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