Genetic engineering can be used to induce male sterility in plants by expressing genes that disrupt pollen development. Researchers have successfully transformed tobacco and oilseed rape plants with the barnase gene, which encodes an RNAse enzyme that destroys tapetal cells, preventing pollen formation. Restoration of fertility was achieved by co-expressing the barstar gene, which inhibits barnase. Similarly, expressing the argE gene in rice under a pollen-specific promoter induces male sterility when activated by an inducer, allowing hybrid seed production. Genetic engineering offers possibilities for more efficient hybrid seed systems in crops where traditional methods have not generated usable male sterility.
The term balanced tertiary trisomic has three words of which (1) “trisomic” indicates the presence of extra chromosome, (2) “tertiary” indicates that the extra chromosome is a trans-located chromosome, and (3) “balanced” refers to the breeding behaviour of the trisomic.
Ramage defined the BTT as a tertiary trisomic constructed in such a way that the dominant allele of a marker gene, closely linked with the translocation breakpoint of the extra chromosome is carried on the extra chromosome, and the recessive allele is carried on the two normal chromosomes that constitute the diploid complement. The dominant marker gene may be located on the centromere segment or the trans-located segment of the extra chromosome.
Heterotic group “is a group of related or unrelated genotypes from the same or different populations, which display similar combining ability and heterotic response when crossed with genotypes from other genetically distinct germplasm groups.”
The term balanced tertiary trisomic has three words of which (1) “trisomic” indicates the presence of extra chromosome, (2) “tertiary” indicates that the extra chromosome is a trans-located chromosome, and (3) “balanced” refers to the breeding behaviour of the trisomic.
Ramage defined the BTT as a tertiary trisomic constructed in such a way that the dominant allele of a marker gene, closely linked with the translocation breakpoint of the extra chromosome is carried on the extra chromosome, and the recessive allele is carried on the two normal chromosomes that constitute the diploid complement. The dominant marker gene may be located on the centromere segment or the trans-located segment of the extra chromosome.
Heterotic group “is a group of related or unrelated genotypes from the same or different populations, which display similar combining ability and heterotic response when crossed with genotypes from other genetically distinct germplasm groups.”
The presentation was done as part of the course STAT 504 titled Quantitative Genetics in Second Semester of MSc. Agricultural Statistics at Agricultural College, Bapatla under ANGRAU, Andhra Pradesh
FERTILITY RESTORATION IN MALE STERILE LINES AND RESTORER DIVERSIFICATION PROG...Rachana Bagudam
1. FERTILITY RESTORATION IN MALE STERILE LINES AND RESTORER DIVERSIFICATION PROGRAMMES.
2. CONVERSION OF AGRONOMICALLY IDEAL GENOTYPES INTO MALE STERILES.
3. GENERATING NEW CYTONUCLEAR INTERACTION SYSTEM FOR DIVERSIFICATION OF MALE STERILES.
Marker Assisted Gene Pyramiding for Disease Resistance in RiceIndrapratap1
Why marker assisted gene pyramiding?
For traits that are simply inherited, but that are difficult or expensive to measure phenotypically, and/or that do not have a consistent phenotypic expression under specific selection conditions, marker-based selection is more effective than phenotypic selection.
Traits which are traditionally regarded as quantitative and not targeted by gene pyramiding program can be improved using gene pyramiding if major genes affecting the traits are identified.
Genes with very similar phenotypic effects, which are impossible or difficult to combine in single genotype using phenotypic selection, can be pyramided through marker assisted selection.
Markers provides a more effective option to control linkage drag and make the use of genes contained in unadapted resources easier.
Pyramiding is possible through conventional breeding but is extremely difficult or impossible at early generations..
DNA markers may facilitate selection because DNA marker assays are non destructive and markers for multiple specific genes/QTLs can be tested using a single DNA sample without phenotyping.
CONCLUSION:
• Molecular marker offer great scope for improving the efficiency of conventional plant breeding.
• Gene pyramiding may not be the most suitable strategy when many QTL with small effects control the trait and other methods such as marker-assisted recurrent selection should be considered.
