Download Effect of silver nitrate on in vitro root formation of Gentiana lutea

ACRomanian Biotechnological Letters
Copyright © 2011 University of Bucharest
Vol. 16, No.6, 2011, Supplement
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ORIGINAL PAPER
Effect of silver nitrate on in vitro root formation of Gentiana lutea
Received for publication, September 15, 2011
Accepted, October 26, 2011
MARIYA PETROVA1, ELY ZAYOVA1 AND ANTONINA VITKOVA2
1
Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences
2
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences
Correspondence to: Mariya Petrova
E-mail: [email protected]
Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences Acad. G. Bonchev
Street, Bldg. 21, Sofia 1113, Bulgaria
Abstract
Gentiana lutea (Gentianaceae) is a valuable, protected species, included in the Red Book of
Bulgaria. Microbial contamination is one of the most critical and often encountered problems in plant
tissue cultures. The influence of silver nitrate (AgNO3) on in vitro rooting of G. lutea was examined.
Considerable limitation of late bacterial contamination and improved in vitro rooting of plants were
achieved on half strength MSR2 rooting medium supplemented with IBA (1 mg/l) and AgNO3 (1 mg/l).
The optimization of this medium provided a high degree of rooting (90%) and lack of bacterial
contamination. A maximum plant height (2.5 cm), number of roots/plant (4.5) with mean length 1.5
cm were obtained on ½ MSR2 medium after four weeks of culture. The effect of different mixture
substrate during acclimatization stage was also studied. Best response (65%) was obtained in peat:
perlite: sand (2:1:1 v/v/v), after eight weeks of transplantation in ex vitro conditions. This technique
can be effectively used for in vitro rooting of G. lutea.
Key words: Gentiana lutea, silver nitrate, bacterial contamination, in vitro rooting
Introduction
Gentiana lutea L. (Gentianaceae) is a perennial herbaceous plant. In Bulgaria the
species is distributed mainly on rocky slopes of Balkan range, Rila, Vitosha, Pirin and
Rhodope Mountains, at 1200-2600 m altitude (BONDEV [1]). The plant is included in the
Red Book of Bulgaria in the category of endangered species. Collection of rhizomes from
natural populations is strictly prohibited. The plant contains bitter secoiridoid glucosides
(gentiopicroside, amarogentin), xanthones, di- and trisacharides, pyridine alkaloids etc.
Gentian root has a long history of use for treatment of digestive disorders and is an ingredient
of many medicines. It is also applied as an immunostimulant and exerts antitumor, antivirus,
antibacterial and antioxidant effects (KONDO & al. [2]; MENKOVIC & al. [3]).
G. luteа is propagated by seeds or vegetative means. The seeds of many Gentian
species have a problem with germination (FRANZ & FRITZ [4]; GALAMBOSI & al. [5];
EVSTATIEVA [6]). The vegetative propagation is rather slow, labour-intensive and timeconsuming process. In vitro propagation ensures the availability of plant material throughout
the year. Several micropropagation protocols of G. lutea have been reported
(WESOLOWSKA & al. [7]; VIOLA & FRANZ [8]; SKRZYPCZAK & al. [9];
MOMČILOVIĆ & al. [10]; MENCOVIC & al. [3]). Rhizogenesis of G. lutea plants was
induced by the addition of auxins to the nutrient medium: indole-3-butyric acid (IBA) or αnaphthalene acetic acid (NAA) (MOMČILOVIĆ & al. [10]; ZELEZNIK & al. [11]). In vitro
microbial contamination is an often encountered problem in plant tissue cultures. Usually, the
bacterial contamination is detected late, because some bacteria remained in latent phase for
long periods of time (LEIFERT & al. [12]; CASSELLS, [13]). The presence of contaminants
can result in variable growth, tissue necrosis, and reduction of shoot proliferation and rooting.
53
MARIYA PETROVA, ELY ZAYOVA AND ANTONINA VITKOVA
However, a tissue culture must be maintained clean of contaminantion. Usually antibiotics or
fungicides are added to the nutrient medium (LEIFERT & CASSELLS [14]). Silver nitrate is
well known for its bactericidal action and is widely used in plant tissue culture (KUMAR &
al. [15]).
The objective of present study was to establish the effect of silver nitrate on bacterial
contamination and rooting of G. lutea in vitro propagated plants.
