Expression of Engineered Nuclear Male Sterility Brassica napus
Molecular mechanism of male sterility in plant system
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Transcript of Molecular mechanism of male sterility in plant system
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SeminarOn
Molecular approaches for Genetic Engineering in
Male Sterility
SeminarOn
Molecular approaches for Genetic Engineering in
Male Sterility
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“The greatest advances of civilization, whether in architecture or painting, in science & literature, in industry or agriculture, have never come from centralized government” Milton Friedman (American Economist)
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What is Male Sterility ? Definition : Inability of flowering plants to produce functional pollen.
Male sterility is agronomically important for the hybrid seed production.
Flower of male-fertile pepper Flower of male-sterile pepper
1st documentation: 1763—Kölreuter
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Types of Male Sterility
Genic Male Sterility (GMS)
- caused by the mutation in nucleus genome
- inherited as a recessive trait
- TGMS, PGMS, Transgenic genetic (G.E)
Cytoplasmic Male Sterility (CMS)
- caused by the mutation in mitochondrial genome
- inherited as a maternally transmitted trait
Cytoplasmic Genetic Male Sterility (CGMS)
Chemically induced Male Sterility
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i. Structural male sterility: anomalies in male sex
organs (or missing all together)
ii. Sporogenous male sterility: stamens form, but pollen absent or rare due to microsporogenous cell
abortion before, during, or after meiosis
iii. Functional male sterility: viable pollen form, but barrier prevents fertilization (anther indehiscence, no exine formation, inability of pollen to migrate to stigma
e.g.soybean, peas)
Phenotypic expression classes of M.S (Kaul.1988 )
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a. Autoplasmic
CMS because of spontaneous mutational changes in the cytoplasm, most likely in the mitochondrial genome of within the species.
b. Alloplasmic
CMS from intergeneric, interpecific or occasionallyintraspecific crosses
CMS can be a result of interspecific protoplast fusion
e.g: N.tobacum x N. rapandata
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Based Stamen (anther and filament) and pollen grains are affectedBased Stamen (anther and filament) and pollen grains are affected
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Male sterility(M.S) More prevalent than female sterility:?
i. less protected :Male sporophyte and gametophyte from environment than ovule and embryo sac.
ii. Easy to detect M.S: because a large number of pollen for study vailable
iii. Easy to assay M.S: staining technique (caramine, lactophenol or iodine); female sterility (fst) requires
crossing.
iv. M.S.has propagation potential in nature (can still set seed) and is important for crop breeding, fst does not.
(Kaul, 1988) 8
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Female Fertile Female Sterile
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Molecular basis of male sterility &
Anther development
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T cytoplasm in maize as model system cms (cms-T)
1.Texas (T) cytoplasmic male sterility discovered in 1940s;
2. Highly stable under all environmental conditions.
3. Characterized by failure of anther exertion and pollen abortion.
4. T- cytoplasm, are susceptible to race T of the southern corn leaf blight – (Bipolaris maydis) Race -T exceptionally virulent.
5. Widespread use of T-cytoplasm led to epidemic in 1970 with the wide spread rise of Race T.
6. Male sterility and sensitivity to fungal toxins- both are mediated by the same gene product 11
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T-urf13 is probably cause of male sterility and disease susceptibility 1.Only present in T cytoplasm
2. Encodes a 13 kDa polypeptide (URF13) associated with inner
mitohondrial membrane (Forde et al. 1978) are also sensitive to the insecticide methomyl.
How does URF13 cause cms?
1.Degeneration of the tapetum during microsporogenesis (Wise et al., 1999) 2. Disruption of pollen development leading to male cell abortion Restoration of fertility through Rf1 and Rf2
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CMS-C type: produces 17.5 Kd protein causes male sterility and fertility restored by Rf4 gene
CMS-S type: fertility restored by Rf3 gene
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Fertility restoration in maize
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Maureen R. H., et.al. 2004The Plant Cell, Vol. 16, S154–S169
(urf13 encodes a 13-kD protein;
pcf indicates petunia CMS Red indicates genes for subunits of ATP synthase.
Shades of blue indicate unknown reading frames within CMS regions.
