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Agronomy 13 00730 With Cover
Agronomy 13 00730 With Cover
Review
Mileide dos Santos Ferreira, Anelita de Jesus Rocha , Fernanda dos Santos Nascimento ,
Wanderley Diaciso dos Santos Oliveira , Julianna Matos da Silva Soares,
Tamyres Amorim Rebouças, Lucymeire Souza Morais Lino , Fernando Haddad,
Claudia Fortes Ferreira, Janay Almeida dos Santos-Serejo et al.
https://doi.org/10.3390/agronomy13030730
agronomy
Review
The Role of Somaclonal Variation in Plant Genetic
Improvement: A Systematic Review
Mileide dos Santos Ferreira 1,† , Anelita de Jesus Rocha 1,† , Fernanda dos Santos Nascimento 1 ,
Wanderley Diaciso dos Santos Oliveira 1 , Julianna Matos da Silva Soares 1 , Tamyres Amorim Rebouças 2 ,
Lucymeire Souza Morais Lino 2 , Fernando Haddad 3 , Claudia Fortes Ferreira 3 ,
Janay Almeida dos Santos-Serejo 3 , Jorge Sandoval Fernández 4 and Edson Perito Amorim 5, *
1 Department of Biology, State University of Feira de Santana, Feira de Santana 44036-900, BA, Brazil
2 Department of Biology, Embrapa Mandioca e Fruticultura, Cruz das Almas 44380-000, BA, Brazil
3 Department of Plant Genetic Improvement, Embrapa Cassava and Fruit Growing,
Cruz das Almas 44380-000, BA, Brazil
4 Department of Agriculture, University of Costa Rica, San José 11501-2060, San Pedro, Costa Rica
5 Department of Plant Genetic Improvement, Embrapa Mandioca e Fruticultura,
Cruz das Almas 44380-000, BA, Brazil
* Correspondence: [email protected]
† These authors contributed equally to this work.
Abstract: The instability of in vitro cultures may cause genetic and epigenetic changes in crops called
somaclonal variations. Sometimes, these changes produce beneficial effects; for example, they can be
used in breeding programs to generate new cultivars with desirable characteristics. In this article,
we present a systematic review designed to answer the following question: How does somaclonal
variation contribute to plant genetic improvement? Five electronic databases were searched for
articles based on pre-established inclusion and exclusion criteria and with a standardized search
string. The somaclonal variation technique has been most frequently applied to ornamental plants,
Citation: Ferreira, M.d.S.; Rocha, with 49 species cited in 48 articles, and to the main agricultural crops, including sugarcane, rice,
A.d.J.; Nascimento, F.d.S.; Oliveira, banana, potato and wheat, in different countries worldwide. In 69 studies, a technique was applied to
W.D.d.S.; Soares, J.M.d.S.; Rebouças, evaluate the genetic diversity generated between clones, and, in 63 studies, agronomic performance
T.A.; Morais Lino, L.S.; Haddad, F.; characteristics were evaluated. Other studies are related to resistance to pathogens, ornamental
Ferreira, C.F.; Santos-Serejo, J.A.d.;
characteristics and resistance to abiotic stresses. The application of the plant growth regulators (PGRs)
et al. The Role of Somaclonal
benzylaminopurine (BAP) and dichlorophenoxyacetic acid (2,4-D) was the most common method
Variation in Plant Genetic
for generating somaclones, and randomly amplified polymorphic DNA (RAPD) molecular markers
Improvement: A Systematic Review.
Agronomy 2023, 13, 730. https://
were the most commonly used markers for identification and characterization. Somaclonal variation
doi.org/10.3390/agronomy13030730 has been used in genetic improvement programs for the most economically important crops in the
world, generating genetic diversity and supporting the launch of new genotypes resistant to diseases,
Academic Editor: Sambasivam
pests and abiotic stresses. However, much remains to be explored, such as the genetic and epigenetic
Periyannan
mechanisms from which somaclonal variation is derived.
