Model plant in molecular biology Arabidopsis, rice .

1. WELCOME

2. Course No: MBB-601
Course Title: Plant Molecular Biology
Topic: Model Systems in Plant Biology(Arabidopsis, Rice,
etc.)

3. 1. Scientific classification of Arabidopsis
Kingdom: Plantae
Clade: Tracheophytes
Clade: Angiosperms
Clade: Eudicots
Clade: Rosids
Order: Brassicales
Family: Brassicaceae
Genus: Arabidopsis
Species: A. thaliana

4. Arabidopsis: The Model Plant
• Arabidopsis thaliana is a small dicotyledonous species, a member of the Brassicaceae or
mustard family.
• Although closely related to such economically important crop plants as turnip, cabbage,
broccoli, and canola, Arabidopsis is not an economically important plant.
• It has been the focus of intense genetic, biochemical and physiological study for over 40
years because of several traits that make it very desirable for laboratory study.
• As a photosynthetic organism, Arabidopsis requires only light, air, water and a few
minerals to complete its life cycle.
• It has a fast life cycle, produces numerous self progeny, has very limited space
requirements, and is easily grown in a greenhouse or indoor growth chamber.

5.  It possesses a relatively small, genetically tractable genome that can be manipulated
through genetic engineering more easily and rapidly than any other plant genome.
 Arabidopsis, like all flowering plants, dehydrates and stores its progeny at ambient
temperature for long periods of time.
 This fact of creating gene knockout lines, has made many basic biologists realize
that Arabidopsis may be the best model system for basic research in the biology of
all multicellular eukaryotes.
 All together, these traits make Arabidopsis an ideal model organism for biological
research and the species of choice for a large and growing community of scientists
studying complex, advanced multicellular organisms.

6. Genomics
 Nuclear genome
 Due to the small size of its genome, and because it is diploid, Arabidopsis
thaliana is useful for genetic mapping and sequencing - with about 157 megabase
pairs and five chromosomes, A. thaliana has one of the smallest genomes among
plants.
 It was the first plant genome to be sequenced, completed in 2000 by
the Arabidopsis Genome Initiative.
 The most up-to-date version of the A. thaliana genome is maintained by the
Arabidopsis Information Resource.
 The genome encodes ~27,600 protein-coding genes and about 6,500 non-coding
genes.

7.  Among the 27,600 protein-coding genes 25,402 (91.8%) are now annotated with
"meaningful" product names.
 Uniprot lists more than 3,000 proteins as "uncharacterized" as part of the reference proteome.
 Chloroplast genome
 The plastome of A. thaliana is a 154,478 base-pair-long DNA molecule, a size typically
encountered in most flowering plants.
 It comprises 136 genes coding for small subunit ribosomal proteins, large subunit ribosomal
proteins (rpl, orange), hypothetical chloroplast open reading frame proteins (ycf, lemon),
proteins involved in photosynthetic reactions (green) or in other functions (red), ribosomal
RNAs (rrn, blue), and transfer RNAs (trn, black).

8.  Mitochondrial genome
1. The mitochondrial genome of A. thaliana is 367,808 base pairs long and contains 57
genes.
2. There are many repeated regions in the Arabidopsis mitochondrial genome.
3. The largest repeats recombine regularly and isomerize the genome.
4. Like most plant mitochondrial genomes, the Arabidopsis mitochondrial genome exists
as a complex arrangement of overlapping branched and linear molecules in vivo.

