Development and evaluation of sterile triploids and polyploid breeding methodologies for commercial species of Acacia in Vietnam, South Africa and Australia

A: Seed from 15 elite A. mangium trees from Dong Ha Vietnamese seed orchard was supplied by RCFTI in July 2004. This was the starting material for polyploid induction experiments at UTAS. A further 100g bulk seed from 36 parent trees also from Dong Ha was supplied by CSIRO ATSC in May 2005. CSIR in RSA will have access to improved A. mearnsii seed supplied by ICFR. This has taken considerable negotiation to obtain and at the time of writing has yet to be received.
Optimisation of colchicine induction protocols is taking place at UTAS on seed of A. mangium and optimised protocols will be repeated in RSA on temperate species. A new approach for Acacia, treating germinated seedlings in seedling trays, has been successful in producing tetraploid plants. This technique will be optimised and success benchmarked against traditional seed soaking approaches.
A literature study reviewing technical options for producing sterile Acacia species has been completed by CSIR.

B: Due to delay in signing of the sub-contract by the South African Government, CSIR joined the project in July 2005, 12 months after the start of the project by UTAS. Laboratory work in CSIR will begin after CSIR personnel have completed a training course in Australia scheduled for August 2005, which will provide experience in lab and field practices related to the project.

C: Shell Forestry LTD sent 45 polyploid A. mangium lines in December 2000 to RCFTI. 33 were captured by RCFTI and 32 lines hardened off into hedgerows. 14 lines were forwarded in culture to UTAS in July 2004. Verification of ploidy status has been undertaken by UTAS using flow cytometry and by RCFTI using leaf morphology; stomata size, and flow cytometry. A further 29 lines were forwarded to UTAS as cuttings in September 2004 (9 lines were received both in culture and as cuttings). Multiple ramets of each were rooted by UTAS and the ploidy of every ramet will be assessed using flow cytometry. Results will be examined in conjunction with results from RCFTI to monitor stability and / or reversions to diploids of polyploids and quality control. An easy to use stomatal assay has been established by UTAS and will be assessed for accuracy against flow cytometry results for determining tetraploids. A root squash technique for direct assessment of chromosome numbers in root tips has also been established.
A 4-week training course for 2 members of RCFTI staff and one from CSIR was designed by staff of UNE and UTAS to be undertaken in Australia in August/September 2005.
32 polyploid lines have been genotyped by 4 microsatellite markers by RCFTI. Markers position for quality control of clonal polyploid trials, confirmation of parentage of controlled pollinations, and evaluation of pollen flow in polyploid trials.

D: RCFTI has established 3 trials in three typical locations: Ba Vi (Ha Tay province) in 8/2003, Dong Ha (Quang Tri province) in 12/2003 and Bau Bang (Binh Duong province) in 8/2002. The design was in consultation with staff of CSIRO. Tetraploid A. mangium clones produced by Shell were interplanted with diploid A. mangium and diploid A. auriculiformis. Results from Part C on confirmation of tetraploid status of Shell clones has indicated that, although all Shell clones that were sent to Vietnam were identified as 4N, results so far indicate that 2/3 may have reverted to diploid or have been misidentified as 4N when sent. RCFTI have recognised this and labelled Am clones as 4N or 'not sure' 4N in field trials. Controlled pollinations for production of triploids will only take place on positively identified tetraploids. Phenology assessments are being undertaken by RCFTI to monitor flowering to assist in scheduling of controlled pollinations in addition to understanding the effectiveness of the open pollination production of triploids.

Objectives E and F were not applicable in the first year of the project.

Progress Objectives (objectives relate to Tables of Activities and Objectives in the Project Document)
A: Two approaches using colchicine have been successful in producing tetraploid A. mangium plants from elite seed supplied by RCFTI, Vietnam. Each approach has produced 2N, 4N, mixoploids (plants with both 2N and 4N cells) and mosaics (plants consisting of 2N, 4N and/or mixoploid parts). These plants are being monitored for stability and or reversion to diploid to determine if method of polyploid induction affects plant stability. The preferred methods will be scaled up for production of more polyploid lines for transfer to RCFTI. Due to the complex nature of the expression of ploidy after induction it has been necessary to devise a suite of protocols to efficiently (time and cost) and correctly identify the ploidy of an induced plant. This includes flow cytometry, stomata size and density measurements, and most recently the use of Near Infra Red Spectrometry.

B: CSIR has identified suitable germplasm to begin induction on both clonal and improved seed material. Material was selected on the basis of field performance as well as potential commercial value. Four hybrid clones with E. grandis and two pure E. grandis clones will be used plus a number of pure and hybrid seedlots. A single tetraploid line of CSIR germplasm was sent back to CSIR by Shell Forestry and successfully propagated. This can now be planted out and treated with paclobutrazol to promote flowering and therefore triploid production. It will also enable study of 2N and 4N lines to help with later identification of induced polyploids.