Received: 27 January 2023
Revised: 24 February 2023 Keywords: somaclones; genetic improvement; tissue culture; somaclonal variation; DNA markers
Accepted: 25 February 2023
Published: 28 February 2023
1. Introduction
Therefore, genetic improvement programs seek ways to reduce the impacts caused
by diseases, pests and abiotic stresses on agricultural crops through the development
of resistant or tolerant cultivars. In order to achieve this goal, different strategies are
used. Plant cell and tissue culture are traditionally used for the production, conservation
and improvement of plant resources from an asexual process where clonal multiplication
is expected to generate genetically uniform plants [2,3]. However, Braun [4] made the
first observation and report of variation originated in cell and tissue cultures defined as
somaclonal variation [5]. This has been one of the biggest longstanding problems, i.e.,
obtaining the genetic fidelity of plants from tissue culture in vitro [6]. However, in 1981,
Larkin and Scowcroft identified somaclonal variation as a potential for crop enhancement,
and this was later documented by other researchers [3,7,8].
Since then, new somaclones from different cultures with characteristics useful for
breeding, such as resistance to pathogens, tolerance to abiotic stresses and high produc-
tivity, have been launched [9–13]. Somaclonal variation, in which clones of genetically
identical plants have different phenotypes after regeneration, was observed in most ex-
plants subjected to micropropagation. It is more evident when cells are propagated in
culture for long periods of time and when explants/micropropagated plants suffer several
subsequent subcultures. The first studies involved genetic and epigenetic variations, which
led to the hypothesis that plant growth hormones, such as auxins and cytokinins, could be
responsible for these genetic changes observed in plants [14–16].
Rai [17] discussed the source and genetic basis of somaclonal variation, its detection
methods and the advantages of this tool for agriculture, with the main emphasis on some
useful somaclonal variants released as cultivars. Other studies have reviewed the potential
application of somaclonal variants in the improvement of horticultural crops [18] and
described the current status of understanding the genetic and epigenetic changes that occur
during tissue culture [19]. To summarize the current status of knowledge generated on
somaclonal variation in plant breeding, this article presents a systematic review (SR) of
studies conducted in the last 16 years. The approach presented here makes use of the SR
tool, which provides a summary of all the relevant evidence available on the applications
of this tool in plant breeding. The main countries that work on somaclonal variation,
the somaclones of various cultures generated globally, the purposes of the generated
somaclones, the methods for induction of somaclonal variation, the number of subcultures,
PGRs most used in the induction of somaclonal variation and their doses, the explants
preferentially used, the main phenotypic characteristics observed in the somaclones, the
molecular markers frequently used in the studies to detect somaclonal variation and
information on the gene expression of some somaclones generated are presented.
Table 1. List of questions about the use of somaclonal variation as a tool in the genetic improvement of
agricultural crops to be answered by a systematic review of articles published in the last fifteen years.
Research Questions
Q1. In which cultures has the somaclonal variation technique been applied?
Q2. For what purposes is the somaclonal variation technique applied?
Q3. What PGRs and doses are most used to generate somaclonal variants?
Q4. How many subcultures were made to generate somaclones?
Q5. In which countries is the somaclonal variation technique most often applied?
Q6. Which somaclones have already been generated?
Q7. What are the most frequent changes observed in the phenotypic characteristics of somaclones?
Q8. What molecular tools are used to characterize somaclonal variants?
The execution stage consisted of three phases: research, selection and extraction.
The electronic searches were performed using a search string defined with the following
keywords: “plant breeding” AND “somaclonal” OR “somaclone variation”. This search
string was designed to cover the largest possible number of articles on the subject and
was used to identify articles in five databases: Web of Science (http://apps.isiknowledge.
com) (accessed on 15 February 2020), PubMed (http://www.ncbi.nlm.nih.gov/pubmed)
(accessed on 15 February 2020), Springer (https://www.springer.com/br) (accessed on 15
February 2020), Portal of Journals CAPES (http://www.periodicos.capes.gov.br/) (accessed
on 15 February 2020). and Google Scholar (https://scholar.google.com.br/schhp?hl=en&
as_sdt=0,5) (accessed on 15 February 2020). Each database was searched for articles
published over a period of 16 years. Some documents were considered relevant but were
published after the selection stage, so they were added manually. The results were exported
in the BIBTEX, MEDILINE or RIS formats compatible with Start software.
