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Anilkumar. CPALM 3001

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Unreduced gamete formation

and

Its role in plant breeding

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Introduction

2n gametes

Sources and mechanisms

Detection and induction

Role in plant breeding

Conclusion

In this mother cell…

Male gametogenesis

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Female gametogenesis

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An unreduced gamete has the same chromosome number as the plant that produced it i.e., 2n

They are also called 2n gamets or diplogametes

It is genetically controlled and results of modification of meiotic process

Contributed little to the origin of polyploids

Unreduced gametes

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Sources of 2n gametes

Interspecific hybrids: both 2n egg and pollen are produced

simultaneously by the same hybrid. but, neither of two

parents of the F1 hybrid nor their F2 sexual polyploidy

progenies can produce 2n gamets.

Ex: Lilium ( Barba-Gonzalez et al., 2005)

Wheat (Xu & Joppa, 2000; Zhang et al., 2010)

Citrus (Chen et al., 2008)

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Meiotic mutants

A mutant active gene in meiosis

2n pollen can formed independent from 2n egg cells and

vice versa.

Ex: Potato (Jongedijk et al., 1991; Peloquin et al., 1999),

Red clover (Parrot & Smith, 1984),

Alfalfa (Barcaccia et al., 2003),

Wheat (Jauhar, 2003; Roberts et al., 1999)

Arabidopsis (d’ Erfurth et al., 2008; Yang et al., 1999)

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Odd polyploids :

Crosses with triploids (gametes can be 1x, 2x or 3x) result

in higher ploidy levels of the progeny and mostly act as a

bridge between diploids and tetraploids

(Kohler, et al., 2010) 9

Mechanisms of 2n gamete formation

Mitotic mechanism:

Somatic doubling, either obtained spontaneously or by the

application of chemicals at zygotic, embryonic or

meristematic stage of a plants life cycle.

This will ultimately lead to the production of polyploid tissues

and possibly the generation of polyploid offsprings.

Ex: Citrus Frequency of polyploids in citrus cultivars

(Usman , et al., 2006) 10

Meiotic mechanisms:

Most commonly arise through meiotic defects

Abnormal meiotic processes resulting in gametes that

have the same chromosome number as that of parent

plant

This is mainly due to Meiotic Nuclear Restitution (MNR)

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1. First division restitution (FDR)

2. Second division restitution (SDR)

3. Indeterminate meiotic restitution (IMR)

4. Post meiotic restitution (PMR)

Other cytological aspects-

FDR and SDR

Cytomixis

Pre-meiotic doubling

4 types-

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(Brownfield and Kohler, 2010) 13

Products of meiotic restitutions

(Gómez-Rodríguez, et al.,2012) 14

Indeterminate meiotic restitution (IMR)

Display a mixture of FDR and SDR.

Failure of chromosome pairing

A part sister chromosome move to the same daughter cell.

The parental genomes are present in odd numbers.

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Cytological aspects:

Other than FDR and SDR-

Cytomixis : chromatin from the nucleus extrudes to

cytoplasm of adjacent mother cell through cytoplasmic

connections (Sheidai et al., 2006) .

Pre- meiotic doubling: before meiosis

a doubling of the chromosomes occur

(Woogenvoort et al., 1990).

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Genes involved in 2n gamete formation

1. SWI1/DYAD

2. CYCA1;2/TAM

3. OSD1

4. AtPS1

5. dif1-1

6. PS- potato

7. el - maize

A. thaliana

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(Brownfield and Kohler, 2010) 19

(A) Metaphase I

(B) dif1-1

(C) swi1-2

(D,E) swi1-2/dif1-1 double

mutant meiocytes.

(D) Sister chromatid cohesion is

lost at the end of prophase I.

(E) The resulting 20 free chromatids at

metaphaseI.

(F) Some cells contained 10

condensed univalents.