• With MAS based gene pyramiding, it is now possible for breeder to conduct many rounds of selections in a year.
• Gene pyramiding with marker technology can integrate into existing plant breeding program all over the world to allow researchers to access, transfer and combine genes at a rate and with precision not previously possible.
• This will help breeders get around problems related to larger breeding populations, replications in diverse environments, and speed up the development of advance lines.
For further queries please contact at isag2010@gmail.com
The use of the term cisgenesis is an attempt to distinguish GM plants or other organisms produced in this way from transgenics that is GM plants that contain DNA from unrelated organisms. Schouten et al. (2006) introduced the term cisgenesis and defined cisgenesis as the modification in the genetic background of a recipient plant by a naturally derived gene from a cross compatible species including its introns and its native promoter and terminator flanked in the normal sense orientation. Since cisgenes shared a common gene pool available for traditional breeding the final cisgenic plant should be devoid of any kind of foreign DNA viz., selection markers and vector- backbone sequences. Sometimes the word cisgenesis is also referred to as Agrobacterium-mediated gene transfer from a sexually compatible plant where only the T-DNA borders may be present in the recipient organism after transformation (EFSA, 2012). The cisgenesis precludes linkage drag, and hence, prevents hazards from unidentified hitch hiking genes (Schouten, and Jacobsen, 2008). Compared to transgenesis, one of the disadvantages shared by cisgenesis is that characters outside the sexually compatible gene pool cannot be introduced. Furthermore, development of cisgenic crops involves extraordinary proficiency and time compared to transgenic crops. Therefore, the required genes or fragments of genes may not be readily accessible but have to be isolated from the sexually compatible gene pool (Holme et al., 2013).
On 16 February 2012, European Food Safety Authority (EFSA, 2012) reported the detail study concerning the safety aspects of cisgenic plants and validated that cisgenic plants are secure to be used in terms of environment, food and feed, similar to the traditionally bred plants. However, the present GMO regulation keeps the cisgenic micro-organisms out from its supervision. The first scientific statement of bringing forth a true plant obtained by cisgenic approach was reported in apple through the insertion of the internal scab resistance gene HcrVf2 influenced by their own regulatory genes into the cultivar Gala, a scab susceptible cultivar (Vanblaere et al., 2011). Barley with improved phytase activity was produced successfully by Holme et al. 2011, through cisgenic approach. Late blight resistant potatoes have developed by cisgene stacking of R- gene (jo et al., 2014).
Marker Assisted Selection in Crop BreedingPawan Chauhan
Marker Assisted Selection is a value addition to conventional methods of Crop Breeding. It has been gaining importance in plant breeding with new generation of plant breeders and to get accurate and fast desired result from plant breeding.
A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
Genetic Engineering of Male Sterility for Hybrid Seed Production # Methods of Hybrid Seed Production - Hybridization techniques # Examples of Male Sterile Hybrid Seed
Hybridization between individuals from different species belonging to the same genus or two different genera, is termed as distant hybridization or wide hybridization, and such crosses are known as distant crosses or wide crosses.
Transgenes may be used to produce GMS which is dominant to fertility.
In these cases it is essential to develop effective fertility restoration systems for hybrid seed production.
An effective restoration system is available in at least one case, Barnase/Barstar system
Recombinant DNA techniques have made it possible to engineer new systems of male sterility by disturbing any or number of developmental steps specifically required for the production of functional pollen within the microspore or for the development of any somatic tissues .
The presentation was done as part of the course STAT 504 titled Quantitative Genetics in Second Semester of MSc. Agricultural Statistics at Agricultural College, Bapatla under ANGRAU, Andhra Pradesh
FERTILITY RESTORATION IN MALE STERILE LINES AND RESTORER DIVERSIFICATION PROG...Rachana Bagudam
1. FERTILITY RESTORATION IN MALE STERILE LINES AND RESTORER DIVERSIFICATION PROGRAMMES.
2. CONVERSION OF AGRONOMICALLY IDEAL GENOTYPES INTO MALE STERILES.
3. GENERATING NEW CYTONUCLEAR INTERACTION SYSTEM FOR DIVERSIFICATION OF MALE STERILES.
Marker Assisted Gene Pyramiding for Disease Resistance in RiceIndrapratap1
Why marker assisted gene pyramiding?
