Post on 13-Feb-2016
description
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