Comparison of In Vitro and Traditional Propagation Methods of Rhubarb

(Rheum rhabarbarum) according to Morphological Features and Yield

L. Lepsea
Pūre Horticultural Research Centre
Abavas iela 2, Pūre, Tukuma raj., LV -3124
Latvia

Keywords: micropropagation, field performance, overwintering, yielding

Abstract
Rhubarb (Rheum rhabarbarum) is one of the first vegetables harvested in
Latvia from May till June. Mostly it is propagated by mother plant division. In some
cases it is multiplied by seeds, but as it is known that the progeny in this case is not
homogeneous. Micropropagation has been used successfully for rhubarb propagation
for several decades. Till now it has not been applied for Latvian genotypes. Rhubarb
was propagated in vitro for two years to obtain homogeneous planting material. Three
media compositions were used to obtain the highest proliferation rate: M&S mineral
salt medium, vitamins 1 mg L-1; inozitol 100 mg L-1; 6-benzilaminopurine (BAP) 1, 1.5
and 2 mg L-1; indole-3-butyric acid (IBA) 1 mg L-1 and in one case gibberellic acid
(GA3) 0.5 mg L-1, sucrose 20 g L-1, agar 8 g L-1, pH 5.5. Most of the genotypes had
good proliferation in the MS media with BAP 1.5 mg L-1 and IBA 0.7 mg L-1.
Micropropagated and traditionally propagated plants were planted in the field to
evaluate their morphological and yield features. The following morphological features
were analysed: leaf number, leaf size, petiole length, overwintering. Yield was
evaluated for one year (second year of vegetation). The results of the first two seasons
show that micropropagated plants and seedlings were better developed and yielded
than plants obtained by mother plants dividing.

INTRODUCTION
During the last decade the acreage of rhubarb (Rheum rhabarbarum L.) in Latvia
has decreased. One of the reasons is insufficient amount of planting material. As open
pollinating species rhubarbs are not stable in the inheritage of morphological traits.
Therefore, generative propagation is not giving morphologically homogeneous and stable
progeny. Vegetative propagation by division of mother plants is inefficient and destroys
mother plants. Micropropagation was suggested as the most appropriate and productive
way of propagation of local genotypes of rhubarbs. In 2004, investigations on in vitro
propagation of local genotypes of rhubarb were started. Various methods of micro-
propagation were used according to Roggemans and Boxus (1988) with modifications.
Comparison of micropropagated plants with generatively propagated plants and plantlets
separated from mother plants were carried out in open field conditions with the aim to
clarify the influence of the propagation method on the morphology and yielding
parameters. The evaluation of influence of micropropagation on the overwintering,
morphology and yield capacity of rhubarb plants was the most interesting. According to
the literature (Zao and Crisp, 2003) micropropagated plants have increased amount of
growing points in the first year and produce high amount of thin stems. It was noted that
somaclonal variation caused by in vitro propagation is not observed on rhubarb. Also it
was pointed out in the literature that micropropagation negatively influences over-
wintering. To clarify impact of micropropagation on the Latvian genotypes in Latvian
agroecological conditions investigation was carried out.

a
Email: liga.lepse@puresdis.lv

Proc. IIIrd IS on Acclim. and Establt. of Micropropagated Plants

Ed.: A. Romano 265
Acta Hort. 812, ISHS 2009
MATERIALS AND METHODS

Generative Propagation
Seeds of cultivar Victoria were sown in April. Rhubarb seedlings grown in plastic
tunnel in the spring of 2004 were used as generatively propagated plantlets. In July
plantlets were pricked out in open field. Sorting according to the colour of the stem was
done before pricking out. Plants were grown on the field in beds till the next spring, when
they were planted in the investigation.

Division of Mother Plants

Five years old mother plants were lift out and younger buds with the part of the
roots were separated from them directly before planting in the investigation.

