Ploidy level effects on floral morphology of Acacia
mangiumand its comparison to A.
auriculiformisSupervisors: Rod Griffin Chris Harwood Anthony Koutoulis
Jane Harbard
Nghiem Quynh ChiNghiem Quynh Chi
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
Species to be Species to be studiedstudied
BackgroundBackgroundACIAR project FST/2003/002 - collaboration between Australia,
South Africa, and Vietnam Prof. Rod Griffin – Australian Project Leader
ObjectiveObjective: Develop polyploid breeding methods for : Develop polyploid breeding methods for Acacia Acacia speciesspecies
- Hybrids (2X or 3X) Provide genetically improved material
(hybrid vigour in growth and timber quality, occurs in natural Acacia hybrids)
- TriploidsIncrease 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 …Continued …
32 polyploid A. mangium clones came from cochicines induction on seeds
(from Shell Company)
Ha Tay prov., the North
Quang Tri prov., the Central
Binh Duong prov., the South
3 field trials were established in 2003 with the same design
Propagation
Somatic chromosome number of A.m & A.a: 2X = 2n = 26 (Shukor
et al., 1992)
A.a 2X
A.m 4X
A.m 2X
A.m 4X
- 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
Trial DesignTrial Design
Polyploid A.m: 30 clones
Diploid A.m: 10 clones
Diploid A.a: 10 clones
20 trees per row2.5m
4m
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
Study with microsatellite markers showed that tetraploid A. mangium at Bau Bang had much higher selfing rates than diploid (Tran Duc Vuong, 2009)
Breeding Breeding systemsystem
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
2006 2007 Combined
Diploid
Tetraploid
Ou
tcro
ssi
ng
rat
es
Bau Bang trial
Is there a strong barrier for crossing of 2X and 4X trees within species?
Controlled crosses
Open pollination
3X hybrid (A.a x A.m) seedling
2x A.a
4x A.m
2x A.m
PollinationPollination
Reproductive barriers to Reproductive barriers to
interploid crossesinterploid crosses1. Pre-mating1. Pre-mating
(e.g. flowering time, pollinator behaviour)
2. Post-mating2. 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 My research question
The feasibility of producing Acacia triploids and hybrids
via sexual reproduction
The feasibility of producing Acacia triploids and hybrids
via sexual reproduction
Different species
(A.m x A.a and A.a x A.m)Different species
(A.m x A.a and A.a x A.m)
Different ploidy levels
(4X x 2X and 2X x 4X)Different ploidy levels
(4X x 2X and 2X x 4X)
Floral Floral MorphologyMorphology
and and
Seed numberSeed number(compare 4X & (compare 4X &
2X)2X)
Floral Floral MorphologyMorphology
and and
Seed numberSeed number(compare 4X & (compare 4X &
2X)2X)
Spike & FlowerSpike & Flower- Length of spike - Total number of flowers per spike- The ratio of male to hermaphrodite flowers- Length of flower
Spike & FlowerSpike & Flower- Length of spike - Total number of flowers per spike- The ratio of male to hermaphrodite flowers- Length of flower
Style & StigmaStyle & Stigma- Length of style - Stigma diameter
Style & StigmaStyle & Stigma- Length of style - Stigma diameter
PolyadPolyad- Polyad diameter- Polyad germination
PolyadPolyad- Polyad diameter- Polyad germination
Ovary & OvuleOvary & Ovule- Number of ovules per ovary Ovary & OvuleOvary & Ovule- Number of ovules per ovary
SeedSeed- Number of seeds & full seeds/pod SeedSeed- Number of seeds & full seeds/pod
Materials and MethodsMaterials and MethodsFlowers 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/speciesFlowers 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
A. mangium 2X A. mangium 4X A. auriculiformis 2X
SpeciesPloidy 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)
a 11.2 b(±23.4)
4X 86.6 (±12.0)
b 194.5 (±35.7)
a 20.3 a(±16.8)
A. auriculiformis
2X 57.6 (±10.3)
c 90.4 (±17.7)
b 22.5 a(±26.0)
Length of flower and style
A. mangium 2X A. mangium 4X A. auriculiformis 2X
SpeciesPloidy level
Length of flower(mm)
Length of style (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
A. auriculiformis 2X 4.3 (±0.5) c 3.4 (±0.5) c
Polyad and stigma diameter and number of ovules
A. mangium 2X A. mangium 4X A. auriculiformis 2X
SpeciesPloidy level
Polyad diameter (µm)
Stigma diameter (µm)
Number of ovule
A. mangium 2X 33.2 (±1.6)
c 48.2 c(±2.6)
14.0 (±1.5)
b
4X 42.2 (±2.3)
a 59.7 a(±3.9)
12.9 (±1.4)
a
A. auriculiformis
2X 37.1 (±1.9)
b 49.9 b(±1.8)
15.2 (±1.5)
b
Pollen viability in vitro
A. mangium 2XA. mangium 4X A. auriculiformis 2X
0.0
2.0
4.0
6.0
8.0
10.0
12.0
No
. of
ger
min
ated
po
lyad
s
1 2 3 4 5 6 7 8 9 10 11 12 > 12
No. of pollen tubes per polyad
A. mangium 4X A. mangium 2X A. auriculiformis 2X
Number of seeds and full seeds
SpeciesPloidy level
Number of seeds/pod Number of full seeds/pod
A. mangium 2X 9.7 a(±1.1)
8.7 a(±1.3)
4X 4.3 c(±1.3)
2.8 c(±1.0)
A. auriculiformis 2X 7.9 b(±1.8)
5.3 b(±1.7)
Seed score:- Small and empty
seed: 1- Big and empty
seed: 2- Small and full seed:
3- Big and full seed:
4
11 22 33 44
ConclusionConclusionThere 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 stigmafor 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 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
Stained with aniline blue, viewed under UV light
2 hours
24 hours
72 hours
Enters in ovule
Pollen tube penetration (fluorescence microscopy)
♀Am58 (4X) x ♂ Am68 (2X)
Acknowledgement 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
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