Micropropagation of Rubus spp

Rapid micropropagation of ‘Black Satin’, ‘Thornless Evergreen’ and ‘Bedford Giant’ was achieved by culturing shoot tips in a 1/1 MS. The best medium in the introduction stage contained 2.0 mg/1 BAP. Shoot proliferation occurred with 3.0 mg/1 BAP. Some problems with callus and phenolic compoundsappeared in both introduction and shoot multiplication stages. The plantlets were rooted either in 1/10 MS without hormones or directly in peat. Two months later most of ‘Black Satin’ and ‘Bedford Giant’ and about half of ‘Thornless Evergreen’ were growing steadily. Theoretically it is possible to produce 60,000 plants within a half year by this method. Index words; Rubus spp., micropropagation, shoot tip, callus, phenolics.


Introduction
There is a growing interest towards black- berries, especially varieties that suit small home gardens.The department of horticulture decided to start experiments to find suitable blackberry cultivars.Therefore a research project on rapid micropropagation was carried out at the department.Furthermore we had only one mother plant of one interesting variety, 'Black Satin', but the requirement of plants for future experiments was consider- able.Some growers are also interested in rapid propagation of thornless blackberries, 'Thornless Evergreen'.A third variety, 'Bed- ford Giant', was chosen, because this had been a good performer in the open field.
George and Sherrington (1984) state that because Rubus belongs to Rosaceae family it can be assumed that propagating methods used for apples, roses and strawberries can probably be adapted also for blackberries.

Materials and methods
Three different varieties of blackberry (Rubus spp.), 'Thornless Evergreen', 'Black  Satin' and 'Bedford Giant' were micropropagated.The material was taken at the end of October 1985 directly from the open field, except 'Black Satin' which was growing in the green- house.The leaves were stripped and the shoots were cut into circa 2 cm nodes.All three varie- ties were sterilized for a half minute in 70 % alcohol, 12 minutes in 5 % NaOCl and rinsed three times in deionized, sterile water.The outside leaf primordia were erased from the axillary buds and the small shoot tips (about 1 mm size) were transfered to a nutrient medium.
In the growing room daylength was 16 h with light intensity at 2000 lux and tempera- ture 25-28°C.

Introduction stage
Sterilization in the introduction stage was succesful.Only a few 'Thornless Evergreen' were contaminated.BAP (2.0 mg/1) was ob- viously better than BAP (2.0 mg/1) + NAA (0.1 mg/1).Callus was a problem on those media that included auxin.(Adventitious shoot proliferation from callus was not de- sirable.)'Black Satin' produced considerable callus on the latter medium, which totally pre- vented shoot elongation and proliferation.A few 'Thornless Evergreen' and 'Bedford Giant' were able to form one shoot per shoot tip in spite of callus formation.
For the medium composing only BAP the case was otherwise.Neither 'Black Satin' nor 'Bedford Giant' formed callus.Shoot forma- tion of both varieties on introduction medium was good (Table 1).'Thornless Evergreen', however, produced as much callus as on the other medium.No shoot multiplication oc- curred and only a few shoots could be trans- fered (rescued) to the multiplication medium.

Cultivar
Good multiplication took place for all varie- ties (Table 2).'Thornless Evergreen' gave 11.2 shoots per medium on average (max 25, min shoot length was best for 'Black Satin', where most of the shoots were over 1 cm.On the other hand most of the shoots of 'Thornless Evergreen' were short, about 0.5-1.5 cm. Furthermore there were a lot of small shoots which were impossible to count.Popov and   Shehelkunova (1973) have induced increased shoot length using GA 3 .

Problems with phenolics
All varieties secreted phenolics, which caused browning and coloured the media a deep yellow tone.Broome and Zimmermann   (1978) have reported similar problems.
For 'Thornless Evergreen' this was a real problem, because almost half of the plants on multiplication medium were in a very bad con- dition.Also for 'Bedford Giant' this was a problem about 1/3 died on multiplication medium.'Black Satin' secreted phenolics too, but no plant in the test tube died even if some shoots were dead.With frequent transfer to fresh media it is possible to control this problem (Broome and Zimmermann 1978).
Rooting 'Bedford Giant' and 'Black Satin' were  rooted both in vitro and in vivo (extra vitrum), whilst 'Thornless Evergreen' was rooted in peat only.The percentage of in v/fro-rooting was 27 for 'Bedford Giant' and 80 for 'Black Satin' (Table 2), but only 40 % of the 'Black Satin'-plants had roots longer than 5 mm.Skirvin et al. (1981) have reported about spontaneous rooting on multiplication media.This did not occur in this experiment.
Direct rooting occurred on unsterilized peat in peat pots (Finnpots®), under plastic and without hormone treatment.This succeeded well.About two months later the plants were re-potted.At this moment 80 % of both 'Black Satin' and 'Bedford Giant' and 47 % of 'Thornless Evergreen' were in full vigour (table 2).
'Thornless Evergreen' showed a poorer rooting response than the other varieties.This could partly have been due to the fact the that microcuttings of 'Thornless Evergreen' were smaller than the others.Many scientists have had better rooting results by using larger ex- plants (Donnely and Daubeny 1986).

Conclusions
In spite of difficulties with callus and phenolics, micropropagation of Rubus spp.seems relatively easy and effective.Take 'Black Satin' as an example: A year ago our department had only one mother-plant now it is possible to begin even more extensive experiments.
Theoretically one 'Black Satin' shoot tip on an introduction medium gives 12 shoots.A further four cultivations after each other on multiplication media increases the plant num- ber to about 75,000 shoots.A rooting-% of 80 gives 60,000 plantlets all this within a half year.SNIR (1981) has estimated that 50,000 plantlets/meristem tip/year can be produced.