Cuttings

There was great interest in the rooting of stem cuttings from tree species grown in Britain from the earliest days of the tree improvement programme. A rooting facility was built at Alice Holt and the effect on rooting of factors such as age of the stock plant, the type of rooting medium, the time of taking of cuttings, the size of cuttings and the type of propagation frame were investigated in a very wide range of broadleaved and conifer species. This was in the days before mist systems were available and the results were found to be very variable. Cuttings from some species were found to be relatively easy to root whilst others were much more difficult. The research expanded considerably and by the mid 1950’s there were propagation facilities at Alice Holt, Kennington (near Oxford), Grizedale and Bush.

Patterns began to emerge from the early studies. Cuttings from older trees generally rooted less successfully than those from young trees. The best time to take cuttings varied with species. It was in an attempt to root the more difficult material that a mist unit was built at Alice Holt. This unit did not prove to be wholly successful but it was used to produce very large numbers of rooted cuttings of Leyland cypress and western red cedar through to the mid-1960s. The rooted plants were established in a number of field tests. Some cuttings of Sitka spruce had been rooted in the early days of the research and the interest in this species increased in the mid-sixties. Cuttings were taken from plus trees and rooted under mist and non-mist conditions. Rooting levels were found to be generally poor and those plants which rooted successfully grew plagiotropically.

A new Physiology Branch was created when the Northern Research Station opened and the study of the rooting of cuttings was transferred to this branch with the mandate to develop the methods further. The material produced would be used initially as a uniform research tool which would greatly increase the precision of experiments. It was also thought that they might serve as alternatives to grafts. It was envisaged that the techniques developed would be further improved and used to produce rooted cuttings for forest use. The candidate species selected for intensive study were Sitka spruce and lodgepole pine.

A great deal of precise research was undertaken and it was soon realised that the rooting of cuttings from mature trees was always going to be problematic. Rooting could be improved in some cases but consistently high levels without plagiotropic growth could not be achieved. It was decided to abandon the rooting of cuttings from tested and older trees and instead to concentrate on producing cuttings from juvenile, immature plants from improved families. Hybrid larch was selected for this study and it was found that the large scale vegetative propagation of hybrid larch for forest planting would involve the rooting of first and second generation cuttings taken from ortets under three years old. The cuttings could be taken in summer and rooted under mist with no propagation bed heating and no hormone treatment. Under these conditions, more than 90% success could be achieved.

The research was transferred to Silviculture Branch in the late 1970s and the simple technique devised for hybrid larch was explored further and applied to Sitka spruce with the aim of developing a commercial process. Numerous field trials were established in which it was demonstrated that rooted cuttings taken from ortets grown from improved seed performed better than the standard, unimproved seedling controls. Large scale trials of the commercial process were started with Sitka spruce at Newton nursery where up to 100 000 cuttings could be raised annually. The trials were very successful and it was decided in the mid-80’s to develop vegetative propagation on a commercial scale at the Delamere nursery. It was hoped that this unit might produce 500 000 rooted cuttings annually. Since that time the unit at Delamere has expanded in facilities and introduced refinements in techniques which have led to a current annual production of around seven million rooted cuttings of improved Sitka spruce per annum. In this process, stock plants derived from seed of the best known parents in the breeding programme are used and it has thus proved an indispensable method of making the latest products of tree breeding available to the forest industry.

Tissue culture

Tree species have been shown to be amenable to various tissue culture techniques since the mid 1930s but it has often been demonstrated that conifer species are much more difficult subjects than non-conifers in vitro. It was decided in 1974 to undertake a study, based at Leicester University, into the possibilities of tissue culture techniques in the improvement of conifers. This work, using Sitka spruce and lodgepole pine as test species, demonstrated that adventitious buds could be induced on explants up to two years old and also on callus derived from normal embryos. Shoots derived from these studies were difficult to root in vitro but could be rooted ex vitro using standard horticultural techniques. Unsuccessful attempts were also made to induce the formation of embryogenic tissues in solution as this was thought to be a technique that had direct application to forestry.

The research was transferred to Physiology Branch in 1978 and concentrated on Sitka spruce. It was found that the induction of shoots on callus would have no commercial use and work concentrated on the micropropagation of shoots. It was found that multiplication by axillary budding on hormone-free medium produced the highest rates of multiplication and that this rate was not improved by hormone treatment or by induced vitrification. Rooting was found to be best under non-sterile conditions with high humidity but not under mist. This technique has now become central to the tissue culture studies in that it is used to multiply any shoot tissues that may be formed from such techniques as in vitro rejuvenation or genetic transformation.

The work was transferred to the new Tree Improvement Branch in 1987 and, at this time, the dream of the 1970s of the formation of somatic embryogenic tissue from conifers was beginning to be realised. Research was undertaken and it was quickly realised that all the stages involved in somatic embryogenesis, ie induction, maintenance, maturation and germination, required a considerable amount of research. To date, the problems of induction and maintenance of embryogenic tissues have been largely solved but there remains a lot of research to be done on the maturation of the embryogenic tissues to form somatic embryos, ie mimicking the events involved during the development of the embryo within the seed but in the test-tube, and the germination of the mature somatic embryos to form whole plants. Interest in the technique remains high because of its potential for producing large numbers of clonal plants economically. It is hoped that, when all the problems with the technique have been solved, it can be adapted to use automated bioreactor technology for plant production.

Plant breeders are always faced with the dilemma of physiological maturation. Trials are established as juvenile plants and, by the time the plants are tested, they are too mature to be propagated successfully. Clonal forestry could make use of tested material only if it could be reverted to the juvenile form after test or maintained in the juvenile form while testing occurred. A number of techniques have been tried to investigate the two possible approaches.

Research with various horticultural species has demonstrated that the rootstock can exert a dramatic influence on the scion grafted to it. Mature Sitka spruce scions were grafted onto juvenile rootstocks in an attempt at rejuvenation. There was no rejuvenation of the scion, even after five repeated regrafts. Callus was induced from mature tissues and subcultured repeatedly. There were changes to the callus growth but there were no indications that there was any rejuvenation, ie it proved impossible to induce adventitious shoots on the treated callus. Some limited success has been achieved with the physiological manipulation of shoots from mature trees in vitro.

Attempts have also been made to delay the maturation process. Juvenile shoots have been incubated at low but not freezing temperatures under minimal maintenance but the tissues appeared to degenerate rapidly. Cryopreservation of tissues at the temperature of liquid nitrogen whilst the genotype is tested holds great promise. Following testing, superior tested genotypes could be withdrawn from cryostorage and propagated to supply plants for clonal forestry. Two pilot projects have been undertaken on the cryogenic storage of Sitka spruce tissues.

One of the most exciting of the new technologies that is being developed is that of genetic transformation, a process which involves the introduction of specific genes, normally from a different species. Conifers have proved to be difficult subjects for this new technique. A pilot project was established at Nottingham University in 1994 to determine whether or not Sitka spruce could be genetically transformed. The project was a great success and whole plants were generated that had been transformed with both a reporter gene and a selection gene using the bacterial injection and gene gun techniques.