Is sex still relevant in 21st century propagation?
Michelle Harnett, New Zealand Tree Grower November 2020.
Detailed genetic data, automation and robotics look to be the future of radiata pine propagation. Does this spell the end for pollen wafting on the open breeze?
The first radiata pines in New Zealand got their start in shelterbelts in the 1850s, planted by people trying to farm the Canterbury Plains. They were a surprising success, growing quickly in poor soils and hot, dry windy conditions. In time, trees were milled and the timber was used for packaging as well as for building barns and houses.
‘A really good second-class timber when from 30 to 35 years old’, while for packaging purposes ‘Pinus radiata comes first’ was the conclusion of the 1913 Royal Commission on Forestry required to identify trees which could supply wood to a growing New Zealand. With this tick of approval, nurseries started producing seedlings and large numbers of trees were planted in the 1920s and 1930s. Harvesting started roughly 30 years later.
However, there was a snag. The first plantation forestry trees had many of the wild characteristics of radiata pine from its home territory of the Californian coast. Dense planting and pruning helped, but something else was needed. As it turns out, the clue to this something else was the differences in form and branching, which suggested a lot of genetic variation and the possibility of selectively breeding for desired traits. Over 150 years, the scrappy trees which fight to survive in their natural environment have been transformed into the tall and straight mainstay of our forestry industry.
Hey, good looking
Tree breeders at the then Forest Research Institute went out in the 1950s looking for exceptional trees. Judged by their appearance, or phenotype, about one tree in every 100 hectares made the cut. Seeds and grafts from these ‘plus’ trees were collected and used for controlled genetic crossing and establishing clonal seed orchards in the late 1950s. The first seed was collected 10 years later.
Breeding has concentrated on increasing the diameter at breast height, stem straightness, branching habits, wood density, core wood stiffness and resistance to the disease Dothistroma. Improvements to these traits are expressed as genetic gain. Scion and the Radiata Pine Breeding Company, which has undertaken commercial radiata pine breeding since 2001, have quantified genetic gain using data from more than 50 years of field trials. For example, planting highly improved seeds
can lead to at least a 25 per cent gain in volume and a 10 per cent increase in wood density, compared with unimproved seeds. The actual gains seen are affected by environmental factors and silvicultural management.
It usually takes two to tango
Each pine tree has two parents, with pollen produced by male cones transported by the wind to fertilise female cones. The combination of genes from both parents from natural crossing makes up each individual tree’s genome.
A tree’s final phenotype, how it looks, is affected by its genome and the environment it is growing in.
When it comes to tree breeding, there is oldfashioned sex where pollen finds female cones. With open pollination, an orchard of selected trees is randomly pollinated so the female parent is known, but not the male. With controlled crossing, the female cones in orchard of selected trees are fertilised with pollen collected from a known male parent. This is a relatively slow way to improve trees as it is restricted by the age of selection which is usually around eight years, and then
the wait until the tree selected produces pollen for the next cycle of mating.
Beyond sex, identical copies of selections can be raised from cuttings, or by grafting. For the most genetic gain, copying the best trees using such vegetative propagation is the most effective. The main problems with this are that once most trees have grown to selection age, they are too old to get viable cuttings from.
Going a step further, virtually unlimited numbers of clones can be produced from one seed using somatic embryogenesis − see the May 2020 issue of Tree Grower for more information. Briefly, immature embryos formed after pollination are made to produce masses of cells that contain many tiny embryos. These can be encouraged to form mature embryos which develop into tiny plants. After acclimatising to nursery conditions, they can be planted out.
Planting copies of the best selections, known as varieties, has the advantage that a forest owner or manager can be sure they are growing superior genotypes. However, somatic embryogenesis consumes time, material and labour, which makes it expensive and slow. It takes nearly a whole rotation, around 20 to 30 years, to get improved trees into the field.
The potential of automation and robotics for producing everything from embryos to producing plants in the nursery is being explored at Scion with the help of the Georgia Institute of Technology. Developing a high throughput production system is part of the 21 Century Tissue Culture Programme being funded by Forest Growers Research and the Ministry of Business Innovation and Employment.
The first step has been setting up an automated bio-reactor system for producing the initial cell masses then encouraging embryo formation. The bio-reactors produce healthy cells and embryos with much less manual handling and potential for contamination. The first results indicated that twice as many of the cells which become embryos are being produced in half the time. This process and other proposed automation should enable us to grow seedlings in bulk cost effectively and reduce the time it takes to get new high value tree varieties into the forest from 24 years to less than 10 years.
Radiata pine breeders have mainly relied on paperbased records to keep track of tree pedigrees. However, the records are only accurate up to a point – who is to say if a sneaky pollen grain slipped in where it should not have? But now Scion have the ability to check the genotypes of large number of trees and find out how trees are actually related and to improve the radiata pine breeding database.
Tiny, harmless mutations occur in the DNA of all organisms and these are passed on to the next generation, where other tiny mutations may occur. Tracking these changes, single nucleotide polymorphisms, allows us to plot family relationships.
Every single nucleotide polymorphism is unique. It could be likened to a key which fits into one unique lock. Specialist companies manufacture chips covered with tens of thousands of specific locks or receptors to detect specific single nucleotide polymorphisms. The New Zealand industry is currently using chips that have locks for 30,000 different ones to genotype radiata pine. The first step in obtaining a genotype is extracting DNA from a tree. This is usually from fresh needles.
