1. The Cell Cycle of Rice Gametes
Gaiping Chen
Dept. of Microbiology and Plant Biology
University of Oklahoma
Norman, OK 73019
12/06/2012

2. Outline 1. Isolation of rice female gametes
1.1 Background
1.2 Results
1.3 Summary
2. The cell cycle of rice gametes
2.1 Introduction
2.2 Hypothesis
2.3 Experimental Design
2.4 Results
3. Acknowledgements

3. Part I Isolation of rice female gametes
Background
Results
Summary

4. Background Male gamete Female gamete 7 cells 8 nucleus: 1 egg cell
2 synergid
3 antipodal
1 central cell
(with 2 PN)
(Zhang&1999)

5. Improved Method Prior method: cut the ovule ---15M per cell
---1 egg cell out of 10 ovaries
(Zhang YN et al.,2010)
Improved method: cut the ovary
---3M per cell
---5 egg cells out of 10 ovaries

6. Results Egg cell 1HAP Fertilized egg cell 9HAP Zygote
Sacle bar is 10µm
(HAP: Hour After Pollination)
Note: Cytoplasm in zygotes are denser with more granular structure.

7. DAPI Images of Egg cell and Zygoteb d c
1HAP Egg cell (a,b)
5HAP Zygote (c,d)

8. Central Cell a c PN EC b
a: Central cell, one PN was visible.
b: Central cell, wrapped with EC.
c: Egg cell with two PN.
EC: egg cell; PN: polar nuclei;
Scale bar is 10 µm
Note: Egg cell has polarity with nuclear at the micropylar end.

9. Two Synergid Cells Smaller synergid (45-50 µm)
Larger synergid (65-70 µm)
Note: Two synergid cells are not identical, at least in size.
Scale bar is 10µm

10. Time Staged Antipodal Cells
hap: hours after pollination
AC: antipodal cells

12. Antipodal cells proliferate upon fertilization, then degenerate quickly

13. Summary
Cell isolation rate and efficiency has been greatly improved by cutting ovary directly.
Egg cell has polarity with cytoplasm and nucleus at mycropylar end and larger vacuoles at the chalazal end.
Cytoplasm in zygote is denser with more granular structure than egg cell
Two synergid cells are not identical, at least in size.
Antipodal cells proliferate upon fertilization but then degenerate quickly.

14. Part II The cell cycle of rice gametes
Introduction
Hypothesis
Experimental Design
Results

15. Introduction Cell Cycle: ---Interphase ---Mitosis G1 phase S phase
Cell Cycle:
---Interphase
G1 phase
S phase
G2 phase
---Mitosis

16. Cell Cycle and Cell Fusion
Sperm cell: haploid
Egg cell: haploid
(1C)
Egg Cell (2C) +
Sperm Cell(2C)
4C zygote
(G2 Mode)
(G1 Mode)
Egg Cell (1C) +
Sperm Cell(1C)
2C zygote

17. Two Examples of Cell Cycle
Arabidopsis is in G2 mode: (Fridmen,1999)
Different stages Relative DNA Content
Generative cell nucleus in prophase C
incipient sperm C
sperm – newly formed C
sperm – at anthesis C
sperm – pollen tube in ovary C
sperm – in embryo sac C
Maize is in G1 mode: (Mogensen et al.,1995)
Different stages Relative DNA Content
Unpollinated egg cell C
Newly formed zygote C
Zygote before division C

18. Hypothesis Hypothesis: Rice Plant is in G1 mode.
The cell cycle of rice gametes
G1 Mode
Egg Cell (1C) +
Sperm Cell(1C)
2C zygote
Hypothesis: Rice Plant is in G1 mode.

19. Experimental Design The cell cycle of rice gametes
The cell cycle of sperm cell
Relative DNA content of sperm at different stages
The cell cycle of rice gametes
The cell cycle of egg cell
Relative DNA content of egg cell or zygote
Generative cell
Newly formed sperm cell
Sperm cell at anthesis
Sperm cell in embryo sac

20. Development of Sperm Cells
Pollen Mitosis I Microspore----->Generative cell+ Vegetative cell
Pollen Mitosis II Generative cell > 2 Sperm cells
(ZS Zhang &2005)

21. Microspore Vegetative cell + Generative cell Vegetative nuclear
+2 Sperm cells
Pollen Mitosis I
Pollen Mitosis II a e d c b

22. Relative DNA Content in Rice Sperm Cell
Generative cell--> Newly formed Sperm-->Sperm at anthesis-->Sperm in embryo sac
Different stages Relative DNA content
Generative cell before mitosis C
Newly formed sperm cell C
Sperm cell in embryo sac C or 2C?
Sperm cell at anthesis ?
(It just takes 45M for sperm cell from anthesis to embryo sac)

23. (Friedman, 1999)

24. Index Table for Sperm Cell Development
Filament length(mm)
Male Gametophyte
developmental stages
>1.2mm
2 Sperm cells mm
Generative cell+ 2 Sperm cells
(Mitosis II)
1.0mm
Microspore+ Generative cell
(Mitosis I) mm
Microspore

25. Different Stages of Rice Sperm Cells
(epifluorescence microscopy with DAPI staining)

26. How to Calculate Relative DNA Content
4',6-diamidino-2-phenylindole (DAPI) is a fluorescent stain that binds to minor groove of double-stranded DNA.
The fluorescence intensity is proportional to the nuclear binding, thus, could be used to determine the relative nuclear DNA content.
Image J ( was used to measure nuclear fluorescence intensity.
Measurement of net relative nuclear DNA amount can be calculated by subtracting the cytoplasmic background value.

27. Rice pollen grain
Fixed with Triton-X 100, Stained with DAPI. Observed under an epifluorescence microscope.

28. Integrated density= Area *Mean gray value

29. Results Sample numbers Sperm stages CTCF se 17 Generative cell 1.60
0.04 45
Newly formed sperm
0.79
0.02 79
Sperm at anthesis
0.80
0.01
CTCF = Integrated Density – (selected area * Mean fluorescence of background).
(CTCF: the Corrected Total Cell Fluorescence)

30. The Cell Cycle of Sperm Cell

31. Relative DNA Content in Rice Sperm Cells
Generative cell--> newly formed sperm-->sperm at anthesis-->sperm in embryo sac
In Rice, Relative DNA Content in following Stages:
generative cell before mitosis C
newly formed sperm cell C
sperm cell at anthesis C
sperm cell in embryo sac C
Conclusion: the cell cycle of sperm cell is in G1 mode.

32. Acknowledgements Committee members: Dr. Scott Russell Dr. Marc Libault
Dr. Barbara Safiejko-Mroczka
Dr. Venkatesan Sundaresan
Dr. Laura Bartley
Dr. Preston Larson
Gregory W. Strout
Daniel Jones
Cassie Ehardt
Nina Desianti
Fan Lin
Funding source: National Science Foundation

33. Thank You!