Adapcar workshop report

AdapCAR workshop report
Somatic embryogenesis for future forestry — capturing the genetic gains from the breeding programme and securing elite plant deployment for production and climate adaptability; status, implementation and expected results
Workshop held at Uppsala, 19^th May 2014
Report authors: David Clapham¹, Ulrika Egertsdotter¹, Tuija Aronen<sup>2

1</sup>SweTree Technologies AB, Virdings allé 2, SE-754 50 Uppsala

²Finnish Forest Research Institute (Metla), Finlandiantie 18, FI-58450 Punkaharju, Finland
Abstract
In an AdapCAR workshop in Uppsala, 19th May 2014, representatives from universities, research institutes, industry and ministries in Sweden and Finland discussed the prospects for somatic embryogenic (SE) plants of Norway spruce in practical forestry. Topics included the number of genotypes that should be included in a stand for secure stand performance and for safeguarding evolution of the trees and associated species; whether the clones should be tested, or whether it was more advantageous to use untested clones from tested families in a version of family forestry; the integration of SE into the general breeding programme; the costs of SE in relation to benefits, and advances in the automation of the production of SE plants.
Introduction
Somatic embryogenesis (SE) is a process of asexual reproduction where an embryo is derived from a single somatic cell or group of somatic cells growing in vitro.

For Nordic foresters, SE aroused interest when two groups described it nearly thirty years ago for Norway spruce (Hakman et al. 1985; Chalupa 1985). Zygotic embryos extracted aseptically from seed are placed on solid medium of suitable composition containing growth regulators. SE cultures develop from somatic cells of the embryos, and can proliferate as proembryogenic masses (PEMs) with a doubling or tripling of fresh weight every two weeks. Mature somatic embryos develop from PEMs on medium containing the growth regulator abscisic acid. The embryos after partial drying will germinate on suitable medium (for details of modern laboratory procedures see von Arnold and Clapham 2008) and develop into plantlets that will grow on in peat substrate. SE trees have behaved normally, like trees from seeds of similar genotype, in field trials (e.g. Högberg et al. 1998).

SE Norway spruce is of interest in practical forestry as a method of cloning favoured genotypes and elite populations that offers advantages over cuttings. Bioreactors and automation of the cumbersome hands-on laboratory SE procedures enable large scale plant production. Millions of cloned plants are feasible at competitive costs. Cryopreservation of SE cultures enables the forester to test clones in the field for twenty years or more before multiplying selected genotypes. The AdapCAR conference considered the current state of SE for practical forestry over the next few years. Dr Ola Rosvall, consultant to the Forestry Research Institute of Sweden, moderated the meeting.
Applications and benefits of somatic embryogenesis
Dr Johan Westin outlined a recently started project sponsored by the Kempe Foundation and the Forestry Research Institute of Sweden to investigate the integration of SE into spruce breeding. The SweTree Technologies laboratory in Uppsala are initiating 10 SE cell-lines from each of 15 full-sib families selected from the national breeding programme. Fifteen to twenty plants from each cell-line will be compared in clonal field tests with cuttings from seedlings of the same families. Elite SE clones selected on the basis of the field tests will be available as cryopreserved material for mass propagation. Cuttings as a means of mass propagation suffers from ageing problems; propagation from a given genotype is possible for only about ten years, in contrast to cryopreserved SE clones which have a much longer life. An advantage of mass propagation from selected and tested SE clones, in contrast to mass propagation in outdoor seed orchards from selected clones, is that unwanted pollination from unselected trees outside the seed orchard does not reduce the genetic gain. Furthermore, SE plants can be produced at any time of the year, whereas seed production from a given seed orchard is seasonal and is plentiful only about once in five years, and it takes 15 years of growth for a seed orchard to start producing seed.

