This document discusses unreduced gamete formation and its role in plant breeding. It begins with an introduction and overview of unreduced gametes, also called 2n gametes, which have the same chromosome number as the parent plant. It then covers sources and mechanisms of 2n gamete formation, including interspecific hybrids, meiotic mutants, and odd polyploids. The mechanisms of 2n gamete formation through mitosis and various types of meiotic restitution are explained. Detection methods and factors that influence 2n gamete frequency are outlined. The role of 2n gametes in plant breeding applications such as ploidy level manipulation, inter-genomic recombination, and development of new crop varieties is then

1. 1

2. 2
Unreduced gamete formation
and
Its role in plant breeding

3. 3
Introduction
2n gametes
Sources and mechanisms
Detection and induction
Role in plant breeding
Conclusion
In this mother cell…

4. Male gametogenesis
4

5. Female gametogenesis
5

6.  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
6

7. 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)
7

8. 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)
8

9. 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

10. 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

11. 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)
11

12. 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-
12

13. (Brownfield and Kohler, 2010) 13

14. Products of meiotic restitutions
(Gómez-Rodríguez, et al.,2012) 14

15. 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.
15

16. 16

17. 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).
17

18. 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
18

19. (Brownfield and Kohler, 2010) 19

20. (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

21. Frequency of unreduced gamete production
(Number of individuals producing 2n gametes/total number of individuals examined)
(Bretagnolle and Thompson, 1995) 21

22. 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
22

23. (Mason et al.,2011)
Male unreduced gamete production in Brassica
species under different temperatures
23

24. Pollen viability estimates
(Mason et al.,2011) 24

25. Techniques to increase frequency
25

26. Detection of 2n gametes
1. Pollen morphology
2. Flow cytometry
3. Analysis of sporogenesis
4. Ploidy analysis of the progeny
26

27. 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
27

28. 28(Xue et al., 2011)

29. 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.
29

30. (Chen and Gmitter , 2011) 30

31. • 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

32.  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.
32

33. Ploidy analysis of the progeny
Chromosome counting method
Flow cytometry
33

34. Induction of 2n gametes
(Dewitte et al., 2012) 34

35. (Nukui, et al., 2011) 35

36. (Nukui et al., 2011)
Restoration of fertility in interspecific hybrids by N2O
36
Untreated N2O Treated

37. (Barba-Gonzalez et al., 2006)
UNTREATED N2O TREATED
Germination of 2n pollen
37

38. 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
38

39. Effect of temperature
(Pecrix et al., 2011) 39

40. Hibiscus syriacus Hibiscus paramutabilis
Interspecific hybridisation
All are tetraploids
50% are hexaploids
(Van Laere et al., 2009) 40

41. 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
41

42. ROLE OF 2n GAMETES IN PLANT BREEDING
Polyploids that originate through the functioning of 2n gametes
(Peloquin, et al., 1999) 42

43. Ploidy level manipulations in potato
(Carputo and Barone, 2005) 43

44. Inter-genomic recombination
(Barba-Gonzalez et al., 2006) 44

45. Interspecific Lilium hybrids
(Barba-Gonzalez et al., 2008)
2n gamete X diploid F1
2n gamete X tetraploid F1
45

46. Use of 2n gametes in potato germplasm introgression
(Carputo, et al., 2000) 46

47. 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

48. B. napus
(AnAnCnCn)
X B. carinata
(BcaBcaCcaCca)
AnCn
BcaCcaGametes:
AnBcaCcaCn
Unbalanced tetraploid
X B. juncea (AjAjBjBj)
Gametes: AnBcaCcaCn AjBj
Unreduced gametes
AnAjBjBcaCcaCn
allohexaploids
(Yan and Shyama , 2007) 48

49. Amphihaploids in wheat
(Jauhar, 2007) 49

50. 50

51. 51