Genetic mechanisms in Cole crops and their utilization in Hybrid seed production

Speaker:-Girdhar Gopal Soni M.Sc. 2nd year A-2010-30-54 1

Introduction• India is the leading vegetable producer in the world

occupying an area of 7.98 mha with the production of 133.74 mt ( NHB 2010) and ranks second in vegetable production after China

• In H.P. area under vegetables is 79.8 (in000’HA) with the production of 1390.7 (in 000MT) ( NHB 2010)

• Limited cultivated area, increasing population, and ever increasing pressure on land due to urbanization & industrialization has left us only with one solution i.e. to increase the productivity

2

Crops Area(IN 000 HA)

Production(IN 000 MT)

Productivity(IN MT/HA)

Cabbage 331.0 7281.4 22.0

Cauliflower 347.9 6569.0 18.9

• The productivity of vegetables can be increased by using F1 hybrids along with improved qualities & standardization of agro-techniques

• Area, production and productivity of major Cole crops in India

(NHB, 2010) 3

• The cost of hybrid seeds is comparatively higher, which is one of the major constraints in achieving more rapid adoption of the hybrid vegetable technology

• With the use of genetic mechanisms i.e. male sterility, self incompatibility etc., we can reduce the cost of hybrid seed production

• And these mechanisms will be helpful in economic hybrid seed production and availability of hybrid seed within the reach of poor farmers

4

Genetic mechanisms of hybrid development in Cole crops

Self incompatibility:- SI refers to the inability of a plant to set seed upon self pollination despite male and female gamete is viable, there is no seed set in self pollination

Lewis (1954) has suggested various classifications of self incompatibility

Heteromorphic system• Pin type• Thrum type

Homomorphic system• Sporophytic self incompatibility (SSI)

i.e. (Cole crops)• Gametophytic self incompatibility (GSI)

5

Difference between GSI and SSI

Gametophytic SI Sporophytic SI

The stigma is smooth and wet The stigma is papilate ( hairy) and dry

Pollen tube inhibition in style Pollen tube inhibition take place on the stigmatic surface itself

The pollen-pistil interaction govern by haploid genome of each male gametes and diploid genome of pistil tissue ( Haplo-Diplo)

The pollen-pistil interaction govern by genome of the plant on which the male and female gamete produced ( Diplo-Diplo)

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Gametophytic self incompatibilitySporophytic self incompatibility

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Male sterility:- Male sterility is defined as the deviant condition in normally bisexual plants (monoecious as well as hermaphrodite) when no viable pollen is formed

Kaul (1988) classified male sterility in three major groups

• Genetic male sterility

• Morphological male sterility:- Not found in Brassicaceae

• Environmental genic male sterility (EGMS) TGMS- Thermo sensitive genic male sterility PGMS- Photoperiod sensitive male sterility

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Genetic male sterilityPhenotypic

• Structural male sterility: Abnormalities in male sex organs Tomato (positional sterility (Atanassova 1999) etc.

• Sporogenous male sterility: Stamens form, but pollen absent

• Functional male sterility: Viable pollen form, but barrier prevents fertilization

Eggplant (functional male sterility in eggplant (Phatak and Jaworski l989) )Tomato, cucumber etc.

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Genotypic

• Genic male sterility: Mendelian inheritance due to nuclear gene (Tomato, Capsicum, cabbage, cauliflower, Watermelon, Lima bean, Lettuce, Muskmelon etc.)

• Cytoplasmic male sterility: Non-Mendelian inheritance – cytoplasmic (Tomato, Pepper, Cole vegetables, Onion, Carrot, Radish)

• Gene-cytoplasmic male sterility: Both nuclear and cytoplasmic genes (Onion, Carrot, Sugarbeet, Capsicum, Radish etc. )

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Why male sterility and self incompatibility?

• Reduced the cost of hybrid seed production

• Production of large scale of F1 seeds

• It avoids enormous manual work of

emasculation and pollination

• Speedup the hybridization programme

• Commercial exploitation of hybrid vigour

11

Why hybrids are adopted by farmers?

