Introduction Six Brassica species are cultivated

worldwide in oilseed and vegetable crops,

Brassica Napus (Rape seed) is a
including Raphanus Sativus (Radish) and
predominant oleiferous crop, amphidiploid
Brassicaceae. These Brassicaceae plants
specie originated from interspecific cross
have long been known for showing strong
of Brassica Olaracea (Wild Cabbage) and
heterosis. Plants with oil seeds include
Brassica Rapa (Field Mustard) and
Brassica Napus, and Brassica Juncea
employed in an enormous variety of
(Brown mustard) shows a hybrid force in
studies. It occurs abundantly in Sweden,
terms of yield of seed (up to 200 percent of
Netherland, Gothland and Britain in its
the parent lines) as well as high heterosis
wild form (Tsunoda 1980).
of vegetables such as cod (B. oleracea).
Previous studies provide a critical Hybrid F1 reproduction for all
relationship between Brassica Napus and Brassicaceae plants is also useful. (Tanaka
Male sterility induced by CRISPR cas9
, N., &Niikura, S, 2006)
(clustered regularly interspaced short
palindromic repeats) and chemical To enable F1 hybrid breeding, the
hybridization. CRISPR cas9 reported as an development of F1 must be effective,
effective genome editor fix for a Male secure, and safe, without polluting each
infertility form (Micheal le page, 2019). parent with self-fertilized seeds. Given the
Production of F1 seed is entirely dependent size and structure of Brassicaceae flowers,
on CMS (cytoplasmic male sterility), manual emasculation and pollination of the
which is a trait inherited from maternal hybrid grain production in Cucurbitaceae
parent. CMS encoded by a gene present in crops are unable to implement (Jain. A.,et
mitochondria responsible for disturbed al, 1994).
production of pollens (Hiroshi Yamagishi
The method of self-incompatibility, which
et al., 2014). Expression of the
was primarily developed by Japanese
mitochondrial gene masked by nuclear
breeders, is an effective breeding
fertility, allows the proffering of functional
mechanism for F1 Brassicaceae. But self-
pollens. Interaction between the genes and
incompatibility is not always robust and
mitochondria controls and regulates male
high temperatures and drought will
specificity, restoration of male fertility,
suppress.
and occurrence.
CMS is an inherited trait from maternal
parent of a mitochondrial cell.
Development of pollens in CMS plants is
disrupted, while it does not usually
influence the role of the female organ. Rf
genes mask the expression of genes of
mitochondrial CMS in order to produce
functional pollens (Kitashiba, H., &
Nasrallah, J. B, 2014).
An amalgamation of a nuclear genome
which possess no Rf gene and a genome of
mitochondria that induces CMS results in
nuclear cytoplasm and Cytoplasmic male
sterility (CMS) phenotype. CMS has been
documented in several plant organisms,
both from the functional reproduction and
from the evolutionary point of view and
fundamental genetics (Gabay-Laughnan,
S., & Newton, K. J, 2012).
In the broadest context, plant sterility
applies to the lack of exposed anthers,
usable pollen, and feasible male gametes.
Male sterility has been reported in more
than 610 plant organisms, as
described by the German botanist Joseph
Gotlieb Kolreuter in 1763; cytoplasmic
male sterility (CMS), induced
mitochondrial genes with nuclear genes,
and genetic male cell sterility (GMS),
caused solely by nuclear genes. The
development of sporophytic
gametophytic anther tissue by
sterility mutants can lead to abnormal
developments. By contrast,
development of microspores or pollen
grains mainly influences male mutants
with gametophytes (Hanson, M. R.,
&Bentolila, S, 2004)
Male sterility plants provide essential
breeding tools to harness hybrid vigor or
heterosis in hybrid plants and provide
material for studying stamen, pollen
development, and genomic interactions
with cytoplasmic-nuclear.
Restriction trends of organelle DNA in
rapeseed (Brassica napus, B campestris
here in after referred to as napus and
campestris rapeseed) favor an alloplasmic
rationale for CMS structures in this crop
(Vedel et al., 1982; Vedel and Mathieu
1983; Erickson et al., 1986). Palmer and
al. (1983) proposed a link between the
existence of linear mitochondrial plasmid
and sterility in two CMS lines of Brassica,
one line of campestris with Raphanus
cytoplasm, and the other line of napus
first known as Korean only.
It is intended that the present paper will be
an overall review for the past techniques
by
employed for Brassica Napus to study
cytoplasmic male-sterility by using
CRISPR cas9 and we will discuss its
potential to change the genome,
or
applications in the genome editing and
male
breeding work, and in addition to this the
drawbacks and disadvantages will also be
the
listed based on previous evolutionary
studies and anaylsis.
History of Research on Ogura CMS
Ogura CMS (Fig. 1) was originally
mentioned in radish and is now commonly
used in Brassica Oleracea (Cabbage), and
Brassica Juncea, Brassica Napus. Ogura
CMS was the first reported and researched
CMS that is demonstrated in -depth, both
in terms of their fundamental molecular
structure and in terms of their functional
application inbreeding. The history of
research on this CMS system reflects
breeding science developments over the
last 50 years.
Figure: Ogura cytoplasmic sterile male
(left) and fertile (right) flowers contained
in a Japanese wild radish community.
Arrows display sterile males (left) and
viable anthers (right). Bar Scale = 1 cm
(Bannerot et al., 1977, Bonnet, 1977)
This CMS was introduced into European
radish although not well used in radish
breeding in Japan. Several European
radishes have an Rf gene (Bannerot et al.,
1977, Bonnet, 1977) while Ogura (1968)
found that there is no such gene in
Japanese cultivars. European scientists put
Ogura CMS into B. Napus by intergeneric
hybridization and re-crossing (Bannerot et
al., 1974, Heyn, 1976).
Ogura CMS (orf138) upstream of
orfB(open reading frame) protein was
defined by the Napus line (Bonhomme et
al., 1991, 1992, Grelon et al., 1994), now
recognized as atp8 (Hazelwood et al.,
2003). In male-sterile plants, Orf138 was
co-transcribed with the atp8 (orfB). At
least nine nucleotide sequence variants
were identified for orf138, including one
with the deletion of 39 nucleotides.
It is noted that in mitochondria targeting
and nuclear expressions of ORF138
protein did not result in the induction of
male sterility in Arabidopsis thaliana,
although cause alternation in the
mitochondria of yeast cells and plants
(Duroc et al, 2006). Orf138 is commonly
present in Japanese wild radish whereas
other cultivated radishes have normal type
of cytoplasm (Yamagishi et al., 1994).
Few of the European varieties of radish
lines have an Rf gene for Ogura
(Yamagishi, 1998), while Brassica species
doesn’t have Rf gene in them because Rf
gene is important for the production of F1
hybrids by CMS system for proper setting
of the crops. So, introduction of Rf (Rfk1)
gene is carried out in the Brassica species
by using R. Sativus thorugh protoplast
fusion (Sakai et al. 1996).
This Rfk1 gene is cloned independently
and simultaneously by three
(Brown et al. 2003: Desloire et al.2003:
Koizuka et al. 2003). Lydiate and Bett
reported three Rf genes in radish.
This is important to notice that some CMS
genes do not include the
mitochondrial gene sequences since some
CMS-associated ORFs are comprised of
sequences of putative mitochondrial ORFs.
Furthermore, certain CMS genes (for
example Orf125 in CMS Kos and its
counterpart Orf138 in CMS-Ogura
Brassica) are non-chimeric genes with
single-source sequences (Chen, L., & Liu,
Y. G, 2014).
o Discovery of Ogura CMS in radish
(Ogura 1968)
o Introduction of CMS genes by
intergenic hybridization into
Brassica (Bennerot et al. 1974)
o Use of cell fusion for chloroplast
substitution (Pelletier et al. 1983)
o Molecular cloning
 CMS gene: Orf 138
 Rf genes: Orf 687
Figure: 2 Histories of CMS on Ogura:
It was observed that CMS genes induce
mitochondrial dysfunction by intervening
with the mitochondrial membranes,
reducing the mitochondrial ATP level, and
the reactive oxygen content of organisms.
Among seven plant species, for instance,
groups
RF (Rfk1) among radish and brassica, nine
Rf (Restorer of fertility) genes have been
identified (Chen, L., & Liu, Y. G, 2014).
The majority of the Rf genes described
encode pentatricopeptide
actual (pentatricopeptide repetition) protections,
while Rf genes are also highly selective,
and different pathways of genomic, after
transcriptional, translational, or metabolic
rates for the respective recovery of fertility
in CMS / Rf systems can be introduced.
• Use of CMS for F1 breeding in
European radish
• Establishment of alloplasmic CMS
lines in Brassica
• Establishment of practical CMS lines
in Brassica
• Marker-assisted selection of the
genes
Stable CMS crop lines could be generated
through interspecific/intergeneric
hybridization of the alien cytoplasm’s
from the relatives or through mediation for with B. Tournefortii, the upstream
mitochondrial rearrangements via somatic mitochondrial rearrangement of the atp9
fusion. gene generated a chimeric orf193 co-
transcribed with atp9. But yet another
For Brassica crops, since cytoplasm-
Brassica Napus alloplasmic CMS
inducing sterility identified in a wild
Tournefortii had a chimeric orf263 near
Raphanus sativus population (Ogura
the atp6 by sexual hybridization of the
CMS) has been introduced into Brassica
same species. Recombinant mitochondrial
Napus & Brassica Oleracea (Bannerot et
genomes also have been produced when
al., 1974), a spectrum of alloplasmic CMS
fertile and sterile cytoplasm protoplasts
lines of different origins was obtained by
have been merged (Kang, L. et al., 2014)
combining the Brassica cenospecies
cytoplasm with crop nuclei, especially in Comparative research found that CMS
Brassica Juncea (Yamagishi, H., & Bhat, associated genes were clustered at the edge
S. R, 2014) (Pua, E. C., & Douglas, C. J., of the syntenic sequence fragments, along
2004) with short repeats and overlapped
repetitions from the mitochondrial
Protoplast fusion or somatic hybridization
genomes of CMS samples. A comparative
is a potential solution for gene transfers
study revealed that such repeats
from wild relatives to crops by merging
contributed to the reorganization by
two distantly linked genes, genus and
homogenous recombination of
tribal genomes, with both nuclear and
mitochondrial genomes. When the parental
cytoplasmic genomes. Chloroplasts are
mitochondrial fused and the recombination
typically inherited from one of the parents
occurred, homologous sequences from the
in somatic variants of protoplast fusion,
fusion parents were overlapped (Sanchez‐
whereas mitochondrial DNA (mtDNA) is
Puerto et al., 2015).
reordered and can include both parents'
DNA .High frequency intergenomic The alloplasmic pol CMS network of
mitochondrial recombination was found to naturally occurring mutations in Brassica
occur in different somatic hybrids from the Napus is still widespread in China for the
combinations of different species within cultivation of hybrid rapeseed, but in
Brassicaceae (Leino. M, et al., 2003) Europe and Canada, it is commonly used
in alloplasmic  Ogura CMS-fertility
At the Brassica napus    Tournefortii-
restoration systems. While many CMS
Stiewe CMS produced by protoplast fusion
systems have been published, the
undesirable CMS phenotype and the
absence of fertility menu genes are not
commercially accessible (Du, et al., 2009).
Polima (pol) CMS is divided into basically
three lines discovered by Agriculture
academy in Hunan. These three lines are
divided on the basis of temperature
sensitivity of male-sterility includes high
temperature CMS line, stable CMS line,
low temperature CMS line ( Fu Tingdong
et al.1989). Pol CMS is originated from a
European variety found in Brassica Napus
and its Rf gene was found in European
stock of B.Napus.
Origins of CMS and the mitochondrial
genes that induce male sterility:
When a nuclear genome is combined with
CMS-inducing mitochondrial genome
which doesn’t have an Rf gene, a
phenotypic expression is showed by CMS
or CMS expressed in a phenotypic fashion.
Such combinations can occur
spontaneously by mutating in the
mitochondrial genome, spreading within a
species or artificial hybridizing. The above
involves interspecific or intergeneric
sexual crosses and cell fusions. Within this
segment, Brassicaceae CMS is defined by
its origins.
 intraspecific modifications,
 interspecific or intergeneric
hybridizations of alloplasmic type,
and
 Cell fusion.
1-CMS caused by intraspecific
Modifications
Polima (pol) CMS B. Napus is another
prominent example of male spontaneous
sterility. Because Pol CMS is temperature
responsive, its practice in F1 hybrid
cultivation is limited, but it has been well
established on a molecular basis. The pol
CMS genome includes the mitochondrial
genome of the orf224 protein, a chemical
protein situated at the center, and co-
transcribed with the atp6 gene (Forsberg,
J., Landgren, M., & Glimelius, K., 1994).
The orf224 / atp6 therapy is impaired in
the presence of the Rf gene; transcriptions
may be altered directly, meaning that
orf224 is the source of the pol CMS
mutation.  Orf224 specifically was
integrated into B.  Oleracea through
somatic cell fusion mediated male sterility.
The protein structure encoded through
orf222, which is responsible for nap CMS
(another example of a normal
mitochondrial form in Brassica napus), is
identical to that of the orf224 protein
component, while Brassica Varieties of
napus with male sterility and orf222
location in the mitochondrial genome
varies from orf224 (L'Homme, & Brown.,
1993). Many forms of random CMS form of CMS referred to as DCGMS
include 681A at Brassica Napus came (Dongbu cytoplasm and genic male-
from cv. Xiangyu and Juncea 681A CMS sterility) identified the causal gene as a
maintainer and restorer lines are the same novel chemical element, orf463, consisting
as those for pol, but 681A lines vary in of a partial coxI sequence of 128-bp and an
their mitochondrial genome structure from unknown core sequence of 1261-bp (Wan,
pol lines, indicating that 681A may be a Z. et al., 2008).
subset of pol CMS (Gobron, et al., 2013).
Two dynamically connected accessions of
On the other side, hau cytoplasm restorers
A, Thaliana were hybridized and obtained
have not yet been established and pol or
male-sterile strains whose history of
nap restorers struggle to save hau mediated
inheritance indicated CMS in reciprocal
male sterility.Another innovative gene of
crosses. (Lee, Y et al., 2008) Analysis of
the CMS, orf288, was discovered in hau
the two accessions' mitochondrial genome
CMS, co-transcribed with atp6. Orf288
showed that the causal gene for this CMS
was cytotoxic to E.Coli and Male Sterility
was orf117. The expected peptide ORF117
Induced in A. Thaliana expressed with or
is 56 percent identical and 69 percent
without the mitochondrial targeting
equivalent to the Moricandia
sequences from the nuclear genome
arvensis( Purple mistress) ORF108
(L'Homme, Y. et al., 1997)
peptide, which causes male sterility in
It was further shown that even when not Brassica Juncea ( Park, et al,2013).
fused with mitochondrial targeting pre-
Alloplasmic CMS leading to
sequence, ORF288 protein was targeted
interspecific and cross-generic
towards the mitochondria. Such results
hybridization
indicate that the causal gene for hau CMS
could be orf288. (Jing, B. et al., 2012). In Brassicaceae, several male sterile lines
have been formulated (see Table 1), after
The latest findings on R. Sativus identified
the formation of CMS lines carried out by
CMS forms other than Ogura CMS.
crossing B. Rapa with Diplotaxis muralis
Another was contained in Korean radish
(Hinata and Konno., 1979).
specimens named NWB CMS; the Rf
genes for this CMS have not yet been Cytoplasm of at least eight types cause,

