CP 3x seedlings

We thank FSIV and the Australian Centre for International Agricultural Research (ACIAR) collaboration projects (FST 2003/002 and FST 2008/007) for plant material, and Hong, Xuan and Dzung for field assistance. Funding and in-kind support for N.Q.Chi was provided by a John Allwright Fellowship from ACIAR.

PLOIDY EFFECTS ON REPRODUCTIVE BIOLOGY OF ACACIA MANGIUM

1. Introduction

2. Methods

3. Key results

An Acacia hybridizing seed orchard was established in southern Vietnam. Ramets of clones of colchicine-induced autotetraploid A. mangium (AM-4x) [1], diploid A. mangium (AM-2x) and diploid A. auriculiformis (AA-2x) were planted in alternate rows to promote inter-ploidy pollination.

Despite heavy and largely synchronous flowering and seed production of all three species/ploidy types, the orchard produced very few open-pollinated triploid (3x) seeds. We examined possible barriers to fertilisation and/or successful development of triploids.

References

1School of Plant Science, University of Tasmania, Hobart, TAS. 7001, Australia2CSIRO Ecosystem Sciences, Hobart, TAS. 7001, Australia

3Research Centre for Forest Tree Improvement, Forest Science Institute of Vietnam, Hanoi, Vietnam

Acknowledgements

4. Conclusions

N. Q. Chi1,3, C. Harwood2, R. Griffin1, J. Harbard1, H. H. Thinh3, and A. Koutoulis1

• Hand pollination was conducted following [2]• Floral morphology and pollen tube growth was studied using light and fluorescence

microscopy • Pollen tube growth was studied using a clear squash technique [3]• One ramet of each of 3 different clones of AM-2x, AM-4x, and AA-2x was selected

for CP treatment• Treatment mean comparisons were performed by Tukey-Kramer’s multiple range

test at α = 0.05

• Stigma cup width was greater than polyad diameter for all species/ploidy combinations

[1] Blakesley D, Allen A, Pellny TK, Roberts AV (2002) Natural and induced polyploidy in Acacia dealbata Link. and Acacia mangium Willd. Annals of Botany 90, 391-398.

[2] Sedgley M, Harbard J, Smith RM, Wickneswari R (1992) Development of hybridisation techniques for Acacia mangium and Acacia auriculiformis. ACIAR Proceedings Series, 63-69

[3] Martin FW (1959) Staining and observing pollen tubes in the style by means of fluorescence. Stain Technology 34, 125-128.

[4] Nghiem CQ, Harwood CE, Harbard JL, Griffin AR, Ha TH, Koutoulis A. (2011). Floral phenology and morphology of colchicine-induced tetraploid Acacia mangium compared with diploid A. mangium and A. auriculiformis: implications for interploidy pollination. Australian Journal of Botany 59, 582-592.

3.1 Floral morphology [4]• AM-4x flowers were intermediate between those of AM-2x and AA-2x

Species/ploidy

Spike Flower

Spike length (cm)

Flowers per spike

Flower length (mm)

Style length (mm)

No. of ovules per

ovary

A. mangium 2x 10.8 a 230.4 a 5.1 a 4.2 a 14.2 ab

A. mangium 4x 8.0 b 161.4 b 4.8 a 3.9 b 13.0 b

A. auriculiformis 2x 5.8 c 99.4 c 4.3 b 3.4 c 15.5 a

Mean diameter of stigma cups and polyads. Error bars show critical difference (P<0.05) between treatments.

3.2. Pollen tube growth and ovule penetration (72 h after pollination)

pt

Treatments Normal seeds

Abnormal seedsType I Type II Type III

Intra-ploidy crosses AM-2x X AM-2x 152 0 5 7 AA-2x X AA-2x 62 1 19 21 AA-2x X AM-2x 162 5 17 42

Inter-ploidy crossesAM-2x X AM-4x 8 67 19 18AA-2x X AM-4x 24 180 286 5

Species/ploidy (taxa)

Types of cross-combination (types)

Ovaries with at least one ovule penetrated (%)

Mean no. of ovules

penetrated per ovary

Mean no. of pollen tubes

per style

AM-2x AM-2x X AM-2x 29.7 abc 1.36 bc 6.7 abcAM-2x X AA-2x 17.6 bc 1.64 bc 6.0 abcAM-2x X AM-4x 14.3 bc 1.33 bc 4.2 c

AM-4x AM-4x X AM-4x 20.6 bc 1.00 c 4.6 bcAM-4x X AM-2x 35.9 abc 1.29 bc 5.3 bcAM-4x X AA-2x 35.6 abc 1.33 bc 6.0 abc

AA-2x AA-2x X AA-2x 46.0 ab 1.85 bc 7.8 abAA-2x X AM-2x 59.9 a 2.77 a 8.9 aAA-2x X AM-4x 34.7 abc 1.92 b 7.0 abc

Significance of differences among taxa-by-types ** *** **

Note: ** = 0.001≤ P ≤ 0.01 and *** = P ≤ 0.001. Different letters denote significant (P < 0.05) differences among nine cross-types

Pollen tubes grew well in the style Ovules penetrated in the ovary Ovule development 5 weeks post-pollination

• Differences in floral morphology were not sufficient to prevent interploidy pollination

• No pre-zygotic reproductive barriers were detected for intra- and inter ploidy mating of A. mangium and A. auriculiformis

• However, yields of pods and normal seeds from inter-ploidy crosses were extremely poor, compared to those from intra-ploidy crosses.

• Post-fertilization genic imbalance may limit the production of viable triploid progeny.

pt

pollen tube

AA-2x AM-4x AM-2x

3.3. Normal and abnormal seeds harvested from different cross-combinations

Note: Different letters denote significant (P < 0.05) differences within species/ploidy combinations

AA-2x

pollen tube

AM-2x x AM-4x

0

10

20

30

40

50

60

70

AM-2x AM-4x AA-2x

Dia

met

er (

µm)

Stigma Polyad

AA-2xAM-2x AM-4x

60 m

Mature seed category

ovule penetrated

30 µm

Normal seed5

mmAbnormal seedType I

Type II

Type III

Normal seed

5 mm

Abnormal seed

Type I Type II Type III

AM-2x AM-4x

Style

polyad

stigma

AM-2x x AM-4x

50 m

pollen tubes

polyad

45 m