9. Arabidopsis versus plants of economic significance
 Why Arabidopsis? Why not concentrate our research efforts and resources on a species
that will actually provide food for our world or useful products for industrial uses?
 In order to make the strides necessary to increase crop production in a relatively short
time, we have to be able to move forward quickly and spend the available human and
financial resources as efficiently as possible.
 This is the advantage of a model system: an organism that is easily manipulated,
genetically tractable, and about which much is already known.
 By studying the biology of Arabidopsis, the model plant, we can gain comprehensive
knowledge of a complete plant.
 In the laboratory, Arabidopsis offers the ability to test hypotheses quickly and

10.  With the knowledge we gain from the model plant thus established as a reference system, we can
move forward with research and rapidly initiate improvements in plants of economic and cultural
importance.
 One advantage offered to the plant researcher by Arabidopsis is its relatively small genome size.
 Many crop species have large genomes, often as a result of polyploidization events and
accumulation of non-coding sequences during their evolution.
 Maize has a genome of approximately 2400 Megabase pairs (Mbp) – around 19 times the size of
the Arabidopsis genome – with probably no more than double the number of genes, most of
which occur in duplicate within the genome.
 The wheat genome is 16000 Mbp – 128 times larger than Arabidopsis and 5 times larger than
Homo sapiens – and it has three copies of many of its genes.

11.  The large crop genomes pose challenges to the researcher, including difficulty in sequencing as
well as in isolation and cloning of mutant loci.
 Evidence from the rice genome project suggests that the Arabidopsis genome may be missing
some homologs of genes present in the rice genome.
 Despite this, most of the difference in gene number between Arabidopsis and crop species
appears to result from polyploidy of crop species’ genomes, rather than from large classes of
genes present in crop species that are not present in Arabidopsis.
 Therefore, the genes present in Arabidopsis represent a reasonable model for the plant
kingdom. However, it is clear that Arabidopsis represents a starting point rather than the finish
line for utilizing the full power of genomics for crop improvement.

12. Kingdom: Plantae
Division: Magnoliophyta
Class: Liliopsida
Order: Cyperales
Family: Gramineae
Genus: Oryza
Species: Sativa
Subspecies: Indica
Common name: Paddy
2. Scientific classification of rice

13.  Rice is emerging as a model cereal for molecular biological studies.
 The main reasons for this are as follows:
(a) The complete genome has been sequenced. Although the complete rice genome sequence is the
proprietary information of a private company, an international group is expected to generate
complete genome sequences that will be accessible to anyone in the near future. In addition, a large
expressed sequence tag (EST) database is available to researchers, facilitating a quick identification
of genes of interest.
(b) Tools for functional genomics are available. Transposon- and T-DNA-tagged rice populations exist,
and the microarray technology for studying mRNA expression profiles is available.
(c) Production of transgenic plants is relatively easy compared to that of other major cereals. Efficient
use of Agrobacterium-mediated transformation made routine use of transgenic rice possible for a
variety of research purposes.
Rice : The model plant

14.  Rice has been cultivated as a major crop for more than 7000 years, and it currently sustains
more than half the world population.
 Rice and many other food plants are monocotyledons - such plants are of clear importance, and
yet they are distinct from the dicotyledonous model plant Arabidopsis in many aspects of
development.
 Recent advances in research on rice include efficient transformation, the creation of a highly
saturated molecular map, and the large-scale analysis of expressed sequence tags.
 Indeed, the number of complementary DNAs analyzed in rice is approaching the number
analyzed in Arabidopsis.
 Rice has reached the point where it can be usefully considered a model monocotyledonous
plant.

15.  Rice is an excellent model system for functional genomics studies due to its small genome
size, availability of genetic resources, high transformation efficiency, and greater genomic
synteny with other cereals.
 Therefore, rice has been increasingly used to test the efficiency of different types of genome
editing technologies, to study the functions of various genes and demonstrate their potential
in rice improvement.
 Although several reviews on genome editing and its role in plants have been published in the
recent times, an elaborative review particularly on genome editing of rice is the need of the
hour keeping in view the rapid and large accumulation of case studies on genome editing of
this agriculturally important crop.
 The application of genome editing tools have broaden rice research, bringing in new
opportunities to develop novel varieties with improved productivity and quality.