We used a protocol for the development of the SR, and the search terms were based on
the four PICO inclusion components (i.e., population, intervention, comparison, outcome
and study type) [20] (Table 2).
Table 2. Definition of the PICO terms for the research question addressed in this study of somaclonal
variation over the last 16 years.
Initially, in the selection phase, only the title, abstract and keywords were read, and
the articles that contained the terms defined in the search string within these features were
selected. In the extraction phase, the articles were read in full, and the articles were accepted
according to the predefined inclusion (I) and exclusion (E) criteria: (I) articles that contain
in the title, abstract or keywords the terms plant breeding and somaclonal or somaclonal
variation; (E) articles published in languages other than English; (E) articles that deviate
from the topic; (E) review articles; (E) theses, dissertations and manuals; (E) book chapters;
(E) articles published in annals of events; and (E) articles on the evaluation of plant fidelity
after in vitro multiplication.
In the summarization step, graphs, tables, word clouds and bibliometric maps were
generated to compose an SR. The frequencies of articles were calculated for the questions
Agronomy 2023, 13, 730 4 of 30
described in Table 1. The graphs were generated in R software [21] with the ggplot2
and dplyr packages. The bibliometric analyses were performed using VOSviewer_1.6.17
software [22].
Risk of bias
To evaluate the risk of bias among the articles selected for this SR, we adapted the
Cochrane risk of bias tool protocol [23]. Three authors (MSF, AJR and FSN) evaluated the
quality of the methods used to select the included studies, and the questions used to assess
the risk of bias were the same as those developed for the protocol (found in Table 1). The
studies were classified according to the number of questions answered that contributed to
the SR. Three classifications were adopted:
1. Low risk of bias (low)—articles that answered 100% of the proposed questions.
2. Moderate risk of bias (moderate)—articles that answered up to 60% of the questions.
3. High risk of bias (high)—articles that answered up to 30% of the questions.
In addition, all the PRISMA guidelines were carefully followed; the PRISMA checklist
is available for download at https://doi.org/10.5281/zenodo.7674859 (accessed on 20
February 2020).
3. Results
3.1. Screening of Studies
Figure 1 represents the PRISMA flow diagram used to screen the articles analysed
in this review. The Web of Science was the database that contributed most to this review,
with 1192 articles (27%). PubMed Central contributed 1069 articles (25%), followed by
Google Scholar with 1010 (23%), Springer with 997 (23%) and the CAPES journal portal
with 75 (2%) articles. Eleven important articles were manually added to this review because
they reported the generation and study of somaclones with resistance to diseases, abiotic
stresses and agronomic and molecular aspects [12,13,15,24–31]. In total, 4351 articles were
identified in the databases, of which 882 were duplicates and 3725 were eliminated in
the selection process. In the extraction phase, 629 articles were read in full, and 410 were
excluded because they did not meet the inclusion criteria. A total of 219 articles were
selected for this SR. The manuscripts were stored in an open access digital library available
at https://doi.org/10.5281/zenodo.7641768 (accessed on 22 February 2023).
of the United Nations, were not separated. The other species were classified by cultivation
type: fruits (9 species and 26 articles); forage, grasses and cereals (16 species and 21 articles);
vegetables, roots and tubers (7 species and 17 articles); medicinal (13 species and 15 articles),
condiments and spices (4 species and 9 articles); and ornamental (24 species and 45 articles)
(Table S1). The most studied species were sugarcane (30), rice (18), banana (13), potato (10)
and wheat (11) (Table S1).
Figure 1. PRISMA flowchart. Process of selecting articles for inclusion or exclusion in a systematic
review of the application of the somaclonal variation technique for plant genetic improvement;
n = number of articles.
In India, the largest numbers of studies have been conducted on sugarcane (14),
medicinal plants (8) and forage, grasses and cereals (7); in Pakistan, sugarcane (14) and
potato (5); in China, rice (6) and ornamental plants (5); in Egypt, potato (3), vegetables,
roots and tubers (3) and wheat (3); in Iran, fruits (4); in Brazil, ornamental plants (7) and
fruits (2); in the United States and South Korea, ornamental plants (6, 4); and in Poland,
vegetables, roots and tubers (Figure 3).