Shades of yellow indicate genes for subunits of cytochrome oxidase.
Orange indicates ribosomal protein genes.
Green indicates chloroplast-derivedsequences.
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Photographs courtesy of G. Brown (Brassica),
P. Simon (carrot), R. Wise (maize), and
K. Glimelius (tobacco).S160 The Plant Cell
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a) Normal (N-cytoplasm, restored CMS plants)b) Brown anther CMS (Sa)c) Petaloid CMS (Sp)
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Breakdown in microsporogenesis can occur at a number of pre-or postmeiotic stages
The abnormalities can involve aberration during the process of meiosis,
a)in the formation of tetrads,
b)during the release of tetrad
(the dissolution of callose),
c) at the vacuolate microspore stage or
d) at mature or near-mature pollen stage
Cytological Changes (Kaul, 1988)
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Male sterility accompanied by qualitative and quantitative changes in amino acids, protein, and enzymes in developing anther
Amino acidsThe level of proline, leucine, isoleucine, phenylalanine and valine is reduced,
but glycine, arginine, aspartic acids is increased
Soluble proteinsM.S. anthers contain lower protein content and fewer polypeptide bandsSome polypeptides synthesized in normal stamens were absent in mutant stamens
Biochemical Changes
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(Kaul, 1988)
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Enzymes
Callase required for breakdown of callose that surrounds PMCs and the tetrad. Low callase activity leads to premature or delayed release of meiocytes and microspore
Esterases have also been related to pollen development. The activity of esterase is decreased
The activity of amylases is decreased and it corresponds with high starch content and reduced levels of soluble sugars
Accumulation of adenine due to the decrease of adenine phosphoribosyltransferase (APRT) activity may be toxic to the development of microspores
Biochemical Changes(cont..)
(Kaul, 1988)
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Plant growth substances(PGS) play an important role in stamen and pollen development.
GMS line was related to a change in the concentration of gibberellins (rice), IAA (Mercurialis annua), ABA (soybean), and cytokinin (Mercurialis annua)
Male serility is associated with changes not in one PGS but several PGS
Hormones and male Sterility
(Kaul, 1988)
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Plant growth regulators and substances that disrupt floral development
Plant hormones/hormones antagonistsa. auxins and auxin antagonists (NAA, IBA, 2,4-D, TIBA, MH)
b. Gibberellins and antagonist (GA3, GA4+7, CCC: 2-
chloroethyl-trimethyl ammonium chloride)
c. ABA caused male sterility if applied to plant just prior to or
during meiosis of pollen mother cells (wheat).
Other substancesa. LY195259- It is 5-(aminocarbonyl)-1-(3-methylphenyl)-1H-pyrazole-4-carboxylic-acid
b. TD1123: potassium 3,4-dichloro-5-isothiocarboxylate (Sawhney and Shukla, 1994)
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Robert B. Goldberg et.al.,1993
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Koltunow et al. (1990)
ANTHER DEHISCENCE INVOLVES THE PROGRAMMEDDESTRUCTION OF SPEClFlC CELL TYPES
(1)Fibrous band thickenings on the endothecial cell walls,
(2) Degeneration of the circular cell cluster and merging of the two pollen sacs in each theca into a single locule,
(3)breakdown of the tapetum & connective,
(4) Rupture of the anther at the stomium and pollen release
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Robert B. Goldberg et.al.,199323
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Robert B. Goldberg et.al.,199324
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GENE EXPRESSION IS TEMPORALLY AND SPATIALLY REGULATED
25Koltunow et al. (1990)
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TRANSCRIPTIONAL PROCESSES CONTROLANTHER-SPECIFIC GENE EXPRESSION PROGRAMS
e.g:- the tapetal-specific TA29 Gene is not transcribed detectably in other plant organs, and chimeric genes with TA29 5’ sequences are active only in the tapetum
(Koltunow et al., 1990; Mariani et al., 1990, 1992)
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Genetic Engineering for Male sterility
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a. Pleiotropic negative effect of the CMS on agronomic quality performance
b. Enhanced disease susceptibility
c. Complex and environmentally unstable maintenance of male sterility and/or male fertility restoration
d. Inability to produce commercial hybrid seed economically because of poor floral characteristic
CMS Limitation:
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Why Genetic Engineering?
