to adapt in changing environment may be lost (Ouborg and Treuren 1994). Local landraces are mainly under threat because agriculture products are exclusively market oriented. Crops with high yield are preferred and cultivated in larger areas in comparison to crops that were once culturally important (Tripp and van der Heide 1996). Extinction of local and wild type plants affects future crop improvement programs as they are natural genetic reservoirs. The decrease in variations due to selection of limited traits acts as bottleneck for plant breeding. Selection of limited traits principally affects variation owing to presence of more dominant genotypes or alleles in a population (Temesgen 2021). There is steady decrease in number of cultivated crops every decade. For instance in Asia, modern high yielding rice varieties were cultivated only in 10% of area in 1970s, which increased to nearly 70% of area in 1998. This shows a large portion of landraces are being replaced by genetically identical modern cultivars (Van De Wouw et al. 2010). However, genetic erosion can be managed by various in situ (conserving of landraces in seedbanks, genebanks etc.) and ex situ conservation strategies. Besides, genetic diversity can be improved by identifying its sources. For example, beneficial mutation and recombi-nation are ultimate sources of genetic diversity (Rogers 2004).

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