A way to utilize the advantages of clonal forestry for Norway spruce? Presentation by Dag Lindgren Finland at conference on Vegetative propagation of conifers Website connected to the meeting at

Status Norway spruce It is now technically and biologically possible and the competence exists to multiply juvenile Norway spruce with vegetative propagation with rooted cuttings. It is the standard method for Norway spruce long-term breeding in Sweden. The added cost over conventional seedlings is in the magnitude >10c per plant. The major reason cuttings is not used to a larger extent in operative forestry is the added cost. Juvenility is a problem and cannot be maintained for long using rooted cuttings. Multiplication takes time, juvenility is lost and distance to breeding population increases. Several large scale attempts to make use of tested clones has failed in Sweden the last decades (Hilleshög, Sydsvenska, Mellansvenska). Mass-multiplication of controlled crosses survives in small scale, mainly at Södra odlarna (magnitude plants a year, increasing, prognosis is , and some other small scaled operations. Only one permille of the plant production in Sweden are clones of conifers. It is probably technically and biologically possible with SE, which starts from immature embryos, but the costs and logistics does not make it worth to use SE even in operational long term breeding. That could be argued that in 5 years SE-plants will be industrially possible at an added cost of 30c per plant if the market is 4 million plants/year, as it is done with southern pines in the US (scale 5-10 million plants, but probably with a large subvention and no profit for Cellfor and Aborgen). STT sets up facilities for SE at Umeå now. SE allows fast multiplication and long storage.

Changes the last decade Many Norway spruce plus trees with superior breeding values are available for crossings, opening the option to get genetically outstanding seeds. SE has reached a scale and technological development so commercial use is in reach for some applications. Low diversity forestry has become more acceptable as experiences have accumulated. Swedish forestry is more interested in raising production than before and it has become more politically acceptable.

My “new” is to focus on vegetatively propagated full sibs rather than tested clones Photo: Mats Berlin 2008

Take home message: The gain of vegetative propagation is in the seeds, not in testing the clones!!!! Full sib family forestry – who could ask for anything more? Photo: Anders Fries 2008

Operational success stories with vegetative propagation of crosses utilizing the offspring of the best parents, e.g. Sitka spruce in UK and Ireland Radiata pine in NZ and Australia Hoop pine in Australia Norway spruce in southern Sweden (Södra Odlarna).

Controlled crosses versus elite seed orchard (%Gain over stand seeds) Elite seed orchardCrosses between best clones Selection of parents unrelated parents +2 Selfing 0+1 (no selfing) Pollen Contamination -50 Gain: 1627 When selecting clones with known breeding values, a large gain is made. At least 6% for the first plus tree selection and an additional for selecting tested plus trees with high breeding values. As more selections are required for a seed orchard than a few crosses, the selection gain will be higher for crosses. It is untested crosses, the option to select among tested crosses is not available in this scenario.

Controlled crosses versus elite seed orchard (%Gain over stand seeds) Elite seed orchardCrosses between best clones Selection of parents unrelated parents +2 Selfing 0+1 (no selfing) Pollen Contamination -50 Gain: 1627 Parents to stand seeds often grow close to each other and are related. Controlled crosses or seed orchards recruit parents over landscapes and break the isolates, parents will not be related.

Controlled crosses versus elite seed orchard (%Gain over stand seeds) Elite seed orchardCrosses between best clones Selection of parents unrelated parents +2 Selfing 0+1 (no selfing) Pollen Contamination -50 Gain: 1627 Selfing occurs at wind-pollination in stands and seed orchards. Selfing is eliminated by controlled crosses.

Controlled crosses versus elite seed orchard (%Gain over stand seeds) Elite seed orchardCrosses between best clones Selection of parents unrelated parents +2 Selfing 0+1 (no selfing) Pollen Contamination -50 Gain: 1627 A major thief of genetic gain in seed orchards is the non seed orchard contaminating pollen. Half of all pollinations come from unimproved trees, thus the selection gain is reduced by ¼ in seed orchards. Controlled crosses eliminate pollen contamination.

Controlled crosses versus elite seed orchard (%Gain over stand seeds) Elite seed orchardCrosses between best clones Selection of parents unrelated parents +2 Selfing 0+1 (no selfing) Pollen Contamination -50 Gain: 1627 This was if efforts started simultaneously, but there are timining differences. There are too few spruce seed orchards, the alternative to crosses is stand seeds, and the gain by crosses will be 27% till a new seed orchard get full production at age 20. The superiority will then sink to 11%. The next cycle of long term breeding will generate 8% better clones, and crosses among them will be 19% superior, when the seed orchard is 30 years. A new better seed orchard will be growing then, but has not replaced the earlier one.

