Adaptation of the obtained in vitr o Gentiana lutea L. plants to ex vitro and in sit u conditions

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Experimental articles
UDK 582. 923.1.+ 58. 084
doi: 10. 15 407/biotech8. 06. 077
ADAPTATION OF Gentiana lutea L. PLANTS OBTAINED in vitro TO ex vitro AND in situ CONDITION
О. Yu. Mayorova
L. R. Hrytsak Hnatiuk Ternopil
N. М. Drobyk National Pedagogical University, Ukraine
E-mail: majorova@i. ua
Received 21. 10. 2015
The aim of the research was to develop the technology of introduction of the Gentiana lutea L. plants obtained by microclonal propagation into conditions in situ. Methods of cultivation of plant objects in vitro were used. There were chosen optimal conditions for rooting G. lutea shoots obtained through microclonal propagation in vitro: МS/2 medium with twice decreased concentration of NH4NO3without vitamins and sucrose supplemented with 3 g/l of mannite and 0. 05 mg/l kinetin, and agar (4 g/l) in combination with perlite (16 g/l) used as a maintaining substrate- or the nutrient medium (MS/2 without vitamins and smaller concentration of NH4NO3) with gradual decrease of carbohydrates from 10 g/l to 2 g/l, and further rooting experimental shoots in tap water. Rooted plants were adapted to conditions ex vitro through planting them into flowerpots with soil and gradual changing hothouse regime for exposed one. The share of adapted to in situ conditions plants (21% after a year of planting) proves the used method to be resultative and promising. Thus, there was suggested the most efficient technology for revival of disturbed G. lutea populations that includes repatriation of rooted and adapted to ex vitro conditions plants obtained through microclonal propagation in vitro.
Key words: Gentiana lutea L., rooting in vitro, adaptation еx vitro, repatriation in situ.
Within a concept of preserving biodiversity of plant and animal world, the investigations aimed at finding ways to stabilize the number of disturbed populations and revival of destroyed natural populations of rare plant species are of current relevance. While protecting such plant species, it is necessary to apply not only passive preservation methods (inventory, including into local lists of rare plants and Red Books of various ranks, conserving on reserved territories), but use active methods (growing in botanical gardens, increasing number of rare species in natural conditions by seeding and planting of obtained in vitro cultures) too [1].
Rare plant species, requiring protection and revival, include medicinal Gentiana lutea L. The complexity of G. lutea seed restoration, excessive pastoral load and recreation have led to reducing areas and violated structures of this species population. G. lutea value for conventional and nontraditional medicine, determined by the pharmacological properties and potential need in raw material, has caused
the necessity to estimate the species resources in Ukraine for creating fundamental principles of preservation and developing new approaches to its population revival.
The recent years being marked by the developed methods of G. lutea microclonal propagation in vitro [2, 3], the repatriation of propagated in vitro plants into natural conditions has become of great importance. The essential condition for successful repatriation is the use of individuals grown in most approximated to natural conditions, and therefore minimally changed genetically [4].
The objective of the investigation was to develop a scheme of reintroducing G. lutea plants developed by microclonal propagation into in situ conditions.
Materials and Methods
The object of the investigation was G. lutea — a perennial with indefinitely long life cycle (over 50 years) and lasting pre-generative period (5−10 years) [5]. The initial seed
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material was gathered during expeditions in the Ukrainian Carpathians in mountainous populations on Petros Marmarosky, Pozhyzhevska, Sheshul-Pavlyk, Menchul Kvasy mountains and Lemska and Rogneska mountainous valleys.
For choice of rooting G. lutea shoots in vitro we took into consideration biological and ecological peculiarities of the species and the results of G. lutea introduction in vitro culture, obtained in the laboratory of ecology and biotechnology of Volodymyr Hnatiuk Ternopil National Pedagogical University. For rooting, we used G. lutea shoots obtained by microclonal propagation and then grown for 1. 5−3 months until they became 15−20 mm high with 3−5 pairs of leaves. Each testing variant included three samplings with 8−10 shoots.