• Earliness• Greater productivity• Better adoptability to variable

environments• Better tolerance against diseases

and pests• Uniform produce• Better market acceptability• Better nutritional quality

12

Self incompatibility and its utilization in hybrid seed production

13

History of self-incompatibility

• First discussion on self-incompatibility by Darwin (1877)

• The term self incompatibility was given by Stout (1917)

• Bateman (1952, 1954, 1955) gave explanation on incompatibility in three Brassicas plants namely, Iberis amara L., Raphanus sativus L. and Brassica campestris L.

  14

Sporophytic self incompatibility• SI phenotype of the pollen is

determined by the genotype of the sporophyte (pollen producing parent)

• It is controlled by single s-locus with multiple allele but also reported more than one loci, more than 80 s-alleles reported in Brassica family ( On the basis of population)

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This system was confirmed :-

Kale ( Thompson 1957)

Radish ( Sampson 1957)

Broccoli ( Sampson 1957 and Odland 1962)

Cabbage ( Adamson 1965)

Cauliflower ( Hoser-krauze 1979)

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Genetics of self-incompatibility

• Bateman (1955) described the control of SI in family Brassicaceae by a single Mendelian locus, the S (Sterility) locus, which exists as multiple alleles

The number of S-locus alleles is usually large:-

22 in Iberis (Bateman 1955)

34 in Raphanus (Sampson 1957)

30 in B. rapa (Nou et al. 1993)

50 in Brassica oleracea (Brace et al. 1994)

17

• A classical genetic analysis has grouped the Brassica S-alleles into two categories based on their phenotypic effect on self-incompatibility characteristics

First group of alleles (high-activity)

Second group of alleles (low-activity)

The first group of alleles (high-activity) are placed relatively high on the dominance scale and exhibit a strong self-incompatible phenotype in which an average of 0 to 10 pollen tubes develop per self-pollinated stigma

The second group of alleles (low-activity) demonstrate a weak or leaky self incompatible phenotypic effect in which 10 to 30 pollen tubes develop per self-pollinated stigma and they are considered to be recessive

(Nasrallah et al. 1991) 18

The compatible or incompatible reaction of pollen and stigma depends

• Genotypes (homozygous/heterozygous) of male and female plant at S locus

Interactions between the two S alleles Dominance (S1 > S2) Co-dominance (S1 = S2) Mutual wakening (no action by either allele) Intermediate gradation (1-100% activity by each

allele)

19

Molecular mechanism of SSI in Brassicaceae

SI recognition is controlled by a multiallelic gene complex

at a single locus, termed the S-locus

The S-locus consists of three genes

SRK (S-locus receptor kinase); (Stein et al.1991)

SP11(S-locus protein 11)/SCR (S-locus cysteine rich);

(Suzuki et al.1999; Takayama et al. 2000)

SLG (S-locus glycoproteins )

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Female determinant

SLG and SRK exhibit a number of characteristics that would be expected for the female determinant of SI

They are predominantly produced in the stigma papilla

cells, which come into direct contact with pollen

Their expression occurs just prior to flower opening

They exhibit allelic sequence diversity among all of the

S-haplotypes21

Male determinant

SP11/ SCR is the male determinant and is predominantly expressed in the anther tapetum

Immunohistochemical studies suggest that the SP11/SCR protein is secreted in a cluster from the tapetal cells into the anther locule and translocated to the pollen surface

The cloning and sequencing of the S-locus region using fluorescent differential display, succeeded in identifying the male determinant genes, which were named SP11/ SCR

22

(Takayama and Isogai 2005)23

Upon pollination, SP11 penetrates the papilla cell wall and binds SRK in an S-haplotype-specific manner

This binding induces the autophosphorylation of SRK, triggering a signaling cascade that results in the rejection of self-pollen

MLPK (M-locus protein kinase) localizes papilla cell membrane and may form a signaling complex with SRK

ARC1(Armadillo repeat-containing 1) identified through protein interaction with SRK

The proteasomal degradation of these substrates could result in pollen rejection

24

Assessment of SI

Number of seed set after each specific self- or cross-

pollination

The fluorescent microscopic observations on pollen

ability to penetrate style (within 12-15 hr) (Dyki 1978)

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Problems in exploiting self Incompatibility

