This document summarizes two studies on in vitro pollen germination in citrus plants. The first study examined the optimal temperature for pollen germination and stigma receptiveness in three orange cultivars. It found that 25C was the optimal temperature for pollen germination, and that two cultivars could be manually pollinated at the balloon stage while one cultivar was best pollinated at the open flower stage. The second study assessed pollen viability and germination in seven lemon varieties using TTC staining and in vitro culture. It determined that 20-25% sucrose concentration optimized pollen germination, and that staining tests only provide a rough estimate of viability while culture allows determining exact viable pollen amounts.

1. In vitro Pollen Germination in Citrus
PRESENTED BY: NOORANI GUL NABI KHAN, M2
DATE: 28-5-2015
ADVISOR: DR. CHITOSE HONSHO

2. Outline
 Section 1: Introduction
 Section 2: Receptiveness of the stigma and In vitro germination of orange pollen, submitted to different
temperature
• Knowledge gape
• Material and Methods
• Results and discussion
• Conclusion
 Section 3 : Assessment of pollen viability and germination in seven varieties of lemon
• Knowledge gape
• Material and Methods
• Results and discussion
• Conclusion
 Section 4: Questions and Discussion
 References

3. Section 1: Introduction
Good quality,
delicious taste
and efficiency in
consumption.
How to produce Seedless Citrus fruits ?
By Breeding approaches
Interploidy Sexual Hybridization
Required Control (Manual Pollination) followed by
fertilization to set fruits.
One factor that affects fruit setup is Pollen Viability

4. Pollen viability section 1
Pollen viability, tube growth and morphological homogeneity are important properties of quality pollen,
useful for breeder, geneticists and breeders (Bolat and Pirlak, 1999).
Advantages:
 Increase the breeding efficiency
 Help in selection of suitable pollinizer for established orchards.
How to test the pollen viability in fruit crops ?

5. Pollen viability by staining tests SECTION 1
Many researchers investigated different
staining tests to determine pollen viability of
fruit crops. They are given below
Acetocarmine or Alexander’s stain test
Propone carmine
Aniline blue
IKI (Iodine potassium Iodide
FDA (Fluorescent Diacetate)
NBT (p-nitro blue tetrazolium)
MTT (2, 5-diphenyletetrazolium bromide)
TTC (2, 3, 5-triphenyletetrazolium chloride)
Advantages:
• Faster and easier compare to in vitro pollen germination
test but in some cases different results may be obtained
(Bolat, and Pirlak, 1999).
•For accurate pollen viability, germination test is
necessary.

6. In vitro Pollen Germination Section 1
Pollen of many flowering plants can be germinated on artificial media consisting of aqueous solutions of
mineral salts (Brewbaker and Kwack, 1963).
But The rate of pollen germination depending on the chemical concentration of medium and cultivar (Bolat,
and Pirlak, 1999).
Thus, suitable germination medium should be investigated for each cultivar of same species in fruit crops.

7. Basic Component of In Vitro Pollen germination Medium Section 1
Generally, pollen germination require
1. Sucrose (Tunistra and Wedel, 2000; Bair and Loomis, 1941 and DeBruyn, 1966a)
2. Boric acid (Bair and Loomis, 1941 and DeBruyn, 1966a)
3. Calcium ions (DeBruyn, 1966b)
Additional component for pollen germination
1. Agar ( Provide stability )
2. K+ ( Stimulatory effect on pollen germination and growth of tube) and so on
Amount of the chemicals components can be vary, depending on the plant species
(Cook and Walden, 1967; Sahar and Spiegel, 1984 and Taylor and Hepler, 1997).
<PH of media, temperature and incubation time can also affect the pollen germination rate>

8. RECEPTIVENESS OF THE STIGMA AND IN VITRO GERMINATION OF ORANGE POLLEN,
SUBMITTED TO DIFFERENT TEMPERATURES (Pio et al., 2004)
Section 2
Knowledge gape: This study was conducted to find out the suitable temperature for in vitro pollen
germination and stigma receptiveness in three orange cultivars (Valencia, Pera and Natal)

9. Material and Methods
For stigma receptiveness experiment:
w
3% Oxygenated
(H2O2) water, 3 min
The release of oxygen
bubbles indicate the
stigma is receptive
Flower collection in
three different
growth stages

10. Experiment for germination temperature
Dehiscence (26 C for 24 h )
100 g/L Sucrose, 10g/l agar,
200mg/l boric acid, 800mg/l
calcium nitrate, PH, 6.5
BOD chamber, 23, 24, 25, 26 C for 12h light
period10 ml media

11. Results and discussion
The enzymatic activity of
peroxidase enzyme was
greater in open flowers

12. Size, color and water droplet on stigma

13. Temperature

14. Conclusion
1. Valencia and Pera cultivars, manual pollen can be performed at the balloon stage
2. Natal cultivar, receptivity was greater in open flowers. Therefore, it can be pollinated during open
stage of flowers when there is no risk of cross pollination.
3. The optimum temperature was 25C
4. Valencia has greater percentage of pollen germination.

15. Section 3
ASSESSMENT OF POLLEN VIABILITY AND GERMINATION IN SEVEN
VARIETIES OF LEMON (Demir et al., 2015)
Knowledge Gape:
To determine the pollen viability by TTC and In vitro germination percentage of seven different lemon
cultivars.

16. Materials and Methods Section 3
Dehiscence (23°C for 24 h )
One or two drop of TTC were dropped on a
clear slides, and pollen were spread on it.
Red stained pollen= Viable
Light red= semi-viable
Colorless= non-viable
BATEM Sarisi, BATEM Pinari,
Interdonato, Italyan Memeli, Kutdiken,
Lamas, and Meyer.
Control conditions for 3-4h
(Norton, 1966).
TTC Stain test for pollen viability

17. In vitro pollen germination on agar-plate Section 3
Five different conc. Of
sucrose 5,10,15,20, 25%
Adding 1% agar, 0.1% boric
acid and incubated 21°C
for 24h in dark

18. Results and Discussion Section 3
Pollen Viability

19. Pollen germination on agar-plat
Pollen germination among
cultivars were significant at all
sucrose concentration
The optimum sucrose
concentration was
20% and 25%

20. Conclusion Section 3
1. Sucrose concentration affected the pollen germination of lemon varieties
2. The optimum sucrose concentrations were 20% and 25% in line with Ateyyeh (2005).
3. Staining tests provide only a rough estimate of pollen viability.
4. The exact amount of viable pollen maybe determine by in vitro pollen germination.

21. References
•Demir, G., Turgutoglu, E. & Kurt, S., 2015. Assessment of pollen viability and
germination in seven varieties of lemon, TURKEY: Journal of Crop Breeding and Genetics .
•Pio, L. A. S. et al., 2004. RECEPTIVENESS OF THE STIGMA AND IN VITRO
GERMINATION OF ORANGE POLLEN, SUBMITTED TO DIFFERENT TEMPERATURES, s.l.:
Univeristy of Lavras.

22. Section 4: Questions and Discussion
The end
Thank you so much
for your kind attention