Fourteen variants of media were tested (tables 1, 2). In eight tested variants, the cultivated in vitro shoots were planted in Murashige and Skoog medium (1962) with half concentration of macro- and microsalts (MS/2) without sucrose and vitamins (table 1). There were experimented two MS modifications media: MS/2 (variants I, II, Ill, IV) — and MS/2 with twice decreased concentration of NH4NO3 (variants V, VI, VII, VIII), as the decrease of macro- and micro-elements and nitrogen concentration are known to promote rooting wild strawberry, apple-tree and other plant cuttings [6−9]. In addition, the research of the component soil composition from natural G. lutea areas showed that the amount of both available and general nitrogen in them was comparatively low. Growth regulators are known to negatively influence adaptation of plants to ex vitro conditions [9], so kinetin (Kin) concentration in all media was reduced to 0. 05 mg/l.
For each modification of nutrient medium, we used various maintaining substrates such as agar (8 g/l) (I, V) — agar (4 g/l) and perlite (16 g/l) (IV, VIII) — agar (4 g/l) and ground perlite (16 g/l) (III, VII) — foam (II, VI) — vermiculite (IX). We also attempted to enroot shoots in a sterilized soil from natural G. lutea habitats combined with peat and perlite in correlation 1: 1:1 (X).
There was tested nutrient medium MS/2 without vitamins with twice decreased concentration of NH4NO3 which was added with 2 g/l sucrose (as the main СО2 source) (XI) — two variants with different concentrations of sucrose and mannite in correlation 1:1 (XII, XIII) — a variant with mannite only (XIV) with its concentration 3 g/l (table 2). Agar (4 g/l) combined with perlite (16 g/l) was used as
a maintaining substrate. Adding mannite was caused by its being osmotically active substance providing antioxidant protection and promoting plant survival in stress [10, 11]. Plants were cultivated for 120 days on the media mentioned above.
G. lutea was cultivated in jars with ventilatory covers. The plants, infected during relocation or not forming roots on nutrient media, were placed into settled tap water. Rooted specimens were planted into flowerpots with soil, having their roots previously washed with distilled water to relieve them from the medium remains.
To avoid dehydration of the plants and create the greenhouse effect the flowerpots were covered with glass. Every day the plants were sprayed and once a week they were watered with settled tap water. Air expositions were used to make them adapt to ex vitro conditions. In 1−1.5 months the flowerpots were definitely opened, watering was done in dependence on soil drying (1−2 times a week), and spraying was daily.
In early June (after 8−10 months of the experiment beginning) the adapted to ex vitro conditions and then grown for 3−4 months G. lutea were planted in situ conditions in the places of bare soil near adult gentian individuals.
General state of plants in ex vitro and in situ conditions was estimated by morphological values (plant height, number of shoots, pairs of leaves and number of internodes per plant). The obtained data were processed statistically [12].
Results and Discussion
The adaptation efficiency of the obtained in vitro plants to ex vitro conditions depends in the first place on their successful rooting, as the existence of well-developed root system provides better adaptation of plants to growth in soil and unstable conditions of unsterile environment (fluctuations of humidity, temperature, etc.). Thus, the research included three stages: rooting obtained by microclonal propagation plants in vitro, adaptation of rooted plants to ex vitro conditions, transfer of adapted to ex vitro plants to conditions in situ.
Rooting G. lutea sprouts obtained by microclonal propagation. The results of the research show that both the composition of nutrient medium and the type of maintaining substrate (table 1) influenced G. lutea rooting. G. lutea shoots planted in agarized media (8 g/l) (I, V) and in vermiculite (IX) necrotized on the
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20−30th day of cultivation, and those placed in media with agar (4 g/l) in combination with ground perlite (16 g/l) (Ill, VII) — on 20−40th day. Evidently, the consistency of nutrient medium decelerates absorbing of nutrient substances by plant roots that leads to their death. This result is confirmed by scientific literature data, pointing to the fact that growth and development of roots in vitro are dependent on aeration of nutrient medium that, in its turn, is dependent on agar concentration. Rooting plant cuttings is decelerated- the development of secondary roots does not take place [7].
The shoots planted into the nutrient media with foam substrate and decreased concentration of agar (up to 4 g/l) in combination with perlite (16 g/l) survived and rooted in dependence on medium composition. The individuals cultivated in MS/2 without vitamins and sucrose (II, VI), died on the 20th (II) and 30th (VI) day. Twice decreased concentration of NH4NO3 in MS/2 medium (VI, VIII) was more efficient. As a result, the share of viable specimens on the 50th day of cultivation constituted 45% (VI) and 66% (VIII), and on the 120th day, it was 10% and
15% respectively. Obviously, the composition of nutrient medium serves as a limiting factor in such a combination of nutrient medium and maintaining substrate.
