In vitro shoot culture and cryopreservation (CP) are techniques essential for the ex situ preservation of genetic resources and the production of plant propagation material of clonally propagated horticultural crops. Changes in plant-associated microbiota diversity and composition induced by in vitro cultivation and CP treatment could have a negative effect on the growth and ex vitro adaptation of the in vitro propagated shoots. Therefore, the aim of the present study was to assess changes in endophytic bacteria diversity in domestic apple tissues induced by in vitro cultivation and CP treatment and to investigate the potential of the bacterial inoculum to improve the rooting and ex vitro acclimatisation of the propagated shoots. Metataxonomic analysis revealed a variation in the endophytic bacteria diversity and taxonomic composition between the field-grown tree dormant bud and the in vitro propagated or CP-treated shoot samples of apple cv. Gala. Whereas Sphingobacteriaceae, Sphingomonadaceae, Pseudomonadaceae, and Beijerinckiaceae families were the most prevalent families in the bud samples, Enterobacteriaceae, Bacillaceae, and Lactobacillaceae were dominant in the in vitro shoots. The bacterial inoculum effect on rooting and ex vitro acclimatisation was assessed using four isolates selected by screening the endophytic isolate collection. Bacillus sp. L3.4, B. toyonensis Nt18, or a combined inoculum resulted in a 21%, 36%, and 59% increase in cumulative root length and a 41%, 46%, and 35% increase in the biomass accumulation of ex vitro acclimatised plantlets, respectively. Root zone microbiota functional diversity analysis implied that growth stimulation was not related to improved nutrient uptake but could involve a pathogen-suppressing effect. The results demonstrate that the application of plant growth-promoting bacteria can potentially improve the performance of the in vitro propagated germplasm.

The scientific research on strawberries (Fragaria spp.), blueberries and cranberries (Vaccinium spp.), and currants and gooseberries (Ribes spp.) have increased worldwide in the last decades. Most molecular studies on berries are focused on the immediate needs of plant breeding, including genetic diversity evaluation, fingerprinting of wild and cultivated genotypes, genetic mapping, and the development of tools for marker-assisted selection. Targeted breeding requires understanding the mechanisms of trait inheritance, plant genetics, and appropriate gene mining, the main directions of which are the development and application of molecular markers, identification of important trait-encoding genes, genetic mapping, and transcriptome analysis. Different ploidy levels and heterozygosity in strawberry (Fragaria spp.) and blueberry (Vaccinium spp.) pose a challenge for breeders in the development of new cultivars with unique genetic traits (e.g., disease resistance, flowering time, fruit aroma quality, male/female sterility). The uniqueness of Ribes spp. is the lack of crossbreeding barriers, which resulted in the most modern blackcurrant cultivars being complex multi-stage interspecific hybrids, which makes the identification of species composing the cultivar relatively difficult and, in some cases, complicates research and breeding of Ribes spp. Therefore, DNA-based molecular markers are reliable and powerful tools for identifying different cultivars in berries and assessing genetic diversity among genotypes quickly enough without environmental influences. The evaluation of molecular variation in a germplasm set through marker-assisted breeding is essential for the faster development of new cultivars. We present the achievements to date for each of these crops in allelic mining and suggest research areas that will be needed to solve over the coming decades to guide future efforts to improve the crops.

Abstract Background In vitro cultivation and cryopreservation techniques are essential tools for genetic diversity conservation and pathogen-free plant propagation of horticultural crops. The optimisation of cryopreservation protocols typically focuses on minimising the negative effects of pretreatment with cryoprotectors (CPs), cryogenic freezing (CF) treatment, and recovery procedures on explants. However, the impact of in vitro and CF techniques on plant-associated microbiota remains poorly understood, and their potential to improve plant adaptation after cryopreservation is underexplored. The aim of the present study was to investigate in vitro shoot culture and cryopreservation-induced changes in the endophytic bacterial diversity of two sweet cherry cultivars and to assess the potential of an inoculum of bacterial isolates to improve the growth of shoot culture after CF. Results Cultivars ‘Sunburst’ and ‘Mindaugė’ showed different responses to cold hardening preconditioning as well as different survival and regrowth rates after cryopreservation. Metataxonomic analysis revealed variation in the abundance and taxonomic composition of bacteria assigned to 35 families in samples of field-grown tree leaves, dormant buds, and in vitro shoot culture before and after CF treatment. Bacillaceae and Enterobacteriaceae bacteria were predominant in the leaf samples of both cultivars. For ‘Sunburst’, Pseudomonadaceae and Sphingomonadaceae bacteria were dominant in dormant buds and in vitro shoots, respectively, while Burkholderiaceae was largely predominant in the shoots following CF treatment. Conversely, ‘Mindaugė’ tissues exhibited more consistent colonisation by Bacillaceae and Enterobacteriaceae across the experimental groups, except for in vitro shoots where Mycobacteriaceae prevailed. A pure bacterial isolate inoculum was applied to the ‘Mindaugė’ shoot culture to counter the CF treatment-induced suppression of shoot growth (~ 40%). Cocultivation with Brevibacterium sp. S1-2, Bacillus cereus S1-3, or B. toyonensis Nt18 increased the shoot leaf area from 48 to 75%. Conclusions This study revealed that endophytic bacterial diversity is significantly reduced under in vitro conditions, often leading to a genotype-specific increase in the abundance and dominance of bacteria attributed to a single bacterial family. Moreover, shoot cocultivation with endophytic bacterial isolates has potential for improving the recovery of in vitro shoots after cryopreservation.