The largest number of somaclones has been generated for sugarcane (16), followed by
ornamental plants (14), banana (9), medicinal plants (6), wheat (6) and rice (5) (Figure 4).
Other crops generated a lower number of somaclones, such as millet (4), strawberries (4),
pineapples (3), cactus (3) and potato (3) (Figure 4). A smaller number of somaclones was
generated for other crops. —
Agronomy 2023, 13, 730 6 of 30
Figure 2. Biometric maps of manuscripts in the last 16 years regarding somaclonal variation in plant
genetic breeding. Frequency of keywords (A); Frequency of Scientific Journals that published the
most (B).
Figure 3. Number of articles published on somaclonal variation around the world in the last 16 years
and main plant species studied. The countries shown in the light green colour have a lowest number
of articles about somaclonal variation. The medium to intense green colours represent countries with
approximately 21 studies on somaclonal variation, and the red colour represents countries with a
higher number of studies on somaclonal variation.
Figure 4. Number of generated somaclones separated by culture in the studies included in a system-
atic review of the application of somaclonal variation in plant genetic improvement.
Agronomy 2023, 13, 730 8 of 30
Figure 5. Number of articles separated by the reported purpose for generating somaclones. The
data were generated for a systematic review of the application of somaclonal variation in plant
genetic improvement.
40 acetic α
Figure 6. Most commonly used PGRs and the purpose of inducing somaclonal variation. benzy-
laminopurine (BAP); Dichlorophenoxyacetic acid (2,4-D);D); αα-naphthalene acetic acid (NAA); kinetin
(KIN/KT); idolacetic acid (IAA); indole-3-butyric acid (IBA); thidiazuron (TDZ). The data were
obtained from the study of 219 articles included in an SR of the application of somaclonal variation in
plant breeding.
There was high variation between the doses of the PGRs applied in the different
manuscripts, varying from 0.01 mg/L to 16 mg/L (Figure 7). In general, the most reported
doses of PGRs varied between the PGRs, whereas BAP presented the highest number of
different doses applied per manuscript followed by 2,4-D and NAA (Figure 7). The most
applied doses for the BAP were 1 mg/L (23), 2 mg/L (21), 0.05 mg/L (17) and 3 mg/L (9). For
the 2,4-D, the most applied doses were 2 mg/L (26), 1 mg/L (18) and 3 mg/L (10) (Figure 8).
The most applied doses for the NAA were 1 mg/L (13), 0.05 mg/L (11), 2 mg/L (9) and
0.1 mg/L (8). The KIN was mostly applied in doses of 0.05 mg/L (8), 1 mg/L (8) and 2 mg/L
(7); IAA was preferably applied in doses of 2 mg/L (8) and 1 mg/L (5). The most applied
doses for the TDZ and IBA were 1 mg/L (8, 6) and 2 mg/L (4, 5), respectively (Figure 7).
Agronomy 2023, 13, 730 9 of 30
Figure 7. The most commonly used doses of PGRs to generate somaclonal variants in vitro.
Dichlorophenoxyacetic acid (2,4-D); benzylaminopurine (BAP); idolacetic acid (IAA); indole-3-butyric
(IBA); kinetin
acid (IBA); (KIN/KT);
kinetin α α-naphthalene acetic acid (NAA); thidiazuron (TDZ). The data were ob-
(KIN/KT);
tained from the study of 219 articles included in a systematic review on the application of somaclonal
variation in plant breeding.
Agronomy 2023, 13, 730 10 of 30
Figure 8. Pie charts summarizing the data of subculture time and number of subcultures in published
articles in the last 16 years recognized in the systematic review: The role of somaclonal variation in
plant genetic improvement: a systematic review.
Of the articles inserted in this SR, 17 referred to the time of subculture in months
or years, ranging from one month to 40 years. In this case, five studies reported that the
subcultures were carried out for one month and some subcultures for two months, four
months and two years; both reported in three articles. The other subculture times were
reported in only 1 article, such as 40, 14 and 10 years and 8 months (Figure 8). The studies
that made clear the number of subcultures totaled 38; within these studies, the highest
number recorded was 25 subcultures, and the lowest was only 2 subcultures (Figure 8).
The number of subcultures recorded in most articles were three (7), four (5) and five (5).
