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
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:
92,000
A.m:
154,000
A.m x A.a hybrid:
232,000
(Ministry of Agriculture and Rural Dev. 2009)
Acacia plantation in Vietnam
Background
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)
… Continued …
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
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
A.m 4X
A.m 2X
A.m 4X
4m
2.5m
20 trees per row



Polyploid A.m:
Diploid A.m:
Diploid A.a:
30 clones
10 clones
10 clones
Breeding system
1.2
Study with microsatellite
markers showed that
tetraploid A. mangium at
Bau Bang had much higher
selfing rates than diploid
(Tran Duc Vuong, 2009)
Outcrossing rates
1
0.8
Diploid
0.6
Tetraploid
0.4
0.2
0
2006
2007
Combined
-0.2
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
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Polyploidy in angiosperms is frequently associated with increased selfcompatibility. 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
Pollination
2x A.a
2x A.m
4x A.m
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
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)
My research question
The feasibility of producing
Acacia triploids and hybrids
via sexual reproduction
Different species
Different ploidy levels
(A.m x A.a and A.a x A.m)
(4X x 2X and 2X x 4X)
Spike & Flower
- Length of spike
- Total number of flowers per spike
- The ratio of male to hermaphrodite flowers
- Length of flower
Style & Stigma
Floral Morphology
and
Seed number
(compare 4X & 2X)
- Length of style
- Stigma diameter
Polyad
- Polyad diameter
- Polyad germination
Ovary & Ovule
- Number of ovules per ovary
Seed
- Number of seeds & full seeds/pod
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)
Spike parameters
Species
Ploidy
level
Length of
Spike (mm)
A. mangium
2X
99.6
Total no. of
flowers/ spike
a
(±12.7)
4X
86.6
2X
57.6
(±10.3)
A. mangium 2X
a
(±41.9)
b
(±12.0)
A. auriculiformis
205.0
Percentage of male
flowers/ spike
194.5
90.4
(±17.7)
A. mangium 4X
b
(±23.4)
a
(±35.7)
c
11.2
20.3
a
(±16.8)
b
22.5
a
(±26.0)
A. auriculiformis 2X
Length of flower and style
Species
Ploidy
level
Length of flower
(mm)
A. mangium
2X
5.1 (±0.3)
a
4.2 (±0.3)
a
4X
4.8 (±0.3)
b
3.8 (±0.3)
b
2X
4.3 (±0.5)
c
3.4 (±0.5)
c
A. auriculiformis
A. mangium 2X
A. mangium 4X
Length of style
(mm)
A. auriculiformis 2X
Polyad and stigma diameter and number of ovules
Polyad diameter
(µm)
Stigma diameter
(µm)
Number of ovule
Species
Ploidy
level
A. mangium
2X
33.2
48.2
14.0
c
(±1.6)
4X
42.2
(±2.6)
a
(±2.3)
A. auriculiformis
2X
37.1
(±1.9)
A. mangium 2X
c
59.7
(±1.5)
a
(±3.9)
b
49.9
(±1.8)
A. mangium 4X
b
12.9
a
(±1.4)
b
15.2
b
(±1.5)
A. auriculiformis 2X
Pollen viability in vitro
No. of germinated polyads
12.0
10.0
8.0
6.0
4.0
2.0
0.0
1
2
3
4
5
6
7
8
9
10
11
12
> 12
No. of pollen tubes per polyad
A. mangium 4X
A. mangium 4X
A. mangium 2X
A. auriculiformis 2X
A. mangium 2X
A. auriculiformis 2X
Number of seeds and full seeds
Number of seeds/pod
Number of full seeds/pod
Species
Ploidy
level
A. mangium
2X
9.7
8.7
a
(±1.1)
4X
4.3
(±1.3)
c
2.8
(±1.3)
A. auriculiformis
2X
7.9
2
3
c
(±1.0)
b
5.3
(±1.8)
1
a
b
(±1.7)
4
Seed score:
-
Small and empty seed:
Big and empty seed:
Small and full seed:
Big and full seed:
1
2
3
4
Conclusion
There are significant differences in floral morphology between 4X & 2X
A. mangium and 2X A. auriculiformis
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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
Controlled pollination has been carried out
1. Thinning and emasculation
3. Applying pollen
2. Put sugar solution on stigma
4. Bagging
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)
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2X x 2X (controlled pollination)
2X x 4X
4X x 2X
4X x 4X
Pollen tube penetration (fluorescence microscopy)
♀Am58 (4X) x ♂ Am68 (2X)
2 hours
24 hours
Stained with aniline blue, viewed under UV light
72 hours
Enters in ovule
Acknowledgement
1. 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
Thanks for your attention