Sib-incompatibility is weak in certain inbreds

Continuous inbreeding may lead to complete loss

of the inbred lines

Pseudo-incompatibility

Hybrid seeds would be expensive if the self-

incompatible lines are difficult to maintain

26

Characteristic of the superior self incompatible lines

Stable self incompatibility

High seed set of self pollination at bud stage

Favorable and uniform economic characters

Desirable combination ability

27

Basic steps in the use of SSI

• Identification of self-incompatible plants in diverse

population/genotypes

• Development of homozygous self-incompatible lines

• Identification of S-alleles in the homozygous self-incompatible

lines

• Establishment of inter-allelic relationships among the S-alleles

• Identifying the best combining lines

• Maintenance of parental self-incompatible lines

• Commercial hybrid seed production28

Maintenance of homozygous SI inbreds

• Bud pollination / Sibmating

• Treatment with CO2 gas (CO2 enrichment) (Jirik 1985) or

sodium chloride ( Kucera 1990)

Other methods:-

• Electronic aided pollination (EAP); (Roggen et al. 1972)

• Steel brush method ( Roggen and Dijik 1972 )

• The pollen washing ( Roggen 1974)

• Thermally aided pollination (TAP); (Roggen and Dijik 1976)29

Use SI line for hybrid production

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Types of crosses

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Public sector hybrids developed by utilizing SI in Cole crops

Crop Name of Hybrid Type of GeneticMechanism (Parentage)

Developing Institution

Cabbage KGMR-1, BRH-5 Self-Incompatibility(KGMR-1=83-1-621 x GA-

111)

IARI regional station,Katrain

Cabbage H-43, H-44 Self-Incompatibility(H-43=S2S2 x Pusa

Mukta)(H-44=S2S2 x Cornell 83-6

IARI regional station,Katrain

Cauliflower Pusa Hybrid-2 (Novmaturing, Group-II), Pusa

Kartik Sankar (group-I)

Self-Incompatibility(Pusa Hybrid-2=CC x 18-

19)(Pusa Kartik Sankar= CC

14 x 41-5)

IARI, New Delhi

Cauliflower Xiahua 6 ( heat-resistant ) self-incompatibility X iamen Agricultural Research Institute of Sciences, China, 2006

32

Male sterility and its utilazation in hybrid seed production

33

History of male sterility Genic male sterility has been reported in cabbage

(Rundfeldt 1960), cauliflower (Nieuwhof 1961)

Male sterility systems have been also developed through

genetic engineering (Williams et al. 1997) and protoplast

fusion (Pelletier et al. 1995)

Male sterility were artificially induced through mutagenesis

(Kaul 1988)34

Manifestations of male sterility • Absence or malformation of male organs

• Failure to develop normal microsporogenous tissue- anther

• Abnormal microsporogenesis deformed or inviable pollen

• Abnormal pollen maturation

• Non dehiscent anthers but viable pollen, sporophytic control

• Barriers other than incompatibility preventing pollen from

reaching ovule

Mehdi and Anwar 2009 35

More prevalent than female sterility

• Male sporophyte and gametophyte less protected from environment than ovule and embryo sac

• Easy to detect male sterility, because a large number of pollen for study available

• Easy to assay male sterility:- Staining technique (caramine, lactophenol or iodine) Female sterility requires crossing

• Male sterility has propagation potential in nature36

Male sterility in Brassica

Genic male sterility

Wide occurrence in plants

Mostly governed by a single recessive nuclear gene , ms

Male sterile alleles arise spontaneously or may be artificially induced

37

Cytoplasmic male sterility (CMS) systems

Determined by the cytoplasm

It is the result of mutation in the mitochondrial genome

(mt-DNA)

CMS transfer easily to a given strain

Most CMS associated genes are chimeric mitochondrial

sequences (Schnable and Wise 1998)

Chemically induced male sterility

Engineering male sterility38

Cytoplasmic male sterility Ogura CMS system (Intergeneric crosses)

Ogura male sterility was reported in Japanese radish, (Ogura 1968)

It was introduced in cabbage nucleus into the cytoplasm of Ogura

male sterile radish (R-cytoplasm) by repeated back crossing

(Bannerot et al. 1974)