16.  In the present review, we focus on the different genome editing strategies and their
applications in rice improvement using specific case studies.
 We have also highlighted the emergence of CRISPR/Cpf1 system and base editing as a
suitable alternative to traditional CRISPR/Cas9 system for rice improvement.
 Furthermore, review also focusses on the major challenges and future implications of genome
editing the in rice improvement.

17. THANK YOU…

18. Forward Genetics
 Forward genetics is a molecular genetics approach of determining the genetic basis
responsible for a phenotype. Forward genetics provides an unbiased approach because it
relies heavily on identifying the genes or genetic factors that cause a particular
phenotype or trait of interest.[1]
 This was initially done by using naturally occurring mutations or inducing mutants with
radiation, chemicals, or insertional mutagenesis (e.g. transposable elements).
Subsequent breeding takes place, mutant individuals are isolated, and then the gene
is mapped.
 Forward genetics can be thought of as a counter to reverse genetics, which determines
the function of a gene by analyzing the phenotypic effects of altered DNA sequences.

19.  Mutant phenotypes are often observed long before having any idea which gene is
responsible, which can lead to genes being named after their mutant phenotype
(e.g. Drosophila rosy gene which is named after the eye colour in mutants).
Techniques used in Forward Genetics:
 Forward genetics provides researchers with the ability to identify genetic changes
caused by mutations that are responsible for individual phenotypes in organisms.
 There are three major steps involved with the process of forward genetics which
includes: making random mutations, selecting the phenotype or trait of interest, and
identifying the gene and its function.
 Forward genetics involves the use of several mutagenesis processes to induce DNA
mutations at random which may include:

20. A. Chemical mutagenesis
B. Insertional mutagenesis
C. Radiation mutagenesis
D. Post mutagenesis
A)Chemical mutagenesis:
 Chemical mutagenesis is an easy tool that is used to generate a broad spectrum of mutant
alleles.
 Chemicals like ethyl methanesulfonate (EMS) cause random point mutations particularly
in G/C to A/T transitions due to guanine alkylation.
 These point mutations are typically loss-of-function or null alleles because they generate
stop codons in the DNA sequence.

21.  These types of mutagens can be useful because they are easily applied to any organism
but they were traditionally very difficult to map, although the advent of next-generation
sequencing has made this process considerably easier.
 Another chemical such as ENU, also known as N-ethyl-N-nitrosourea works similarly to
EMS.
 ENU also induces random point mutations where all codons are equally liable to change.
These point mutations modify gene function by inducing different alleles, including gain
or loss of function mutations in protein-coding or noncoding regions in the genome.

22. B) Radiation mutagenesis:
 Other methods such as using radiation to cause large deletions and chromosomal
rearrangements can be used to generate mutants as well.
 Ionizing radiation can be used to induce genome-wide mutations as well as chromosomal
duplications, inversions, and translocations.
 Similarly, short wave UV light works in the same way as ionizing radiation which can
also induce mutations generating chromosomal rearrangements.
 When DNA absorbs short wave UV light, dimerizing and oxidative mutations can occur
which can cause severe damage to the DNA sequence of an organism.

23. C) Insertional mutagenesis:
 Mutations can also be generated by insertional mutagenesis.
 Most often, insertional mutagenesis involves the use of transposons, which introduces
dramatic changes in the genome of an organism.
 Transposon movements can create random mutations in the DNA sequence by changing
its position within a genome, therefore modifying gene function, and altering the
organism’s genetic information.
 For example, transposable elementscontaining a marker are mobilized into the genome at
random. These transposons are often modified to transpose only once, and once inserted
into the genome a selectable marker can be used to identify the mutagenized individuals.
Since a known fragment of DNA was inserted this can make mapping and cloning the
gene much easier.

24. D) Post mutagenesis:
 Once mutagenized and screened, typically a complementation test is done to ensure
that mutant phenotypes arise from the same genes if the mutations are recessive.
 If the progeny after a cross between two recessive mutants have a wild-type
phenotype, then it can be inferred that the phenotype is determined by more than one
gene.
 Typically, the allele exhibiting the strongest phenotype is further analyzed.
 A genetic map can then be created using linkage and genetic markers, and then the
gene of interest can be cloned and sequenced. If many alleles of the same genes are
found, the screen is said to be saturated and it is likely that all of the genes involved
producing the phenotype were found.