Cryopreservation features of Lithuanian garlic (Allium sativum L.) cultivars ‘Žiemiai’ and ‘Dangiai’ were investigated. The survival rate and state of explants, depending on the type of explant, and dehydration conditions were evaluated in the experiment. Explants isolated from cloves and bulbils were dehydrated with a plant vitrification solution 3 (PVS 3) containing 50% w/v glycerol and 50% w/v sucrose in liquid MS medium. Three different exposure durations of 1.5, 2, and 3 h in PVS3 solution were applied at 0 °C. Unloaded stem domes were planted in Petri dishes with MS medium supplemented with 1 mg L−1 naphthaleneacetic acid (NAA) and 6–dimethylallylamino purine (2-iP) on 0.8% plant agar. The obtained results showed that the cryopreservation in liquid nitrogen reduced the survival rate of explants by 20–40%. The average number of surviving explants after freezing reached 64.3% for both cultivars. Cultivar ‘Žiemiai’ showed a 24% higher capability for survival than ‘Dangiai’. The total number of surviving explants of ‘Žiemiai’ reached 76.1%. Explants from bulbils were 23% more effective for cryopreservation compared to cloves. Evaluation of the effect of dehydration duration showed that the survival rate of the explants from bulbils of both cultivars was highest after treatment with PVS3 for 3 h and reached 91%. Treatment in PVS3 solution for 2 h was sufficient for survival of explants from cloves. Obtained results confirm that the efficiency of garlic cryopreservation depends on complex factors.

ABSTRACT The goal of this research was to evaluate the effect of light, temperature, sucrose and PEG on the growth of Fragaria vesca in vitro and the expression of regulatory Myb10, WD40 and enzyme-coding genes CHI, CHS, DFR, EGL, F3H and UFGT , which are essential for anthocyanin biosynthesis. We observed plants’ response to osmotic stress, the decrease in growth and microshoot weight. A change in the expression of the investigated genes was evident under the suboptimal concentration of sucrose. The addition of PEG to the medium caused a decrease in microshoot weight and gene expression. Blue + red lights of the LED lighting system significantly affected microshoot growth in vitro . Red and blue + red + UV lights slightly reduced microshoot weight and caused a reddish colour of petioles, which indicate increased anthocyanin synthesis. Moreover, most of the studied genes’ expression tended to increase when shoots were exposed to blue, blue + red and blue + red + UV lights. A temperature of 15°C (vs 22°C) significantly reduced the mean fresh weight of microshoots while increasing CHI and CHS gene expression and decreasing WD40 gene expression. Exposure to a higher temperature (30°C) induced the vitrification of microshoots, although the fresh weight did not differ from that of the control. Gene expression also depended on the duration of exposure. In the case of CHS , gene expression remained the same or increased after exposure for 1 week and then decreased after exposure for 4 weeks.

Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial communities. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation; however, the effect of antibiotic treatment on the diversity of indigenous microbial populations and the consequences on the performance of tissue culture is not completely understood. Therefore, the aim of this study was to assess the effect of antibiotic treatment on the growth and stress level of tobacco ( Nicotiana tabacum L.) shoots in vitro as well as the composition of the plant-associated microbiome. The study revealed that shoot cultivation on a medium supplemented with 250 mg L −1 timentin resulted in 29 ± 4% reduced biomass accumulation and a 1.2–1.6-fold higher level of oxidative stress injury compared to the control samples. Moreover, the growth properties of shoots were only partially restored after transfer to a medium without the antibiotic. Microbiome analysis of the shoot samples using multivariable region-based 16S rRNA gene sequencing revealed a diverse microbial community in the control tobacco shoots, including 59 bacterial families; however, it was largely dominated by Mycobacteriaceae . Antibiotic treatment resulted in a decline in microbial diversity (the number of families was reduced 4.5-fold) and increased domination by the Mycobacteriaceae family. These results imply that the diversity of the plant-associated microbiome might represent a significant factor contributing to the efficient propagation of in vitro tissue culture.