Thus, one can assume that using MS/2 medium with twice decreased NH4NO3 concentration and maintaining substrates such as foam and agar (4 g/l) combined with perlite (16 g/l) positively influences rooting shoots. As the results of shoots survival on conditions of using foam and agar with perlite did not sufficiently differ, one can use both variants for further cultivation. However, referring to the objective of the research, we find the use of the second variant more efficient as semisolid maintainig substrate will enable better adaptation of plant root system to growth conditions in soil. Other researchers prove positive effect of using agar combined with perlite as a maintaining substrate: plants of wild strawberries, apples, pears, ashes, lilac formed a well-developed root system on such a substrate [7].
On conditions of rooting G. lutea shoots in sterilized soil with adding peat and perlite in correlation 1: 1:1 all the tested shoots died on 20−30th day. Evidently, soil sterilization
Table 1. Rooting of G. lutea shoots in vitro
Percentage of viable shoots, %
Days MS/2 medium without vitamins and sucrose supplemented with 0. 05 mg/l Kin MS/2 medium without vitamins and sucrose- with twice decreased concentration of NH4NO3, supplemented with 0. 05 mg/l Kin Vermiculite
Maintaining substrate
Ag F Ag+gPr Ag+Pr Ag F Ag+gPr Ag+Pr
I II III IV V VI VII VIII IX
10 51±3.8 84±3.5 42±2.4 100 49±3.2 100* 100* 100* 100
20 0 0 0 27±1.8 0 100* 18±1. 4* 93±5. 4* 43±3. 2
30 0 0 0 0 0 100* 44±2. 8* 93±5. 4* 0
40 0 0 0 0 0 85±5. 4* 0 71±4. 7* 0
50 0 0 0 0 0 45±3. 6* 0 66±4. 2* 0
60 0 0 0 0 0 30±3. 1* 0 48±3. 9* 0
90 0 0 0 0 0 10±1. 5* 0 43±3. 3* 0
120 0 0 0 0 0 10±1. 5* 0 15±1. 8* 0
Notes: Ag, F, Ag + gPr, Ag + Pr — maintaining substrates-
Ag — agar (8 g/l), F — foam, Ag + gPr — agar (4 g/l) combined with ground perlite (16 g/l), Ag + Pr — agar (4 g/l) combined with perlite (16 g/l) —
* - marked variants of the experiment in which the parameters of share of viable shoots during use of modified variants MS/2 media with twice decreased concentration of NH4NO3 reliable (Р & lt- 0. 05) differ from the results on nutrient media MS/2 with full NH4NO3 concentration.
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leads to death of mycorrhizal organisms that negatively affects growth and development of G. lutea, which is a mycorrhizal species [13].
An attempt to enroot G. lutea shoots on the medium suggested by Petrova and colleagues [14] and which was efficient for rooting and further adaptation of obtained in vitro G. lutea plants to ex vitro conditions, and then to in situ in Natural Park of Vitosha (Bulgaria) failed to have any positive results in our case. None of the cultivated in vitro shoots formed roots that is probably caused by genotype peculiarities and heterogeneity of natural habitats of plants.
The shoots cultivated on the medium without sucrose and additional supply of СО2 died probably because of the complexity of adaptation to autotrophic type of nutrition, little photosynthetic activity and scarcity of oxygen. Therefore, for further cultivation, we used nutrient medium MS/2 without vitamins with twice decreased concentration of NH4NO3 and added carbohydrates (XI, XII, XIII, XIV) (Table 2).
The shoots cultivated on XI, XII and XIII variants of the medium were viable on the 30th day, but they did not form roots. That is why all the specimens for rooting were planted in tap water, covered with caps, which were
gradually opened to adapt the plants to ex vitro conditions. On the 20th day after planting into water 38% of specimens demonstrated rhizogenesis, and on the 90th day 94% of the specimens formed roots. The G. lutea shoots grown on XIV variant of the medium also formed roots (Fig. 1), the percentage of adaptation on the 120th day of cultivation was 97 (Table 2).