Are not tested clones better? Elite seed orchard ≈ 20%, vegetative propagation of offspring to crosses gives additional ≈19%, tested clones additional ≈7% (discussed later), thus ≈ 3/4 of the gain by vegetative propagation is in using cross seeds, less is added by using tested clones.

Other advantages of controlled crosses Genetic variation under better control than with seed orchard seeds Adaptation is under better control than with seed orchard seeds Small batches and shorter distance between decision and delivery offer better flexibility to meet variable demands and desires and to explore new opportunities.

Caveats (not said) Exact figures may be discussed and will depend on particular circumstances for the actual case. Sweden believe in a “kick” effect of seed orchard seeds (after-effects? Epigenics?), this was neglected. There may be “after-effects” with vegetative propagation also (like more sturdy plants better able to survive insects attacking plants). No naturals in plantations assumed. Over Sweden, naturals contribute ≈ 1/5 of the production in planted stands, thus reduce gain by 1/5, but less on the best spruce sites.

ATTACK OF THE CLONES New episode: Diversity

Absence of genetic variation may offer advantages Homogeneity is an advantage for the market, for the forest manager and for the plant producer. If the rotation time is short, and the land controlled by the company owning one large mill. The most pessimistic risk scenarios seem not supported by accumulating experience and if rotation time is short clones can be changed when found susceptible. Photo: Dag Lindgren 1977

But the advantages of no genetic variation may be small for a long rotation species like Norway spruce The end use of the harvest is unpredictable. A felled stand is a small unit for the end user, thus homogeneity of individual stands is not important. The advantage for the manager is not uncontroversial. A clone forest in boreal conditions may not appear uniform even when its genetics is uniform. Vegetative propagation with rooted cuttings is a cause of variation in nursery crop, not a remedy. The environment of plantations sites is variable and unpredictable The crop stays for a century, a mistake with genetics remains longer than the geneticist.

Genetic variation got advantages! Different genotypes will utilize the different niches on a site better together Diseases and pests are likely to make less harm in a diverse stand Diversity makes it possible to expand on what becomes the best share of the trees when they become older. The environments are variable and unpredictable, genetic variation contributes to that at least a share of the trees will be adapted to future conditions and thus give a higher stability. A genetically uniform stand may offer fewer niches for other living beings. Plants which propagate by cloning in nature typically have 3-15 varieties, indicating that there often is an advantage with clone mixtures. A mix of two full sib families is likely to be as variable as a natural stand from most relevant aspects. A genetically variable stand is a better seed source. More socially acceptable; Conclusion: For Norway spruce in Sweden, the main line in the foreseeable future should be diversity, even when vegetative propagation is utilized.

Added gain from selecting tested clones Dominance variation adds to gain if selected clones are planted instead of used as parents. The dominance variation seems to be around a a quarter of the additive, implying that the dominance gives a minor contribution to selection gain, I guess it adds 2% genetic gain to clonal forestry over CP forestry. For the additive gain there is no important difference between CP forestry and clonal forestry. There is a considerable genetic variation within in a full sib family, if the best tested clone is selected a considerable extra gain can be made compared by the corresponding CP (controlled pollination) cross. That variation is used in the long-term breeding and improves parents. If only the best clones were planted, the selection intensity would have been slightly higher for clonal forestry than for parents, but for Norway spruce in the foreseeable future mixtures of several clones are envisaged. Furtheron, it seems that many tested clones will not be found suitable for vegetative propagation. Thus the selection intensity for use as tested clones will not be higher than for use as parents. For long-term breeding of Norway spruce in south Sweden, the progress (made by within family selection) seems to be 8 percent production in a breeding cycle which takes 20 years, of which field testing is 15. It takes some years to get seeds and carry out the vegetative propagation with CP-seeds, for clonal forestry this might be started faster after testing, Thus clonal forestry is closer to the breeding population and that may give an advantage of say 3% (=7-8 years genetic progress in the breeding population). Thus clonal forestry may raise forest production about 5 percent above CP-forestry. CP-forestry requires sexual maturity, in the above I assumed 15 year old tests. If younger tests are used the time advantage is larger, but on the other hand the gain is lower and more doubtful. But considering this and that clonal forestry can be a bit opportunistic in entering material where prospects are best, it may be more fair to estimate the possible superiority to 7%. These comparisons are tested clone vs. CP with best parents, the advantage will be somewhat lower if comparison is made with CP of tested families.