Figure 9. Most frequently used explants for induction of somaclonal variation per culture. The data
were obtained from 219 articles included in the review of the application of somaclonal variation for
plant breeding.
Agronomy 2023, 13, 730 11 of 30
Table 3. Cont.
for sugarcane where phenotypes with colour variation, smaller diameter or increase in
diameter and length were described, and the number of internodes increased (Table 3).
Phenotypic changes in the pseudostem were observed only for banana genotypes
with changes in length increase and colour appearance variations. In relation to leaves, the
alterations were reported mainly in medicinal plant species to increase substances used for
therapeutic and ornamental purposes, where the presence of genotypes with variegation
characteristics or alterations in colour and conformity are commercially desirable. Similarly,
morphological changes in flowers have been documented only in ornamental plants. On
the other hand, changes in fruits and seeds were reported in important food crops, mainly
to increase the number of fruits in tomato and grain yields in rice, sorghum and corn
(Table 3).
Figure 10. Frequency of molecular markers associated with strategies to identify genetic variation
over the last 16 years. The data were obtained from articles included in the systematic review of the
application of somaclonal variation in plant breeding.
As expected for the set of inserted articles, the objective of using each of the different
molecular markers reported is to verify the mechanism related to somaclonal variation
either by methylation in DNA or changes in the sequence of DNA base pairs. Some articles
also evaluate, through markers, the presence of mutations (Table S2).
Among the 219 accepted articles, 12 evaluated the gene expression of the generated
somaclones. Studies of expression of genes related to disease resistance, ornamental traits,
protein expression and other molecular mechanisms are described in detail in Table S3.
Agronomy 2023, 13, 730 14 of 30
A word cloud was made to identify the relevant genes analysed in somaclone studies,
where the size of the name of each gene indicates the number of articles that describe the
expression of the gene (Figure 11). The most frequent genes were PMADS4, Expansin
and OP J-06, respectively. PMADS4 genes are considered higher-order protein complexes,
responsible for changes in floral morphology in somaclonal variants. The Expansin gene is
related to cell expansion; in the articles of this review, this gene was related to dwarfism
events in somaclones. The Op J-06 genes are responsible for the Foc (Fusarium oxysporum
f. sp. cubense) resistance response to banana somaclonal variants. Other genes were
also noted in the word cloud, which indicates their expression in many studies of this
review, such as the TDFs genes that are fragments derived from transcription and the RPK2
genes that are involved in signal transduction. These are in addition to NPR1 genes which
function as master regulators of the plant hormone salicylic acid (SA) signalling and play
an essential role in plant immunity (Figure 11).
Figure 11. Word cloud of the frequency of genes with differentiated expression shared in manuscripts
regarding somaclonal variation in plant breeding.
4. Discussion
4.1. Screening of Studies
This SR comprises articles that aimed to generate somaclonal variants or study so-
maclones generated or marketed in the last 16 years. Therefore, many articles were elimi-
nated in the extraction stage (410) because they dealt only with genetic variability without
breeding purposes, where somaclonal variation is labelled in germplasm banks or in
Agronomy 2023, 13, 730 15 of 30
seedlings for field planting as an undesirable characteristic; in these cases, the objective is
to ensure the genetic fidelity of plants. On the other hand, we included in our SR a set of
219 articles that deal specifically with the use of the technique for obtaining somaclonal
variants with desirable characteristics to plant breeding programs. Although our study
includes an extensively large number of articles, which makes it difficult to extract and
discuss in detail all the data, we try to list the main data obtained in summary form to
derive conclusions and tendencies regarding the proposed subject.
Our bibliometric analysis confirmed that the term “somaclone” began to be more
frequent in the last two decades, when studies on the induction of somaclonal variation
began to be developed for genetic breeding purposes (Figure 2). At that time, several
journals that are focused on publications in the areas of tissue culture and biotechnology
began to publish articles with terms related to “somaclonal variation” (Figure 3). However,
in previous years, the changes from in vitro cultivation described in different studies were
tested to evaluate the genetic fidelity of plants in relation to the original plant and did
not have the objective of generating somaclones to be applied in the genetic improvement
of crops. Thus, the term “somaclones” becomes more frequent in recent years for this
purpose [17,91,92]).