The analyzed results show that efficiency of rooting G. lutea shoots can be provided only by combination of optimal composition of nutrient medium and favourable for rooting maintaining substrate. The optimal medium among those tested proved to be MS/2 medium with twice decreased concentration of NH4NO3 without vitamins and sucrose supplemented with 3 g/l mannite and 0. 05 mg/l Kin and agar (4 g/l) combined with perlite (16 g/l) used as a maintaining substrate. Gradual diminishing carbohydrates from 10 g/l to 2g/l in the nutrient medium (MS/2 without vitamins and twice decreased concentration of NH4NO3) with further rooting of the shoots in tap water proved equally efficient (Fig. 1).
Adaptation of the rooted plants to ex vitro conditions. Repatriation in disturbed populations of plants obtained through microclonal propagation in vitro first requires solving the problem of their adaptation
Table 2. Rooting of G. lutea shoots
Days Percentage of viable shoots, % / Percentage of rooted plants, %
MS/2 medium with twice decreased concentration of NH4NO3 without vitamins, supplemented with 0. 05 mg/l Kin, and agar (4 g/l) in combination with perlite (16 g/l) used as a maintaining substrate
2 g/l sucrose 2 g/l sucrose, 2 g/l mannite 1 g/l sucrose, 1 g/l mannite 3 g/l mannite
XI XII XIII XIV
10 100/0 100/0 100/0 100/0
20 100/0 100/0 100/0 100/0
30 100/0 100/0 100/0 100/54±3. 2
Shoots planted into tap water
40 100*/0 97±4. 7*/97±4. 7
50 100*/38±2.8 97±4. 7*/97±4. 7
60 94±5. 3*/72±6.1 97±4. 7*/97±4. 7
90 94±5. 3*/94±5.3 97±4. 7*/97±4. 7
120 94±5. 3*/94±5.3 97±4. 7*/97±4. 7
Note: * - marked variants of the experiment in which the parameters of the share of viable shoots, on conditions of using modified variants of MS/2 media with twice decreased concentration of NH4NO3 supplemented with carbohydrates reliable (Р & lt- 0. 05) differ from the results on the nutrient MS/2 medium with twice decreased concentration of NH4NO3 without carbohydrates (Table 1, VIII variant of nutrient medium).
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Fig. 1. Rooting G. lutea shoots in vitro:
A — rooting in the medium MS/2 with twice decreased concentration of NH4NO3 without vitamins and sucrose, supplemented with 3 g/l mannite and 0. 05 mg/l Kin, the maintaining substrate is agar (4 g/l)
combined with perlite (16 g/l) — B — rooting in tap water
to ex vitro conditions. According to the research literature data [15−17], this process is labour intensive, as specific conditions of in vitro culture in many cases cause the formation of microshoots with defective physiological processes, morphological and anatomical structure. After transplantation from cultivation-vessels to ex vitro conditions such plants may be damaged because of altered cultivation conditions and therefore they require adaptation.
For adaptation G. lutea to ex vitro conditions the plants which formed roots in tap water and in the medium with twice decreased concentration of NH4NO3 and mannite were planted in soil (Fig. 2) To avoid drying of plants and their better adaptation to ex vitro conditions the flowerpots with planted specimens were covered by glass. Air expositions were done for 4−6 weeks, the duration and frequency of openings were gradually increased.
Percentage of adaptation for plants from various experimental variants somewhat differed (Fig. 3). Thus, on the 30th day the share of adaptation for plants which previously formed roots in water was higher compared to the plants cultivated in nutrient medium (XIV). However, the percentage of viable plants from both variants did not significantly differ hereafter and on the 150th day constituted 72. 4% (rooted in tap water) and 70. 8% (rooted in nutrient medium).
To estimate the condition of G. lutea individuals, we determined some morphometric parameters: height of plants (to clarify their adaptive capacity), number of pairs of leaves (leaves are the main organs of photosynthetic activity), and the number of internodes (to show adaptive changes of plants to ex vitro conditions, as internodes formation is the peculiarity of G. lutea plants cultivated in vitro).
