Biofertlizer Lumbrical improves the growth and ex vitro acclimatization of micropropagated pear plants

In vitro micropropagation of plants is highly useful for obtaining large quantities of planting material with valuable economic qualities. However, plantlets grow in vitro in a specific environment and the adaptation after the transfer to ex vitro conditions is difficult. Therefore, the acclimatization is a key step, which mostly determines the success of micropropagation. The aim of this investigation was to study the effect of the biofertlizer Lumbrical on ex vitro acclimatization of micropropagated pear rootstock OHF 333 (Pyrus communis L.). Micropropagated and rooted plantlets were potted in peat and perlite (2:1) mixture with or without Lumbrical. They were grown in a growth chamber at a temperature of 22±2 °C and photoperiod of 16/8 hours supplied by cool-white fluorescent lamps (150 μmol m-2 s-1 Photosynthetic Photon Flux Density, PPFD). The plants were covered with transparent foil to maintain the high humidity, and ten days later, the humidity was gradually decreased. Biometric parameters, anatomic-morphological analyses, net photosynthetic rate and chlorophyll a fluorescence (JIP test) were measured 21 days after transplanting the plants to ex vitro conditions. The obtained results showed that the plants, acclimatized ex vitro in the substrate with Lumbrical, presented better growth (stem length, number of leaves, leaf area and fresh mass) and photosynthetic characteristics as compared to the control plants. This biostimulator could also be used to improve acclimatization in other woody species.


Introduction
In vitro micropropagation of plants is highly useful for obtaining large quantities of planting material with valuable economic qualities. However, woody plants are often recalcitrant to in vitro cultivation and this process is highly genotype dependent. Pear rootstocks and varieties are often difficult to cultivate in vitro, although some micropropagation protocols have been published (Chevreau et al., 1992;Bell, Reed, 2002;Nacheva et al., 2009;Reed et al., 2013). During in vitro cultivation, plantlets grow under specific conditions: in small tightly-closed vessels; with high air humidity, low gas exchange and, thus, a CO 2shortage during almost the whole photoperiod; ethylene production and relatively low light intensity; in a culture medium with a large concentration of sugar (Ziv, 1991). These special conditions result in the formation of plants with abnormal morphology, anatomy and physiology. During acclimatization, the adaptation of the plant to new environmental conditions is essential (Apóstolo et al., 2005;Ďurkovič et al., 2009). According to Brainerd and Fuchigami (1982), the low survival rate of plants when they are removed from in vitro culture is associated with poor stomatal functioning and excessive water loss. During ex vitro acclimatization, many changes can occur to the morphological and physiological state and photosynthesis due to differences in the environmental conditions. However, studies on this aspect of acclimatization are still limited (Shina et al., 2014). Natural light shading, antitranspirant treatment for reducing plant transpiration and application of different plant growth-promoting substances are often used to increase plant survival rate after transplanting. The benefits associated with inoculation of in vitro -raised plantlets with selected N 2 -fixing bacteria and/ or arbuscular mycorrhizae fungi (AM) have been reported in several horticultural, fruit, ornamental and forest species (Rai, 2001;Kapoor et al., 2008;Singh et al., 2012).
Vermicompost is an organic fertilizer that is a result of a bio-oxidative process of organic waste, which is done by earthworms and microorganisms, with significant effects in the improvement of the soil fertility, in the crop yield and in the contribution to the agro-ecological sustainability (Broz et al., 2016;Xu, Mou, 2016). Vermicompost is a rich source of nutrients and plant growth regulators that could increase plant production (Singh et al., 2008;Lim et al., 2015).
The vermicompost Lumbrical is a product of the activity of cultivating worms of Lumbricus rubellus. This red California worm excreta is extremely rich in humus, containing all the substances necessary for plants. A significant number of works have shown that Lumbrical improves the growth and yields of many crops in particular vegetables: lettuce (Steffen et al., 2010), tomatoes (Gutiérrez -Miceli et al., 2007;Masheva et al., 2009), potatoes (Mrinal-Saikia et al., 1998, watermelon (Pelizza et al., 2013), as well as ornamentals, such as petunia, gladiolus (da Cruz et al., 2018), etc. It was shown that vermicompost is a suitable planting substrate for hardening of in vitro regenerated plants of Tylophora indica (Rani, Rana, 2010;Kaur et al., 2011) and banana (Fernández et al., 2016) to their field transfer.
The aim of this research was to study the effect of biofertilizer Lumbrical on ex vitro acclimatization of micropropagated pear rootstock OHF 333 (Pyrus communis L.).