identified. One was contained in CMS in B. Juncea, the most important

Uzbekistan's radish. An analysis of the full oilseed crop in South Asia (Banga, et al.,

mitochondrial genome sequence of this 2003).

 TABLE 1: Combination of cytoplasm
and nucleus in alloplasmic CMS to
produce CMS plants by cell fusion
a :Confers CMS in several species of
Brassicaceae
b: used for the production of CMS
plants through cell fusion
Brassica Juncea, and Brassica Napus
possess orf108 upstream of atpA. Also,
CMS was acquired in Brassica Juncea
arising from interspecific hybridization of
Brassica Rapa et Brassica Juncea; later
this CMS was transferred to the mustard
leaf, mustard tuber (Brassica juncea var.
tumida) and mustard stem (Brassica

As seen in Table 1, five distinct organisms, juncea) (Kumar, et al., 2012).

D. Muralis, D. Sifolia, B. Oxyrrhina, and Three cytoplasms that grant CMS to B.

B. Turnefortii, and Enarthrocarpus lyratus Juncea (D. berthautii, D. Catholica, and
are effective as male-sterile cytoplasm D. erucoides) typically possess orf108
donors for more than one cultivated upstream of atpA as described earlier and
species (Deol, et al., 2003). co-transcribe the two proteins analyzes of

Male sterility in species B. rapa, speech proposed orf108 as the causal gene

B.Olaracea, and B. Napus is induced by in some CMS origins. Orf108 has also

cytoplasm of D.Muralis. The cytoplasms been used in sterile B. Juncea for males

that confer CMS in Brassica Rapa, (Yang, et al., 2005).