It was shown that within the period from the 1st to 30th day the accretion of plants rooted
Fig. 2. G. lutea planted in soil:
vegetation duration 30 (A), 60 (B), 90 ©, 120 (D) days
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Fig. 3. Adaptation in soil for plants rooted in vitro:
I — percentage of adaptation for plants which
formed roots in tap water-
II — percentage of adaptation for plants rooted in
the nutrient medium MS/2 with twice decreased concentration of NH4NO3 without vitamins and sucrose, supplemented with 3 g/l mannite and 0. 05 mg/l Kin, the maintaining substrate is agar (4 g/l) combined with perlite (16 g/l)
in the nutrient medium was 1.6 times higher compared with the plants which formed roots in water (Table 3). This phenomenon can be explained by the available reserve of nutrient substances stored in the organs of plants from the nutrient medium. From 30th to 90th day, the results were the opposite: accretion of the rooted in water plants was twice bigger that may testify to faster adaptation to ex vitro conditions. From 90th to 120th day the accretion of plant height was 1.3 times bigger for specimens rooted in the nutrient medium.
During growing in soil, the rooted in tap water plants formed on average 4−5 pairs of leaves and those rooted in the nutrient medium — 2−3 ones. That proves more intense photosynthetic activity, better viability and adaptation to ex vitro conditions. The number of internodes was the same in both variants. Besides, merome formation decreased in the process of cultivation and on 150th day it stopped (Table 3). Evidently, in ex vitro conditions G. lutea acquire morphological structure of intact plants, as the formation of radical rosette is representative for this species in nature [18].
Thus, the percentage of adaptation and accretion of plants rooted in tap water and in
the nutrient medium did not practically differ. For rooting and further adaptation to ex vitro conditions, G. lutea can be planted in both water and medium MS/2 with twice decreased concentration of NH4NO3 without vitamins and sucrose, supplemented with 3 g/l mannite and 0. 05 mg/l Kin. It is reasonable to use agar (4 g/l) combined with perlite (16 g/l) as a maintaining substrate.
Transplantation of adapted ex vitro plants into conditions in situ. At the beginning of June all viable and adapted to ex vitro conditions individuals were planted in natural conditions on Pozhyzhevska mountain (territory of the Carpathian National Natural Park) (Fig. 4). The area is located on the slope of northern-western exposition with steepness 20−40° 1450 m high above sea level.
According to literature data [19, 20], which were confirmed by our research, germination and taking to root of undergrowth for Gentiana species, including G. lutea, take place best in conditions of disturbed gramineous sodding. That is why the individuals were planted in places of bare soil to reduce the level of interspecies competition and provide optimal conditions for G. lutea adaptation and growth.
On the 3rd day of growth in natural conditions, we observed the loss of turgor in 15 ± 1. 2% plants- on the 30th day the share of adapted plants constituted 97 ± 2. 5%. The plants were viable, 2. 5−7.5 cm high, with 6−16 pairs of leaves (Table 4). Evidently, their adaptation in situ was favoured by weather conditions of highland, as it rained almost every day in June-July, 2013. On the 60th day of growing the share of viable plants was 51 ± 1. 8%. However, almost 40% of individuals died, having been eaten by small animals (rodents, lizards). Next year the share of viable plants equaled 21%.
General accretion of G. lutea height after 60 days of growing in natural conditions was insignificant (5.8 mm) that is probably caused not only by the complexity of adaptation, but biological peculiarities of the species too. Particularly, in the first years of ontogenesis G. lutea are known to grow slowly (2−5 cm/ per year), forming 5−8 pairs of leaves [5]. The accretion of plants height from 30th to 60th day compared with the same value from 1st to 30th day was almost 3 times bigger that proves the successful adaptation. From 1st to 30th day the number of leaves was observed to increase, and from the 30th to 60th day — to decrease (Table 4). Probably the lack of rains in the first half of August resulted in drying out lower pairs of leaves.