This CMS in cabbage had the problem of chlorosis at low

temperature (below 120C), it replace by protoplast fusion (Radish

chloroplasts with Brassica oleracea) ; (Robertson et al. 1987)

Ogura-CMS is stated to be a mitochondrial DNA encoded male

sterility39

CMS induced by interspecific cross B. oleracea with rutabaga – B. napus (Chiang, Crete 1985,

1987) transfer resistance against Plasmodiophora brassicae

Radish and B.oleracea L. var. italic (broccoli) and later into Cabbage (Pearson 1972)

Other sources of CMS:- B. nigra sterile cytoplasm and sterile ‘Anand’ cytoplasm

from B. rapa in which it was originally derived from the wild species, B. tournefortii (Swarup 2006)

One such CMS system 'tour' which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp.(Arumugam et al. 1996)

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Origins of CMS

Intergeneric crosses

Interspecific crosses

Intraspecific crosses

Mutagens (EMS, EtBr)

Antibiotic (streptomycin and Mitomycin)

Spontaneus

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Advantages of CMS

• Highly stable and not influenced by environmental conditions

Limitations of CMS

• Not use where seed is the economic product

• CMS line has inferior agronomic performance

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Maintenance of CMS line

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Basic steps in the use of CMS

• A-line (female parent) of desired genetic background (S-

msms)

• B-line (maintainer): This is an isogenic line (genetically N-

msms)

• C-line (male parent): This is a male parental line and also

the best specific combiner with A line.

• Maintenance of A, B and C lines

• Hybrid seed production

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45

Public sector hybrids developed by utilizing CMS in Cole crops

Crop Name of Hybrid Type of GeneticMechanism (Parentage)

Developing Institution

Cabbage KCH-5 Ogura CMS IARI regional station,Katrain

Cabbage H-11, H-46 CMS(H-11=Cornell 83-23 x golden acre), (H-46=Cornell 83-23 x golden acre)

IARI regional station,Katrain

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Genetic male sterility(GMS)

• GMS has been reported in about 175 plant species (Kaul 1988) including important vegetable crops

Salient points• Usually recessive & monogenic • GMS does not have any undesirable agronomic

characters

Limitations of GMS• Less Stable to temperature and photoperiod • 50% of the fertile plants to be removed from the field • Availability of marker gene- closely linked with ms gene  

47

Origin of GMS

Spontaneous mutation

Mutation by ionizing radiation

Chemical mutagens such as ethyl methane sulphonate

(EMS) and ethyl imine (EI)

Genetic engineering

Protoplast fusion

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Maintenance of GMS

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Use GMS for hybrid development

LINE A X LINE C

Male sterile Male fertile

msms MsMs

COMMERCIAL HYBRID

Msms (AXC)

(All male fertile)

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Male-sterility through genetic engineering

(Goldberg et al. 1993)51

Dominant Nuclear Male Sterility (Barnase/barstar system (Goldberg et al. 1993 ))

Barnase is extracellular RNase ( from bacterium Bacillus

amyloliquefaciens)

Barstar is inhibitor of barnase (B. amyloliquefaciens; barstar to

protect itself from barnase)

Fuse the barnase and barstar genes to TA29 promoter (TA29 is

a plant gene that has tapetum specific expression)

Plants containing the TA29–barnase construct are male sterile

Those with TA29–barstar are not affected by the transgene

Barstar is dominant over barnase

Mehdi and Anwar 200952

Induced GMS

Promoter which induces transcrip-tion in

male reproductive specifically

Gene which disrupts normal function of

cell

Agrobacterium-me-diated transforma-

tion

regenera-tion

male-ster-ile plant

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Engineering genic male sterility using barnase(A) and restoring fertility to F1 plants using barstar (B)

Vinod 200554

Using engineered male sterility in F1 hybrid seed production

The barnase-barstar male sterility-fertility restoration system was identified in Cauliflower and Tomato ( Banga and Raman 1998)55

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 (-/-)

glufosinate

X C (R/R)

Fertile F1 (SH/-, R/-)

Fertile F1 (-/-, R/-)

