Maataloustieteellinen Aikakauskirja
Vol. 58: 193—196, 1986

RESEARCH NOTE

Micropropagation of Rubus spp.

ÄKE FINNE
Department of Horticulture, University of Helsinki,
SF-00710 HELSINKI, Finland

Abstract. 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 prob-
lems with callus and phenolic compoundsappeared in both introduction and shoot multiplica-
tion 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 departmentof horticulture
decided to start experiments to find suitable
blackberry cultivars. Therefore a research pro-
ject 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 differentvarieties of blackberry (Ru-
bus spp.), ‘Thornless Evergreen’, ‘Black
Satin’ and ‘Bedford Giant’ were micropropa-
gated.

The material was taken at the end of Octo-
ber 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-

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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.

The nutrient medium was full strength MS
(Murashige and Skoog 1962) containing vita-
mins (niacin 0.5 mg/1, thiamine 0.4 mg/1,
pyridoxine 0.5 mg/1, glycine 2.0 mg/1, meso-
inositol 100 mg/1 and adenine sulphate 80
mg/1) and 0.7 % agar and 3 % sucrose (pH
5.7). In stage I hormones were either 2.0 mg/1
BAP plus 0.1 mg/1 NAA (Skirvin et al. 1981)
or 2.0 mg/1 BAP. In stage II 3.0 mg/1 BAP
was used. The plantlets were rooted either in
1/10 MS without hormones or directly in peat.

In the growing room daylength was 16 h
with light intensity at 2000 lux and tempera-
ture 25—28°C.

Results and discussion

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.

Table 1. Differences between the media BAP and
BAP + NAA for ‘Black Satin’ (BS), ‘Bedford
Giant’ (BG) and ‘Thornless Evergreen’ (TE).

Introduction medium 1Cultivar

Callus 2Number of shoots
per medium

BAP BAP + BAP BAP +

(max/min) NAA NAA

0112BS ccc
(24/1)

06 1
(16/2)

BG

TE

cc

0.5 3 0.5 3

(2/0)
ccc ccc

10 media/variety (6 BAP + NAA & 4 BAP)
2 callus occurrence was marked by following scale: 0 = no

callus, c = little (o<2 mm), cc = some (2<o<s mm),
ccc = much callus (0 > 5 mm)

3 only a few could be rescued from »callus death»

Table 2. Multiplication rate and rooting response for
‘Black Satin’ (BS), ‘Bedford Giant’ (BG) and
‘Thornless Evergreen’ (TE).

Cultivar Multiplication medium Rooting

Number of shoots/
medium 1

In In
vitro vivo

x max/ phenolic
min death

9.3 19/3 > 0% 2 80 % 80%
14.2 41/0 >3O % 27 % 80 %

11.2 25/2 >50% — 3 47 %

BS
BG
TE

10 media/variety
2 none dead because of phenolics, but on average 10

(about halO of the shoots/test tube were dead
3 TE’ was not rooted in vitro

Shoot multiplication

Full strength MS containing 3 mg/1 BAP
was used as a multiplication medium. How-
ever some scientists, for example Pyott and
Converse (1981), have had fine results with
more dilute nutrient concentrations.

Good multiplication took place for all varie-
ties (Table 2). ‘Thornless Evergreen’ gave 11.2
shoots per medium on average (max 25, min
2 shoots/medium), ‘Bedford Giant’ made 14.2
(max 40, min 0) and ‘Black Satin’ made 9.3
(max 19, min 3) shoots/medium. Increase in

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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 GA3 .

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 avery 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 prob-
lem (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 phe-
nolics, micropropagation of Rubus spp. seems
relatively easy and effective. Take ‘Black
Satin’ as an example: A year ago our depart-
ment had only one mother-plant now it is
possible to begin even more extensive experi-
ments.

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
50,000plantlets/meristem tip/year can be pro-
duced.

Acknowledgements. I thank Tapani Haapala and Sei-
ja Häivä for their valuable assistance.

References

Broome, O.C. & Zimmermann, R.H. 1978. In Vitro
Propagation of Blackberry. HortSci. 13(2): 151—153.

gation by Tissue Culture. Handbook and Directory of
Commercial Laboratories, p. 458—459. England.

Donnely, D.J. & Daubeny, H.A. 1986. Tissue culture of
Rubus species. Acta Hortic. 183: 305—314.

Murashige, T. & Skoog, F. 1962. A revised medium for
rapid growth and bio assays with tobacco tissue cul-
tures. Phys. Plant. 15: 473—497.George, E.F. & Sherrington, P.D. 1984. Plant Propa-



Popov, Y.G. & Shchelkunova, S.E. 1973. (The regen-
eration of Rubus idaeus shoot apices in vitro in rela-
tion to the presence ofgrowth substances in the nutrient
medium.) Botanichskii Zhurnal 58 (10): 1515—1520.
In George and Sherrington 1984.

Pyott, J.L. & Converse, R.H. 1981. In vitro Propaga-
tion of Heat-treated Red Raspberry Clones. HortSci.
16 (3): 308—309.

Snir, I. 1981. Micropropagation of red raspberry. Scien-
tia Hortic. 14: 139—143.

Skirvin, R.M., Chu, M.C. & Comez, E. 1981. In vitro
Propagation of Thornless Trailing Blackberries. Hort-
Sci. 16 (3): 310—312.

Ms received September 19, 1986.

SELOSTUS

Karhunvatun (Rubus spp) mikrolisäys

Äke Finne

Puutarhatieteen laitoksella on lisätty mikrolisäysmene-
telmällä kolmea karhunvattulajiketta, (Rubus spp)
‘Thornless evergreen’, ‘Black Satin’ ja ‘Bedford Giant’.
Paras aloitusalusta oli 1/1 MS, joka sisälsi 2.0 mg/1
BAP:a. Jakoalustassa käytettiin 3.0 mg/1 BAP:a. Kallus-
ja fenoliongelmia esiintyi hiukan kummallakin alustalla.
Mikrokasvit juurrutettiin joko 1/10 MS-alustalla ilman

hormooneja tai ne pistettiin suoraan turvealustalle. Noin
kaksi kuukautta myöhemmin suurin osa sekä ‘Black
Satin’- että ‘Bedford giant’- ja noin puolet ‘Thornless
Evergreen’-lajikkeiden taimista kasvoivat hyvin. Teoreet-
tisesti tällä menetelmällä voidaan puolessa vuodessa tuot-
taa 60.000 tainta.

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