Conventional breeding for sterility can be difficult, long term, and in some cases, impossible.
Introduce more than one gene at a time.
Genetic engineering preserves original traits of the plant.
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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
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The first transgene designed to confer NMS was reported by Mariani et al. in 1990.
Tapetal-specific transcriptional activity of the tobacco TA29 gene.
barnase from Bacillus amyloliquefaciens
RNAse-T1 from Aspergillus oryzae
RNase genes selectively destroyed the tapetal cells during anther development and prevented pollen formation
herbicide glufosinate-ammonium resistant gene
Dominant NMS linked to a selectable marker
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1st successful Expt. in transgenic for M.S
by: Mariani et.al.,1990Crop : tobacco
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Bacillus amyloliquefaciens Aspergillus oryzae
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Selection by Herbicide Application
TA29 Banase NOS-T
TA29 Barstar NOS-T Gene for a RNase from
B. amyloliqefaciens
Tapetum-specitic
promoter
35S PAT NOS-T
Gene for glufosinate resistance from S.
hygroscopicus
Gene for inhibitor of barnase from
B. amyloliqefaciens
fertile33
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Mariani et al. in 1990. 34
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Selection by Herbicide Application
pTA29-barnase : S (sterility)p35S-PAT : H (herbicide resistance)pTA29-barstar : R (restorer)
SH/-
SH/-
-/- SH/-
SH/-
-/- SH/-
-/-
SH/-
-/-
-/- SH/-
-/- SH/-SH/-
-/- -/-
-/-SH/-SH/-
-/- -/-
-/- -/-
-/--/--/-
-/- -/-
A (SH/-) X B (-/-)
glufosinateX C (R/R)
Fertile F1 (SH/-, R/-)
Fertile F1 (-/-, R/-)
35Mariani et al. in 1990.
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Other strategies have been developed
(1) The use of a chimeric tapetal-specific glucanase gene to prematurely disrupt microspore development (Worrall et al., 1992),
(2) Antisense inhibition of flavonoid biosynthesis with in tapetal cells to disrupt pollen development (van der Meer et al., 1992),
(3) overexpression of a chimeric cauliflower mosaic virus 35s atp9 that disrupts anther development (Hernould et al., 1993).
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Inducible Sterility Male sterility is induced only when inducible chemical is applied.
Glutamate Glutamine
NH4+
N-acetyl- L-phosphinothricin (non-toxic)
Glufosinate (toxic)N-acetyl-L-ornithine
deacetylase (coded by argE)
Male sterilityaccumulationin tapetal cell
Plants of male sterile line were transformed by a gene, argE, which codes for N-acetyl-L-ornithine deacetylase, fused to TA29 promoter.
Induction of male sterility can occur only when non-toxic compound N-acetyl-L-phosphinothricin is applied.
Kriete G, et.al., 1996. Male sterility in transgenic tobacco plants induced by tapetum-specific deacetylation of the externally applied non-toxic compound N-acetyl-L -Phosphinothricin. The Plant J. 9: 809-818
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2nd successful Expt. by: Kriete G, et.al., 1996
Crop : tobacco
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Inducible Sterility
Sterile parent X Fertile parent
fertile
selfing
Plants transformed by TA29-argE
fertile
Fertile F1 plant
N-acetyl-L-phosphinothricin
Plants transformed by TA29-argE
Kriete G, et.al., 1996. 38
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Deacetylase system Kriete G, et.al., 1996. 39
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Male sterility is generated by the combined action of two genes brought together into the same plant by crossing two different grandparental lines each expressing one of the genes.
Each grandparent has each part of barnase.
Two proteins which are parts of barnase
Two proteins can form stable barnase
The Plant Journal (2002) 31(1), 113-125
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3rd successful Expt. by: Burgess et.al.,2002Crop : tobacco,Tomato
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Two component barnase system (Cont..)