Epigenetic changes are responsible for phenotypic changes observed in somaclones, and
these changes, such as loss of DNA methylation, may be reversible [15].
DNA methylation in the form of 5-methylcytosine (5mC) is an important epigenetic
marker involved in gene expression and plays an important role in plant regulation and devel-
opment [116]; in plants, it usually occurs in cytosine bases in all sequence contexts [92,117,118].
Although not recorded in our data, genetic and/or epigenetic changes that occur in vitro can
also generate chimeras (mosaics). In chimeras, the variations affect the function of chloroplasts
in different regions in the plant tissues of the same plant. This event occurs through variations
in their plastomas, i.e., the region responsible for governing the expression of genes related to
photosynthesis, with this change resulting in an albino phenotype [16]. These changes are
responsible for presenting altered morphological characteristics in micropropagated plants.
In our study, we described the phenotypic changes in different parts of microprop-
agated plants in vitro to obtain somaclones (Table 3). In general, our data demonstrate
that the adoption of in vitro micropropagation methods with the use of PGRs BAP, 2,4-D,
NAA, TDZ, IAA and IBA at different doses together with successive cultivation has the
potential to cause desirable modifications to the genetic improvement of various crops of
agricultural and commercial importance.
Many results showed that supplementation with high concentration of 6-benzylaminopurine
(4.0 mg/L BAP) alone or combined with indole-butyric acid (IBA) produces a higher percentage
of dwarf variants [32–37]. Thus, plants with the dwarf phenotype have been reported for some
crops; it serves as a marker for the presence of variations or as an important characteristic to
facilitate cultural treatments and management in monocultured species, or as characteristics of
ornamental interest [36,37]. In pineapple culture, useful mutants were identified with less spiny
leaves that are easier to manage in the field and hence, represent another dwarf phenotype with
ornamental value [35]. In wheat crop, a new strain of buckwheat, AS34, was developed by somatic
variation and will be useful in wheat breeding programs, particularly because the modification of
high commercial varieties reduces the risk of tilting; this is one of the most important agronomic
characteristics of wheat [34].
The morphological alterations were seen more in plants of ornamental and medicinal
interest. The SVT14 variants of Caladiums (Caladium × hortulanum Birdsey) presented
rounder and thicker leaves and, in Chrysanthemum (Dendranthema grandiflora), changes
were described in relation to the number of flowers, flower size, flower weight, leaf weight,
stem weight or plant size, as well as a reduction in flowering induction time [68,78,79].
In the tobacco crop (Nicotiana tabacum), promising somaclones were developed with
variations in the increase in length, width and number of leaves that can contribute to
higher productivity of the crop [59]. Morphological changes in fruits and seeds were
also found. Our results showed that tomato crop stood out with studies that obtained
somaclones with changes in the number of fruits, i.e., an agronomic characteristic of great
importance for this crop [80,85,90].
Our results showed promising results for obtaining improved cultivars in relation to
grain yield, which is a target characteristic for the genetic improvement of large agricultural
crops, such as corn, rice and wheat [86,87,89].
economical, and by the ease of application in a less technical laboratory considering that
the studies inserted in this SR are since 2007.
In addition, the use of RAPD markers depends on genetic markers located in parts of
the DNA sequence, and large amounts of DNA are not required to locate the sequences.
These markers are polymorphic and express genetic variations in band imprinting, thus
making it possible to perform genetic mapping to indicate genetic diversity in parental
genotypes; this is very useful for identifying variants among genotypes in germplasm banks
with genetic characteristics that differ from clones of genotypes stored in banks [120,121].
However, we indicate that there may be a tendency to use improvements in the RAPD
technique, such as Sequence Characterized Amplified Region (SCAR), DNA amplification
fingerprint (DAF) and sequence-related amplified polymorphism (SRAP).
The ISSR marker is a low-cost and highly efficient method that detects very small
genetic variations and is widely used in studies of plant genetic diversity and to determine
genetic relationships. Similar to RAPD markers, ISSRs are dominant markers and do not
require prior sequencing. One of the advantages of the AFLP technique, besides being
a low-cost technique, is the detection of a larger number of loci and providing a wide
coverage of the genome. AFLP markers are capable of detecting genetic variations such as
chimeras and identification of mutants [122–124].