Plant material and experimental conditions
The experiment was carried out on micropropagated plantlets of pear rootstock OHF 333 (Pyrus communis L. 'Old Home' x 'Farmingdale'). Well-rooted plantlets were potted in plastic form pads (528 x 308 x 60 mm), filled with peat-perlite (1:1, v/v) -control or with the same substrate enriched with the organic fertilizer Lumbrical (1:16,v:v). At the beginning of the experiment, the plants were fully covered with transparent polyethylene to prevent drying and ten days later, the humidity was gradually reduced. The plants were kept in a growth chamber at 22±2°C under 16/8 h light/ dark photoperiod (150 μmol m −2 s −1 Photosynthetic Photon Flux Density, PPFD). After 20 days in ex vitro conditions, plant growth analysis, physiological and biochemical analysis were performed.

Chemical analysis on the peat-perlite substrate and biofertilizer
Before the experiment, chemical analysis was performed both on the peat-perlite substrate and on the biofertilizer.
The analyses were performed according to established methods, described by Tomov et al. (1999). The total nitrogen was determined titrometrically, after burning in sulphuric acid and subsequent distillation on a Parnas-Wagner apparatus. The total content of phosphorus was determined colourimetrically using the method of Egner-Riehm. The total amount of potassium was determined in a hydrochloric acid extract (2 N HCl) using the modified method of Milcheva (Tomov et al., 1999) and the measurement was made using a flame photometer.
On the 20 th day after the transfer to ex vitro conditions, the content of the mineral elements (N, P, K) in the leaves of the plants was recorded.

Growth parameters
The fresh weight (FW) and leaf area were determined immediately after removing the plants from the soil. The dry weight (DW) of the plants was measured after drying the material at 80° С for 48 h (Beadle, 1993).

Physiological and biochemical parameters Gas-exchange analysis
The gas-exchange analysis was performed on the youngest fully-developed leaves of five randomly selected plants of the control and plants treated with Lumbrical. Measurements were taken with LCpro + portable photosynthesis system (ADC, UK) at a light intensity of 180 µmol m -2 s -1 PPFD and a temperature of 25 °C. Net photosynthesis rate (A, µmol CO 2 m -2 s -1 ), transpiration intensity (E, mmol H 2 O m -2 s -1 ) and stomatal conductivity (g s , mol H 2 O m -2 s -1 ) were determined.

Photosynthetic pigments
The photosynthetic pigments (chlorophyll a, chlorophyll b and total carotenoids) were extracted in 85% acetone. The extracts absorbance was determined spectrophotometrically. The content of pigments (mg g -1 FW) was calculated according to the formulae of Lichtenthaler, Wellburn (1983).

Chlorophyll a fluorescence
Chlorophyll a fluorescence analysis was performed using a Handy PEA fluorimeter (Handy Plant Efficiency Analyzer, Hansatech Instruments Ltd., UK) on the youngest native fullydeveloped leaves of five representative plants of the respective variant. The measured spots of the leaves were kept in darkness in a special clip for 40 minutes just before measurement. Induction curves of rapid chlorophyll a fluorescence (JIP test) were recorded for 1 s with 3000 µmol m -2 s -1 PPFD. The primary data processing was done using the PEA Plus Software (V1.10, Hansatech Instruments Ltd., UK). The parameters measured and calculated from this test (Table 1) were interpreted and normalised according to Strasser, Strasser (1995) and Goltsev (2016).

Anatomical and morphological analysis of the stomata
The anatomical and morphological analysis of the stomata was performed with a scanning electron microscope (SEM -FEI Quanta 200) at the Dendrology Laboratory in Przelewice, Poland. Samples for analysing the anatomical structure were taken from the middle part of fully developed 1 st to 4 th leaves from the top to the base of the shoots. The measurements were performed on fresh plant material with out pre-treatment. The plant material was placed on an aluminium holder in a SEM chamber for measuring at magnification (1000x). For each sample, ten measurements of the stomata were made.

Statistical analysis
For each experimental treatment, three replications, each containing 40 plants, were tested. The experiment was performed twice. For growth parameters, ten representative plants were studied. For gas-exchange and chlorophyll a fluorescence analysis, at least five measurements on different plants were performed.
Data of different parameters were analysed statistically using one-way ANOVA in SPSS statistical software (version 13 for Windows) at a significance level between the means and the evaluated of P≤ 0.05 (Tukey test).