An alternative to the use of tested clones is what is called family forestry (see Lindgren 2008); the mass propagation of untested individuals or clones from tested families. Family forestry exploits only superior general combining ability, not specific combining ability, but saves years in waiting for the results of tests. Dr Göran Örlander from Södra forest company favoured incorporating clonal forestry, by rooted cuttings and/or SE-technology, into family forestry. This saves at least 20 years from tree breeding to practical implementation, with less need to establish seed orchards. Dr Karl-Anders Högberg of the Swedish Forestry Institute presented data on initiation and regeneration of plants. As an example, starting SE with 30 full-sib families and 30 explants per family, 164 SE cell-lines were initiated from 25 families; plants were regenerated from 99 clones from 23 families. Dr Högberg emphasized, in order of increasing importance, the loss of families, the uneven distribution of clones among families, and the further selection of a limited number of clones having good plant production capacity. But one can argue that many families are of nearly equal quality, and that it is unnecessary for them to be equally represented among the SE-clones; and what evidence is available suggests that the SE process does not select for or against economic characters (Högberg et al. 1998). If the forest owner plants with altogether 25 untested clones distributed over 6 tested families, the average performance of the SE-trees in the field is expected to be close to the average for the families based on trees from seeds.

Potential risks with somatic embryogenesis
Some of the speakers, including Professor Pär Ingvarsson of Umeå University, considered the risks of planting SE trees. From the point of view of the forest owner, risks include:

(i) Reliance on a small number of clones that have not been adequately tested in field conditions, or that prove to have undesirable properties, such as susceptibility to a newly arising disease, or to be poorly adapted to climatic change. This risk is reduced by planting with a mixture of clones; 25 clones from about 6 tested families (see Lindgren 2009).

(ii) Currently unknown risks that the limited genotypic variability – and reduced phenotypic variation – of clonal forestry leads to unfavourable competitive interactions, or unpredictable genotype-environment interactions. These valid concerns are perhaps unlikely to cause sleepless nights. In a monoclonal stand the phenotypic variance among trees is 80% of that in a seedling forest for a typical economic trait with a heritability of 0.2, and in a mixture with a sample of ten clones the corresponding figure is 98% (Sonesson et al. 2001). Planting with several SE-clones should therefore reduce the possible risks of limited genotypic variability to negligible proportions. A conference participant mentioned that varieties of most cereals are genetically almost uniform, without causing serious competitive or other complications, as are clonally propagated trees such as poplar and willow.

(iii) Uncertainty as to the demand for Finnish or Swedish timber at the time of felling, 60, 90 or 120 years in the future; of course, this risk is not confined to SE trees, but applies to forest plantation in general. For the forest owner, this is surely a far greater risk than either of the first two. The current downturn in the demand for paper, for example, was hardly foreseeable until about twenty years ago. For reasons of this kind, economists discount profit at 1-2% for each year before the profit is realized.

From the point of view of the forest regulator, the naturalist and the evolutionary geneticist, a possible risk with really large scale clonal forestry is that genetic variation is reduced to a point where the future evolution of Norway spruce is no longer safeguarded. Additional concerns raised by Dr Sanna Black-Samuelsson of the Swedish Forest Agency (Skogsstyrelsen) are the negative effects of plantation forestry replacing more natural forest; the often depressing appearance of dense forests; the threatened existence of red-listed species of plants and animals associated with forests. But the effect of plantation forestry on the occurrence of red-listed species depends greatly on how intensively the forest is managed; plantation forests that are not heavily fertilized are often home to red-listed species (Strengbom et al. 2011) and clonal forests do not have to be heavily fertilized, even from economic considerations.

How can these environmental and conservational risks be minimized? The Swedish Forest Agency requires that at most 5% of the forest land unit (roughly the area of an individual owner) is down to clonal forestry; 20 hectares are allowed within a unit. This reduces environmental risks from clonal forestry to an acceptable level. Furthermore, Professorr Ingvarsson mentioned that ten unique clones of Norway spruce harbour the same amount of genetic diversity as offspring generated with these trees as parents; and cereal breeders testify to the astonishing breadth of variation in the F2 from a cross between two cereal varieties. Clonal forestry in Sweden can therefore hardly be a threat to the evolutionary future of spruce. In Finland, the area down to clonal forestry within a forest unit is not currently restricted, but as Kari Leinonen from the Finnish authority, Evira, explained, the number of planting stock per single clone is restricted and depends on the category of the material. In the qualified category only clonal mixtures consisting of at least 11 clones are allowed, with the maximum number of one million copies of a single clone. In the tested category, the minimum number of clones in mixture is four, but also single clones can be marketed, with no restrictions to the amount of plants. He indicated that the regulations in Finland are from twenty years ago and could be revised.

In principle, seed orchards of Norway spruce, and in the future when the technology has developed, Scots pine, might be built from SE plants, or cuttings or grafts from SE plants/trees. This straight-forward, minimally controversial application has not been attempted as yet in Nordic countries; see Lindgren (2009), Rosvall (2011).