The IRAP and REMAP markers are based on retrotransposons. Retrotransposons
move through an RNA molecule, are dispersed throughout the plant genome and can
contain thousands of copies, thus contributing to size, structure, diversity and variation in
the genome which may affect gene function. The IRAP and REMAP markers are, therefore,
considered very efficient molecular markers to investigate genetic variability in plants [125].
Such markers were used to study genetic variation induced by tissue culture in date palms
(Phoenix dactylifera L.) and alkaligrass (Puccinellia chinampoensis Ohwi) [126,127]. Other
studies have demonstrated the efficacy of these markers to evaluate genetic diversity and
stability in crops such as beans [128], Egyptian barley [129] and date palm [130].
Single nucleotide polymorphism (SNP) can be applied to characterize allelic variation,
genome-wide mapping and as a tool for marker-assisted selection. In the last decade, the
identification of SNPs plays an important role in molecular genetics providing a better un-
derstanding of genetic architecture and the identification of several economically important
characteristics in various crops [131–134].
Some articles addressed the gene expression of the generated somaclones, providing
information about the genes involved in the expression of morphological and genetic traits
(Table S3). Analysis of the expression of genes involved in resistance to Fusarium oxysporum
f. sp. cubense tropical race 4 (TR4) Guijiao 9, a somaclonal variant of banana belonging to
the Cavendish subgroup, revealed that during the onset of infection by Foc TR4, resistant
Guijiao 9 showed a higher number of differentially expressed genes (DEGs) than the
susceptible Williams cultivar. Multiple resistance pathways were activated in Guijiao 9,
and the DEG genes were involved in plant-pathogen interactions, signal transduction,
secondary metabolism and other processes. This suggests that the pathogen response is
regulated by multigene networks of DEG genes related to resistance [102].
In the study of Lee et al. [77], gene expression analysis was used to evaluate levels of
endoreduplication in the variants of Phalaenopsis WP, an ornamental species. The study
indicated that the high levels of endoreduplication in these variants are associated with
changes in the normal growth of petals and leaves. In addition, high expression levels of
the HPY2 gene are associated with endoreduplication only in some cases, indicating that
additional genes are involved in the induction of polyploidy in Phalaenopsis WP variants.
However, the PMADS4 gene studied was highly expressed in the petals of normal plants
compared to those of somaclones, indicating its normal function in the development of
floral parts. Hsu et al. [135] also studied gene expression in somaclones and found five
sequences that showed higher expression levels in the wild plant than in Phalaenopsis
Hsiang Fei cv. HF. These genes correspond to sequences encoding casein kinase, isocitrate
dehydrogenase, cytochrome P450, EMF2 and an unknown protein. Two other sequences
Agronomy 2023, 13, 730 19 of 30
found in this study, whose roles were unknown, were expressed at a higher level in the
somaclone plant than in the wild-type plant. The authors concluded that mosaic colour
patterns and aberrant flower shapes may be caused by these genes in somaclonal variants
of Phalaenopsis Hsiang Fei cv. HF. Further studies on the gene expression of somaclones
are needed and may provide a more complete view of the genes involved in the changes
that occur in somaclones. Understanding the mechanisms of somaclonal variation, as well
as the expressed genes, may provide an alternative to generate somaclones of all cultures
using previously described genes.
5. Conclusions
A total of 219 articles published between 2007 and 2022 were included in this review,
encompassing a large number of studies in which somaclonal variants of various cultures
were generated. The in vitro genetic diversity created in several plant species and agricul-
tural crops has led to the emergence of characteristics related to resistance to biotic factors,
improved agronomic performance and tolerance to abiotic stresses. Somaclonal variation
has been used in genetic improvement programs of several crops worldwide, generating
genetic diversity and providing the launch of new genotypes of important agricultural
crops, such as sugarcane, wheat, rice, potato, banana and ornamental and medicinal plants,
among others, with resistance to diseases, pests and abiotic stresses.