For traits that are simply inherited, but that are difficult or expensive to measure phenotypically, and/or that do not have a consistent phenotypic expression under specific selection conditions, marker-based selection is more effective than phenotypic selection.
Traits which are traditionally regarded as quantitative and not targeted by gene pyramiding program can be improved using gene pyramiding if major genes affecting the traits are identified.
Genes with very similar phenotypic effects, which are impossible or difficult to combine in single genotype using phenotypic selection, can be pyramided through marker assisted selection.
Markers provides a more effective option to control linkage drag and make the use of genes contained in unadapted resources easier.
Pyramiding is possible through conventional breeding but is extremely difficult or impossible at early generations..
DNA markers may facilitate selection because DNA marker assays are non destructive and markers for multiple specific genes/QTLs can be tested using a single DNA sample without phenotyping.
CONCLUSION:
• Molecular marker offer great scope for improving the efficiency of conventional plant breeding.
• Gene pyramiding may not be the most suitable strategy when many QTL with small effects control the trait and other methods such as marker-assisted recurrent selection should be considered.
• With MAS based gene pyramiding, it is now possible for breeder to conduct many rounds of selections in a year.
• Gene pyramiding with marker technology can integrate into existing plant breeding program all over the world to allow researchers to access, transfer and combine genes at a rate and with precision not previously possible.
• This will help breeders get around problems related to larger breeding populations, replications in diverse environments, and speed up the development of advance lines.
For further queries please contact at isag2010@gmail.com
The use of the term cisgenesis is an attempt to distinguish GM plants or other organisms produced in this way from transgenics that is GM plants that contain DNA from unrelated organisms. Schouten et al. (2006) introduced the term cisgenesis and defined cisgenesis as the modification in the genetic background of a recipient plant by a naturally derived gene from a cross compatible species including its introns and its native promoter and terminator flanked in the normal sense orientation. Since cisgenes shared a common gene pool available for traditional breeding the final cisgenic plant should be devoid of any kind of foreign DNA viz., selection markers and vector- backbone sequences. Sometimes the word cisgenesis is also referred to as Agrobacterium-mediated gene transfer from a sexually compatible plant where only the T-DNA borders may be present in the recipient organism after transformation (EFSA, 2012). The cisgenesis precludes linkage drag, and hence, prevents hazards from unidentified hitch hiking genes (Schouten, and Jacobsen, 2008). Compared to transgenesis, one of the disadvantages shared by cisgenesis is that characters outside the sexually compatible gene pool cannot be introduced. Furthermore, development of cisgenic crops involves extraordinary proficiency and time compared to transgenic crops. Therefore, the required genes or fragments of genes may not be readily accessible but have to be isolated from the sexually compatible gene pool (Holme et al., 2013).
On 16 February 2012, European Food Safety Authority (EFSA, 2012) reported the detail study concerning the safety aspects of cisgenic plants and validated that cisgenic plants are secure to be used in terms of environment, food and feed, similar to the traditionally bred plants. However, the present GMO regulation keeps the cisgenic micro-organisms out from its supervision. The first scientific statement of bringing forth a true plant obtained by cisgenic approach was reported in apple through the insertion of the internal scab resistance gene HcrVf2 influenced by their own regulatory genes into the cultivar Gala, a scab susceptible cultivar (Vanblaere et al., 2011). Barley with improved phytase activity was produced successfully by Holme et al. 2011, through cisgenic approach. Late blight resistant potatoes have developed by cisgene stacking of R- gene (jo et al., 2014).
Marker Assisted Selection in Crop BreedingPawan Chauhan
Marker Assisted Selection is a value addition to conventional methods of Crop Breeding. It has been gaining importance in plant breeding with new generation of plant breeders and to get accurate and fast desired result from plant breeding.
A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
Genetic Engineering of Male Sterility for Hybrid Seed Production # Methods of Hybrid Seed Production - Hybridization techniques # Examples of Male Sterile Hybrid Seed
Hybridization between individuals from different species belonging to the same genus or two different genera, is termed as distant hybridization or wide hybridization, and such crosses are known as distant crosses or wide crosses.