Results and discussion
The substrate used for the experiment had low to medium nitrogen content, medium phosphorus content and a high content of potassium (Tomov et al., 1999; Table 2). Unlike the control substrate, Lumbrical biofertilizer contains a sufficient amount of mineral compounds necessary for plant growth and development.  Strasser, Strasser (1995) and Goltsev (2016).

Chlorophyll Fluorescence Parameter Description
Measured parameters and basic JIP-test parameters derived from the OJIP transient Relative variable fluorescence at the J-step Quantum yields and probabilities ψ EO = 1 -V J Probability (at t = 0) that a trapped exciton moves an electron into the electron transport chain beyond QA - Quantum yield (at t = 0) for electron transport from QAto plastoquinone Efficiency/ probability (at t = 0) with which an electron from the intersystem carriers moves to reduce end electron acceptors at the PSI acceptor side

Performance indexes
PI ABS Performance index of PSII based on absorption PI total = PI ABS x δRo/(1 -δRo) Performance index of electron flux to the final PSI electron acceptors, i.e., of both PSII and PSI We recorded that the pear plants acclimatized ex vitro with the Lumbrical biofertilizer were better development than the control plants (Table 3, Fig. 1). The plants acclimatized to ex vitro condition on the substrate with biofertilizer were distinguished by their higher stem length, the number of leaves, leaf area, fresh and dry biomass.
The elements nitrogen, phosphorus and potassium in the plants, grown on substrate enriched with the Lumbrical biofertilizer, were higher than in the control plants (Table  4). The values were close to the optimal ones, which in pears according to Hanson (1993) are 1.8-2.5 % (on a dry mass basis) for nitrogen, 0.12-0.3 % for phosphorus, and 1.0 -2.0 % for potassium. Other authors (Sainz et al., 1998;Tejada et al., 2007) have found similar results with vermicomposting.
The results of the gas-exchange analysis fully confirmed the data from the anatomical and morphological analysis ( Table 5). The net photosynthetic rate of plants grown in a substrate with a biofertilizer was about 28% higher compared to control plants. No significant differences were found in the transpiration and stomatal conductance of the two studied groups of plants.
Although there was a tendency for a higher content of photosynthetic pigments in the plants grown with Lumbrical, the difference with the control plants was not statistically significant (Table 6).
Chlorophyll a fluorescence is another indicator of the functional activity of the photosynthetic apparatus of plants and along with the intensity of the photosynthesis. The    analysis of the induction curves of rapid chlorophyll fl uorescence (OJIP test) links the structure and functionality of the photosynthetic apparatus. It allows for rapid assessment of plant viability, especially in stress conditions (Strasser et al., 2000(Strasser et al., , 2004. In the two studied variants, the rapid chlorophyll fl uorescence curves had a typical OJIP shape from F 0 to F M level with clearly separated J and I phases (Fig. 2), indicating that the pear plants, acclimatized to ex vitro conditions, were photosynthetically active (Yusuf et al., 2010). In both studied groups of plants, the maximal (F M ) fl uorescence of two studied variants was not signifi cantly diff erent (Table 7).
Although the quantum yield (Yield = F v / F M ) of plants acclimatized with Lumbrical, that refl ected the potential photochemical activity of photosystem II (PS II), was signifi cantly higher than that of control plants (0.814 and 0.772, respectively), these values corresponded to normal values (0.750-0.830) for healthy, unstressed leaves (Bolhar-Nordenkampf, Oquist, 1993). Th is indicated that a normally-developed photosynthetic apparatus was functioning. However, a more in-depth analysis of the JIP test parameters revealed some characteristic features of the potential of the photosynthetic apparatus in plants, acclimatized with Lumbrical and in the control plants. Th e Fv / Fo ratio (Table  7.) was lower in control plants (3.38) than in those acclimatized with the Lumbrical biostimulator (4.386). According to Strasser et al. (2010), the Fv / Fo ratio refl ects the effi ciency of excitation energy use in PS II. Moreover, the parameter ψ EO refl ects the probability of electron transport outside QA. Plants acclimatized ex vitro with soil enriched with Lumbrical were characterised by higher ψ EO as compared to the control plants. Th e performance index represents an absorbance basis and is used to quantify the PS II behaviour and shows the functional activity of the PS II relative to the absorbed energy (Kalaji et al., 2014a). Two times higher PI ABS was found in plants, grown with Lumbrical (1.913) in comparison to the control plants (0.851), which unequivocally showed that in these plants a better structured photosynthetic apparatus functioned.    