Nowadays, the mass reproduction of ornamental plants using biotechnological methods holds a prominent place in the industrial floriculture in the leading countries of the world (Kushnir et al. 2005). This is due to a number of advantages of plants obtained in vitro compared to traditional methods: they are virus-free, genetically homogeneous and healthy. In addition, in vitro plant tissues are successfully used as a donor material to improve the existing varieties and to create new ones with specified quality parameters via cell and genetic engineering methods, multiplication of rare and endangered plants (George 1993; Sriskandarajah et al. 2007; Chornobrov et al. 2019).
Plants of
The authors investigated the regenerative capacity of Schlumbergera genus tissues in vitro, selected optimal conditions of cultivation, studied somatic embryogenesis, and have transferred the target genes into plant material using the Agrobacterium tumefaciens vector system (Amir 2001; Al-Ramamneh et al. 2006; Sriskandarajah et al. 2004, 2007; Deeksha Raj et al. 2020). In particular, Perez et al. (1999) cultured
Deeksha Raj et al. (2020) observed that direct regeneration was observed in MS media containing different concentrations of growth regulators BAP with constant 0.1 mg/l NAA. Early shoot proliferation was found in longitudinally sliced segment section and late response was found in transversely sliced segment section. The highest number of shoots was recorded in media containing 3 mg/l of BAP with constant 0.1 mg/l NAA.
The regenerative capacity of tissues in vitro is affected by a number of factors (physiological, genetic, hormonal and physical), which determines the need for careful selection of cultivation conditions and nutrient media components for microclonal propagation of each Schlumbergera genotype individually.
In our previous publications, we have described the particular microclonal propagation of S. truncata by direct morphogenesis (Chornobrov and Bilous 2016). The next stage of the study was obtaining callus culture, induction of indirect morphogenesis, initiation of shoots growth, their proliferation and multiplication in vitro and mass production of plants.
The purpose of the study was to determine regeneration peculiarities of indirect morphogenesis of S. truncata from segment section for microclonal propagation.
Phylloclade explant (S = 0 .5−0.8 cm2) isolated from
Obtained sprouts from callus were transferred on MS medium supplemented with growth regulators: 3 -in-dolyl butyric acid (1.0 mg/l IBA), 6-benzylaminopurine (0.5, 2.0 mg/l BA) and 6-furfurylaminopurine (0.25, 0.5 mg/l). kinetin). The medium was added 100 mg/l of myo-inositol, 30 g/l of sucrose and 7.0−7.3 g/l of microbiological agar were added to the modified nutrient media. 2.0 g/l of activated carbon was added to some variants of the nutrient medium. The acidity value of the medium (pH) was brought to the level of 5.7−5.9. The growth characteristics of plant sprouts capable of regenerating were determined on day 60 of in vitro cultivation.
The plant material was cultured in a light room and a thermostat (without illumination) at 25±1°C and illumination 2.0–3.0 klx with a 16-hour photoperiod and a relative air humidity of 70–75%. The hormone-free MS nutrient medium was used as a control. The studies were conducted over the period 2016–2018. The replication of experiments is 3–5 times. In the current study, the following biotechnological methods were used: plant tissue culture in vitro, microclonal reproduction and callus culture. MS Excel software package was used to process the experimental data, the mean and its standard error were calculated. To analyse the influence of the nutrient medium composition and the light regime on the frequency of callus formation, a two-way analysis of variance (ANOVA) with replication was performed. One-way analysis of variance was performed to analyse the influence of the nutrient composition on the micropropagation coefficient and the length of micro sprouts. In results, we used the following abbreviations: F – calculated F value, F1 – critical F value.
To neutralize the exogenous microbiota of phylloclade explant of
The intensity of dedifferentiation of S. truncata plant explants was highly dependent on the composition of the nutrient medium, in particular the concentration and the ratio of growth regulators of auxin and cytokinin modes of action, and cultivation conditions (Tab. 1).
The effect of growth regulators and conditions of illumination on the induction of callus formation in
Variants | The composition of the nutrient medium | Conditions of illumination | |
---|---|---|---|
Light room (2.0−3.0 klx) callus formation frequency (mean ± standard error), % | Thermostat (without light) callus formation frequency (mean ± standard error), % | ||
C | Control | 0 | 0 |
1 | MS + 2.0 mg/l 2.4-D | 0 | 33.3±8.8 |
2 | MS + 1.0 mg/l BA + 0.5 mg/l NAA | 90.0±5.8 | 93.3±3.3 |
Notes: 1C − control (hormone-free MS nutrient medium).