25.  Reverse genetics:
 Reverse genetics is a method in molecular genetics that is used to help understand the
function(s) of a gene by analysing the phenotypic effects caused by genetically
engineering specific nucleic acid sequences within the gene.
 The process proceeds in the opposite direction to forward genetic screens of classical
genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait,
reverse genetics seeks to find what phenotypes are controlled by particular genetic
sequences.
 Automated DNA sequencing generates large volumes of genomic sequence data relatively
rapidly.
 Many genetic sequences are discovered in advance of other, less easily obtained,
biological information.

26.  Reverse genetics attempts to connect a given genetic sequence with specific effects on
the organism.
 Reverse genetics systems can also allow the recovery and generation of infectious or
defective viruses with desired mutations.
 This allows the ability to study the virus in vitro and in vivo.

27. Techniques used:
 In order to learn the influence a sequence has on phenotype, or to discover its
biological function, researchers can engineer a change or disrupt the DNA.
 After this change has been made a researcher can look for the effect of such
alterations in the whole organism. There are several different methods of reverse
genetics:
A. Directed deletions and point mutations
B. Gene silencing
C. Interference using transgenes

28. A) Directed deletions and point mutation:
 Site-directed mutagenesis is a sophisticated technique that can either change
regulatory regions in the promoter of a gene or make subtle codon changes in
the open reading frame to identify important amino residues for protein function
 Alternatively, the technique can be used to create null alleles so that the gene is not
functional. For example, deletion of a gene by gene targeting (gene knockout) can
be done in some organisms, such as yeast, mice and moss. Unique among plants,
in Physcomitrella patens, gene knockout via homologous recombination to
create knockout moss (see figure) is nearly as efficient as in yeast.[4] In the case of
the yeast model system directed deletions have been created in every non-essential
gene in the yeast genome.[5] In the case of the plant model system huge mutant
libraries have been created based on gene disruption constructs.[6] In gene knock-
in, the endogenous exon is replaced by an altered sequence of interest.[7]
 In some cases conditional alleles can be used so that the gene has normal function
until the conditional allele is activated. This might entail 'knocking
in' recombinase sites (such as lox or frt sites) that will cause a deletion at the gene
of interest when a specific recombinase (such as CRE, FLP) is induced.

29.  Cre or Flp recombinases can be induced with chemical treatments, heat shock treatments or be restricted
to a specific subset of tissues.
 Another technique that can be used is TILLING. This is a method that combines a standard and efficient
technique of mutagenesis with a chemical mutagen such as ethyl methanesulfonate (EMS) with a
sensitive DNA-screening technique that identifies point mutations in a target gene.
 In the field of virology, reverse-genetics techniques can be used to recover full-length infectious viruses
with desired mutations or insertions in the viral genomes or in specific virus genes. Technologies that
allow these manipulations include circular polymerase extension reaction (CPER) which was first used
to generate infectious cDNA for Kunjin virus a close relative of West Nile virus.[8] CPER has also been
successfully utilised to generate a range of positive-sense RNA viruses such as SARS-CoV-2,[9] the
causative agent of COVID-19.

