SeminarOn

Molecular approaches for Genetic Engineering in

Male Sterility

SeminarOn

Molecular approaches for Genetic Engineering in

Male Sterility

1

“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

1

6

5

4

2

7 3

3

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

4

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

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 )

6

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

7

Based Stamen (anther and filament) and pollen grains are affectedBased Stamen (anther and filament) and pollen grains are affected

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

Molecular basis of male sterility & 

Anther development

10

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

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

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

Fertility restoration in maize

13

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.

14

Photographs courtesy of G. Brown (Brassica),

P. Simon (carrot), R. Wise (maize), and

K. Glimelius (tobacco).S160 The Plant Cell

15

a) Normal (N-cytoplasm, restored CMS plants)b) Brown anther CMS (Sa)c) Petaloid CMS (Sp)

 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)

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

17

(Kaul, 1988)

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)

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)

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)

Robert B. Goldberg et.al.,1993

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

22

Robert B. Goldberg et.al.,199323

Robert B. Goldberg et.al.,199324

GENE EXPRESSION IS TEMPORALLY AND SPATIALLY REGULATED

25Koltunow et al. (1990)

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)

26

Genetic Engineering for Male sterility

27

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:

28

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

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

30

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

31

1st successful Expt. in transgenic for M.S

by: Mariani et.al.,1990Crop : tobacco

32

Bacillus amyloliquefaciens Aspergillus oryzae

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

Mariani et al. in 1990. 34

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.

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

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

37

2nd successful Expt. by: Kriete G, et.al., 1996

Crop : tobacco

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

Deacetylase system Kriete G, et.al., 1996. 39

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.,2002Crop : tobacco,Tomato

Two component barnase system (Cont..)

41

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

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

43

4th successful Expt. by: Oscar NR et.al.,2005

Crop : tobacco,

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

44

Chloroplast Transformation

pLDR-5’UTR-phaA-3’UTP vector construction

fertileTransformation by

Particle bombardment

45

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

Reversibility of Male Fertility

Acetoacetyl-CoA

Acetyl-CoA

Malonyl-CoA Fatty acid

Acetyl-CoA carboxylase

Illumination for 8 ~ 10 days

Male fertility

β-ketothiolase

47

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

48

5th successful Expt.,2010

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

49

- Fruit in F1 of a cross between a male-sterility plant (B3) and a

Cre-expressing plant (R63).

50

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

51

Female sterility

52

53

54

55

CASE STUDIES IN DIFFERENT CROPS

Brassica juncea Sesamum indicum.L

Poplars Nicotiana tobacum.L

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

56

Case study-1

57

Arun jagannath,200258

59

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]

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

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.

60

Genetic Engineering for male sterility in tree, ornamental &

Hort.crops

61

62

63

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.

• Increase flower longevity and number

• Eliminate nuisance fruit• Increase vegetative growth• Reduce allergic reactions• Prevent sexual propagation

and crossing• Eliminate invasiveness

64

Why Engineer Sterility?

Strategies for Reducing Invasiveness in Horticultural Crops with Engineered Sterility

Nicole GardnerAlan G. Smith

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.

65

Limiting fertility or seed dispersal of established invasive plants is impossible. therefore

The introduction of sterility into ornamental crops before introduction prevents invasive.

Fruits of Lantana Birds aid seed dispersal

interpretation:• New ornamentals are potentially invasive.• Male sterility a new tool to eliminate plant

invasiveness.• Female sterility greatly reduces seed set.

67

Identify new ornamentals where sterility would be most useful.

Introduce sterility gene into known invasive ornamentals.

Develop gene introduction methods.

Future prospectus

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

70

Tree sps

Thank U