Juncea produced by the fusion of cells

Nucleus Cytoplasm Recipient Donor
with Brassica Juncea, M. Arvensis (Mint).
Source Source
The CMS Brassica Juncea axis of somatic
Brassica B. B. Napus A.
Napus tournefortiiab, thaliana, hybridization with M. Arvensis kept the
D. Muralisa, B. mitochondrial genome unchanged from M.
D. Siifoliaa Tournefo- Arvensis which contains orf108 (Prakash
rtii et al., 1998). The finding is the first

B.Juncea B. oxyrrhinaaa Diplotaxis demonstration of a common molecular

B. tournefortia, Catholica mechanism that underlies CMS in different
D.Catholicab lines of origin (Kumar, et al., 2008).
B.Olerace D. Muralisa, B.Oleracea Arabidop
a M. Arvensisab -sis In comparison to the organisms described,
thaliana orf108 has also been detected in many
B.rapa Dilpotaxis B.rapa Raphanus
wildlife populations including Brassica
Muralisa, Sativus
Oxyrrhina (Coss), Sinapis Alba (White
B.Oxyrrhniaa,
Moricandia
Arvensisab
Raphanus B.Maurorama
Sativus
Mustard), En. Lyratus, D. Tenuisilque ARVENSIS & R. Sativus, which were used
indicative of its broad distribution. Orf108 in sexual hybridization as cytoplasm
was missing at D. Muralis, Siifolia& D. donors, cytoplasm’s of A.thaliana,
Cretacy. Hence, orf108 tends to be of orychophragmus violaceus, B. oleracea
maternal origin but is missing or changed Sinapisarvensis and trachystomaballii
in certain lineages (Landgren, et al., were shifted to B. Napus, B. Juncea by
1996).The latest discovery in A, somatic cell fusion in order to get CMS
thaliana of    CMS-associated orf117 with (Yamagishi, and Landgren, 2002).
a 69% homologation to orf108 (Forsberg
Whereas alloplasmic CMS lines acquired
et al., 1994), the opinion is further
via sexual hybridization inherit the female
endorsed. The causal gene in CMS lines
parent's mitochondrial genome, somatic
Brassica was presumed to be Orf263. Both
hybridization that gives rise to novel
Mitochondria of D. Muralis, which
recombined mitochondrial genomes
induces CMS in three species of Brassica
derived from both parental organisms,
(Table 1), a novel chimeric gene, orf72,
creates complication in the identification
located downstream of rps7 and
of causal CMS genes. Up to now, two
comprising an atp9 fragment, was
genes exhibiting the property of
described. A comparison of gene
identifying the contrasting features in
expression and pollen fertility shows that
relation to the CMS genes on alloplasmic
orf72 was correlated with CMS (Shinada,
lines have been established (Dieterich, et
et al, 2006).
al., 2003).
2-CMS plants obtained through cell-
Progeny obtained from somatic hybrid
fusion
between M. Arvensis and B. Juncea
Cell fusion does not involve fertilization, showing that CMS had mitochondrial
as compared to sexual hybridization, and genomes and chloroplast genomes similar
therefore a significantly broader variety of to those of M. Arvensis, without
hybridization organisms may be utilized. recombination. This line of CMS had
Wild species that were not used in orf108 as the causal gene upstream of
interspecific and intergeneric sexual atpA transcripts, similar to alloplasmic
hybridization were used to create male plants obtained through sexual
sterile lines for cell fusion (Yamagishi, hybridization. Somatic hybrid progeny
and, Nakagawa, 2008). Together with B. between B. Tournefortii and B. Napus had
D. Turnefortii and Catholica, M.
upstream of atp9, orf193 (Kirti, et al.,
1995).
Cell fusion and sexual hybridization
culminated in separate genes of CMS,
orf193, and orf263, of the same parental
mix (Carlsson, et al, 2007).
An expansion of a male-sterile line at B.
Juncea was confirmed to be attributed to
mitochondrial recombination. Plants of the
cytoplasm of Ogura and B. Juncea nucleus
had petaloid anther flowers and reduced
reproduction in females (Leino, et al.,
2004). A completely female-fertile,
anther-sterile line of CMS was produced
through somatic hybridization. It has been
proposed that a difference in the
mitochondrial coxI-2 gene induces CMS in
a cell fusion between B. Juncea& D.
Catholica, though this has not been
proven. There was found a recombined
mitochondrial genome in Brassica
olarecea CMS napus lines derived from a
somatic hybridization. CMS lines are
defined by contrasting the structure of the
mitochondrial genome and gene
expression in restored lines of male
sterility, maintainer, and fertility ( Leino,
et al., 2003) A mitochondrial gene
interaction with CMS is suspected because
the gene is expressed only in the male-
sterile plant but is silenced in the restored
fertility plant bearing a nuclear Rf allele.
(Table 3) lists the causal genes of CMS in
the crops identified to date in
Brassicaceae. Twelve genes from ten
groups of initial donor cytoplasm are
cloned. Among the genomes, eight have a
chimerical composition composed among
parts of identified mitochondrial gene and
uncertain reference sequences (Teixeira, et
al., 2005).
3-The CMS Rf genes
The F1 hybrid will be male fertile to
ensure a suitable collection of seeds as
CMS is introduced for the F1 hybrid
breeding of oil-seed crops (B. juncea and
B. napus) (Heyn, 1976). Therefore, CMS
needs to discover Rf genes which are
capable of supplying male fertility to CMS
plants. Rf genes are usually found among
the germplasm lines of this species
intraspecific cytoplasmic variants. Rf
genes are not commonly present in
recipients organisms in alloplasmic line
CMSs resulting from sexual or somatic
hybridization and must be obtained from
animals serving as cytoplasm donors
(Kirti, et al., 1998).
For the CMS-inducing cytoplasm
including Ogura (Heyn, 1976), Rf genes
were successfully transferred from other
organisms into cultivated Brassica habitat
(Ballii (1997 Kirti et al.), (M. Kirti et al.,
1998).
Fertility regeneration is sporophytic on by pol CMS and nap CMS genes have
most CMS lines (i.e. 100 percent pollen- identical structures, as described above.
fertile F1 hybrid plants) (Bhat, S. R., The nap (Rfn) and pol (Rfp) Rf genes are
Prakash, S., Kirti, P. B., Kumar, V. D., & allelic. And both influence mRNA
Chopra, V. L, 2005) however, in CMS transcription of the CMS genes by causing
lines (M. ARVENSIS, D. Erucoides& D. the elimination of the 5′-end transcript
Berthautii), the restoration of reproduction sequences (Chamola, et al., 2013).
is gametophytic, i.e. only Rf gene-carrying
Throughout alloplasmic B, Rf genes of
pollen is usable and thus F1 hybrid plants
diverse sources have a shared role. As
are 50% pollen-fertile. Therefore, male
already mentioned in B. Juncea, four CMS
sterility and reproduction do not
lines have a shared gene for CMS (orf108
distinguish the self-progeny of
in Table 2) and share a similar gene for
gametophytically restored plants.
restoration. The bi-cistronic orf108-at
transcript is cleaved only downstream of
the start codon of orf108 in the presence of
the restore, which results in the
development of mono-cistronic atpA
transcript (Ashutosh et al., 2008, Kumar et
al., 2012). Such results are remarkable
because they demonstrate that the rising
genetic pathways of CMS and restore
Pollen reproduction recovers sporophytic
fertility have developed and are shared by
(left) and gametophytic (right) forms.
different organisms and genera. For Ogura
R;Active allele restorer, r; Non-functional
CMS, orf687, the Rf gene does not
allele restorer.
influence the transcription of theorf138,
Relevant from researching co-evolution nor controls translation of orf138 mRNA.
between mitochondrial and nuclear In wild Japanese radishes, where Rf genes
genomes are the interactions between the are popular for Ogura CMS, the
mitochondrial CMS genes and their occurrence of orf687 plants is small.
counterpart Rf genes. These tests were Another Rf gene (Rft), contained in most
carried out for pol and Brassica napus wild radishes in Japan, controls the
CMS, B sections with alloplasmic CMS. production of orf138 mRNA. This
Juncea and Ogura CMS, proteins encoded discovery suggests that Rf genes
developed in radish with various molecular mitochondrial genomes.  Incompatible
pathways to suppress the expression of plastids that induce leaf chlorosis in CMS
orf138, the causal gene of Ogura CMS. have a negative impact on B. Rapa and B.
Juncea and B. Ogura napus, B.
Table: 2
Oxyrrhenia, M. Arvensis cytoplasm, and
Genes Adjacent Origin chloroplast replacement have already been
genes discussed above as an approach to solving
Orf138 atp8 R.Sativus this issue.
(Kosena)
Orf222 nad,5c,orf13 B.Napus(nap) CMS lines of B. Juncea with the Ogura,
9 D. Catholica, and T. ballii cytoplasm
Orf125 atp8 R.Sativus shows low female fertility (Teixeira, et al.,
(Ogura) 2005). Expression levels of meristem
Orf224 atp6 B.Napus(pol)
identity and homeotic genes are modified
Orf72 rsp7 D.Muralis
by nuclear–mitochondrial interactions in
Orf108 atpA D.erucoides,
alloplasmic male sterile lines of Brassica
D.catholica,
napus (The Plant Journal, 42(5), 731-
M.Arvensis (by
742). For certain instances, only certain
cell fusion)
mitochondrial genes were analyzed that
Orf193 atp9-2 B.Tournifortii
are specifically correlated with male
Orf263 atp6 B.Tournifortii
sterility. While the CMS lines often
Orf220 atpA B.Rapa
modify the expression patterns of several
Orf288 atp6 B.Rapa
other mitochondrial genes, the
implications of these modifications and the
4-Development of cytoplasm, retrograde function of Rf genes in the repair of certain
manipulation, and polymorphic plant defects have barely been studied. The
phenotypes connection drag associated with the
introgressed Rf gene is another
Availability of alien CMS systems in
consequence that may come into play in
Brassicaceae crops poses the issue of
alien CMS systems.
adverse effects correlated with alien
cytoplasm, as the implementation of CMS While CMS was linked to similar
for commercial hybrid production will mitochondrial genes, numerous floral
entail the absence of any such effects that phenotypes induced by the same
may be attributed to plastid or cytoplasm have not been thoroughly
investigated in different organisms. For continue to be elucidated. Studies
instance, even if orf108 causes CMS in B. mentioned above indicate that CMS can
Juncea lines which carry D. Catholica, D. have common molecular structures in lines
Berthautii and   D. erucoide or M. arvensis of varying origin. There may also be a
mitochondria and all of these CMS lines close similarity in the Rf genes (Yang, et
are restored by a popular restore, D. al., 2008)
Catholica cytoplasm results in petaloid
On the other side, several means of
anther, multi-locular silica, and low female
restoring fertility by the development of
fertility while others result in
various Rf genes are often apparent (as in
gametophytic pollen sterility only (Dong,
the case of Ogura CMS). The
et al., 2013).
developmental interactions between CMS
(Yang et al., 2008) showed that CMS and Rf genes are required to be explained
phenotypes can be partly mimicked by in more comprehensive and integrated
treating plants with mitochondrial electron research. These studies will also help to
transport chemical inhibitors. Hence, develop effective hybrid F1 breeding
owing to the expression of CMS-inducing systems in crops from Brassicaceae
mitochondrial genes in different nuclear (Primard-Brisset, et al)
contexts, polymorphic phenotypes are
Hybrid reproduction and male sterility
likely to arise from variations in the energy
state of cells. In the 1860s, Charles Darwin first
identified heterosis in maize, and by
While novel mitochondrial orfs have been
crossing different strains of pea plants
discovered causing male sterility, their
Gregor Mendel developed a genetic
particular mode of evolution and
foundation for inherited traits.
importance beyond male sterility has not
Nevertheless, maize heterosis started to be
been critically examined. For example, the
utilized only in the 1920s and the first
Texas CMS often allows plants susceptible
hybrid crop farm sold was maize.
to Southern corn leaf blight pathogen
Cochliobolus heterostrophus in cotton, and The hybrid reproduction systems of other

