This document summarizes a seminar on molecular approaches for genetic engineering of male sterility. It begins by defining male sterility as the inability of flowering plants to produce functional pollen. It then describes different types of male sterility including genic, cytoplasmic, and chemically-induced sterility. The document discusses the molecular basis of male sterility and anther development, using the T cytoplasm in maize as a model system. It also outlines several genetic engineering approaches that have been used to induce male sterility in crops like tobacco, including the use of ribonuclease genes, a deacetylase system, a two-component barnase system, and engineering chloroplast-induced sterility.

1. Seminar
On
Seminar
On
Molecular approaches for
Genetic Engineering in
Male Sterility
1

2. “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)
2

3. 1
2
3
4
5
6
7 3

4. What is Male Sterility ?
 Definition : Inability of flowering plants to
produce functional pollen.
 Male sterility is agronomically important
for the hybrid seed production.
1st documentation: 1763—Kölreuter
Flower of male-fertile pepper Flower of male-sterile pepper
4

5. 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
5

6. Phenotypic expression classes of M.S (Kaul.1988 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)
6

7. Based Stamen (anther and filament) and ppoolllleenn ggrraaiinnss aarree aaffffeecctteedd
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 occasionally
intraspecific crosses
CMS can be a result of interspecific protoplast fusion
e.g: N.tobacum x N. rapandata
7

8. 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

9. Female Fertile Female Sterile
9

10. Molecular basis of male
sterility &
Anther development
10

11. 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

12. 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 Restoration of fertility throu tgoh m aRlfe 1c eall nadbo Rrtifo2n
12
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

13. Fertility restoration in maize
13

14. (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-derived
sequences.
Maureen R. H., et.al. 2004
The Plant Cell, Vol. 16, S154–S169
14

15. a) Normal (N-cytoplasm, restored CMS
plants)
b) Brown anther CMS (Sa)
c) Petaloid CMS (Sp)
Photographs courtesy of
G. Brown (Brassica),
P. Simon (carrot),
R. Wise (maize), and
K. Glimelius (tobacco).
S160 The Plant Cell
15

16. Cytological Changes (Kaul, 1988)
 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

17. Biochemical Changes
(Kaul, 1988)
 Male sterility accompanied by qualitative and quantitative
changes in amino acids, protein, and enzymes in developing
anther
 Amino acids
The level of proline, leucine, isoleucine, phenylalanine and
valine is reduced,
but glycine, arginine, aspartic acids is increased
 Soluble proteins
M.S. anthers contain lower protein content and fewer
polypeptide bands
Some polypeptides synthesized in normal stamens were
absent in mutant stamens
17

18. Biochemical
Changes(cont..)
 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
(Kaul, 1988)

19. Hormones and male
Sterility
 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
(Kaul, 1988)

20. Plant growth regulators and substances
that disrupt floral development
Plant hormones/hormones antagonists
a. 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 substances
a. 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)

21. Robert B. Goldberg et.al.,1993

22. ANTHER DEHISCENCE INVOLVES THE PROGRAMMED
DESTRUCTION 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
22
Koltunow et al. (1990)

23. Robert B. Goldberg et2.3al.,1993

24. Robert B. Goldberg et2.4al.,1993

25. GENE EXPRESSION IS TEMPORALLY AND SPATIALLY
REGULATED
Koltunow et al. (1990) 25

26. TRANSCRIPTIONAL PROCESSES CONTROL
ANTHER-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)
26

27. Genetic Engineering
for Male sterility
27

28. CMS Limitation:
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
28

29. 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.
29

30. Induced GMS (Transgenic male sterility)
Promoter which
induces transcription
in male
reproductive
specifically
Agrobacterium-mediated
transformation
Gene which disrupts
normal function of cell
regeneration
male-sterile
plant
30

31. Dominant NMS linked to a selectable marker
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
31
1st successful Expt. in
transgenic for M.S
by: Mariani et.al.,1990
Crop : tobacco

32. 32
Bacillus amyloliquefaciens Aspergillus oryzae

33. 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
fertile
33

34. Mariani et al. in 1990. 34

35. Selection by Herbicide Application
SH/-
A (SH/-) X B (-/-)
-/- SH/-
-/- -/-
-/- -/- -/-
SH/-
-/-
SH/-
-/- SH/-
-/-
SH/-
pTA29-barnase : S (sterility)
p35S-PAT : H (herbicide resistance)
pTA29-barstar : R (restorer)
-/-
-/- SH/-
-/- SH/- SH/-
-/- -/-
SH/- SH/- -/-
glufosinate
X C (R/R)
Fertile F1 (SH/-, R/-)
Fertile F1 (-/-, R/-)
Mariani et al. in 1990. 35

36. 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).
36

37. 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
Inducible Sterility
 Male sterility is induced only when inducible chemical is applied.
NH4
+
accumulation Male sterility
in tapetal cell
Glutamate Glutamine
N-acetyl-
L-phosphinothricin
(non-toxic)
Glufosinate
N-acetyl-L-ornithine (toxic)
deacetylase
(coded by argE)
 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.
37
2nd successful Expt.
by: Kriete G, et.al., 1996
Crop : tobacco