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Table 3. Morphometric parameters of G. lutea planted in soil
Morphometric parameters of plants rooted in tap water
Parameters at the time of planting 30th day 60th day 90th day 150th day Total accretion
PH, mm X± Sx 29±3.2 34±2.5 42±3.4 44±4.0 53±6.2 —
Accretion — 5±0. 4* 8±0. 6* 2±0. 2* 9±0. 6* 24±0. 7
NPL, pieces X± Sx 7. 8±0. 77 9. 3±0. 82 10. 1±0. 75 10. 3±0. 76 12. 0±0. 89 —
Accretion — 1. 5±0. 2* 0. 8±0.1 0. 2±0.1 1. 7±0. 2* 4. 2±0. 3
NI, pieces X± Sx 3. 1±0. 35 4. 2±0. 33 4. 3±0. 33 4. 5±0. 43 4. 5±0. 37 —
Accretion — 1. 1±0.1 0. 1±0. 04 0. 2±0. 05 0 1. 4±0. 2
Morphometric parameters of plants rooted in the nutrient medium1
PH, mm X± Sx 43±4.3 51±6.4 56±7.9 57±8.1 69±6.8 —
Accretion — 8±0.5 4±0.4 1±0.2 12±0.7 25±0. 8
NPL, pieces X± Sx 8. 8±0. 73 9. 5±0. 80 10. 2±0. 84 10. 5±1. 10 10. 8±1. 16
Accretion — 0. 7±0.1 0. 7±0.1 0. 3±0. 05 0. 3±0. 05 2. 0±0. 2
NI, pieces X± Sx 3. 9±0. 83 4. 7±0. 99 5. 1±0. 96 5. 3±0. 30 5. 3±0. 30 —
Accretion — 0. 8±0.1 0. 4±0. 09 0. 2±0. 06 0 1. 4±0. 2
Notes: PH — plant height, NPL — number of pairs of leaves, NI — number of internodes- 1 — the medium MS/2 with twice decreased concentration of NH4NO3 without vitamins and sucrose, supplemented with 3 g/l mannite and 0. 05 mg/l Kin, the maintaining substrate is agar (4 g/l) combined with perlite (16 g/l) —
* - marked variants of the experiment in which the morphometric parameters of plants rooted in the nutrient medium MS/2 with twice decreased concentration of NH4NO3 without vitamins and sucrose, supplemented with 3 g/l mannite and 0. 05 mg/l Kin reliable (Р & lt- 0. 05) differ from the parameters of plants rooted in tap water.
Fig. 4. G. lutea planted in natural conditions: on 3rd (A), 30th (B), 60th © days of vegetation and a year after planting (D)
Within 60 days' period of G. lutea growing in natural conditions the length and width of lamina increased 7.8 and 2.9 times respectively. The formation of internodes stopped, and on the 60th day sporadic individuals had 1−2 internodes left, that proves that the plants acquired characteristic for the species habitus [18].
Thus, the conditions for rooting G. lutea shoots obtained by microclonal propagation have been chosen. It has been established that the biggest share of rooting shoots was provided by optimal combination of nutrient medium composition and supporting substrate. The medium MS/2 with twice decreased concentration of NH4NO3 without vitamins and sucrose, supplemented with 3 g/l mannite
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Table 4. Morphometric parameters of G. lutea planted in situ (Mt. Pozhyzhevska, 1450 m a.s.l.)
Parameters Time of measurement Increase of morphometric parameters
1st day 30th day 60th day 1−30th day 30−60th day 60−90th day
Plant height, mm Х 41.4 42.9 47.2 1.5 4.3 5. 8
Sx 1.3 1.3 0. 35
Sx'- 1. 84 1. 63 1. 70
Min 20.0 25.0 30. 0
Max 70.0 75.0 75. 0
Number of internodes per plant, pieces Х 4. 19 2. 23 0.6 -2. 67 -1. 63 -4. 30
Sx 1. 12 0. 41 0. 02
Sx'- 1. 47 0. 58 0. 13
Min 1.0 0 0
Max 10.0 4.0 1. 0
Number of pairs of leaves per plant, pieces Х 8. 54 9. 16 8. 75 0. 62 -0. 41 0. 21
Sx 2. 49 2. 38 0. 71
Sx'- 2. 69 2. 64 2. 97
Min 4.0 6.0 4. 0
Max 13.0 16.0 14. 0
Length of lamina, mm Х 7.8 8.2 10.9 0.4 2.7 3. 1
Sx 0. 08 0.1 0. 13
Sx'- 0. 26 0. 35 0. 39
Min 5.0 5.0 7. 0
Max 12.0 15.0 20. 0
Width of lamina, mm Х 3.6 4.7 6.8 1.1 2.1 3. 2
Sx 0. 02 0. 04 0. 05
Sx'- 0. 09 0.1 0. 17
Min 1.5 3.0 5. 0
Max 4.5 7.0 10. 0
and 0. 05 mg/l Kin and agar (4 g/l) combined with perlite (16 g/l) as a maintaining substrate proved to be the most efficient among all tested variants. Quite effective was also gradual diminishing of carbohydrates from 10 g/l to 2 g/l in the nutrient medium (MS/2 without vitamins and decreased concentration of NH4NO3) and further rooting of shoots in tap water.