Transgenes may be used to produce GMS which is dominant to fertility.
In these cases it is essential to develop effective fertility restoration systems for hybrid seed production.
An effective restoration system is available in at least one case, Barnase/Barstar system
Recombinant DNA techniques have made it possible to engineer new systems of male sterility by disturbing any or number of developmental steps specifically required for the production of functional pollen within the microspore or for the development of any somatic tissues .
It is a presentation prepared to tell people more about male sterility in brief. I have also included one case study to explain and help you. Hope you like it. Thanks!
1. STABILITY OF MALE STERILE LINES - ENVIRONMENTAL INFLUENCE ON STERILITY - EGMS - TYPES AND INFLUENCE ON THEIR EXPRESSION, GENETIC STUDIES.
2. PHOTO SENSITIVE GENETIC MALE STERILITY AND ITS USES IN HETEROSIS BREEDING
3. TEMPERATURE SENSITIVE GENETIC MALE STERILITY AND ITS USES IN HETEROSIS BREEDING
Similar to Genetic Engineering for Male Sterility in Plants (20)
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
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In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
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Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
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2. What is male sterility?
• Male sterility is a situation where the male reproductive parts of a plant are either
absent, aborted, or nonfunctional, and hence they fail to participate in the process of
natural sexual reproduction.
• This situation can arise due to any developmental defect at any stage of
microsporogenesis or release of pollen grains.
• First observed by Koelrouter in 1763-anther abortion within intraspecific hybrids of
tobacco.
• Darwin (1877) recognized the importance of this phenomenon and hypothesized that the
loss of reproducing ability of plant helps evolutionary processes in enhancing adaptation
through gene transfer from various related and unrelated individuals through cross-
pollination.
Sterile Fertile
3. What about female sterility?
• Occurs rarely in nature.
• Not useful in Plant Breeding.
• Very difficult to detect when compared to male sterility (large
no: of pollens produced).
• Does not have the ability of self propagation(seed set).
• Cannot be stained for preparing assays as that of male
sterility.
5. Classification of male sterility
1) Based on the type of malfunctioning of the androecium,
(i) Structural (absence or deformity of anthers)
(ii) Sporogenous (defective microsporogenesis) and
(iii) Functional (failure of mature pollen to germinate) male sterility
2) On the basis of genetic control mechanisms,
(i) Genetic Male Sterility (GMS)
(a) Environment insensitive GMS
(b) Environment sensitive GMS
(ii) Cytoplasmic Male Sterility (CMS) and
(iii) Cytoplasmic Genetic Male Sterility (CGMS)
3) Artificiallly induced male sterility
(i) Chemically induced male sterility and
(ii) Genetically engineered male sterility
7. Environment sensitive Genetic male Sterility(EGMS)
• Induction of male sterility in response to fluctuations in
environmental conditions.
• Particularly
Photoperiod(PGMS) and
Temperature(TGMS).
• Eg:Rice
• Controlled by nuclear genes.
• No need to use maintainer line.
• Two-line hybrid system-Only male sterile line(female parent) and
Pollinator(male parent) present.
8. • PGMS lines(Rice) : Nong-Ken 58 S,Zennong s,X-88.
• TGMS lines(Rice):Annong S,Hennong S,Novin PL 12,IR 68945.
• Any genotype can be used as a Pollinator parent.
INHERITANCE OF EGMS
Contd…
9. Origin of male sterility systems
• Selection from natural variation
• Integration of cultivated genome in to alien cytoplasm (using wild sp.
as female parent)
• Intergeneric and interspecific hybridisation
• Selection from recombinant populations
• Induced mutations (physical and chemical mutagens)
10. Chemical induced Male Sterility
• The chemical which induces male sterility artificially called as
male gametocides are used.
• These chemicals are also known as Chemical hybridizing agents.
• It is rapid method but the sterility is non-heritable.
• In this system A, B and R lines are not maintained.