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Use of chemical hybridizing agents (CHA) for inducing male sterility

Various terms have been used to describe chemicals that induce male sterility in plants

Gametocide Selective gametocide Pollen suppression Male sterilant Selective male sterilant Pollen suppressant Pollenocide Androcide

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• First demonstrated in maize (MH) (Moore 1950; Naylor 1950)

Following CHA which are using for inducing male sterility

GA, Gibberellins synthesis, CCC inhibitors, ABA (Abscisic acid), FW-450 (Mendok), Dalapon, TIBA (Triiodobenzoic acid), NAA, Ethephon, Dalapon and -chloropropionate

,

Vegetable Applied chemicals

Remark(s) Reference

Colevegetables

GA Reported promising for

utilization

Van Der Meer and Van Dam

1979

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Chief features of an ideal CHA

• It must be highly male or female selective• It should be easily applied and economical in use• The time of its application should be as flexible as

possible• It must not be mutagenic

• It must not be carried over in F1 seeds

• It must consistently produce complete (95% or more) male sterility

• It must cause a minimum, preferably zero, reduction in seed set

• It should not be hazardous to the environment 59

Environmental genic male sterility (EGMS)

• EGMS is more popularly termed as “Two line Hybrid Breeding” as against “Three Line Hybrid Breeding” in case of CMS system

• Determination of critical environment for sterility and fertility expression (Usually temperature or photoperiod)

Vegetable Mutant Temperature Reference

Cabbage TGMS, PGMS <100C Rundfeldt 1961

Brussels sprouts TGMS <100C Nieuwhof 1968

Broccoli TGMS, PGMS 100-110C Rick 1948; Sawhney 1983

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Mechanism of male sterility

Expression of male sterility trait is associated with

Morphological changes

Histological changes

Cytological changes

Biochemical changes and

Molecular changes

61

Morphological changes

• Variable (complete absence of male reproductive organs)

• Male sterile flowers are commonly smaller in size in comparison to the fertile

• The size of stamens is generally reduced

62

Histological changes Role of tapetum

• Premature breakdown of tapetum

• Abnormal development of tapetum in male sterile plant first report by

(Monosmith 1926)

Role of callase

• Early or delayed callase activities have been found to be associated with

male sterility Role of esterase

• Decreased activity of esterase in male sterile plants has been observed

in tomato (Bhadula and Sawhney 1987) & in radish (Zhou and Zhang

1994) Role of PGR’s

• Reduced level of cytokinins and increased level of abscisic acid associated with GMS and CMS plants

Mehdi and Anwar 200963

Cytological Changes

In both GMS and CMS systems, male sterility is the consequence of breakdown of tightly regulated pollen development and fertilization processes at any of the pre- or post-meiotic stages i.e.

During the formation of tetrad

During the release of the tetrad

At the vacuolated microspore stage or at pollen dehiscence

stage

Wu and Yang 200864

Biochemical Changes

Amino acids• Reduced the level of proline, leucine, isoleucine,

phenylalanine and valine • Increased the level of asparagine, glycine, arginine,

aspartic acids

Soluble proteins• Male sterile anthers contain lower protein content and fewer

polypeptide bands

Enzymes• Mistiming of callase activity • Decreased the activity of esterase and amylases

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Molecular changes in CMS CMS resulted from an inability of the mitochondria to meet

the energy demands of male gametogenesis

Male organs shown lower levels of ATP production and high levels of alternative oxidase activity

Mitochondria play a central role in premature programmed cell death (PCD)

Pelletier and Budar 200766

Constraints in the spread of hybrid varieties

Lack or steady supply of seed

Lack of extension work

Exorbitant cost of hybrid seed

Poor marketing facilities of vegetables

Improper quality attributes

67

Conclusion

Hybrid technology offers tremendous potential for the much needed second Green Revolution

Area expansion not possible thus need is to increase production/unit areas

Cost effective hybrid seed production (SI, MS etc)

68

Future prospects

To fulfill the demand of vegetables in our country

To identify the stable SI and CMS lines

There is need to acquire deeper knowledge about these two important mechanisms for developing location/ environmental specific SI and MS lines

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THANK YOU

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