Micropropagation
Initiation of rhubarbs in the culture was started in the May of 2004. Usual
disinfection solutions (Roggemans and Boxus, 1988) were used for plant material
disinfection. Plant material was of two sizes: bud together with small segment of the root
(1-2 cm2); bud with removed outer layer and very small segment of root (0.5 cm2). Apical
tips of 500-1000 µm size were cut from non-sprouting buds for culture establishment.
Initiation media composition was used according to Roggemans and Boxus (1988) and
Roggemans and Claes (1979), but explants of all genotypes were not similarly initiated.
Therefore medium composition was modified. In all variants Murashige and Skoog
(1962) medium (MS) was used supplemented with different growing hormones: 1) BAP 1
mg L-1 and IBA 1 mg L-1; 2) BAP 1.5 mg L-1 and IBA 1 mg L-1; and 3) BAP 2 mg L-1,
IBA 1 mg L-1 and 0.5 GA3. For proliferation media the following compositions were
used: MS supplemented with BAP 1 mg L-1 and IBA 1 mg L-1, or supplemented with BAP
1.5 mg L-1 and IBA 0.7 mg L-1. Rhizogenesis was induced using MS medium containing
0.1 mg L-1 IBA. Adaptation of microplants to ex vitro environment was conducted by
planting them in peat substrate and ensuring high air moisture and temperature
(respectively 90% and 22°C).

Field Trials
Field trials were established on August of 2005 in biologically certified field, with
loamy sandy soil, in 4 replications. Three variants of plantlets were used: generatively
propagated, plantlets separated from mother plants and micropropagated plants. Nine
genotypes were included in the investigation – 8 local clones (1PR10031, 1PR10032,
1PR10022, 1PR10006, 1PR10016, 1PR10004, 1PR10007) cultivar Tukuma, and cultivar
Victoria. As cv. Tukuma is derived by clonal selection from cv. Victoria both of them
were suggested as genetically close (Baumane, 1960, 1967). In 2006 following
parameters were registered: overwintering (scored from 1 to 5), leaf number, leafs size
and petiole length (measured in m). In 2007 also yield per 20 plants was registered. Data
statistical analyse was performed by using dispersion and ANOVA.

RESULTS

Generative Propagation
Seed germination was quite low (in average 77%). Phenotypical splitting of
seedlings in the variant of generative propagation according to the petiole colour was
observed. Three percent of seedlings had untypical colour for variety – green.

Division of Mother Plants

In average 5 plants from each mother plant were obtained. Plantlets had thick root
- in 3-5 cm diameter and 15-20 cm in length and one vegetative bud. Such plantlets were
considered as appropriate for planting in the field investigation.

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Micropropagation
In comparison of 2 disinfection methods of plant material, the best results were
obtained with he second variant, where the smallest segment of root and peeled bud were
disinfected. The percentage of infected explants in the 1st variant was 55.53% and in the
2nd– 27.50%. The highest percentage of alive explants for local genotypes was registered
in the 3rd variant of the initiation media (Table 1). Nevertheless, it was not similar for all
local genotypes. The highest proliferation rate was obtained in the 2nd proliferation media
(Table 2). This media showed significantly highest proliferation rate, especially for local
clones. For ‘Victoria’ type cultivars it was similar in both variants. Successful
rhizogenesis was induced using MS medium containing 0.1 mg L-1 IBA.

Field Trials
Evaluation of leaf amount per plant in the 1st year of growing shows that
significantly highest number of leaves was registered for generatively propagated
‘Victoria’ type plants. In the same time in average for all genotypes plants derived by
mother plant division had significantly more leaves in comparison to micropropagated
plants (Table 3). Micropropagated plants in the 1st year of vegetation had no significant
differences in leaf number between genotypes. If comparing all 3 propagation methods,
micropropagated plants had significantly less amount of leaves, but significantly biggest
leaf size (Fig. 1). There was significant difference between propagation methods in the
petiole length in the 1st vegetation season. The shortest petioles (average 0.13 m) had
vegetatively propagated plants, but tallest ones (0.18 m) - generatively propagated plants.
Significant difference between plants in the overwintering in the 1st winter after planting
also was stated. Plants derived by mother plant division survived very bad during winter
(scored by 2.3). The best overwintering was found for micropropagated plants (score 3.5).
Significant differences in yield, petioles weight and length between both genotypes and
propagation methods in the 2nd vegetation season were not found (Fig. 2). But there is a
tendency that in the first year of propagation, generatively propagated plants had the
lowest yields while vegetatively propagated, the highest.