India, Pakistan, China, Egypt, Iran and Brazil have the largest numbers of studies
on somaclonal variation in the world. Studies on sugarcane, ornamental plants and fruit
plants have been the most common over the last 16 years. Studies involving the induction
of somaclonal variation focused on the identification of molecular genetic variation, the
selection of useful agronomic traits, resistance to pathogens, tolerance to salinity and
tolerance to water deficit. Studies evaluating somaclones with tolerance to abiotic stresses,
such as lead tolerance, toxic metal tolerance and copper tolerance, were also cited. This
indicates that the induction of somaclonal variation has been explored in recent decades
from several perspectives.
PGRs and frequent subcultures are the most commonly used techniques for the in-
duction of somaclonal variation according to the results of this review. The PGRs BAP
and 2,4-D with doses of 0.5 mg, 1 mg and 2 mg/L were the most commonly used. The
use of subcultures and PGRs, and the concentrations of these PGRs to induce somaclonal
variation, does not require very sophisticated techniques; this makes them accessible for
studies of somaclonal variation in breeding programs. In addition, the launch of new
cultivars derived from somaclonal variation is not a bureaucratic process and is considered
inexpensive; it differs from the development of cultivars derived from other methods, such
as genetically modified (GM) crops, which face major social and ethical obstacles.
It is observed that techniques for inducing somaclonal variation have been applied to
a variety of crops. With the success of these techniques, many cultivars with agronomic
characteristics useful for agriculture, such as nutrient quality, yield, disease resistance and
tolerance to abiotic stress, should be included in different genetic improvement programs,
and future studies may provide relevant information. Each year, new cultivars are launched,
and many are being studied and evaluated for marketing purposes.
There is still a broad expectation that increasing the understanding of the mechanisms
involved in somaclonal variation, the expression of genes of the generated somaclones and
information about the biochemical and molecular pathways involved in the selection of
somaclonal variants needs to be further explored. Future molecular research may help
in the identification of somaclonal variants through polymorphic fragments involved in
the process of somaclonal variation and selection of some genes associated with unique
characteristics of somaclones. The expansion of knowledge on the genetic and epigenetic
mechanisms of somaclonal variation will increase its use in crop breeding.
Agronomy 2023, 13, 730 20 of 30
Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/agronomy13030730/s1. Table S1: Plant species studied in articles on
the application of the somaclonal variation technique in plant breeding in the last 16 years. Table S2:
Molecular markers associated with strategies to identify genetic variation in various crops. The data were
obtained from articles published in the last sixteen years on the application of somaclonal variation in
plant breeding. Table S3: Genes associated with strategies to identify molecular changes in different crops.
The data were obtained from articles published in the last 16 years on the application of somaclonal
variation in plant breeding. Table S4: Assessment of the bias risk of the 219 articles included in the SR of
the application of somaclonal variation for plant breeding over the last 16 years [135–269].
Author Contributions: M.d.S.F. and E.P.A. conceived the idea that formed the topic of this article.
M.d.S.F., A.d.J.R., F.d.S.N., J.M.d.S.S., C.F.F., J.A.d.S.-S. and E.P.A. developed the initial protocol for the
review. M.d.S.F., A.d.J.R., F.d.S.N. and J.M.d.S.S. assisted in the use of the software and performed the
literature search. M.d.S.F. wrote the article. M.d.S.F., F.d.S.N., A.d.J.R., J.M.d.S.S., T.A.R., W.D.d.S.O.
and L.S.M.L. processed the data, developed the methods for analysing the data and prepared the
results and discussion. E.P.A., C.F.F., F.H., J.S.F. and J.A.d.S.-S. provided technical guidance and
research supervision and critically reviewed the study. All authors collaborated in the review and
editing portion of the writing process. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by IITA/The Bill and Melinda Gates Foundation—Accelerated
Breeding of Better Bananas, ID OPP1093845.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank the Graduate Program in Biotechnology (PPGBiotec) of the State
University of Feira de Santana, as well as Fapesb (Bahia Research Foundation) for granting DSc. scholar-
ships to M.d.S.F., J.M.d.S.S. and A.d.J.R.; and CNPq (National Council for Scientific and Technological
Development) for the research productivity grants for E.P.A. and C.F.F.; CAPES (Coordination for the
Improvement of Higher Education Personnel) for granting DSc. scholarships to F.d.S.N.
Conflicts of Interest: The authors declare no conflict of interest.
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