The DNA is then chopped up into tiny pieces − imagine running over a phone book with a lawnmower, repeatedly. The solution containing the chopped-up DNA is then washed over the chip. Where the tiny DNA keys find a match if they are present, the receiving lock will change colour. The resultant pattern of colours shows which single nucleotide polymorphisms are present. The pattern is recorded and saved as a tree’s genotype. A comparison of genotypes allows geneticists
to work out family relationships.
So far, the genotypes of more than 10,000 trees used for breeding radiata pine in New Zealand have been characterised. The amount of variation in the pedigree records has been reduced to less than five per cent and the accuracy of breeding values for some populations have nearly doubled.
When a tree’s physical characteristics and genotype are known, geneticists can start to look for similar patterns to predict traits of trees in tissue culture, rather than waiting for trees to mature. So far, genotype patterns have been used to successfully predict tree diameter and wood density. Scion scientists believe that, using genomic selection, increases of around 14 mm for diameter at breast height and 22 kilogrammes a cubic metre for wood density should be possible after about nine years, or one generation.
The choice of plantation forestry species is not limited to radiata pine and many owners of small to medium sized forests are growing other species for environmental, economic and social reasons. The same propagation techniques used for radiata pine can be applied to these species. Scion has active breeding programmes for Douglas-fir, some eucalypt species and some cypress.
Trials of clones and a provenance and progeny trial are under way for coast redwood but there is no current breeding programme at Scion.
Douglas-fir can be sensitive to its environment and prone to the disease swiss needle cast. As a result, Douglas-fir breeding has stopped and started a few times and breeding is only in the second generation. Specific targets include developing resistance or tolerance to swiss needle cast and increasing wood stiffness. Up to 30 per cent genetic gains for growth are available from local Douglas-fir seed orchards. Seed from selections with superior wood stiffness are also available.
Eucalyptus nitens, E. regnans and E. fastigata have all had at least three generations of genetic improvement.
• Eucalyptus nitens is currently undergoing fourth generation re-selection and is being re-measured for growth, form and wood quality. Increases in height, diameter at breast height and volume have all increased, and internal checking has been reduced by 30 per cent. Some 700 trees have been genotyped.
• Eucalyptus regnans has undergone fourth generation re-selection. Height and diameter at breast height have increased.
• Eucalyptus fastigata is in its third cycle of selection, made up of a mix of second and third generation material. Height, diameter at breast height and health have all increased, and malformation has decreased by 15 per cent.
Scion has recently selected trees across a range of provenances from two stringybark species, E. muelleriana and E. pilularis. These selections are now being grafted for the establishment of new seed orchards. The stringybark eucalypts are valued for their stable timber with naturally durable heartwood. They are slightly slower growing than E. fastigata and more susceptible to frost so need to be sited carefully.
The aim of the cypress breeding programme is to develop canker-resistant trees with durable wood which will meet the specifications of the furniture or cladding markets. Individual breeding programmes for Cupressus macrocarpa and C. lusitanica are continuing to select for growth, form, branching, tolerance to canker and where possible, heartwood durability.
A cypress hybrid breeding programme has crossed Chamaecyparis nootkatensis to add durability Cupressus arizonica and the more canker tolerant C. guadalupensis with the best C. lusitanica and C. macrocarpa specimens So far, six new Ovensii and six new Leyland hybrids have been released to market, with many more yet to be evaluated.
Work to match genotypes and environments for optimal productivity is continuing. Rainfall and maximum site temperatures have the greatest effects on productivity. Coast redwood performs very well in Northland, as well as the coastal line along the east coast of North Island. However, a larger sample size tested across more environments is needed for a more detailed exploration of genotype and environment interactions.
If a coast redwood breeding programme were to be developed, more consistent wood durability and increasing wood density could be expected while maintaining or improving growth rates. Additionally, a choice of diverse, favourable genotypes suitable for planting across a range of sites, with resistance to pathogens and climate change would increase the resilience of coast redwood in New Zealand.
The best of both worlds
Plant breeders need to be able to choose the parents of the next generation from a genetically diverse population. Growing only a few different clones limits genetic diversity in the field and may make plantation forests vulnerable to attacks from new pests or pathogens. And inbreeding can cause loss of vigour and negative traits to come to the fore.
Scion’s tree archive is a resource rich in genetic diversity. Copies of many of the good looking radiata pines first selected for their growth and form in the 1950s are growing happily in the Scion genetic archive in Rotorua. Every 30 years or so, new grafts are taken from the trees to preserve their valuable genetic material, and the information it contains about traits that have not yet been a focus of the breeding programme. These include resistance to new pathogens and the ability to flourish in a wide range of environments, including those that might be expected with climate change.
The current phenotyping programme, where methods such as remote sensing are being used to find outstanding trees in all environments, shows the search for new genes is not over. Instead of a slow selectionbased process, integration of quantitative and molecular genetics and automation will speed up the production of genetic gains across multiple traits, including productivity, health and wood quality.Acknowledgments
We would like to thank the Forest Growers Levy Trust, the Ministry of Business Innovation and Employment, the Scion Strategic Science Investment Fund and the Radiata Pine Breeding company for support, advice, guidance and supply of