Breeding Polyploid Acacia mangium for wood production

Jane L. HarbardSchool of Plant Science, University of Tasmania

Tetraploid A. mangium in RCFTI Trial, Vietnam

What is polyploidy?• having three, four, or more sets of chromosomes

A. mangium 2n=2x=26• 40-70% of plant taxa are polyploid• including many tree species

Populus tremula, Fraxinus americana,Ulmus americana, Quercus robur, Betula pubescens, Leucaena leucocephala, Sequoia sempervirens

Casuarina littoralis 2x, 4x A. cowleana 4x

A. holosericea 2x, 4x, 6x A. aneura 3x, 5x

A complicated business - why should we bother?

• Australian Acacia are important plantation species in SE Asia including Vietnam • adaptable across sites-growing well on poorer soils,

nitrogen fixers• short rotation crop producing pulp wood – 6 years and

saw logs at 8-9 years

Plantation Area Vietnam (ha)A. auriculiformis: 92,000 A. mangium: 154,000 A.m x A.a hybrid: 232,000 (Ministry of Agriculture and Rural Dev. 2009)

• but they are prolific seed producers – can be invasive

Established polyploid breeding programs

• Polyploid breeding is a staple of diverse agricultural species such as wheat, sugar cane, hops, cassava, watermelon, citrus

• Traits of interest include• seedlessness via seedless triploids• increase in chemical compounds of interest• increased biomass• larger fruits

Tetraploid (4x) and triploid (3x) traits of interest

• Gigantism characteristics• longer wood fibres (good for papermaking)• adaptations to wider range of environments ?

• Pure species and hybrid triploids - 4x X 2x = 3x• faster growth than 2x (?)

demonstrated in commercial triploid hybrid leucaena for fodder production

• sterile …less weedy • biomass reallocated from reproductive to vegetative

growth (increased wood production?)

Outline of the program – an Australia/Vietnam collaboration

Polyploid lines to RCFTI Vietnam from Shell Forestry

LTD. UK 2001

32 lines captured, bulked up in TC and weaned for mother

hedge plants Vietnam

Bau Bang hybridising orchard in south of Vietnam- planted 2003

Test OP progeny from 4x parents Clone and breed

from best trees

2x Aa 4x Am 2x Am

Generating new 4x genotypes using a range of colchicine concentrations and exposure timesI. Application of colchicine to apical meristem

of germinated seedlingsII. Seed immersion in aqueous colchicine

solutionIII. Seed germination on colchicine saturated

filter paper

• Treatments applied to bulk seedlots of A. mangium seed supplied by RCFTI seed orchards in Vietnam

Application of colchicine to apical meristem of germinated seedlings

• “Drop method”• 5µl of colchicine applied to

apical meristem at seedling emergence at midday for 3 days

• labour intensive, not all seed treated

• 8.9% 4x with 1.5% colchicine

Seed immersion in aqueous colchicine solution

• seed immersion in colchicine solution impaired shoot and root growth– not successful for A. mangium

Germinants following immersion in 0.01% colchicine solution for 16 h, 27 days after transfer to Petri dishes for germination

Seed germination on colchicine saturated filter paper

• 0.02% colchicine soaked filter paper, 16 hours at 29 C

• 7% 4x

Seeds following treatment with 0.02% colchicine on saturated filter paper for 16 h and transfer to fresh Petri dish for germination. Germinants 4 days after transfer showing normal root growth

Flow Cytometry• must sample min.2 phyllodes/plant• phyllode sample + modified Galbraith Buffer • Pisum sativum cv. Torstag – internal standard• filtered through a BD Falcon 40 µm nylon cell

strainer• propidium iodide stain (20µg/ml) • Coulter Elite ESP Cell Sorter • known 2x and 4x included each run• 5000 or10000 counts / sample

FCM - output

2x 4xmixoploid

2C DNA amounts – P. sativum (9.10 pg) 2x A. mangium (1.3pg)4x A. mangium (2.6pg)

Induction 2010-2011• 250 seed x 5 families

from Elite Vietnam genotypes

• 0.02% colchicine imbibed on filter paper 29C for ↑24 hours

Plants aged 8 months

8x

2x/4x chimera

2x control

4xmixoploid

Ploidy types following colchicine induction

Results

• 225 seedlings tested (FCM) - age 5-6 months• 73 putative tetraploid plants or 32%

Alternative ploidy identification methods

Polyad measurements– 4x polyads 20% larger

Can only be used on matureflowering trees

Stomatal measurements– 4x stomates 12% larger

2x 4x 4x(av length 24.3µm)

2x(av length 20.9µm)

Is there a high throughput, accurate ploidy identification

method with no sample preparation??????

NIRANear Infrared Analysis

• NIR analysis works when light usually from a halogen bulb is reflected onto the sample and the energy released from the vibration of specific bonds is converted to a spectra.

• Spectral data are combined with multivariate analysis techniques such as principal components analysis to enable quantitative or qualitative analysis.

• Requires expertise

InstrumentationBruker MPA Fourier Transform NIR analyser• FT instrument scans all wavelengths

coincidentally,• higher resolution• no sample preparation required

Dispersive eg Polychromix Phazir™

• dispersion instrument scans the sample one wavelength at a time

• resolution lower• no sample preparation required• portable

NIRA calibration flow chartDiverse Population

set is collected

Spectra are measuredA calibration set

selectedA validation set

selectedFCM analysis for all samples

Calibration model created using Multivariate Statistics

Validation testing of the model

Routine Analysis

Are check

samples accurate

?

Are check

samples outliers?

Check wet chemistry

Add “outlier”

samples to Calibration

set

Ploidy models

Bruker MPA Fourier Transform NIR analyser

Dispersive Polychromix Phazir™

Ploidy score plots

Bruker MPA Fourier Transform NIR analyser

Dispersive Polychromix Phazir™

Polyploid identification methods 1. Flow Cytometry

– expensive, slow sample preparation, assesses all layers, definitive result 2. Stomata measurements

– accuracy and cost depends on access to computer software for measurements, low throughput

– only assesses epidermis3. Polyad measurements

– only available on mature flowering trees4. Near Infra Red Analysis

– expensive, calibration requires expertise– fresh material, can efficiently do more samples/plant– high throughput– is model robust?

5. Chromosome counts– requires actively growing roots, harvest must not damage plant,

chromosomes very small, low throughput

Conclusion

• Flow cytometry although labour intensive provides definitive result for ploidy analysis• becomes expensive however when more than one

phyllode per plant must be tested and when follow-up testing is required to confirm stability of ploidy inductions

AcknowledgementsIn Australia• School of Plant Science –

UTAS– Anthony Koutoulis, Rod

Griffin, Aina Price, Alvin Lam, Susan Foster, Clare Brooker

• Mark Cozens – Menzies Research Institute , UTAS

• Thomas Rodman – Central Science Lab, UTAS

• Chris Harwood – Ecosystem Sciences, CSIRO

In Vietnam• Dr Thinh (Director RCFTI)-

lab and field staff• Ms Duong Thanh Hoa, Mr

Do Huu Son, Mr Le Son, Ms Nghiem Chi, Mr Tran Duc Vuong, Mr Tran Huu Bien, Mr Mai Trung Kien