(Mercier et al., 2003) 20

Frequency of unreduced gamete production

(Number of individuals producing 2n gametes/total number of individuals examined)

(Bretagnolle and Thompson, 1995) 21

Factors affecting frequency of unreduced gamete production

Stimulated by environmental factors such as temperature,

wounding, water and nutrient stress……

Formation of giant pollen in Brassica inter specific hybrids

more viable than normal.

frequency is high under cold temperatures

these are two times greater than parents in terms of

magnitude

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(Mason et al.,2011)

Male unreduced gamete production in Brassica species under different temperatures

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Pollen viability estimates

(Mason et al.,2011) 24

Techniques to increase frequency

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Detection of 2n gametes

1. Pollen morphology

2. Flow cytometry

3. Analysis of sporogenesis

4. Ploidy analysis of the progeny

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Pollen Morphology

• Banana, rose (Rosa) and sweet

potato 30% larger

• Chinis jujube (Ziziphus jujube) 1.5

times larger

(Xue et al., 2011)

Search for large pollen in the population

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28(Xue et al., 2011)

FLOW CYTOMETRY

Direct quantification of pollen

nuclear DNA

-practiced in Lilly, tobacco, maize

and in rape –Pan et al., 2004

Compares the DNA content of pollen nuclei to the DNA content of somatic leaf tissue.

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(Chen and Gmitter , 2011) 30

• Complex outer exine layer on the pollen surface is the main

obstacle in releasing the nuclei from pollen.

Bohne et al., 2003

Difficulties and solutions

• Bead beating

Roberts, 2007

• Chopping of pollen grains

Laere et al., 2009

• Nuclear isolation protocol which only releases nuclei from

germination tubes

Dewitte et al. (2006,

2009)31

Alternative method to confirm the presence of 2n pollen

Analysis of microsporogenesis

Provide insight in the mechanisms (FDR, SDR) behind 2n

gamete formation.

Molecular cytological techniques like GISH, FISH or AFLP

markers can be used

Draw back: does not provide any information about pollen viability.

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Ploidy analysis of the progeny

Chromosome counting method

Flow cytometry

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Induction of 2n gametes

(Dewitte et al., 2012) 34

(Nukui, et al., 2011) 35

(Nukui et al., 2011)

Restoration of fertility in interspecific hybrids by N2O

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Untreated N2O Treated

(Barba-Gonzalez et al., 2006)

UNTREATED N2O TREATED

Germination of 2n pollen

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Trifluralin treatment

• Induced 2n pollen by submerging flower buds of Begonia in

a trifluralin solution.

Dewitte et al. (2010)

• Spraying maize tassels with a trifluralin solution before

flowering

Kato, (1999)

• Induction of both 2n pollen and 2n egg cells

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Effect of temperature

(Pecrix et al., 2011) 39

Hibiscus syriacus Hibiscus paramutabilis

Interspecific hybridisation

X

F1 All are tetraploids

F2 50% are hexaploids

(Van Laere et al., 2009) 40

Tools to engineer 2n gamete formation

Isolation of genes involved in 2n gamete production

Site-directed mutagenesis

Ethyl methane sulphonate (EMS)

Random insertional mutagenesis or irradiation of seeds or buds

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ROLE OF 2n GAMETES IN PLANT BREEDINGPolyploids that originate through the functioning of 2n gametes

(Peloquin, et al., 1999) 42

Ploidy level manipulations in potato

(Carputo and Barone, 2005) 43

Inter-genomic recombination

(Barba-Gonzalez et al., 2006) 44

Interspecific Lilium hybrids

(Barba-Gonzalez et al., 2008)

2n gamete X diploid F1

2n gamete X tetraploid F1

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Use of 2n gametes in potato germplasm introgression

(Carputo, et al., 2000) 46

AA

CCBB

AABB AACC

BBCC

B. rapa

B. oleraceaB. nigra

B. juncea B. napus

B. carinata

AABBCC

Super Brassica

Breeding “Super Brassica” cultivars

(Yan and Shyama , 2007) 47

B. napus (AnAnCnCn)

X B. carinata (BcaBcaCcaCca)

AnCn BcaCcaGametes:

AnBcaCcaCn

Unbalanced tetraploidX B. juncea (AjAjBjBj)

Gametes: AnBcaCcaCn AjBj

Unreduced gametes

AnAjBjBcaCcaCn

allohexaploids

(Yan and Shyama , 2007) 48

Amphihaploids in wheat

(Jauhar, 2007) 49

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Conclusion…

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