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Two-Component System
X
F1 (Bn3/-)
A (Bn5/Bn3)
A2 (Bn3/Bn3)fertile
A1 (B5/B5)
fertile
fertile
fertile
sterile
X A2 (Bn3/Bn3)fertile
A1 (B5/B5)fertile
B (- -)
A1 (Bn5/Bn5)
A1 (Bn5/Bn5)
X
F1 (Bn5/-)fertile
A (Bn5/Bn3)sterile
selfing selfing
Bn3 : 3’ portion of barnase geneBn5 : 5’ portion of barnase gene42
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Engineering CMS via the Chloroplast Genome
CMS is induced by the expression of phaA gene in chloroplast. Fertility is restored by continuous illumination. Non-transgenic plants are used as the maintainer for the propagation of male sterile plants.
Plant Physiology, July 2005, Vol. 138, pp. 1232–1246
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4th successful Expt. by: Oscar NR et.al.,2005
Crop : tobacco,
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Reactions for the synthesis of PHB
PHB synthase
fertileAcetoacetyl-CoA
reductase
Glucose
CS-CoACH3
O
CCH3
O
CS-CoACH2
CH3
HO
CH
O
CS-CoACH2
O
C
CH2
CH
CH3
O
O
C
CH3
CH
CH3
O C
O
O -
CoASH
NADPHNADP+
OAcetyl-CoA
Acetoacetyl-CoA
(R)-3-Hydroxybutyryl-CoA
Polyhydroxybutyrate (PHB)
n
(phaA gene)
( phaB gene )
(phaC gene)
β-ketothiolase
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Chloroplast Transformation
pLDR-5’UTR-phaA-3’UTP vector construction
fertileTransformation by
Particle bombardment
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Mechanism for CMS
Pollens of untransformed plant
Pollens of transgenic plant
Microspores and surrounding tapetal cells are particularly active in lipid metabolism which is especially needed for the formation of the exine pollen wall from sporopollenin.
High demand for fatty acid in tapetal cells cannot be satisfied because of the depletion of acetyl-coA. 46
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Reversibility of Male Fertility
Acetoacetyl-CoA
Acetyl-CoA
Malonyl-CoA Fatty acid
Acetyl-CoA carboxylase
Illumination for 8 ~ 10 days
Male fertility
β-ketothiolase
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Inbred/ pure line (E-8) with ‘cre’ gene by transformation
Inbred/ pure line (E-8) with Barnase gene flanked by loxP
The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase
a novel method was designed to create an MS line with Barnase
and a restore line with Cre/loxP there by substituting Barstar.
Eggplants transformed with either Cre or Barnase under the control of the promoter TA29 flanked by 2 identical loxp sites
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5th successful Expt.,2010
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Stigmata, anthers and flowers of a Barnase transgenic plant and a non-transgenic plant.
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- Fruit in F1 of a cross between a male-sterility plant (B3) and a
Cre-expressing plant (R63).
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a. The STIGI gene is developmentally regulated & expressed specifically in the stigmatic secretory zone.
b. Pistils of transgenic STIGI barnase plants have normal development,but lack the stigmatic secretory zone and are female sterile.
c. Application of stigmatic exudate from wild-type pistils to the ablated surface increases the efficiency of pollen tube germination
d. Demonstrate the importance of the stigmatic secretory zone in the pollination
e. The cytotoxic STIGI-barnase gene was then transferred Via Agrobacterium
M.H.S.Goldman', R.B.Goldberg and C.Mariani
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Female sterility
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CASE STUDIES IN DIFFERENT CROPS
Brassica juncea Sesamum indicum.L
Poplars Nicotiana tobacum.L
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B.Juncea cv.varuna
B.Juncea is predominantly self pollinated crop
B. oxyrrhina, B.polima, B.tournefortii, B.maricandia studied for their sterility
3 barnase line x several single copy barstar lines
Out of 30 different cross combinations only one restored fertility in among F1 progeny (Heterozygous for BarN & Bar S)
F2 analysis showed stable inheritance of BarN and BarS
Current science 82(1) 2002
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Case study-1
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Arun jagannath,200258
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The engineered M.S gene barnase together with the mark gene bar were transformed to isolated cotyledon of a sesame variety Yuzhi 4 by microprojectile
The transformed cotyledon cultured in darkness for 12 days then converted into dark-light alternative conditions on the culture media MS+5mg/L BAP+1mg/L IAA+1mg/L ABA + 5mg/L AgNO3.