• Some of the male gametocides used are Gibberellins (Rice,
Maize), Sodium Methyl Arsenate (Rice) and Maleic hydrazide
(Wheat, Onion).
• Could be used in the large scale commercial production of
hybrid seed (Hybrid wheat in UK, Germany).
11.
12. Induced GMS (Transgenic male sterility)
Promoter which
induces transcription
in male reproductive
specifically
Gene which disrupts
normal function of cell
Agrobacterium-
mediated
transformation
regeneration
male-sterile
plant
14. • The first transgene designed to confer GMS was reported and were
used to transform Tobacco and oilseed rape plants.
• Tapetal-specific transcriptional activity of the tobacco TA29 gene.
• Upstream regulatory elements of TA29 gene used to drive the
expression of transgenes (extracellular RNAses from bacteria). Two
genes were used:
barnase from Bacillus amyloliquefaciens
RNAse-T1 from Aspergillus oryzae
• RNase genes selectively destroyed the tapetal cells during anther
development and prevented pollen formation
• Herbicide (bialophos) resistant gene (bar) used as selectable marker
Abstract
17. Gene expression is temporally and spatially regulated
17
Koltunow et al. (1990)
18. Fig 1. RNA gel blot analysis of organ-specific gene expression
Results…
19. Fig 2. Floral morphology of male sterile (transgenic) and untransformed plants
20. Fig 3. Tissue abnormalities in male sterile tobacco
and oilseed rape anthers (Bright field microscopy)
Fig 4. SEM micrographs of pollen grains produced by
male sterile tobacco and oilseed rape anthers
E- epidermis; C- connective tissue; F- filament;
PS- pollen sac and T- tapetum
22. • Barstar gene codes for an intracellular bacterial protein which is an
inhibitor of barnase.
• Oilseed rape plants were transformed with a binary vector containing
barstar gene along with bar gene as selectable marker using
agrobacterium mediated transformation.
• When the transgenic plants containing barstar were crossed with the
male sterile transgenic plants (barnase), the F1 progenies segregated
in the ratio of 2:1 (male fertile : sterile) (viable progenies) after
selection using herbicide spray at the seedling stage.
• Around 25% male sterile plants produced in the F1 will be lacking bar
gene and they will die.
• The plant transformed with the barstar gene serves as the restorer of
fertility.
Abstract
23. Fig 1. Restoration of male fertility by crossing oilseed rape plants
containing the TA29-barstar and TA29-barnase genes
Note: ms, rf and hr refer to the
hemizygous chromosomal loci that
lack the TA29- barnase, TA29-barstar
and bar genes respectively
24. Fig 2. Oilseed rape flowers and anther cross sections
Male fertile plants containing the TA29-barstar gene
Male sterile plants containing the TA29-barnase gene
Male fertile plants restored to fertility containing
both the TA29-barstar and TA29-barnase gene
E-epidermis; En-endothecium; PG- pollen grain; PS- pollen sac and T-tapetum
Results…
25. Fig 3. Scanning electron micrographs of oilseed rape pollen grains and dehiscing
anthers
Dehiscing anther from
an untransformed plant
Dehiscing male sterile
anther from a plant
containing TA29-barnase
gene
Dehiscing anther from a
plant restored to male
fertility containing both
the TA29-barstar and
TA29-barnase gene
Pollen grains –
untransformed anthers
Pollen grains – anthers
containing both the TA29-
barstar and TA-29 barnase
genes – restored to fertility
26. Fig 4. Presence of barnase and barstar mRNAs in anthers of oilseed rape
plants restored to male fertility
27. Fig 5. Presence of barstar and barnase proteins in oilseed rape anthers
restored to male sterility
MS plants (TA29-
barnase gene)
Anther proteins from
wild type plants
MS/RF plants (TA29-
barnase+TA29-barstar)
Barstar and barnase
proteins circled in the
immunoblot
Purified barstar-barnase
complexes (denatured,
fractionated by 2D gel
electrophoresis)
30. • L-ornithinase (argE) gene of E.coli was fused to the OSIPA promoter
sequence which is known to function specifically in the pollen grains.