C: RCFTI forwarded 34 polyploid lines of Shell Forestry Ltd A. mangium clones as cuttings or as tissue culture to UTAS and 22 were captured with a range of 1 to 23 ramets per clone captured. A total of 115 ramets were screened for their ploidy using flow cytometry and stomatal assays. All lines were originally sent to RCFTI as tetraploids. Results show that 8 lines have reverted to pure diploid, 7 clones have ramets of different ploidy levels, 1 clone is mixoploid and 5 clones are tetraploid. Of these five 4N clones only one ramet was captured. This highlights another restraint in production, which is the greater difficultly in rooting tetraploid over diploid clones. These results reflect the findings in current UTAS experiments where there has been incomplete transformation to 4N and reversions to 2N of experimental plants. Practical management of these biological complexities is a major focus for the project.
Q3 2005 was dedicated to organising and then conducting an intensive four-week training course for two scientists from RCFTI and one from CSIR. This was conducted by staff at UNE, Armidale and UTAS staff , Hobart in August/September 2005. Topics covered included, lecture series on acacia floral development, reproductive biology, hybridisation, polyploidy, sterility, pollen storage, guest lectures on 'Drought tolerance in Acacia', 'Use of polyploidy in hop breeding', and 'Timelines for operational deployment of polyploid acacia clones and potential genetic gain'.

Practical work covered pollen viability testing, UV microscopy and flower preparation, micro techniques for thin sections, chromosome counts, stomata preparations and measurements, colchicine experiments, flow cytometry preparations, herbarium visit. Field trips to Smithton, NW Tasmania for management of native blackwood (Acacia melanoxylon) forests and to Washpool National Park, NSW.

D: Three field trials of polyploids interplanted with diploid A. mangium and A. auriculiformis were established in 2002/2003 by RCFTI. The southern most trial had limited flowering in December 2005 and open pollinated seedlots from 13 trees were harvested in May 2006 for testing for triploid status. Flowering in December 2006 will be closely monitored and a scientist from UTAS will visit to help implement control pollinations and collect 4N flowers and pollen for monitoring of polyploid pollen viability and flower/pollen size variations.
A visit by ENSIS staff established the set up of a mini-cutting propagation system at FSIV, Hanoi. This will be essential when propagating 4N lines and may be the quickest path to optimise number of converted plants by taking cuttings and rooting parts of the plant that has been identified as 4N. Advice was also supplied on optimising nutrition of the mobile polyploid seed orchard. Following Field inspections in August 2005 the Project Leader provided notes on the work program and recommendations for thinning and nutrition of field trials and priorities for DNA fingerprinting. These recommendations were confirmed in a follow-up visit by ENSIS staff, suggestions/comments were also recorded on the set up of the hydroponic min-cuttings system.

Objectives E and F were not applicable in the second year of the project.

Progress Objectives (objectives relate to Tables of Activities and Objectives in the Project Document)
A: Due to the complex nature of the expression of ploidy after induction it has been necessary to devise a suite of protocols to efficiently (time and cost) and correctly identify the ploidy of an induced plant. This includes flow cytometry and stomatal size and density measurements. Near Infra Red Spectrometry has now been added to these suite of tools. NIR is essential in identifying an induced plant as stable tetraploid, mixoploid or chimeric. Once identified if the plant is coppiced then all coppice material must subsequently be retested and ploidy established. Using NIR we will scale up induction experiments in 2007/2008 as ploidy identification is now cheaper and quicker. Stem material from 4N and 2N glasshouse plants were pulped with 4N fibres 15% longer than 2N. Fibre measurements will be repeated on Ba Vi field material in 2007/2008.

B: Project scientists from CSIR visited UTAS for discussions on experimental procedures for CSIR. Visit report attached in Appendix 2. CSIR has applied colchicine induction treatments to both clonal and improved seed material. Growth after both seed treatment and treatment of buds on potted clonal material has been poor. This is not unexpected or unusual and is an observed response in many plant species. A single tetraploid line of CSIR germplasm was sent back to CSIR by Shell Forestry to be propagated. This was undertaken by commercial nurseries but was unsuccessful. The mother plants at each nursery were tested for ploidy using both flow cytometry and stomatal analysis. Only one plant was verified as possible 4N with the remainder either reverted to 2N or mislabelled in the nursery. It will be used to investigate other tools for polyploid screening.

C: Verification of both ploidy and fingerprinting of Shell 4N clones in Vietnam has continued into 2007. Stomatal assays were prepared by RCFTI in Vietnam and assessed at UTAS on selected trees at two field sites. Trees were selected for testing if they had been used as CP mothers, sources of OP seed or selected for wood sampling. Fingerprinting at RCFTI to confirm clone identity has been unsuccessful possibly due to type of equipment used. A project scientist from Vietnam, awarded a John Allwright Fellowship for study at UTAS, will continue this work as part of his Masters research beginning in July 2008.

D: Three field trials of polyploids interplanted with diploid A. mangium and A. auriculiformis were established in 2002/2003 by RCFTI. The southern most trial had limited flowering in December 2005 and open pollinated seedlots from 13 trees were harvested. Ploidy of progeny was assessed using flow cytometry at UTAS, 2 clones had 4N progeny and 9 clones had 2N progeny, 2 clones had few seed that did not germinate. No open pollinated triploids were found, but in this instance flowering was limited in the orchard and selfing is probable. 4N progeny are stable and will be planted out as soon as possible in Vietnam for selection. Flowering in December 2006 was accompanied by a scientist from UTAS to implement controlled pollinations and collect 4N flowers and pollen for monitoring of polyploid pollen viability and flower/pollen size variations. Detailed microscopic examination of flower morphology will be essential to establish whether biological barriers to inter-ploidy crossing exist. Field visits by the Project Leader in March 2007 also identified management/thinning regimes to be implemented.
Full details in Visit reports attached in Appendix 2.

E: No OP triploid seed has yet been identified from the polyploid field trials but testing will continue into 2007 following the harvest from December 2006 flowering. Additional work is planned to investigate the apparent reproductive barriers to production of triploids.

F: A final workshop is not required for this project but all research will be submitted for publishing.