Production of SE plants

As Matti Kallio, Managing Director of Siemen Forelia, and other speakers emphasized, SE production has to be cost effective. The laboratory procedures, with many hands-on moments, are being automated with bioreactors, imaging instruments that sort embryos according to quality, and automatic planting machines, as Professor Ulrika Egertsdotter and Dr Mats Johnson, CEO SweTree Technologies, illustrated in their presentations. Dr Susanne Heiska, from the Finnish Forest Research Institute, also took up bioreactors and temporary immersion systems, as well as the use of LED light systems, and the importance of effective cryopreservation. One of the Institute’s projects is with ornamental Norway spruce, where the final cost of the product is probably less critical. The presentation from Dr Harald Kvaalen (NFLI, Norway) concerned the effects of temperature during proliferation and maturation on budburst and bud set in the regenerated young trees. High temperatures during development favour a longer critical nightlength for bud set, presumably an epigenetic effect (Kvaalen and Johnsen 2008). This is a phenomenon to be recognized and perhaps exploited.

With forestry developing in countries with a climate favouring much higher yields than is possible in the north, Nordic forestry may come to depend more on specialized high technology products. Genetically modified wood products, from trees deriving from transformed SE-cultures, may become acceptable. This controversial topic, however, was not discussed at the conference.

Conclusions

Thirty years after the discovery of somatic embryogenesis in Norway spruce, and more than a decade after the promising results from comprehensive tests in the field, the next step is to exploit the emerging automated procedures and scale up to a production of 100,000 and to a million plants. This will enable realistic costing, and correction of any procedural problems. The plants will be delivered to nurseries and planted out in the field. By adapting the principles of family forestry and planting out on the unit area a total of 25 clones initiated from around 6 tested families, the risks to the forest owner and to the environment are essentially removed. In the future, by the continual initiation of SE clones from elite families as they are evaluated within the general breeding programme, SE activities will keep up with breeding progress. In Finland, however, the law does not at present allow untested clones to be planted, so scaling up must await results from field tests. Also in Sweden, tested clones when available can be included among the 25-30 planted out on the unit area.

References
Chalupa V. 1985. Somatic embryogenesis and plantlet regeneration from cultured immature and mature embryos of Picea abies L. Karst. Communicationes Instituti Forestalis Cechoslovaca, 14: 65–90.

Hakman I, Fowke LC, von Arnold S, Eriksson T. 1985. The development of somatic embryos of Picea abies (Norway spruce). Plant Sci 38:53–59.

Högberg K-A, Ekberg I, Norell L, von Arnold S. 1998. Integration of somatic embryogenesis in a tree breeding programme: a case study with Picea abies. Can. J. For. Res. 28:1536-1545.

Lindgren D. 2008. A way to utilize the advantages of clonal forestry for Norway spruce? http://www-genfys.slu.se/staff/daglMeetings/Finland08/VegPropFinland08.htm

Lindgren D, 2009. Working papers of the Finnish Forest Research Institute 114: 08-15. In: Proceedings of the Nordic meeting, Vegetative propagation of conifers for enhancing landscaping and tree breeding, 10-11 Sept 2008, Punkaharju, Finland.

Kvaalen H, Johnsen Ö. 2008. Timing of bud set in Picea abies is regulated by a memory of temperature during zygotic and somatic embryogenesis. New Phytol. 177:49-59.

Rosvall O. 2011. Review of the Swedish tree breeding programme. Skogforsk, Uppsala, Sweden. http://www.skogforsk.se/PageFiles/57629/Tree%20breeding.pdf

Sonesson J, Bradshaw R, Lindgren D, Ståhl P. 2001. Ecological evaluation of clonal forestry with cutting-propagated Norway spruce. Forestry Research Institute of Sweden, Report No. 1. 2001.

Strengbom J, Dahlberg A, Larsson A, Lindelöw Å, Sandström J, Widenfalk O, Gustafsson L. 2011. Introducing intensively managed spruce plantations in Swedish forest landscapes will impair biodiversity decline. Forests 2:610-30

Von Arnold S, Clapham D. 2008. Spruce embryogenesis. In: Suarez MF, Bozhkov PV (eds) Plant embryogenesis: methods in molecular biology, Human Press, Totowa, NJ, 427:31-47.