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

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: 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 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)

… 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

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

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

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

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 akdsdkand

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

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) Total no. of flowers/ spike Percentage of male flowers/ spike A. mangium 2X 99.6 (±12.7) a 205.0 (±41.9) 11.2 b (±23.4) 4X 86.6 (±12.0) b 194.5 (±35.7) 20.3 a (±16.8) A. auriculiformis 57.6 (±10.3) c 90.4 (±17.7) 22.5 a (±26.0) A. mangium 2X A. mangium 4X A. auriculiformis 2X

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

Polyad and stigma diameter and number of ovules Species Ploidy 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. auriculiformis 37.1 (±1.9) 49.9 b (±1.8) 15.2 (±1.5) A. mangium 2X A. mangium 4X A. auriculiformis 2X

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

Number of seeds and full seeds Species Ploidy 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 2.8 c (±1.0) A. auriculiformis 7.9 b (±1.8) 5.3 b (±1.7) 1 2 3 4 Seed score: Small and empty seed: 1 Big and empty seed: 2 Small and full seed: 3 Big and full seed: 4

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

Controlled pollination has been carried out 3. Applying pollen 1. Thinning and emasculation 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) 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 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

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

Thanks for your attention