The total performance index (PI total ) reflects the functional activity of the PS II, PS I and the electron transport chain between them (Strasser et al., 2000). PI total is closely related to the overall growth and survival rate of plants under stress conditions and has been described as a very sensitive parameter for the JIP test (Strasser et al., 2004). The decrease in the values of PI ABS and PI total observed in the control plants (C) compared to the plants cultivated with Lumbrical corresponded to the lower FW, DW, stem length, leaf area and P N of the control plants and could be indicative of the negative effects of culture conditions on PSII and PSI activity (Yusuf et al., 2010). The acclimatization of pear plants in soil enriched with the biofertilizer Lumbrical contributed to the more active development and structuring of the photosynthetic apparatus, which is a prerequisite for more intensive photoassimilation and biomass accumulation ( Fig. 1 and Tables 3, 5). These results also indicated that chlorophyll fluorescence parameters could be a reliable non-destructive method for early diagnosis of disorders in PS II functionality and growth. Martins et al. (2015) studied these parameters of JIP test to improve growth and acclimatization of micropropagated Neoregelia concentrica under different day light regimes.
The anatomical and morphological analysis revealed a significantly higher density of the stomata (per mm 2 ) in plants grown on the enriched substrate: 184.9 per mm 2 , which is higher than the control (106.0 per mm 2 ) by 74.4 % (Table 8 and Fig. 3). At the same time, the stomata length of the control plants was greater (25.9 µm) compared to that of the plants treated with the Lumbrical biofertilizer (20.9 µm). The greater number but smaller stomata in the plants grown in the substrate with Lumbrical may be a prerequisite for a more intensive gas exchange.
There is a specific course of water vapour diffusion through very small openings, such as the stomata. According to Stefan's law (Kerin et al., 2011), the amount of water vapour that diffuses through small holes within a definite period of time is proportional to the perimeter instead of the total pore area. Alternatively, with a greater number of smaller pores (which is observed in plants grown on soils with Lumbrical) the diffusion will be more intense, as the relative share of the total length of the boundary peripheries increases and therefore the evaporation is more intense.
During the acclimatization to ex vitro conditions, plants are forced to switch to autotrophic carbon assimilation. For this reason, the adaptation of the plant to new environmental conditions is essential. Because of that, the acclimatization is a key stage in micropropagation (Apóstolo et al., 2005;Ďurkovič et al., 2009). The transfer of in vitro grown plantlets to ex vitro conditions is often accompanied by water stress and/ or photoinhibition (Semorádová et al., 2002;Carvalho et al., 2001Carvalho et al., , 2006. Therefore, the application of approaches to stimulate photosynthesis are extremely important for the success of acclimatization. Vermicompost is a very useful growth medium for most crops because of the high content of many available nutrients and plant growth promoters (Arancon et al., 2004). According to Ricci et al. (1995), vermicompost provides P, Ca, Mg and S similarly to inorganic fertilizers. Additionally, several researchers have documented the presence of plant growth regulators such as auxins, gibberellins, cytokinins of microbial origin (Muscolo et al., 1999) and humic acids (Atiyeh et al., 2002;Arancon et al., 2004) in vermicompost in considerable quantities. Positive effects of vermicompost have been reported for many crops (Arancon et al., 2004;Gutierrez-Miceli et al., 2007;Berova et al., 2013) and also have been observed in forest species, such as pine trees (Lazcano et al., 2010). Possibly due to better physical properties, higher microbial and enzymatic activity and higher content of available nutrients, the vermicompost could be used as a natural fertilizer having a number of advantages over chemical fertilizers (Venugopal et al., 2010). Our results showed that Lumbrical improved the growth and photosynthetic ability of micropropagated pear plants during ex vitro acclimatization and they are similar to the findings obtained by other authors, e.g. to results reported by Vasane et al. (2010), concerning the increased survival of banana plants after their transfer to ex vitro in medium containing soil: PMC:vermicompost in a ratio of 1:1:1 (v/v/v). In Table 8. Anatomic-morphological analysis of pear plants on day 20 after transfer to ex vitro conditions. The first fully-developed leave was examined (4 th leaf from top to bottom)

Conclusions
In conclusion, environmental conditions during transfer to ex vitro conditions influenced the growth and acclimatization of in vitro propagated pear plants. The biofertilizer Lumbrical improved the vegetative growth, photosynthetic ability and ex vitro acclimatization of micropropagated pear plants. These results showed that the Lumbrical biofertilizer could be useful in acclimatizing other woody species and largescale commercial production of high-quality plants.