the restorer gene does not rectify this crop species have recently been introduced

adverse impact. Likewise, molecular such as rice ( Oryza sativa), garlic

aspects of the sorting by the nuclear Rf (Hordeum vulgare) and Rapeseed

genes of CMS-associated transcripts or (Brassica napus), which contribute

proteins in the Brassicaceae crops tremendously to increases in farm yield,

because hybrid plants have superior agricultural practice to increase the quality
characteristics such as improved of hybrid seed production and the
adaptability, increased standardization, and productivity of crops.
improved abiotic and biotic stress
Restoration of Fertility and CMS lines
tolerance. The usage of heterosis
contributed to an rise in the production of Cytoplasmic male sterility (CMS) is

wheat in the region of around 3.5–15 phenotypically represented as

percent 11% of wheat, 55% of corn, 47% nonfunctional pollen grain development

of typical bean (Proteus vulgaris), 68% of when paired with the absence of a

foxtail millet (Setaria italica) and 200% of functional male fertility restorer (Rf)

Brassica oils. Cross pollination of parental gene(s) by a CMS cytoplasm (Yamagishi

lines with genetic substance is essential to et al. 2014). The CMS phenotype

generate hybrid seed. Diversity is also a inheritance occurs in mothers because the

precondition for the usage of male-sterile CMS mitochondrial genome has a

female lines for self-pollining plant plants. sequence-different fragment of the DNA

Additionally, for large-sized usage as a of the normal genome. CMS was present

male sterile crossing partner the male in more than 150 plant organisms and is a

sterile plants have to be stored and result of random or unnatural mutations

propagated. Furthermore, in an F1 hybrid (Kim, Y. J et al. 2018).

generation, male plant sterility should be At the same time, diverse Rf loci were
overcome in order to achieve greater yield found in different plants, each with a
for seeded crops. certain type of sterile cytoplasm repressed

Although many methods have been by Rf. 38 CMS lines have been studied in

utilized to establish male-sterile feminine 28 inbred contexts for the restoration of

lines, including emasculation and chemical fertility, and are categorized into CMS-C,

therapy, the usage of natural male-sterile CMS-S and CMS-T in maize by their

lines is best used for large-scale hybrid reaction to specific restaurant genes

seed growth. This is an update on the (Bosacchi, M. et al. 2015). In many of the

molecular regulation of male fertility used cultivated plant species, the three-line

in the development of hybrid seed with the hybrid breeding system (the maintainer

goal of applying fundamental knowledge line, the CMS line, and the restorer line)

of plant biology on this subject and employing the CMS line has been

offering more tools for improving established successfully for hybrid seed
production in the CMS series and the normal functioning of mitochondria such
restorer line containing the RF gene(s) as the tricarboxylic acid cycle and the
(Figure). generation of ATP. The identified CMS
DNA sequences encode small, 10–35 kDa-

Maintainer Restorer CMS sized and cytotoxic transmembrane

proteins. Accumulation of Spatiotemporal-
rf rf rf rf Rf Rf
specific aggregation inside the tapetum of
cytotoxic CMS proteins, the innermost

Male somatic portion of the anther wall or

Fertile Fertile
sterile microspores also induce altered
CMS F1 Hybrid
mitochondrial activity, such as ATP
rf rf rf Rf synthesis and abnormal programmed cell
death (PCD) in sporophytic or

Male sterile Fertile gametophytic cells, contributing to male

Previously, numerous
mitochondrial DNA sequences in CMS
lines and distinct Rf genes in the restaurant
lines have been cloned and functionally
defined in crop plants such as maize, corn,
brassica and sunflower
annuus) (reviewed in). Recent research has
been based on the latest identification of
CMS / Rf genes in rice, wheat and brassica
and their evolutionary implications are
addressed.
sterilities during microspore growth ( Chen
L, et al; Yamagishi et al. 2014) .
mutated
Among 13 crop species, 28 CMS genes
have been identified so far. Seven forms of
CMS lines occur in rice: CMS- BT (Wang,
Z. et al.2006), HL, LD, CW, WA, RT102,
and RT98, of which CMS-WA was
(Helianthus
commonly used for breeding. Chemeric
mitochondrial open reading frames (ORFs)
responsible for male sterility were
identified using mitochondrial
transcriptomic and proteomic and genetic
approaches, including orf79 from CMS-
BT (Wang, Z. et al.2006) and CMS-LD

Mitochondrial Genes as CMS (Kazama, T. et al.2016), orfH79 from

controllers: CMS-HL (Wang, K. et al. 2013), orf352

from CMS-RT102 (Okazaki M. et
Mitochondrion is a semi-autonomous
al.2013), and WA352 from CMS-WA
organelle containing its own genome
(Luo, D. et al 2013).
encoding genes that are essential for
Orf79 and its orfH79 version encode a
minimal protein (79 amino acids) that
includes a COX1-like N-terminus and is
co-transcribed with the upstream ATP6
gene encoding part of the mitochondrial
ATPase complex (Kazama, T. et al.2008).
More than 20 times the toxic orf79 protein
aggregation in CMS-BT relative to orf79
in CMS-LD induces extreme
retained mitochondrial sequences, as well
as by substoichiometric shifts and
sequence adjustments.
Orf352 (WA352a) and WA352c have five
single nucleotide polymorphisms (SNPs)
and contain the dangerous protein 352
amino acid. Orf352 is regarded as being
pollen Generation of Orf352 is carried out by the

abortion in CMS-BT, indicating a link recombination of orf284 with O.

between the amount of orf79 protein and Rufipogon (Brown bread rice) and some

the CMS phenotype (Kazama, T. et unknown sequence of another species of

al.2016). The transmembrane cytotoxic Oryza.

proteins encoded by these mitochondrial Figure: 3 (Kim, Y.-J., & Zhang, D.

chimeric ORFs can interact with (2018)
mitochondrial protein complexes such as
COX11, which induce mitochondrial
dysfunction by impairing the respiration of
oxygen and generating ATP in anther
tissue (Wang, K. et al. 2013: Luo, D. et al
2013: Ding, X. et al.2016)), .