Material and Methods
The micropropagated shoots, obtained from seedlings of G. lutea were used for in
vitro rooting. The seeds were collected from the natural population of the species in the region
of Vetrovala 2001, Nature Park “Vitosha”, Bulgaria. The shoots were cultivated on half
strength MS (MURASHIGE & SKOOG [16]) medium containing IBA at different
concentrations (Table 1). The rooting medium provided appropriate conditions for
development of saprophyte bacteria after prolonged sub-cultivation of G. lutea plants. Silver
nitrate was added to half strength MS medium containing 1 mg/l IBA for rhizogenesis, to
limit the bacterial contamination (Table 2). Sucrose and agar concentrations were constant 2% and 0.6%, respectively. Each treatment involved 20 plants. The following parameters
were determined: percentage of rooted plants, average number of roots per plant and root
length after 4 weeks of cultivation. All cultures were placed in growth room at 22±2 °C, 16/8
hours photoperiod and illumination 40 μmol m-2s-1. The medium pH was adjusted to 5.7
before autoclaving at 1 atm. (120 °C for 20 min).
For acclimatization, the rooted plants were washed carefully with running tap water
and planted in small plastic pots (7 cm diameter). Initially four different potting mixes were
used for adaptation of the plants (Table 3). To maintain high humidity, the pots were covered
with transparent polyethylene. The plants were incubated at 22±2 °C with 16 hours
photoperiod. After two weeks, covers were removed and the plants were exposed to external
moisture levels gradually. The plants were transferred to bigger plastic pots (14 cm diameter)
containing soil and perlite (2:1 v/v). The elongation of transplanted shoots and emergence of
new leaves after 12 days was observed. The survival rates of plants grown on different potting
mixes were calculated after 8 weeks. Data of all results were statistically analyzed using
Sigma Stat computer package (Sigma Stat 3.1, Systat Software, San Jose, California, USA).
Results and Discussion
In vitro rooting of plants: The existence of well-developed and healthy root system of
in vitro plants was a very important step for subsequent acclimatization in conditions ex vitro.
Initiation of roots emerged 14 days after planting. The shoots formed roots on ½ MS nutrient
media containing IBA at all tested concentrations (Table 1). It was observed that in vitro shoots
grown on ½ MS medium containing 0.5 mg/l IBA produced 3.08 roots per explant (Fig. 1a).
High root number per explant was obtained on ½ MS nutrient medium containing 1 mg/l (Fig.
1b) or 2 mg/l IBA. The highest number of roots per shoot (6.66) and higher root growth (1.95
cm) were achieved on the medium containing 3.0 mg/l IBA (Fig. 1c). The root growth and
development was positively influenced by all tested IBA concentrations, but at 1 mg/l IBA the
roots were suitable for subsequent adaptation to conditions ex vitro. This medium was
assessed as most appropriate for rooting and was efficiently used in further passages. For in
vitro rooting of G. lutea plants, the addition of auxins to the medium was necessary
(MOMČILOVIĆ & al. [10]).
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Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)
Effect of silver nitrate on in vitro root formation of Gentiana lutea
Table 1. Effect of different concentrations of IBA on in vitro rhizogenesis of G. lutea micropropagated shoots
Nutrient medium
№
MS0
MS1
MS2
MS3
MS4
Concentration
of IBA, mg/l
0
0.5
1
2
3
Rooted
plants, %
No roots/
plant
(х ± SE)
Root length
(х ± SE)
20
80
85
90
95
1.14 ± 0.45
3.08 ± 0.64
4.71 ± 1.05
6.45 ± 1.24
6.66 ± 0.97
1.23 ± 0.42
0.95 ± 0.11
0.96 ± 0.10
1.53 ± 0.14
1.95 ± 0.17
Fig. 1. In vitro rooted G. lutea plants on half strength MS medium supplemented with: а) 0.5 mg/l IBA; b) 1
mg/l IBA and c) 3 mg/l IBA.
Effect of silver nitrate on the plant rooting: During the rooting of G. lutea plants, which
were maintained through several passages, a bacterium-like contaminant was found (Fig. 2a).
This is a serious problem and hampered further plant rooting. The plants retarded their
development; necroses appeared on leaves and on the basis of explants and percentage of
rooting considerably decreased. The registered bacterial contamination was an obstacle to
achieve maximum effect of the rooting medium, which was characterized with very good
balance of nutrient substances and induction of great number of roots. The results showed that
application of silver nitrate stimulated root induction. The response varied depending on the
applied silver nitrate concentration (Table 2). The highest percentage (90%) of root formation
was observed on ½ MSR2 rooting medium supplemented with 1 mg/l AgNO3 (Table 2; Fig.