30. B) Gene silencing:
 The discovery of gene silencing using double stranded RNA, also known as RNA
interference (RNAi), and the development of gene knockdown using Morpholino oligos,
have made disrupting gene expression an accessible technique for many more
investigators. This method is often referred to as a gene knockdown since the effects of
these reagents are generally temporary, in contrast to gene knockouts which are permanent.
 RNAi creates a specific knockout effect without actually mutating the DNA of interest.
In C. elegans, RNAi has been used to systematically interfere with the expression of most
genes in the genome. RNAi acts by directing cellular systems to degrade target messenger
RNA (mRNA).
 RNAi interference, specifically gene silencing, has become a useful tool to silence the
expression of genes and identify and analyze their loss-of-function phenotype. When
mutations occur in alleles, the function which it represents and encodes also is mutated and
lost; this is generally called a loss-of-function mutation.[10] The ability to analyze the loss-
of-function phenotype allows analysis of gene function when there is no access to mutant
alleles.[11]

31.  While RNA interference relies on cellular components for efficacy (e.g. the Dicer
proteins, the RISC complex) a simple alternative for gene knockdown
is Morpholino antisense oligos.
 Morpholinos bind and block access to the target mRNA without requiring the activity of
cellular proteins and without necessarily accelerating mRNA degradation. Morpholinos
are effective in systems ranging in complexity from cell-free translation in a test tube
to in vivo studies in large animal models.

32. C) Interference using transgene:
 A molecular genetic approach is the creation of transgenic organisms that overexpress a
normal gene of interest. The resulting phenotype may reflect the normal function of the
gene.
 Alternatively it is possible to overexpress mutant forms of a gene that interfere with the
normal (wildtype) gene's function. For example, over-expression of a mutant gene may
result in high levels of a non-functional protein resulting in a dominant
negative interaction with the wildtype protein. In this case the mutant version will out
compete for the wildtype proteins partners resulting in a mutant phenotype.
 Other mutant forms can result in a protein that is abnormally regulated and constitutively
active ('on' all the time).

33.  This might be due to removing a regulatory domain or mutating a specific amino
residue that is reversibly modified (by phosphorylation, methylation,
or ubiquitination). Either change is critical for modulating protein function and often
result in informative phenotypes.

34. WELCOME

35. Course No: MBB-601
Course Title: Plant Molecular Biology
Course Teacher: Dr. M. P. Moharil
Topic: Cytoplasmic Male Sterility

36. CYTOPLASMIC MALE STERILITY
 Cytoplasmic male sterility is total or partial male sterility in hermaphrodite
organisms, as the result of specific nuclear and mitochondrial interactions.
 Male sterility is the failure to produce functional anthers, pollen, or male gametes.
Such male sterility in hermaphrodite populations leads to gynodioecious
populations (populations with coexisting fully functioning hermaphrodites and
male-sterile hermaphrodites).
 Cytoplasmic male sterility, as the name indicates, is under extranuclear genetic
control (under control of the mitochondrial or plastid genomes).
 It shows non-Mendelian inheritance, with male sterility inherited maternally. In
general, there are two types of cytoplasm: N (normal) and aberrant S (sterile)
cytoplasms. These types exhibit reciprocal differences.

37.  CMS is one case of male-sterility, but this condition can also originate from nuclear
genes.
 In the case of nuclear male sterility (when male sterility is caused by a nuclear
mutation), the transmission of the male sterility allele is cut in half, since the entire
male reproductive pathway is canceled.
 CMS differs from the latter case (nuclear male sterility) because most cytoplasmic
genetic elements are only transmitted maternally.
 This entails that for a cytoplasmic genetic element, causing male sterility doesn't affect
its transmission rate since it is not transmitted via the male reproductive pathway.

38.  Inactivation of the male reproductive pathway (sperm production, production of male
reproductive organs, etc) can lead to resource relocation to the female reproductive
pathway, increasing the female reproductive capabilities (female fitness), this phenomenon
is referred to as Female Advantage (FA).
 The female advantage of many gynodioecious species has been quantified (as the ratio
between male-sterile's female fitness and hermaphrodites' female fitness) and is mostly
comprised between 1 and 2.