Forming new mitochondrial CMS genes in

plants may facilitate outcrossing and
improve fitness but it remains widely
unclear how the mitochondrial CMS genes
develop and evolve. (Tang H. et al. 2017)
studied recombinant genomic
mitochondrial structures associated with
WA352c (or WA352) in wild and
cultivated rice, and indicated that WA352c Interestingly, two non-functional but

may be produced in the wild rice expressed protogenes were demonstrated

mitochondrial genome (Oryza rufipogon) and thought to have that much potential of

by numerous rearrangements between evolving through sequence diversification

into functional CMS genes. These genes (Murai, K. et al. 2002). The PCMS is
evolved from sequence variation might regulated by long-term conditions (> 15 h)
have experienced selection during the at the flower differentiation stage which
course of evolution by allowing the indicate homeotic stamina transformation
WA352c-related proteins to perform into pistilloid structures, which may be
interactions with nuclear encoded COX11, induced by unprocessed mitochondrial
mitochondrial-localized proteins which orf25, and the phenotype may be restored
can cause premature male sterility and on chromosome7B by the Rfd1 locus
tapetel PCD (Kim, Y. J et al. 2018) New (Murai, K. et al. 2002). It also remains to
CMS genes may also be developed be known that LD facilitates the
through genome evolution, duplication, development of the floret gene(s) and
protogenic development, multi- CMS genes.
recombination and more versatility
Compared to the finding of monocotype
through incremental systemic
plants, the CMS lines from dicotype plants
diversification, sequence, and copy
such as sunflower, brassica, and carrot
number (Tang H. et al. 2017) (Figure 3).
(Daucuscarota) were shown to be induced
The problem of the absence of effective by chimeric mitochondrial ATP8 genes
fertility-restoration genes and the toxicity ( Chen L, et al 2014), like orf138,
and selectiveness of chemical hybridizing identified by CMS-Ogu and commonly
agents arises with the use of the used in the hybrid breeding of Brassica
conventional CMS and male genes for crops.
hybrid wheat breeding. In order to
Orf288 of mustard (B. juncea) CMS-Hau,
overcome these limitations, (Murai, K. et
orf224–atp6 of CMS-Pol and orf222 of
al. 2016) proposed the development,
CMS-Nap, orf463 of radish (Raphanus
through the use of photo-opera sensitive
sativus) CMS-Don, and orf725 of onion
cytoplasmic male-sterile (PCMS), of two-
(Allium cepa) CMS-S findings indicate
line hybrid seed production systems in
that plant CMS phenotypes are caused by
common wheat (Triticum aestivum). In
multiple rearrangements of the
contrast to rice the cytoplasm of a related
mitochondrial genome resulting in the
wild grass, goat grass, (Aegilops species)
formation of functional CMS genes.
show photoperiodic sensitive phenotyping
Overall, the emergence of CMS genes
of CMS lines, several CMS wheat lines,
from both monocotype and dicotype plants
like CMS-Norin (Murai, K. et al. 2016:
is supposed to establish distinct heritage
through foreign pollination which
enhances genetic changes (Kim, Y. J et al.
2018).
Rf genes of Brassica Napus:
In Brassica Napus, two types
endogenous male cytoplasm are present
named as nap and pol. Number of studies
are carried out that reveals that nuclear
restoration of polima CMS is regulated by
a gene known as Rfp which is also used as
modifier for modification of pol-CMS
transcripts associated with orf224/atp6
mtDNA (Li, X, Q. et al. 1998). A number
of Brassica Napus species possess nap
cytoplasm which is male-fertile due to the
presence of restorer gene for nap CMS
represented as Rfn (Fan, Z.et al. 1986).
Nap nuclear restorer gene is quite similar
to Methionine S-methyl transferase (Mmt)
which is a gene that governs
So, if a comparison is carried out of CMS
in Brassica species and other plants, it can
be easy to restore different forms of
Brassica CMS by alleles having a single
nuclear locus. In a survey reported by (Li,
of X, Q. et al. 1998) some species of Sinapsis
and Brassica failed to show any evidence
regarding Rfn(Mmt) in fertility restored,
nap cytoplasm which means that in
Brassica napus Rfn(Mmt) arose with nap
cytoplasm.
A number of Brassica Napus species lack
restorer gene for Polima CMS (Fan, Z.et
al. 1986). The table represented below
represents a relationship between
cytoplasm of restorer genes of Brassica
Napus and their sterility and fertility
Table:
Restorer genotype and fertility status
the Cytoplasm rfn,rfp Rfn,rfp rfn,Rf

modification of different transcripts from p

Nap Sterile Fertile Sterile
mtDNA regions, includes one nap CMS Pol Sterile Sterile Fertile
associated DNA and contain orf222, and a
chimeric gene. In some varieties of plants different types

So, researchers found that Mmt is allelic to of CMS are present and can be

Rfp which means restorer genes for both differentiated by their nuclear Rf genes,

cytoplasms epitomize different alleles of a and novel mitochondrial ORFs which

single locus by restriction fragment length restore their fertility.

polymorphism mapping. This study clearly Rf Genes for CMS lines:

reveals that Rfp and Rfn map on the same
Rf genes from the lines of the reinstate are
position of chromosome (Li, X, Q. et al.
nuclear genes that encode proteins located
1998).
to mitochondria which play a reactionary
regulatory role by suppressing CMS
deformities. Until now, nine crop plants
have isolated 14 Rf genes. Probably half of
the identified Rf genes encode
Pentatricopeptide repetition (PPR)-
proteins located to mitochondria
plastids (Chen L, et al 2014). The
detrimental effect of CMS proteins on the
implementation of tapetals and pollen can
be saved by the interaction between CMS
and Rf members via different mechanisms
(Chen L, et al 2014). At the post -
translational modifications step,
products may also change the transcripts of
CMS genes in mitochondria by gene
editing, splicing, polyadenylation, and/or
cleavage. In CMS-BT rice, RF1A attaches
and scatters dicistronic transcripts ATP6–
orf79 in the intercistronic region and
RF1B strongly affects degradation of the
transcript ATP6–orf79 (Wang, K. et al.
2013).
Likewise, Rf4 in CMS-WA and Rf1b in
CMS-BT work to lower rates MRNA
(Tang H. et al. 2014). WA352c in CMS-
HL rice, atp6–orfH79 transcript cleavage
by RF5 (also named RF1A) and RF6
provide multiple stimuli, i.e. the glycine-
rich protein GRP162 and OsHXK6,
respectively (Huang, W.C. et al. 2014: Hu,
J. et al. 2015). The transcript cleavage of
orf224 in CMS-Pol and orf222 in CMS-
Nap is constantly delivered by Rfp and
Rfn (Liu, Z.et al.2016: Menassa, R. et al.
1999).
A non-PPR Rf gene, Rf2, encrypts a
glycine-rich domain-targeted
mitochondrial protein that encourages
or
fragmentation of orf79 RNA in both CMS-
LD and CMS-BT lines (Chen L, et al
2014). Translational or post-translation
inhibition may also recover the vitality of
many CMS processes. Rf0 (also named
Rfk1) encrypts PPR-B (or ORF687) which
links and suppresses the translation of
Rf
orf138 mRNA in Brassica's CMS-Ogu and
radish's CMS-Kos. Rf3 for CMS-WA rice
does not impact the WA352c mRNA
stage, but does obstruct its protein
aggregation (Luo, D. et al 2013).
Another non-PPR type Rf, BvORF20 in
sugar beet, encodes an OMA1-like
mitochondrial protein, a membrane-bound
metallopeptidase from yeast and other
eukaryotic organisms, and communicates
with the preSATP6 CMS protein to
modify its transcriptional architecture.
PreSATP6's 250 kDa homo-oligomer,
harmful to meiotic anther, detaches in the
anther in the presence of BvORF20 and
this can reestablish fertility (Kim, Y. J et
al. 2018).
The first Rf gene separation, corn Rf2 fully
recovered CMS-T, which encrypts an
aldehyde dehydrogenase detoxification
alcohols and toxins, is the metabolic
transformation of CMS lines. Amusingly,
Rf5 and Rf6, many also recognized from
CMS-HL tincture, have been able to
rescue other types of CMS rice. Rf5 can
restore CMS-BT rice fertility, as can Rf1a
from CMS-BT, and Rf6 can restore
fertility in CMS-BT lines as well, implying
that preserved underlying mechanisms for
Rf male fertility reconstruction actually
happened in different lines prior to
interbreeding (Kim, Y. J et al. 2018).
In both Rf5 and Rf6 hybrid plants, male
fertility in CMS-HL is recovered more
stable than plants that carry only one Rf-
gene. The hybrid line Rf6 restoration is
stated to be more reliable in CMS under
conditions of extreme heat
Restoration of the hybrid liner Rf5
This discovery suggests that Rf5 and Rf6
use specific pathways in male fertility
restoration, which is compatible with the
hypothesis that multiple interacting
pathways have been formed between the
mitochondrion and nucleus to restore
fertility of CMS lines. Recently there were
evidence of sequence duplication and
homologous recovery in Rf-like PPR
clusters of Oryza chromosome suggested
rapid spread and diversification of PPR
genes. Additionally, some miRNAs were
predicted for rice, mustard and soybean
regulate Rf-like genes to modulate CMS
recovery, although further clarity is
needed, the utilization of a three-line
hybrid breeding facility therefore relies on
continuous identification in each targeted
species of CMS lines comprising recessive
Rf alleles and a subsequent Rf allele-
containing restorer line. Efficient and
reliable lines of restorers are yet to be
defined for other plants, such as wheat. In
fact, CMS genes and the related nuclear Rf
genes appear to have been coevolved,
raising a daunting problem of how
intracellular – nuclear genomes coevolve
(Kim, Y. J et al. 2018)..
CRISPR/Cas9 Mediated genome editing
in Brassica Napus:
In 1763 a German botanist named Joseph
Gottlieb observed male sterility in 610
species of plants (Letian Shan et al., 2013).
Abnormal development of sporophytic and
gametophytic anthers reported in the past.
The presence and absence of
mitochondrial plasmids confirm the
abnormal growth in the anthers. Cas9 is
highly specific in genome editing and
cutting through adenoassociated virus
(AAV) to induce targeted mutations. A
chemical hybridization agent basically
causes male sterility. A wide range of
reproductive abnormalities such as
degenerate anthers, aborted pollens,
carpelloid, and petaloid stamens was
observed in cytoplasmic male phenotypes
in the previous studies (chase, 2007).
CRISPR cas9 mediated genome editing
has been widely applied as a most potent
tool for the modification of preferable
endogenous genes. Restriction patterns of
various organelles in Brassica Napus
support an alloplasmic explanation for the
cytoplasmic male sterility system (CMS)
in this crop. (L Erickson et al. 1985) have
proposed a correlation between the
presence of a linear mitochondrial plasmid
and sterility in two CMS lines of Brassica.
Among them, a compestris line with
Raphanus cytoplasm and the other is a
Napus line identified only as being from
Korea. The plasmid occurs in a typical
Brassica Napus cultivar, which plays no
known role in any cms system and once
widely grown (L Erickson et al. 1985). In
the above conditions, the male
reproductive system fails to produce
dehiscent anthers and viable gametes, as
explained earlier.
(Jannia Bratz et al.2017) studied CRISPR
for Brassica Napus in order to transform
teraploid Brassica Napus with a Cas9
construct targeting two homologs named
as ALCATRAZ (ALC) as ALC is used for
the development of margins of valve so in
seed shattering it plays a vital role. In
Brassica ALC enhances the shatter
resistance in order to decrease seed loss in
mechanical harvesting. (Jannia Bratz et
al.2017) create a transgenic plant with 4
mutants alleles of ALC by using a single
target sequence and didn’t find any off-
target effects (Jannia Bratz et al. 2017).
Gene pool of Brassica Napus possesses
low genetic diversity (Bus et al. 2011). In
order to acquire desired novel mutations
introduction of CRISPR-Cas9 is observed
in the previous studies as its nuclease has
the ability to be easily programmed for
inducing double-stranded beaks within ant
target sequence (Doudna & Charpentier,
2014). CRISPR Cas9 system is reported as
system for target mutagenesis used for
reducing yield loss in Brassica Napus
because natural dispersal of B.Napus seeds
involves siliques (shattering of dry seeds
and fruits) (Jannia Bratz et al.2017).
Brassica Napus pods which are fully
matured are prone to open against any
internal or external force which results in
shattering of seeds. Another study carried
out by (Qamar Uz. Z. et al 2019) in
Brassica Napus revealed the role of
CRISPR-Cas9 in carrying out multiplex
genome editing on five homologs for
controlling the pod shattering resistance.
Pseudo seeds were generated and then
divided in to two halves but these mutants
didn’t have the ability to generate any
productive seeds in next generations and
mutated Brassica Napus by using JAG
genes which provide an initiative to carry
out mutations in other Brassicaceae
species (Qamar Uz. Z. et al.2019).
Male sterility mutants possess the ability to
cause uneven development of anther
tissues and functional pollens.
Incorporating male sterility into hybrid
seed production lowers its cost and also
ensures varietal purity. CRISPR cas9 was
employed in several cases reported in the
past to disrupt a putative strictosidine
synthase gene. Male sterility provides a
route toward crucial breeding to harness
hybrid vigor or heterosis in cytoplasmic-
nuclear genomic interactions. Hybrid vigor
or heterosis is a phenomenon in which the
progeny attained from a cross of two in
hybrid lines outperform the parent line
because they can produce 15-50% yields,
and it requires emasculation.
CRISPR cas9 system is now generated to
for genome editing to yield the desired
hybrid lines of brassica napus that is a
technique employed to introduce mutations
in DNA either by inserting or deleting and
making changes in the target sequence
(Hakim Manghwar et al., 2019).
Incorporation of male sterility in the
production hybrid seed was proved as a
great source of cost reduction and assures
the purity with high yield. Despite this
fact, male steriles were still not used as
good source for the production of Brassica
Napus hybrid seeds (Minmin Du et al.,
2020). Many of the researchers focused on
using the biotechnology-based breeding
technique to produce hybrid seeds via the
use of male sterility.
(Hong yang et al. 2017) used CRISPR
mediated genome editing for examining
the mutation efficiency, heritability and
specificity of the Brassica Napus genes to
create specific mutations at multiple loci
with an average frequency of 65.3%. This
study reported that CRISPR-Cas9 serve as
a potent tool for the production of targeted
genome modifications in Brassica Napus
(Hong yang et al. 2017).
In order to this a novel maele-sterile
Brassica Napus line was generated by
clustered interspaced CRISPR cas9
protein. A number of scientist reported that
a transgenic maintainer, transformed by
male-sterile plants by restorating the
fertility gene is able to link to a seedling-
colour gene (Minmin Du et al., 2020).
Recently, more enticing genetic
manipulation devices have been
established to alter particular locations of
selected genes. Of these, the FAD2-1A and
FAD2-1B genes were tried to knock out by
TALEN (transcription activator-like
effector nuclease) to modify the
concentration of soybean oil. Moreover,
the CRISPR (regularly linked short
palindromic made a significant change
method is a revolutionary genome editing
device that can alter any section of the
genome of any organisms with higher
sensitivity and specificity.
The CRISPR program, which can insert
mutations explicitly and successfully into
the genomes of model plants, arabidopsis,
corn, and tobacco, has been developed
exponentially to a large array of main
crops and vegetable varieties, including
such maize, wheat, tomato, and cucumber.
In addition, the T-DNA area of genome-
edited variant plants can be effectively
eliminated in subsequent generations by
personality-pollinating or ferocious-type
mixing (WT) of most species of plants.
It is where we send the CRISPR / Cas9
framework implementation to B. Napus to
establish a novel altered allele
BnaA.FAD2.a (referred as FAD2 Aa),
with premature stop codon or amino acid
substitution to boost the amount of oleic
acid; Agrobacterium-mediated
modification was carried out with the
vector CRISPR / Cas9 having RNA guide
developed for FAD2 Aa, and
transgenic plants with modified FAD2 Aa
alleles were produced. The concentration
of fatty acids was evaluated in the mutant
genotypes which did not carry the
transgene CRISPR / Cas9. Requires Agro-
bacterium mediated
Limitations of using CRISPR/ Cas9: transformation for
mutant plants
production
Although CRISPE/Cas9 system is
considered as best choice for genome
editing in Brassica Napus for now but it
has some limitations as well that restricts
its widespread applications. A number of
researchers has been focusing on
modifying the CRISPR system with
emerging variants of CRISPR/ Cas such as
spCas9-NG, xCAS9 base editing etc
(Hakim Manghawar et al., 2019). Certain
limitations of CRISPR CAS9 are
discussed. Editing efficiency of
CRISPR/CAs9 hinders due to its large size
and it is also not suitable for packaging
into viral vectors for delivery to somatic
tissues. In order to carry out efficient
genome editing small size of CRISPR
CAs9 is required. 5ʹ-NGG-3ʹ PAM is
required for SpCas9 immediately present
adjacent to a 20-nt DNA target sequence
where it only recognizes the NGG PAM
site and this can limit its effectiveness
compared with new CRISPR/Cas variants.
Target efficiency of xCas9 is greater and
has high DNA specificity with broad
two compatibility such as with GAT, NG, and
GAA and low off-target activity. CRISPR
Cas9 has the ability of introducing
multiple random off-target mutations in
genome (Zhang, X.H et al. 2015).
It introduces Requires Agro- imposed by regulatory systems for the
multiple and random bacterium mediated
mutations. transformation for field release of genetically modified
mutant plants
organisms (Hakim Manghawar et al.,
production
2019).