38. Inducible Sterility
Sterile parent X Fertile parent
Plants transformed
by TA29-argE
fertile
selfing
fertile
Fertile F1 plant
N-acetyl-L-phosphinothricin
Plants transformed
by TA29-argE
Kriete G, et.al., 1996.
38

39. Deacetylase system Kriete G, et.3a9l., 1996.

40. 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
40
3rd successful Expt.
by: Burgess et.al.,2002
Crop : tobacco,Tomato

41. Two component barnase system (Cont..)
41

42. Two-Component System
X
A (Bn5/Bn3)
F1 (Bn3/-)
A2 (Bn3/Bn3)
fertile
A1 (B5/B5)
fertile
fertile
fertile
sterile
selfing selfing
X A2 (Bn3/Bn3)
fertile
A1 (B5/B5)
fertile
B (- -)
A1 (Bn5/Bn5)
A1 (Bn5/Bn5)
X
F1 (Bn5/-)
fertile
A (Bn5/Bn3)
sterile
Bn3 : 3’ portion of barnase gene
Bn5 : 5’ portion of barnase gene 42

43. Plant Physiology, July 2005, Vol. 138, pp. 1232–1246
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.
43
4th successful Expt.
by: Oscar NR et.al.,2005
Crop : tobacco,

44. Reactions for the synthesis of PHB
Acetoacetyl-CoA
reductase
fertile
PHB synthase
Glucose
C
CH S-CoA 3
O
C
CH3
O
C
CH S-CoA 2
HO
CH
CH3
O
C
CH S-CoA 2
O
C
CH2
CH3
CH
O
O
C
CH3
CH3
CH
O C
O
O -
CoASH
NADPH
NADP+
O
Acetyl-CoA
Acetoacetyl-CoA
(R)-3-Hydroxybutyryl-CoA
Polyhydroxybutyrate
(PHB)
n
(phaA gene)
( phaB gene )
(phaC gene)
b-ketothiolase
44

45. Chloroplast Transformation
pLDR-5’UTR-phaA-3’UTP
vector construction
Transformation by
Particle bombardment
fertile
45

46. 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

47. Reversibility of Male Fertility
Acetoacetyl-CoA
Acetyl-CoA
Malonyl-CoA Fatty acid
Acetyl-CoA
carboxylase
Illumination
for 8 ~ 10 days
Male fertility
b-ketothiolase
47

48.  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
The Barnase-coding region was flanked by loxP recognition sites
for Cre-recombinase
Inbred/ pure line (E-8) with ‘cre’ gene by transformation
Inbred/ pure line (E-8) with Barnase gene flanked by loxP
48
5th successful Expt.,2010

49. Stigmata, anthers and flowers of a Barnase transgenic plant and
a non-transgenic plant.
49

50. - Fruit in F1 of a cross between
a male-sterility plant (B3) and a
Cre-expressing plant (R63).
50

51. M.H.S.Goldman', R.B.Goldberg and C.Mariani
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
51
Female sterility

52. 52

53. 53

54. 54

55. CASE STUDIES IN DIFFERENT CROPS
55
Brassica juncea Sesamum indicum.L
Poplars Nicotiana tobacum.L

56. Current science 82(1) 2002
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
56
Case study-1

57. 57

58. 58
Arun jagannath,2002

59. Transformation of engineered male sterile gene and establishment
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 plants
confirmed stable integration of both barnase and bar genes into
nuclear DNA. The transfomation efficiency reached 1.46%.
59
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 Yongru
Henan 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]

60. 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.
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: ncbisht@nipgr.res.in, ncbisht@gmail.com
60

61. Genetic Engineering for male
sterility in tree, ornamental &
Hort.crops
61

62. 62

63. MODIFICATION OF FLOWERING IN TRANSGENIC
63
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.

64. Strategies for Reducing Invasiveness in
Horticultural Crops with Engineered Sterility
Why Engineer Sterility?
Nicole Gardner
Alan G. Smith
• Increase flower longevity
and number
• Eliminate nuisance fruit
• Increase vegetative growth
• Reduce allergic reactions
• Prevent sexual propagation
and crossing
• Eliminate invasiveness 64

65. 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.
Limiting fertility or seed dispersal of established invasive plants
is impossible. therefore
The introduction of sterility into ornamental crops before
introduction prevents invasive.
65

66. Fruits of Lantana Birds aid seed dispersal

67. interpretation:
• New ornamentals are potentially invasive.
• Male sterility a new tool to eliminate plant
invasiveness.
• Female sterility greatly reduces seed set.
 Identify new ornamentals where sterility would be
most useful.
 Introduce sterility gene into known invasive
ornamentals.
 Develop gene introduction methods.
67
Future prospectus

68. 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.

69. conclusions
69

70. 70
Tree sps
Thank U