1. Ploidy level effects on floral morphology of Acacia mangium and its comparison to A. auriculiformis
Nghiem Quynh Chi
Supervisors: Rod Griffin Chris Harwood Anthony Koutoulis Jane Harbard

2. Acacia plantation in Vietnam
Species to be studied
1. Acacia mangium Willd (A.m)
2. Acacia auriculiformis A.Cunn ex Benth (A.a)
- adaptable to a wide range of site conditions
- produce pulp logs and small sawlogs in period of 7 to 10 years
Together with Eucalyptus and Acacia hybrid, they are the main species for short -rotation tree plantations in Vietnam
Plantation Area (ha)
A.a: ,000
A.m: ,000
A.m x A.a hybrid: ,000
(Ministry of Agriculture and Rural Dev. 2009)
Acacia plantation in Vietnam

3. Background Prof. Rod Griffin – Australian Project Leader
ACIAR project FST/2003/002 - collaboration between Australia, South Africa, and Vietnam
Prof. Rod Griffin – Australian Project Leader
Objective: Develop polyploid breeding methods for Acacia species
- Hybrids (2X or 3X)
Provide genetically improved material
(hybrid vigour in growth and timber quality, occurs in natural Acacia hybrids)
- Triploids
Increase woody biomass
(triploid hybrids of white Poplars was used in China to obtain heterosis in growth and
resistance (Li S et al. 2004; Kang X et al. 2000))
Reduce invasive weeds in exotic environments (sterility)

4. … Continued … Propagation
Ha Tay prov., the North
32 polyploid A. mangium clones came from cochicines induction on seeds
(from Shell Company)
Quang Tri prov., the Central
Propagation
Binh Duong prov., the South
Somatic chromosome number of A.m & A.a: 2X = 2n = 26 (Shukor et al., 1992)
3 field trials were established in 2003 with the same design

5. Trial Design
- rows of a mix of polyploid clones interplanted with rows of a mix of A.m clones and a mix of A.a clones
- single trees plots/ 4 replicates
A.a 2X
20 trees per row
2.5m 4m
A.m 4X
A.m 2X
A.m 4X
Polyploid A.m: 30 clones
Diploid A.m: 10 clones
Diploid A.a: 10 clones

6. Breeding system
Outcrossing rates
Study with microsatellite markers showed that tetraploid A. mangium at Bau Bang had much higher selfing rates than diploid
(Tran Duc Vuong, 2009)
Polyploidy in angiosperms is frequently associated with increased self-compatibility. Chromosome doubling often produces new allelic interactions in pollen that lead to a breakdown of self-incompatibility (de Nettancourt, 2000).
Barrier to pollination between diploids and tetraploids
Insect behaviours

7. 3X hybrid (A.a x A.m) seedling
Pollination
2x A.a
4x A.m
2x A.m
Controlled crosses
Open pollination
Bau Bang trial
Is there a strong barrier for crossing of 2X and 4X trees within species?
3X hybrid (A.a x A.m) seedling

8. Reproductive barriers to interploid crosses
1. Pre-mating
(e.g. flowering time, pollinator behaviour)
2. Post-mating
a. Pre-zygotic barriers
(e.g. flower structure or preventing pollen tube penetration in stigma or style)
b. Post-zygotic barriers
(e.g. embryo abortion)

9. My research question
akdsdkand

10. Spike & Flower Style & Stigma Polyad Ovary & Ovule Seed
- Length of spike
Total number of flowers per spike
The ratio of male to hermaphrodite flowers
Length of flower
Style & Stigma
- Length of style
Stigma diameter
Floral Morphology
and
Seed number
(compare 4X & 2X)
Polyad
Polyad diameter
Polyad germination
Ovary & Ovule
Number of ovules per ovary
Seed
Number of seeds & full seeds/pod