CMS in other plants:

Limitations of
CRISPR/Cas9 CMS is also reported in the studies of
hybrid seed production of other plants as
well. There are several crops reported to
use CMS in their hybrid seed production
Limited PAM sites Hard to
commercialize the includes Medicago Sativa (Alfalfa),
Bigger Protein size
transgenic crops Allium cepa (Onion), Daucus Carota
(Carrot), Lollium Perenne (Perenial Rye
grass), Zea Mays (Maize), Pennisetum
Variants of CRISPR/Cas have improved Glaucum (Millet), Schinus Molle (Pepper),
their genome editing efficiency by Secale Cereal (Rye), Sorghum bicolor
recognizing various versatile PAMs of (Sorghum), Helianthus (Sun flower),and
target bases found in the sequence of sugar and table beets etc.
interest. CRISPR /cas9 has introduces
Medicago Sativa (Alfalfa):
some mutations at similar non-specific
It is an autotetraploid legume mainly
loci, similar in homology to target sites but
produced for ruminant animals as forage.
not identical.
Development of alfafa populations is done
In order to create a mutant plant
by carrying out interpollination among
CRISPR/Cas9 requires an Agrobacterium
superior plants, in history. Seventy hydbrid
mediated transformation system which is
crops of alfalfa is studied by (Pedersen and
resource and time consuming and costlier.
stucker, 1970) and they demonstrated
Potential improvements to efficiency can
combining abilities among them. The
be achieved by the use of tissue culture-
CMS plants increase outcrossing,
free genome editing system. Difficulties
contributing to superior forage yields by
exist for the commercialization of
reduced inbreeding.
transgenic crops expressing CRISPR/Cas9
Allium Cepa (Onion):
in various countries, primarily because of
the development costs and constraints
In Japanese, European and North America recessive genes located as a single locus
hybrid onion cultivars is dominating in the (Tatlioglu and Wricke, 1988). While in
market, although still some specific case of Japanese bunching onion CMS is
regions are cultivating OP (Open conditioned by recessive alleles located at
Pollinated) populations for markets such as two restorer nuclear loci and male-sterile
Walla Walla sweets in Washington. cytoplasm (Moue and Uehara, 1985).
Argentina, Australia, Turkey, New Instead of confirmation that these sources
Zealand, and India also grow OP cultivars are used for producing Japanese bunching
because the seed prices are quite low or and hybrid onion cultivars, still the
that adapted indigenous populations information about Allium prevalence of
possess exclusively or predominantly the hybrid versus OP cultivars was not
male-sterile cytoplasm, making the accomplished.
extraction of maintainer lines difficult to
Cytoplasm of A. galanthum is used as
impossible (Havey, 1993). Commercially,
alloplasmic source of CMS was transferred
for hybrid onion production two types of
to shallot, onion, and Japanese bunching
sources are mainly used. One is S
onion (Havey, 1999; Yamashita and
cytoplasm and the other is T (texas)
Tashiro 1999; Yamashita et al., 1999)
cytoplasm (Berninger, 1965) which is used
because A. galanthum CMS system have
for producing the hybrids in Japan and
several advantages such as nuclear
Europe (Harvey, 2000). S cytoplasm
restoration of male-fertility loci found to
founds more uses in producing majority of
be non-existent and rare (Havey, 1999)
onion hybrids as it was available in short
which indicates that many populations can
and long-day germplams and in 1951 it
be used for maintaining this CMS source.
was the first onion CMS source reported
(Goldman et al., 2000). CMS has been Daucus Carota (Carrot):

reported in A. fistulosum L (Japanese In case of carrots two types of CMS

bunching onion) and Schoenoprasum L sources are used for the production of
(Chive) and exploited for production of Hybrid seeds. One is the petaloid male-
hybrid seeds in both crops. sterile cytoplasm which is predominant

In Chive a unique sensitivity was observed and in this replacement of anthers is

towards tetracycline that is a male fertility carried out by whorl of petals (Eisa and

restorer (Tatlioglu, 1986) and the Wallace, 1969).