Rest. calli were selected on herbicide Basta 2mg/ L. 11 anti-Basta plants were obtained. Southern blot of 3 well-developd green plantsconfirmed stable integration of both barnase and bar genes into nuclear DNA. The transfomation efficiency reached 1.46%.
Transformation of engineered male sterile gene and establishment of transgenic plants in sesame (Sesamum indicum L.)
Chen Zhankuan, Zhi Yubao, Yi Minglin, Wang Jinlan, Liang Xiuyin, Tu Lichuan, Fu Rongzhao, Cao Guangcheng, Shi Yanhong, Sun YongruHenan Academy of Agricultural Sciences.Zhengzhou 450002,Henan;China;;Institute of Genetics,Chinese Academy of Science.Beijing 100101;China,Act Agriculturae Boreali-Sinica [1996, 11(4):33-38]
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Bisht NC, Jagannath A, Gupta V, Burma PK and Pental D. A novel method for obtaining improved fertility restorer lines for transgenic male-sterile crop plants and a DNA construct for use in said method.
US Patent 7741541 (granted on 22.06.2010); Indian Patent 238973(granted on 03.03.2010); European Patent 1644506 (granted on 09.09.2009).
Dr. Naveen Chandra Bisht Staff Scientist III
Ph.D. Genetics, University of Delhi South Campus, India Tel: 91-11-26735183 Fax: 91-11-26741658
Email: [email protected], [email protected]
Retransformation of a male sterile barnase line with the barstar gene as an efficient alternative method to identify male sterile-restorer
combinations for heterosis breeding.Bisht NC, Jagannath A, Burma PK, Pradhan AK, Pental D.
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Genetic Engineering for male sterility in tree, ornamental &
Hort.crops
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MODIFICATION OF FLOWERING IN TRANSGENIC TREES
Richard Meilan*, Amy M. Brunner, Jeffrey S. Skinner, and Steven H. Strauss (2001)Forest Science Department, Oregon State University, Corvallis, Oregon, U.S.A, 97331-5752;
Sterility can reduce genetic pollution from plantations, promote vegetative growth, and eliminate nuisance tissues.
Flowering control should also allow for shorter breeding cycles.
Engineering sterility has advantages, but which technique is best ?
variety of techniques, such as tissue-specific ablation; dominant negative mutations; and post-transcriptional gene silencing, including RNA interference. Using the first approach, Arabidopsis gene APETALA3 promoter has directed expression of reporter and cytotoxin genes. arabidopsis, tobacco, and poplar.
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• Increase flower longevity and number
• Eliminate nuisance fruit• Increase vegetative growth• Reduce allergic reactions• Prevent sexual propagation
and crossing• Eliminate invasiveness
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Why Engineer Sterility?
Strategies for Reducing Invasiveness in Horticultural Crops with Engineered Sterility
Nicole GardnerAlan G. Smith
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Invasive Plants
• Introduced plants have the potential to become invasive and disrupt natural ecosystems
• The introduction is usually irreversible
• Mostly these were originally introduced for ornamental and landscape purposes.
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Limiting fertility or seed dispersal of established invasive plants is impossible. therefore
The introduction of sterility into ornamental crops before introduction prevents invasive.
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Fruits of Lantana Birds aid seed dispersal
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interpretation:• New ornamentals are potentially invasive.• Male sterility a new tool to eliminate plant
invasiveness.• Female sterility greatly reduces seed set.
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Identify new ornamentals where sterility would be most useful.
Introduce sterility gene into known invasive ornamentals.
Develop gene introduction methods.
Future prospectus
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Advantages of CMS Engineering
Male sterile parent can be propagated without
segregation. Transgene is contained via maternal inheritance. Pleiotropic effects can be avoided due to subcellular compartmentalization of transgene products. Non-transgenic line can be used as maintainer.
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conclusions
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Tree sps
Thank U