11. Materials and Methods Flowers and pollen were sampled in Vietnam
Spikes
10 clones x 1 ramet x 3 spikes = 30 spikes/species
Flowers
10 clones x 1 ramet x 1 spike x 30 flowers = 300 flowers/species
Polyad diameter and no. of ovules/ovary
10 clones x 1 ramet x 30 polyads & ovaries = 300 polyads & ovaries/species
Seed number
10 clones x 1 ramet x 30 pods = 300 pods/species
Flowers fixed in 3:1 methanol:acetic acid solution and preserved in 70% alcohol
Observation using light microscope, fluorescence microscope with UV light, and scanning electron microscope (SEM)

12. Spike parameters Species Ploidy level Length of Spike (mm)
Total no. of flowers/ spike
Percentage of male flowers/ spike
A. mangium 2X
99.6 (±12.7) a
205.0 (±41.9) b
(±23.4) 4X
86.6 (±12.0) b
194.5 (±35.7) a
(±16.8)
A. auriculiformis
57.6 (±10.3) c
90.4
(±17.7) a
(±26.0)
A. mangium 2X
A. mangium 4X
A. auriculiformis 2X

13. Length of flower and style
Species
Ploidy level
Length of flower
(mm)
Length of style
A. mangium 2X
5.1 (±0.3) a
4.2 (±0.3) 4X
4.8 (±0.3) b
3.8 (±0.3)
A. auriculiformis
4.3 (±0.5) c
3.4 (±0.5)
A. mangium 2X
A. mangium 4X
A. auriculiformis 2X

14. Polyad and stigma diameter and number of ovules
Species
Ploidy level
Polyad diameter (µm)
Stigma diameter (µm)
Number of ovule
A. mangium 2X
(±1.6) c c
(±2.6)
(±1.5) b 4X
42.2 (±2.3) a a
(±3.9)
12.9 (±1.4)
A. auriculiformis
37.1 (±1.9) b
(±1.8)
15.2 (±1.5)
A. mangium 2X
A. mangium 4X
A. auriculiformis 2X

15. Pollen viability in vitro
A. mangium 4X
A. mangium 2X
A. auriculiformis 2X

16. Number of seeds and full seeds
Species
Ploidy level
Number of seeds/pod
Number of full seeds/pod
A. mangium 2X a
(±1.1) a
(±1.3) 4X c c
(±1.0)
A. auriculiformis b
(±1.8) b
(±1.7) 1 2 3 4
Seed score:
Small and empty seed: 1
Big and empty seed:
Small and full seed:
Big and full seed:

17. Conclusion
There are significant differences in floral morphology between 4X & 2X A. mangium and 2X A. auriculiformis
spike length
total number of flowers/spike, the ratio of male to hermaphrodite flower/spike
flower and style length
polyad and stigma diameter
number of ovules/ovary
number of seeds & full seeds/pod
However, the size of the stigma cup is always greater than that of polyad regardless of whether they are diploids or tetraploids
This allows at least one polyad
to be accommodated on the stigma
for pollination
Style
Stigma
Polyad

18. Controlled pollination has been carried out
3. Applying pollen
1. Thinning and emasculation
2. Put sugar solution on stigma
4. Bagging

19. To answer the question
“Whether we can produce triploids of A.m and acacia hybrid (A.m x A.a) by sexual reproduction ”
We need to compare the penetration of pollen tubes in different crosses of two species (A.m & A.a)
2X x 2X (controlled pollination)
2X x 4X
4X x 2X
4X x 4X

20. Pollen tube penetration (fluorescence microscopy)
♀Am58 (4X) x ♂ Am68 (2X)
2 hours
72 hours
24 hours
Here are some pictures to show my work with microscope
Enters in ovule
Stained with aniline blue, viewed under UV light

21. Acknowledgement Funding support
ACIAR ( Australian Centre for International Agricultural Research)
Collaborative research projects
John Allwright Fellowships
FSIV (Forest Science Institute of Vietnam)
Field trials and personnel assistance
2. Assistance
Prof. Rod Griffin
Dr. Chris Harwood
Ass. Prof. Anthony Koutoulis
Ass. Prof. Rene Vaillancourt
Ms. Jane Harbard
Dr. Ha Huy Thinh

22. Thanks for your attention