susceptibility if tetracycline is governed by

The second is brown anther and in this
shriveled anthers are produced by
complete flowers with no pollens (Welch
and Grimball 1947). Male-fertility
restoration for these sources of CMS is
complexly inherited with up to five loci
affecting this trait (Peterson and Simon,
1986).
Hybrid carrots with petaloidy as the CMS
source for production of hybrid seeds
represents 50% of the world market. The
remaining hybrid seeds are produced by
brown anthers. The production carried out
by petalloidy CMS is preferred because
less revision occurs to male fertility while
the seeds produced by brown anthers give
generally higher yields (Peterson and
Simon, 1986).
Lollium Perenne (Perenial Rye grass):
Development of two CMS sources is
carried out in case of Lollium Perenne
(Perenial Rye grass). One is by carrying
out interspecific crossing between L.
perenne and L. multiflorum (Wit, 1974).
The second is by crossing L. perenne and
Festuca pratensis intersgenerically
(Connolly and Wright-Turner, 1984).
However there is no ryegrass reported
commercially at this time.
Zea Mays (Maize):
Zea Mays (Maize) was the first reported
hybrid crop that is produced on the large
scale. In several countries such as New
Zealand, Australia, Japan, Europe, and US
the sweet corn production is done by
hybrid cultivars only. In sweet corn
specific hybrids produced through
emasculation have short life span of 3 to 5
years which is not enough to convert the
inbreds in to CMS through backcrossing.
When hybrid seeds are produced by CMS
S and C type cytoplasm are commonly
produced while T is rare. This is due to the
dependence of different CMS cytoplasm
on stability of male sterility over
environment and specific population.
Number of hybrids does not carry alleles
of male-fertility restoration in maize and
this is why they are male sterile. Thus,
blending of hybrid seeds with N-
cytoplasmic emasculated hybrid seeds is
carried out which is male fertile and able
to provide maximum pollen in the
production field. Most maize breeders
reported that the blended hybrids are 50%
CMS with 50% N-cytoplasmic plants. The
recent incorporation of transgenes in maize
hybrids has decreased the use of CMS to
produce hybrid seed, because of the time
requirement to backcross sources of CMS
into transgenic lines (Michael Havey,
2004).
Pennisetum Glaucum (Pearl-Millet):
In US the hybrids of pearl-millet prevailed production (Dr. Thomas Miedaner,
at a percentage of 100 to 50 % in India and University of Hohenheim, Germany,
in African region the prevalence is personal communication). This crop
reported to be very low. In 1950, CMS possesses outcrossing ability naturally due
was firstly discovered pearl-millet (Burton, to its gametophytic self-incompatibility.
1958; Menon, 1959). A number of sources Some plants that are self-sterile have been
of CMS in pearl millet were identified by reported which allows development of
Burton but they were not considered as inbred line.
stable enough for commercial usage and
P (Pampa) cytoplasm of CMS is used to
were difficult to find nuclear restorer
produce hybrid rye (Geiger and Schnell,
(Smith and Chowdhry, 1989). A4
1970). P cytoplasm finds huge applications
Cytoplasm type is used for producing
due to its stable male sterility across
forage hybrids which do not use male
environments (Geiger and Miedaner, 1996;
fertility restoration.
Miedaner et al., 2004). A second potent
Schinus Molle (Pepper): source of CMS was also developed in the
German Democratic Republic University
Hybrid cultivarsis dominating the
of Nebraska named as G cytoplasm though
production market of some classes of
this source and its relationship with the
peppers.
hybrid crops is still unclear.
For example, 100% bell peppers in the
Sorghum bicolor (Sorghum):
market are hybrid as compared to
Jalapenos. Other types of pepper are OP Hybrid sorghums prevails the US and
(Open pollinated)(Paul Bosland, New world markets.
Mexico State University, personal
Hybrid sorghum predominates the US and
communication). Only a single source for
world markets. Hybrid sudangrass
producing CMS hybrid pepper cultivated
cultivars also exist, however OP
is reported (Peterson, 1958).
populations still represent a significant
Secale Cereal (Rye): component of production. CMS is
routinely used to produce hybrid seed of
It is a vital grain of great importance in
sorghum and 630 MICHAEL J. HAVEY
Poland, Russia and Germany. Hybrid
sundangrass. Although several sources of
cultivars of rye are now cultivating in
CMS have been described (A1 through
Poland at 5-10% of production and in
A4), the predominant male-sterile
Germany reported to have 60% of
cytoplasm is the A1 (milo) cytoplasm
(Holland and Stephans, 1954; Pring et al.,
1995; Schertz et al., 1997). Fertility
restoration of the A1 cytoplasm requires
the action of two complementary nuclear
male-fertility restorers (Rf1 and Rf2),
similar to T-cytoplasmic maize. There was
some effort in producing male sterile
sorghum x sudangrass hybrids in systems
with fewer fertility restorers. However the
emergence of sorghum ergot (Claviceps
africana) in the Western Hemisphere in the
1990s caused abandonment of this effort.
The infection site for sorghum ergot is the
unfertilized stigma and it was feared that
large populations of CMS sudangrass
hybrids could serve as an inoculum source
for other sorghums (Dr. Jeff Peterson,
University of Nebraska, personal
communication).
Sugar and table beets:
CMS is used to produce hybrids of both
table and sugar beets. Sugar-beet cultivars
are almost exclusively hybrids in the US
and Europe, with some open-pollinated
cultivars grown in regions of the world
with lower inputs such as Morocco and
Egypt (Mitch McGrath, USDA, personal
communication). Both diploid and triploid
sugar-beet hybrids are widely grown.
Approximately 50% of table-beet cultivars
are hybrid; OP cultivars are still produced
with the advantage of cheaper seed (I.
Goldman, personal communication). The
sole source of CMS used to produce
hybrid beets was described by Owen in
1945. In beets, male sterility is conditioned
by the interaction of the S cytoplasm with
recessive alleles at two nuclear loci (xx
zz). Owen (1945) recognized that the
approach of Jones and Clarke (1943) could
be used to produce beet hybrids. A second
alloplasmic source of CMS from Beta
aritime has been described (Boutin et al.,
1987), but has not been used to date to
produce commercial beet hybrids.
Helianthus (Sun flower):
Hybrid sunflower dominates many of the
word’s production areas, including 100%
in Argentina, Australia, Europe, India,
South Africa, Turkey, and USA. Other
countries, such as China, have significant
percentage of (>35%) hybrid production.
Hybrid sunflower is produced using the
PET1 cytoplasm, which was developed by
transferring the cytoplasm of Helianthus
petiolaris to H. annuus (LeClerq, 1969).
Although other sources of CMS in
sunflower can be distinguished by
organellar polymorphisms (Crouzillat et
al., 1991), PET1 is the only cytoplasm
used to produce sunflower hybrids. The
predominance of the PET1 source of CMS
is due primarily to its stable expression
and complete male-fertility restoration by
one dominant locus (Rf1) (Jerry Miller,
USDA, personal communication).
Other techniques for hybrid seed
production of Brassica:
We have reviewed the Cytoplasmic male
sterility in detail and now we will discuss
about further techniques which can be used
to produce hybrid seeds in brassica
species. There are a number of techniques
available and a few among them are
discussed below.
Self-Incompatibility:
A number of Brassica rapa species have
self-incompatibility in them naturally
which restrict the crops from self-
pollination. This technique is specially
used in producing the hybrid seeds in
Brassica vegetables such as cabbages and
cauliflower. This system is tested and
suggested for Brassica Napus
incompatibility pollination (Goring 1992,
Gowers 1989, and Rahman 2005). In some
environments SI inbred lines tend ro self-
pollinate that resukts in lower hybridity in
hybrid seeds. Reports have shown that
significant heterosis is carried out as well
to get agronomic characters such as leaf
area, plant height and yield in B. Napus.
In Brassica species S-alleles controls the
self-incompatibility and pollen and stigma
of the plants influence the SI (Hodgkin,
1976). Wild brassica species hare self-
incompatible. Different crosses are
performed in diploid Brassica species
followed by doubling of chromosomes
yeilds tetraploid species that have strong
selective advantage and possess self-
compatibility. Tetraploids having self-
compatibility assist in the isolation of plant
from the parental specie (Palmer, 1962).
Diploid species of B. Napus, B.Rapa, B.
Nigra, B. Alba, Raphanus Sativus, and B.
Olaracea require cross pollination for
yielding better results. Majority of Species
are self-compatible such as yellow sarson,
B. Napus, B.Rapa, B. Olaracea, broccoli,
some cabbages and caulifolowers (Yarnell,
1956; Davey, 1959; Watts, 1968; Olsson,
1960; Downey et al., 1980; Chen et al.,
1999; Okuda et al., 2000).
Three-line system:
Hybrid seed production in Brassica Napus
involves cytoplasmic-line multiplication
system and F1 hybrid seed production as
well. These lines include genetic male-
sterile line (A line), Maintainer line (B
line) and a restorer line (R line) (Ayaz
Latif 2019).
Two-line system:
Production of hybrid seed utilizes photo-
period sensitive genetic male sterile
(PSMS). Any line can act as a restorer.
Emasculation or Pollination:
Pollination or emasculation can be carried
out chemically or it occurs naturally as
well. Insects serve as efficient pollinators
or some other pollinators such as honey
bees (Megachile rotundata) can also
enhance outcrossing. Female plants
provide better yields to produce hybrids.
Male lines are destroyed after pollination
takes place by chopping, cutting, tilling,
using various herbicides or other toxic
substances. Certain chemicals are able to
sterilize the stamen without affecting the
normal functioning of pistil of plants can
be used as emasculator to emasculate
female parent for producing hybrid
Brassica Napus seeds. There is no need of
developing special male sterile lines and
also no no need to develop restorer lines.
Male gametocide 1 and Male gametocide 2
are the chemical emasculators reported in
China and successfully used for hybrid
seed production. In this type of
emasculation, physiological sterility in
males is artificially created by spraying
brassica plants with the chemicals
mentioned above without causing any
harm to the pistil of the stamen of plants to
induce stamen sterility.
Genetic Male Sterility:
A number of methods are available for
conferring genetic male sterility such as
multiple mutant genes present at separate
locations within the genome, chromosomal
translocations, nuclear male sterility which
incorporates identification of a gene
critical for male fertility. After that
silencing of that gene occurs by removing
the native promoter and replacing them
with inducible promoters, inserting this
genetically engineered gene back into the
plant; and thus creating a plant that is male
sterile because the inducible promoter is
not "on" resulting in the male fertility gene
not being transcribed. Fertility is restored
by inducing, or turning "on", the promoter,
which in turn allows the gene that, confers
male fertility to be transcribed.
Importance of hybrid seed production
in plants:
In United Kingdom round 60% of
rapeseed oil is generated by growing
hybrid seeds. Now the demand of
producing hybrid seeds has been increased
as the farmers has realized the benefits of
hybrid breeding as it allows greater genetic
diversity in plants and enhances the levels
of production of plants with desirable
agronomic traits. It has been reported that
in Germany, hybrid seeds accounts for
more than 84% of rapeseed oil, 99% in
North America and about 77% in France.
Average yield is growing upwards to 5t/ha
due to modern hybrids production.
 A number of surveys conducted among
farmers reveals that a hybrid has the ability
to produce around 350-500 kg/ha in
comparison to the conventional variety.
Profitability of overall crop is increased as
the oil content with bonuses is now being
paid 1.5% rate of the contract price for
each 1% extra oil over 40% as oil content
is the stability characteristic for any plant.
The reliability of seed yield is also
increased by DSV hybrid.
In studies of hybrid production it is
clearly elaborated that kin general hybrid
seed are more vigorous than the
conventional ones which means that
hybrid seeds possess the ability of
compensation in the fields growing under
difficult conditions. Vigours produced in
autumn helps in establishment and closure
of crop gaps in order to reduce the amount
of landing areas for pigeons and recover
them from weather hazards after the winter
season. In spring vigor fast growth rate
takes plants so in that case plants are less
prone to damage by pests and show better
recovery from weather damage after
winters. If herbicide efficacy is calculated
or examined as poor, vigor helps the plants
to compete the weeds. Hybrids show better
establishment among a wide range of
drilling dates and conditions in comparison
to the conventional varieties. Most hybrids
have a later sowing date which can prove
very important in difficult years and helps
in spreading the work load after harvest.
Hybrids needs low seed rate with a density
of approximately 40 m² to show better
performance because it allows the
establishment of right canopy structure and
lower the population which helps in
eliminating the risks of lodging. Hybrid
rapeseed suffers less environmental stress
as compared to the conventional varieties
and is more useful in case of challenging
situations and less fertile situations. The
hybrid characterizes of the plants enables
them to resist extreme conditions such as
extreme cold and lack of water, but still
they show efficient yields as they adopt
their selves to these unfavorable
conditions. Hybrids possess well
developed network of roots which enables
them to resist against drought conditions
because the extensive root system helps in
uptake of water. Currently the breeders are
working to combine the characteristic that
includes the reduction height of hybrids
and increasing standing power of hybrids
by improving their genome sequencing via
CMS.
Importance of hybrid seed production
via CMS in Brassica Napus:
In World, Brassica Napus is the 2nd most
vital oilseed crop and considered as best
suitable plant for carrying out breeding and
basic research. Rapeseed possesses agriculture importance in the production of
complex polyploidy genome due to its hybrid seed as it has the ability to render
evolution and origin. Recent studies plants male-sterile by preventing them
carried out for the cultivation of F1 Hybrid from undergoing self-pollination (M.W.
seed via CMS revealed that CMS provides Gray, 2013). Another remarkable
an expedient mechanism for the large characteristic of using CMS is that its play
productions of commercial F1 male- sterile a vital role of nuclear genes in creating
seeds. modifying effects. Nuclear genes (restorer
loci) have the ability to reverse the
Male sterility reduces or blocks the process
mitochondrial determinant effect of CMS
of self-fertilization and modifies the flower
(M.W. Gray, 2013) but in some plants like
which enhances pollen access. This
chilli, tomato and melon it has no use as
technique enables the researchers to
the hybrid progeny produced is male
understand the sequencing of
sterile.
corresponding genomes and gives them a
basis for better understanding and Conclusion:
exploitation of the genetic diversity
References:
involved in the major traits of rape seed.
When there is a need for F1 hybrid seed to
set seeds and produce pollen, CMS
(cytoplasmic male sterility) has the ability
to be reversed by nuclear-encoded restorer
of fertility alleles (Michael J Havey, 2004).
CMS can be transferred easily to any strain
by the use of that particular strain as a
recurrent parent (pollinator) in generating
backcrosses successively (R.K. Dhall,
2010). Cytoplasmic male sterility (CMS)
can also be used for the production hybrid
plants in those plants and vegetables in
which the vegetative part has great
economic value such as Carrot, onion,
radish, rapeseed, and cole crops (R.K.
Dhall, 2010). CMS trait has a great