More arguments against single well-tested clones The actual testing of clones is generally based on few plants and unreliable, the value of their parents and the family are generally more accurate. Superior clones will get trade identification and brands and owner rights following legal processes. They are then likely to stay in the market even after they have been genetically outdated and passed their best before date. The market forces will encourage dominance of a few clones used over long time, even if it is not the best option for the forest. Testing of clones and maintaining them constant costs much. If that should be charged on a small market, it may mean high additional costs per plant sold. It is argued that clones can combine characters, I suggest different factors can usually be combined into a single index and mainly dealt with as a single character. Different clones are differently difficult to multiply vegetatively, thus the best tested clone may not be worth the added plant production cost, and genetic gain may be lost. If SE-clones are tested, the cost of starting up a clone is considerable and thus the selection intensity may be considerable lower than with rooted cuttings or long-term breeding and subsequently the genetic gain lower. Maybe only 5 clones among 100 started develop in clones easy to multiply. Somaclonal variation is likely to exist and increase by the vital life time of clones Clones are likely to change performance during testing and use. That includes mutations. Clones quite likely accumulate diseases and genetic scrap. Parallell: the main argument against animal cloning may be animal health rather than the health of human consumers. There are thus argument to minimize the vital life time of clones before they become plants in the forest. A monoclonal stand is unsuitable for seed collection and natural regeneration (also in the neighborhood).. Worries about monoclonal forestry will limit the legally permitted and publically acceptable areas of vegetative propagation in forestry with Norway spruce and easily cause a legal limbo. Family forestry sounds more attractive to public and authorities. In terms of production value of long rotation major species I would recommend withdrawing around 2% of production for monoclonal culture for lack of diversity when making comparisons. Conclusion: Family forestry mixing two families seem unproblematic conpared to a single tested clone from the forestry point of view.

Cross seeds have their problems… Norway spruce is a late, infrequent and unreliable flowerer. Crosses are administratively demanding (staff must be available and instructed, pollen must be managed, equipment must be in place, knowledge and experience is desirable) Cost of around 0.5-1€ per seed Controlled cross seeds used directly for forestry are forbiddingly expensive, logistically impossible and not an option for Norway spruce. But combined with subsequent vegetative propagation it will work!

Controlled crosses in Aborgen taeda seed orchard (Charleston). This company heads for 100% control cross (without vegetative amplification). The extra cost is justified! Photo: Dave Gerwig

Amount of seed and pollen for Norway spruce? Logistically possible to get seeds annually and sufficient pollen Outdoor reasonable mature trees may produce 1000 seeds/tree annually. A reasonable mature seed orchard gives seeds per hectare annually. 10 grafts or 0.04 ha seed orchard can produce seeds annually. 50 per pollination bag, 20 bags a tree gives 1000 cross seeds per reasonable mature tree a reasonable good flowering year. Large amount of crosses can be more manageable if dealing with potted grafts in green house, or grafts outdoors forced to grow horizontally, at least as females. Pollen availability and quantity in Norway spruce is unlikely to be a problem. Pollen appears as early as female flowers do. Pollen production does not vary as much among years as seeds. Pollen can be stored some years waiting for females to appear. Pollen can be harvested from old grafts or trees in trials. A cheap and simple way to get seeds; collect wind-pollination from the best clones in seed orchards. But the genetic gain of those seeds are only a few percent better than the conventional seed orchard harvest. In many particular situations this may still mean say 15% gain compared to the alternative (stand seeds) for some time. Such seeds can be used to start (or fill in) a capacity of cutting production till cross seeds become available, or use at occasions when cross seeds fail. But cross seeds give 10% more gain! Its like accepting an added cost of cutting of 15c for the first 15% gain, but refusing an additional 3c cost for an extra 10% gain!

Vegetative propagation of seeds! Seed cost and crossing trouble shared on many plants. The cross effort will not be a major part of plant cost. Fluctuations in seed availability can be absorbed by plants produced from each seed. Long-term breeding anyway produces some excess seeds or seeds where added cost is low. Rather large and expensive areas or facilities are required for plants to harvest cuttings from (several hectares for an annual production of millions of cuttings on free land).

From seeds to plants… Seeds can be magnified vegetatively by a factor 50->1000 by juvenile cuttings (depending on opinions, methods, genotype, degree of juvenility required, green fingers, luck etc). I suggest 150 as realistic for a well-managed annually harvested cutting producing facility if each seed gives only one donor plant, but vegetative propagation for donor plants can increase the multiplication rate seeds can give some million plants annually may mean each third year. Many seeds to get by crosses, but few plants to sell! This crossing amount is probably feasible, but ten times more would be logistic demanding and probably require special preparation to get manageable mothers. SE can be used to produce mother stocks (used for Sitka on Ireland) when there may be no limits on the multiplication rate from a seed. If SE mother plants cost 1€, it still will only be an insignificant additional cost per cutting. The design of the cutting donor plant (stock plant) facility can be discussed. A hedge orchard in nursery beds with about donor plants on an hectare may support some millions cutting plants annually. Stock plants may spend the first two years as potted plants in green house and when five years in productive hedge orchard. It may be better to have pots on tables to facilitate management. The management would profit from mechanization, but efforts so far during four decades have generally not been very successful. But generally development proceeds and sometime it may come a break-through….