DISCUSSION
In comparison of disinfection methods the average percentage of infected plants
was high (27.5%), but it has be taken into account that explants of rhubarb were taken
from the root zone (underground part of plant) and the infection possibility is very high.
Moreover bud is embosomed by several leaves which are excreting mucous excrements,
and it makes problematic cutting and transfer of meristematic tissue. Mucus is sticky and
it is excellent vector of infection organisms.
Propagation rate observed in our investigation is similar to referred by Lassus and
Voipo (1994) when explants from sprouting buds were used for cv. Victoria.
In evaluation of influence of propagation method on overwintering of plants in the
field investigation it should be noted, that winter after planting of the investigation had a
little snow and quite low temperatures (-25°C). It could cause bad overwintering of plants
derived by mother plant division, because they were bigger and there is high possibility
that growing point was closer to the earth surface and therefore was damaged by low
temperatures.
The evaluation of leaf size and number in the first year was the only vegetative
parameters illustrating the plant vigour and yield potential in the 1st vegetation season in
our investigation. But it should be taken into consideration that not always the high
number of leaves indicates the vigorousity of plant. In our case leaves of plants derived
by mother plant propagation were of small size and numerous and had thin petioles. From
the point of yield such plants are unusable. Zhao and Crisp (2003) mentioned that in vitro
propagated plants have increased number of growing points and consequently thinner
petioles. In our investigations it was not proved completely. Plants derived by mother
plant division had leaves of smaller size, increased number of growing points, thinner
petioles and also overwintering was not so satisfactory. Since Zhao and Crisp (2003)

267
performed their experiments in UK these differences could be explained by different
climatical conditions. As leaves are consider as the main organ of synthesis of nutritional
substances for plant (Lassus, 1994) and therefore influences overwintering, the plants
with small leaves are more endangered in severe winters.
In conclusion, it should be stressed, that micropropagation is considered as an
appropriate method for propagation of rhubarb planting material in Latvia conditions.

ACKNOWLEDGEMENTS
The investigations were carried out with the financial support of the Ministry of
Education of Latvia grants No TOP 04-33 and TOP 06-11.

Literature Cited
Baumane, M. 1960. Rabarberi, Rīga
Baumane, M. 1967. Daudzgadīgie dārzeņi, Rīga
Lassus, C. and Voipo, I. 1994. Micropropagation of rhubarb with special reference to
weaning stage and subsequent growth. Agricultural Science in Finland 3:189-194.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Plant 15:473-497.
Roggemans, J. and Boxus, P. 1988. Rhubarb (Rheum rhaponticum L.). p.339-350. In:
Y.P.S. Bajaj (ed.), Biotechnology in agriculture and forestry. Vol. 6, Crops II. Springer-
Verlag, Berlin Heidelberg.
Roggemans, J. and Claes, M.C. Rapid clonal propagation of rhubarb by in vitro culture of
shoot-tips. Scientia Hort. 11:241-246.
Zao, Y. and Crisp, P.C. 2003. Inadvertent selection for unwanted morphological forms
during micropropagation adversely affects field performance of European rhubarb
(Rheum rhaponticum). Acta Hort. 616:301-307.

Tables

Table 1. Average percentage of alive explants in two groups of genotypes in three variants
of initiation media.

Genotype Media variant

1 2 3
Local clones 20 35 53
‘Victoria’ 0 0 25

Table 2. Proliferation rate in two media compositions.

Genotype BAP 1 mg L-1 + IBA 1 mg L-1 BAP 1.5 mg L-1 + IBA 0.7 mg L-1
Local clones 1.7 2.6
‘Victoria’ 2.5 2.5

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Table 3. Influence of propagation method on the average leaf number per plant in the 1st
vegetation season.

Propagation method Ave. to all clones ‘Victoria’ genotypes

Generative 8.5 12.4
Micropropagation 5.5 4.5
Mother plant division 9.7 11.1
α0.05 = 3.16 α0.05 = 4.51

Figurese

0.60

0.50

0.40
size, m

0.30

0.20

0.10

0.00
generative microclonal mother plant
division
propagation method
lenght width

Fig. 1. The average leaf size of all genotypes in the 1st growing season.

4.0 4.0
3.5 3.5
3.0 3.0
2.5 2.5
kg

2.0 2.0
m

1.5 1.5
1.0 1.0
0.5 0.5
0.0 0.0
generative mother plant division microclonal

propagation method

20 petioles weight yield/plant length

Fig. 2. Morphological and yield parameters in the 2nd vegetation season.

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