39.  In the case of nuclear male-sterility, a female advantage of at least 2 is
required to make it evolutionary neutral (FA=2) or advantageous (FA > 2)
since half of the transmission is cut because of the male-sterility allele.
 Cytoplasmic male-sterility requires no female advantage to be evolutionary
neutral (FA=1), or a small female advantage to be evolutionary advantageous
(FA > 1).
 As far as we know, CMS is much more common than nuclear male-sterility
with results from a study of 49 gynodioecious plants found 17 species (35%)
exhibiting CMS and only 7 (14%) exhibiting nuclear male-sterility (all
remaining species have unknown determinism of male-sterility).

40. Genetic sterility:
 While CMS is controlled by an extranuclear genome, nuclear genes may have the
capability to restore fertility.
 When nuclear restoration of fertility genes is available for a CMS system in any
crop, it is cytoplasmic–genetic male sterility; the sterility is manifested by the
influence of both nuclear (with Mendelian inheritance) and cytoplasmic
(maternally inherited) genes.
 There are also restorers of fertility (Rf) genes that are distinct from genetic male
sterility genes.
 The Rf genes have no expression of their own unless the sterile cytoplasm is
present.
 Rf genes are required to restore fertility in S cytoplasm that causes sterility.
 Thus plants with N cytoplasm are fertile and S cytoplasm with genotype Rf- leads
to fertiles while S cytoplasm with rfrf produces only male steriles.

41.  Another feature of these systems is that Rf mutations (i.e., mutations to rf or no
fertility restoration) are frequent, so that N cytoplasm with Rfrf is best for stable
fertility.
 Cytoplasmic–genetic male sterility systems are widely exploited in crop plants
for hybrid breeding due to the convenience of controlling sterility expression by
manipulating the gene–cytoplasm combinations in any selected genotype..
 Incorporation of these systems for male sterility evades the need for emasculation
in cross-pollinated species, thus encouraging cross breeding producing only
hybrid seeds under natural conditions.

42. n hybrid breeding:
 Hybrid production requires a plant from which no viable male gametes are introduced.
 This selective exclusion of viable male gametes can be accomplished via different
paths.
 One path, emasculation is done to prevent a plant from producing pollen so that it can
serve only as a female parent.
 Another simple way to establish a female line for hybrid seed production is to identify
or create a line that is unable to produce viable pollen.
 Since a male-sterile line cannot self-pollinate, seed formation is dependent upon pollen
from another male line.
 Cytoplasmic male sterility is also used in hybrid seed production. In this case, male
sterility is maternally transmitted and all progeny will be male sterile.
 These CMS lines must be maintained by repeated crossing to a sister line (known as the
maintainer line) that is genetically identical except that it possesses normal cytoplasm
and is therefore male-fertile.

43.  In cytoplasmic–genetic male sterility restoration of fertility is done using
restorer lines carrying nuclear genes. The male-sterile line is maintained
by crossing with a maintainer line carrying the same nuclear genome but
with normal fertile cytoplasm.
 For crops such as onions or carrots where the commodity harvested from
the F1 generation is vegetative growth, male sterility is not a problem.

44.  Cytoplasmic male sterility (CMS), a condition under which a plant is unable to
produce functional pollen, is widespread among higher plants.
 CMS systems represent a valuable tool in the production of hybrid seed in self-
pollinating crop species, including maize, rice, cotton, and a number of vegetable
crops.
 Hybrids often exhibit heterosis, more commonly known as hybrid vigor, whereby
hybrid progeny exhibit superior growth characteristics relative to either of the
parental lines.
 CMS systems can be of considerable value in facilitating efficient hybrid seed
production.

45.  There is growing interest in improving hybrid technology both to help supply
food for the world’s increasing population and to contribute to land
conservation efforts.
 For example, the use of hybrid rice enabled China to reduce the total amount
of land planted to rice from 36.5 Mha in 1975 to 30.5 Mha in 2000 while at
the same time increasing total production from 128 to 189 million tons,
representing a yield increase of 3.5 to 6.2 tons/ha.
 Understanding the molecular basis of CMS, as well as other hybrid
production methods involving self-incompatibility and apomixis, is critical
for continued improvements in hybrid technology