• OSIPA – Oryza sativa indica pollen allergen
• argE gene – involved in arginine biosynthesis in E.coli and also can
deacetylate N-acetyl Phosphinothricin (N-ac-PPT) to yield
phosphinothricin (PPT).
• N-ac-PPT, a non toxic compound, but when converted in to PPT
becomes cytotoxic.
• Phosphinothricin is the active ingredient of the herbicides Basta
(bialophos) and Glufosinate.
• Homozygous transgenic rice (variety BPT 5204) plants (T2) were
obtained with argE gene by selection with hygromycin (hyg) following
Agrobacterium mediated transformation.
Abstract
31. • Transgenic rice plants expressing argE gene became completely male
sterile after application of N-ac-PPT (inducer) due to the pollen
specific expression of argE.
• The argE transgenic plants produced fertile seeds in the absence of N-
ac-PPT treatment.
• Normal fertile seeds were obtained when male sterile argE
transgenics were cross pollinated with untransformed control plants.
• Female fertility male of sterile argE transgenics is not affected by the
N-ac-PPT treatment.
• First report of induction of complete male sterility in Rice.
• This system does not require the use of restorer line.
Contd…
33. Table 2. Effect of N-ac-PPT on pollen fertility of argE rice transformants after treatment
with irrigation (0.2 mg/ml @ 80-90 days, 50ml/plant)
Table I. Effect of N-ac-PPT on pollen fertility of argE rice transformants after topical
application (0.075 mg/ml @ 100-110 days, 25 ml/plant)
34. Fig 2. Induction of pollen sterility in argE transgenic rice plants by N-acetyl-PPT treatment.
(Alexander staining was carried out before and after N-ac-PPT treatment)
Transformed
Untreated anther and gynoecium Treatment with N-ac-PPT
through irrigation
Treatment with N-ac-PPT by
spraying
Control
35. Fig 3. Pollen grains germination ability of UC and argE-transgenic rice plants before
and after N-ac-PPT treatment
Treated with N-ac-
PPT by spraying
Treated with N-ac-
PPT by irrigation
Untreated pollen
grains
Transformed
Control
36. (B) Plants treated with N-acetyl-
PPT through irrigation
Fig 4. Effect of N-acetyl-PPT on male fertility of argE transgenic rice plants
(A) Plants treated with N-acetyl-PPT
by spraying topically
37. (A) Treated with N-ac-PPT by
spraying
Later, they were allowed to self pollinate. UC: Panicle of untransformed control showing normal
seed setting. 9-6 & 25-3: Panicles of two argE transformants showing the failure of seed setting.
Fig 5. Seed setting ability of argE transgenic rice plants after treating with N-acetyl- PPT
(B) Treated with N-ac-PPT through
irrigation
38. Fig 6. Seed setting ability of argE male-sterile rice lines after pollination with UC plants.
(A) Treated with N-ac-PPT by
spraying
(B) Treated with N-ac-PPT
through irrigation
43. Commercial exploitation of GE male sterility
GM Canola – barnase/barstar system
• Aventis has successfully introduced a GE canola hybrid, using the barnase/barstar gene
system in 1996.
• Regulatory agencies in such countries as Canada , USA , Mexico , Europe, Australia and
Japan have approved consumption of this GE canola
44. Issues in developing Genetically engineered hybrid crops – the Indian case
• One of the DMH-11 genes, called the bar gene, made the plant resistant to a herbicide (or weed killer)
brand-named Basta, a product sold by multinational company Bayer Cropscience.
• Two other genes — that weren’t patented in India — called barnase and barstar — were used to make
mustard varieties more amenable to becoming hybrids.
• DMH-11 developed by crossing Indian and East European mustard varieties. 25-30% more yield.
45. • Hybrid varieties have had the greatest impact on increasing the world’s
feed or food resources.
• Usable male sterility systems have not yet been generated through
conventional breeding methodologies for several important crop plants.
• In such a scenario, genetic engineering becomes an efficient and rapid
approach for developing male sterile lines in these crops.
• The creation of new and efficient means of pollination control to
produce better hybrids will soon be the plant biotechnologist’s
contribution to this effort.