2b). At this concentration silver nitrate root emergence was enhanced from the cut ends of
shoots after 12 days and root number per shoot and shoot height were increased. The
significant decline of the visible contamination and the high percentage of rooting are due to
the suppression of the bacterial development. It was found that the nutrient medium
supplemented with 2 mg/l AgNO3 reduced the number of roots per plant and increased the
root length. In addition, from the visual observation, shoot color became dark green and more
vigorous with increasing of AgNO3 concentrations. Maximum root length was recorded on
MSR3 medium (1.81 cm) in comparison with root length (0.25 cm) obtained on MS0
(control). The number of roots decreased and the shoots were shortened on the medium
supplemented with 3 mg/l AgNO3. The visible bacterial contamination was not observed.
The optimum concentration required to induce maximum root growth and to limit bacterial
contamination was found to be 1 mg/l AgNO3. The silver nitrate improved in vitro root
formation of several species: Decalepis hamiltonii (BAIS & al. [17]; REDDY & al. [18]);
Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)
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MARIYA PETROVA, ELY ZAYOVA AND ANTONINA VITKOVA
Vanilla planifolia (GIRIDHAR & al. [19]); Coffee (GIRIDHAR & al. [20]) and Rotula
aquatica Lour (SUNANDAKUMARI & al. [21]).
Table 2. Effect of silver nitrate in half strength MS rooting medium on rhizogenesis of G. lutea micropropagated
shoots
Media
Concentration
Rooted shoots,
of AgNO3, mg/l
%
MSR0
0
55
MSR1
0.5
65
MSR2
1
90
MSR3
2
60
MSR4
3
50
* MSR0 Control without silver nitrate
Plant height,
cm
1.4±0.17
1.6±0.18
2.5±0.23
2.1±0.20
1.7±0.19
No roots/
plant
1.6±0.18
2.2±0.17
4.5±0.36
2.8±0.25
1.9±0.21
Root length,
cm
0.25±0.04
0.83±0.07
1.52±0.14
1.81±0.08
1.26±0.06
Fig. 2. Rooted G. lutea plants: a) on half strength MS medium without AgNO3 and b) on half strength MS
medium with 1 mg/l AgNO3.
Acclimatization of G. lutea plants in conditions ex vitro: The acclimatization of
rooted plants in ex vitro conditions was difficult. The plants with well-developed root system
were transferred to small pots containing four different mixtures. Тhe influence of the peat
mixtures on plant survival rate during adaptation were studied (Table 3). When the plants
were transferred from in vitro to ex vitro conditions, many of them died. The most suitable
proved to be a mixture of peat, perlite and sand in the ratio 2:1:1 (Fig. 3), where the survival
rate was 65% after 8 weeks, followed by the mixture of peat and coco (2:1 v/v). Then, the
plants were transferred to bigger pots containing soil and perlite (2:1 v/v). Vigorous G. lutea
plants were transferred to the fields at high-mountain experimental stations - Beglika,
Rhodope Mts and Zlatni Mostove, Vitosha Mt (1500 m altitude), where survival rate was 2025%. VIOLA & FRANZ [9] reported that in the greenhouse where conditions are more
favorable for plant growth, survived only 25-40% of rooted plants.
Table 3. Effect of different mixture substrates on survival rate of G. lutea plants during ex vitro acclimatization
Peat mixture
Peat: Perlite (2:1 v/v)
Peat: Coco (2:1 v/v)
Peat: Perlite: Coco (2:1:1 v/v/v)
Peat: Perlite: Sand (2:1:1 v/v/v)
56
Number of transplanted plants
30
40
20
40
Survival of plants, %
20
30
25
65
Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)
Effect of silver nitrate on in vitro root formation of Gentiana lutea
Fig. 3. Acclimatized G. lutea plants in conditions ex vitro
Conclusion
The appropriate conditions for in vitro rooting of the protected species G. lutea were
established. The ½ MS nutrient medium supplemented with silver nitrate (1 mg/l) and IBA (1
mg/l) reduced the bacterial contamination, enhanced the development and growth of shoots
and increased the percentage of rooted plants. In this report we provided methodology for
adaptation of G. lutea plants. The developed techniques ensured the planting of plants under
mountain conditions.
Acknowledgements
The authors are grateful for the financial support provided by the Bulgarian National
Science Fund, Ministry of Education, Youth and Science (Project DTK-02/38)
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