Long-term breeding - a good starting point Swedish (and Finnish?) program: Get breeding values of selected plus-trees Mating of plus trees with high breeding value, half of crosses are made between parents with similar breeding value The Swedish breeding program for Norway spruce is at the point there selections are ready to be mated or have been mated for all plus-trees. Plenty of grafts at flowering age exists. Thus Sweden is ready for large scaled matings combining parents with superior breeding value. Cloned full sibs are produced using rooted cuttings and tested for field performance, typically 50 families each with 40 clones each with 14 ramets. Measured typically at age 6 and For some south Swedish populations selections has been initiated, and it would be possible now to initiate a mass-crossing program on the resource basis of clone-tested F1s. After the late measurement crossing is done The share of the long-term breeding program an added vegetative propagation program can utilize, can be considered as sunk cost

Connect to most advanced long- term breeding Cutting harvest propagation can be made with the top from a families, around 100 clones in tests planted in rows on 0.1ha. The area could be harvested for cuttings as long as cuttings are juvenile, when measurements start the ramets are thinned, when juvenility is lost harvest of cuttings stops and the plantation is converted to a crossing archive with say the 6 top clones, which probably become useful for seed production two decades after establishment. A rather long time delay and a rather limited production of cuttings, so this is just one option The best selected clones can be top grafted for fast seed production.

Long term breeding gives the starting point! Long term breeding produces now trees with high breeding values, which can be better exploited by crosses than by seed orchards

This is a genetically thinned seed orchard made from cuttings (Lagan), pruned to a convenient height for crosses. A thinned archive with the best clones preserved for crossings could look the same…. Photo: Anders Fries 2008

Potted spruce grafts for crosses in a green house (Ekebo, photo: Bo Karlsson)

Somatic Embryogenesis (SE) No donor archive needed, simpler, more flexible. Very fast multiplication The clones can be stored The clones can be preserved while testing.

SE for the first cycles? Cross seeds becomes a major problem to get beyond some million cuttings annually. If SE makes the first cycle(s) in multiplication and cuttings just a last amplification by a factor 100, a high cost of each SE-plant is acceptable, the quantitative seed need would not be a problem, seeds can be blown up to any quantity. This is used for Sitka spruce on Ireland, where SE plants are multiplied by a factor 150. SE does not succeed in all clones and some are difficult to propagate. Does not matter, when cross propagation is the important. SE can be stored, that means that clones are always available for mass multiplication.

For Sweden now... Now VINNOVA and forest companies have started generating SE-clones with good parents which are in field testing. It would be outermost idiotic of forestry not to use the opportunity of harvesting most of the gains of this expense immediately, instead of waiting years for test results. Thus I suggest to put hard pressure on STT to make many SE-plants of these. Even if the technique of cheaply manufactured SE plants is not yet ready, it is very good for development to start with a sharp material, and this plants can stand a much higher cost as they will be the basis for a multiplication phase with rooted cuttings.

SE in long term breeding It seems a good idea to routinely make SE lines for some of the best families in each breeding population and test these lines as a part of the ordinary long-term breeding.

It is a good investment… Twenty percent added production capacity can economically motivate an added plant cost of c on good spruce sites by the increased land value. For 20 percent of the Swedish spruce plant market cutting propagation of crosses seems economically justified. Other constraints (environmental, legal, public opinion, lack of sufficient relevant experience, few success stories, time to build capacity) make more than 20 percent before 2025 an unrealistic dream. I guess the legal system will tolerate 20%, if forestry wants it. To accept more additional large scale experience will probably be required.

Other aspects SE cultures, artificial seeds and probably also SE plantlets can be transported over EU borders. Thus an SE-factory may not just support Sweden, but Finland and northern Europe. That gives some advantages of scale for the first SE-factories. Cutting handling and donor archives can be placed where labor is relatively cheap within the EU spruce area, perhaps the Baltic states. Probably outdoors donour plants work better and maintain juvenility longer in the warmer climate in south Sweden or Balticum than in middle Sweden or Finland. If cuttings are used in the north it may still be better to produce them in the south. A risk with production sites in the south may be early lignification, which may speed up maturation.

End