There are chosen conditions for adaptation of rooted in vitro plants to conditions ex vitro. It has been found that plants rooted on both mentioned above variants can be used for this purpose. The obtained initial results of repatriation of G. lutea plants (21%) in natural habitats testify to efficiency of the suggested technology and rationality of its use for revival of disturbed gentian populations.
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BIOTECHNOLOGIA ACTA, V. 8, No 6, 2015
АДАПТАЦ1Я ОДЕРЖАНИХ in vitro РОСЛИН Gentiana lutea L.
ДО УМОВ ex vitro ТА in situ
О. Ю. Майорова Л. Р. Грицак Н. М. Дробик
Тернотльський нащональний педагопчний ушверситет iMeHi Володимира Гнатюка, Укра1на
Е-mail: majorova@i. ua
Метою роботи було розробити технолопю перенесення одержаних шляхом мшрокло-нального розмноження рослин Gentiana lutea L. в умови in situ. Використовували методи куль-тивування рослинних об’екмв in vitro. ПЩбра-но оптимальнi умови для вкоршення отрима-них мiкроклональним розмноженням пагошв G. lutea in vitro: живильне середовище МС/2 з половинним вмятом NH4NO3 без вiтамiнiв та сахарози, доповнене 3 г/л машту та 0,05 мг/л кшетину, з використанням як шдтримуваль-ного субстрату агару (4 г/л) у поеднанш з пер-лiтом (16 г/л) або поетапне зменшення в се-редовишД МС/2 без вiтамiнiв та зi зменшеною концентрацiею NH4NO3 концентраци вуглево-дiв з 10 г/л до 2 г/л iз подальшим укоршенням цих пагонiв у водопровiднiй водь Укоршеш рослини адаптовано до умов ex vitro шляхом висаджування 1х у горщики з Грунтом та по-ступовим переведенням тепличного режиму до вщкритого. Частка адаптованих до умов in situ рослин — 21% через рш шсля висаджування — свщчить про перспектившсть розро-бленого способу культивування. Таким чином, запропоновано один з ефективних способiв вiдновлення ушкоджених популяцш G. lutea, що включае репатрiацiю в них укорiнених та адаптованих до умов ex vitro рослин, отрима-них мшроклональним розмноженням in vitro.
Ключовi слова: Gentiana lutea L., вкоршення in vitro, адаптащя ex vitro, репатрiацiя in situ.
АДАПТАЦИЯ ПОЛУЧЕННЫХ in vitro РАСТЕНИЙ Gentiana lutea L.
К УСЛОВИЯМ ex vitro И in situ
О. Ю. Майорова Л. Р. Грицак Н. М. Дробык
Тернопольский национальный педагогический университет имени Владимира Гнатюка, Украина
Е-mail: majorova@i. ua
Цель работы — разработка технологии перенесения полученных путем микроклонального размножения растений Gentiana lutea L. в условия in situ. Использовали методы культивирования растительных объектов in vitro. Подобраны оптимальные условия укоренения полученных микроклональным размножением побегов G. lutea in vitro: питательная среда МС/2 с половинным содержанием NH4NO3 без витаминов и сахарозы, дополненная 3 г/л маннита и 0,05 мг/л кинетина, с использованием в качестве поддерживающего субстрата агара (4 г/л) в сочетании с перлитом (16 г/л) либо поэтапное уменьшение в среде МС/2 без витаминов и с уменьшенной концентрацией NH4NO3 концентрации углеводов с 10 г/л до 2 г/л с последующим укоренением этих побегов в водопроводной воде. Укоренившиеся растения адаптировали к условиям ex vitro, высаживая их в горшки с почвой, и постепенным переводом тепличного режима в открытый. Количество адаптированных к условиям in situ растений — 21% через год после высаживания свидетельствует о перспективности разработанного способа культивирования. Таким образом, предложен один из эффективных способов восстановления поврежденных популяций G. lutea, состоящий в репатриации в природные места произрастания укорененных и адаптированных к условиям ex vitro растений, полученных микроклональным размножением in vitro.
Ключевые слова: Gentiana lutea L., укоренение in vitro, адаптация ex vitro, репатриация in situ.
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