Thus, the cultivation of explants under illumination conditions on a nutrient medium with the addition of 2.0 mg/l of 2,4-D tissues becomes thicker and swelling at the notch sites without subsequent callus formation.
The use of a similar nutrient medium under the conditions of cultivation of plant material in a thermostat without illumination caused a rather low activity of callus formation in their minor fraction.
A rather high rate of callus formation of explants (over 90 %), followed by significant tissue growth, was observed under the conditions of cultivation on solid MS medium with the addition of 1.0 mg/l of BA and 0.3 mg/l of NAA (Fig. 1b).
According to the results of analysis of variance (ANOVA), it was found that the effect of the interaction of the lighting regime and the composition of the nutrient medium on the callus formation frequency is statistically significant at α = 0.05 (F > F1, F = 7.36, F1 = 5.32, p < 0.05; where F – calculated F value, F1 – critical F value). There is a statistically significant difference between the frequency of callus formation on different nutrient media α = 0.05 (F > F1, F = 184.09, F1 = 5.32, p < 0.05). There was also a statistically significant difference between the frequency of callus formation at different light conditions at α = 0.05 (F > F1, F = 11.00, F1 = 5.32, p < 0.05) (Tab. 1).
The culturing conditions of the explants, in particular the illumination mode, determined the texture of the tissue. When the illumination with intensity of 2.0–3.0 klx was used, a callus of dense consistency, of dark green pigmentation with light green foci was obtained (Fig. 1b); when the thermostat without illumination was used, the callus had loose consistency, dark yellow pigmentation, was hydrated, and disintegrated into separate fragments (Fig. 1c).
The morphometric parameters of aseptic sprouts of plants capable of regenerating after exposure to growth regulators are shown in Table 2.
Growth characteristics of
Variants | The composition of the nutrient medium | The length of one segment section of sprouts (mean ± standard error), cm | Plants multiplication factor (mean ± standard error) | Pigmentation | The presence of the root system |
---|---|---|---|---|---|
C | hormones free | 1.0±0.2 | 2.0±0.3 | light green | + |
1 | 2.0 mg/l BA | 0.5±0.1 | 10.0±0.7 | light green - | − |
2 | 0.5 mg/l kinetin BA + 0.5 mg/l | 1.2±0.2 | 8.8±0.6 | green | + |
3 | 1.0 mg/l IBA | 1.1±0.2 | 1.6±0.2 | green | + |
4 | 0.25 mg/l kinetin | 1.4±0.1 | 3.8±0.4 | green | + |
5 | activated 0.25 mg/carbon l kinetin adsorbent + 2 g/l | 1.5±0.1 | 4.2±0.4 | green | + |
Notes: C– control – medium without hormones; the presence of the root system «+» − present; «−» − absent)
Shoots of
The influence of composition of the nutrient medium is statistically significant at the 5% significance level on the multiplication factor of the explants (F > F1, F = 58.18, F1 = 2.62; p < 0.05) and on the sprout length (F > F1, F = 5.42, F1 = 2.62, p < 0.05). All the suggested modified nutrient media induced the development of the root system of formed sprouts, the exception was Variant № 1 (Tab. 2). Thus, in the case of use of 1.0 mg/l IBA, regeneration of the root system, starting from the periphery of the explants, was registered in more than 90% of the explants (Fig. 1D, E).
MS nutrient medium is basic, universal, balanced with mineral nutrients, so it is often used as a basis in studies with tissue culture in vitro. In our studies, as well as the authors (Sriskandarajah et al. 2004; Al-Ramamneh et al. 2006; Deeksha Raj et al. 2020), we used solid nutrient medium prepared according to MS formula for microclonal propagation of
In our studies, intense callus formation on phylloclade explant
It is known from the literature review that the intensive microclonal propagation of Schlumbergera plant explants occurs on a nutrient medium with the addition of cytokinins (Kinetin, BA, TDZ, Zeatin) (Perez et al. 1999; Sriskandarajah et al. 2004). In particular, in research by Sriskandarajah et al. (2004) active sprouts formation in cultivars Schlumbergera was observed on MS medium with addition of cytokinins (6.1 mg/l BA, 5.9 mg/l TDZ, 5.9 mg/l zeatin). According to our research, a rather active sprouts formation in explants
This experiment shows the natural (genetically determined) ability of