Antibiotics are used in plant in vitro tissue culture to eliminate microbial contamination or for selection in genetic transformation. Antibiotic timentin has a relatively low cytotoxic effect on plant tissue culture; however, it could induce an enduring growth-inhibiting effect in tobacco in vitro shoot culture that persists after tissue transfer to a medium without antibiotic. The effect is associated with an increase in oxidative stress injury in plant tissues. In this study, we assessed changes of reactive oxygen species accumulation, protein expression, and oxidative protein modification response associated with enduring timentin treatment-induced growth suppression in tobacco (Nicotiana tabacum L.) in vitro shoot culture. The study revealed a gradual 1.7 and 1.9-fold increase in superoxide (O2•−) content at the later phase of the propagation cycle for treatment control (TC) and post-antibiotic treatment (PA) shoots; however, the O2•− accumulation pattern was different. For PA shoots, the increase in O2•− concentration occurred several days earlier, resulting in 1.2 to 1.4-fold higher O2•− concentration compared to TC during the period following the first week of cultivation. Although no protein expression differences were detectable between the TC and PA shoots by two-dimensional electrophoresis, the increase in O2•− concentration in PA shoots was associated with a 1.5-fold increase in protein carbonyl modification content after one week of cultivation, and protein carbonylation analysis revealed differential modification of 26 proteoforms involved in the biological processes of photosynthesis and glycolysis. The results imply that the timentin treatment-induced oxidative stress might be implicated in nontranslational cellular redox balance regulation, accelerates the development of senescence of the shoot culture, and contributes to the shoot growth-suppressing effect of antibiotic treatment.

Transgenic plants have a great potential for production of high-value molecules, including peptides, for medical or industrial applications. Cultivation of plant tissues in a contained environment, such as plant tissue culture in vitro , could effectively address safety issues associated with transgenic plants as well as strict manufacturing standards. However, unfavourable in vitro conditions can often lead to elevated stress levels and suppressed growth of in vitro tissues urging the need to improve plant adaptation to in vitro conditions. As the beneficial effect of endophytic bacteria on the growth of in vitro cultures has been described previously, the aim of the present study was to assess the effect of growth-promoting endophytic bacteria isolates of the Bacillus cereus group on the growth of transgenic tobacco shoots in vitro and the accumulation of recombinant protein (RP) construct comprising a green fluorescent protein and a collagen mimicking peptide conjugate. The research revealed that transgenic tobacco shoots had reduced growth and increased oxidative injury levels compared with the non-transgenic parental line. The inoculated bacteria isolates colonised plant tissues and were sustained at densities of 0.6–9.0 × 10 5 CFU g -1 of the shoot fresh weight for at least several passages. Enhanced, 18% to 30% higher, biomass accumulation compared to the uninoculated transgenic tobacco shoots, and 14% to 25% lower levels of the oxidative lipid injury marker malondialdehyde (MDA) were detected for three-week-old shoots co-cultivated with isolates of Bacillus mycoides Nt.10.1, B. toyonensis Nt.18 and B. wiedmannii Nt.3.2. B. wiedmannii isolate Nt.14.2 reduced oxidative stress injury symptoms but had no effect on shoot growth. Co-cultivation with endophytic isolates had no effect on the expression of the recombinant protein. These results suggest that co-cultivation with growth-promoting endophytic bacteria provides a viable solution to improve the growth of transgenic plant tissues used for recombinant protein production in vitro .

Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial community. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation, however, the effect of antibiotic treatment on the diversity of indigenous microbial population and consequences for performance of the tissue culture are not completely understood. Therefore, the aim of this study was to assess the effect of antibiotic treatment on the growth and stress level of tobacco ( Nicotiana tabacum L.) in vitro shoots as well as the composition of plant-associated microbiome. The study revealed that shoot cultivation on medium supplemented with 250 mg L −1 timentin resulted in 29 ± 4% reduced biomass accumulation and 1.2–1.6 fold higher level of oxidative stress injury compared to control. Moreover, the growth properties of shoots were only partially restored after transfer to medium without antibiotic. Shoot microbiome analysis using multi-variable region-based 16S rRNA sequencing revealed abundant microbial community in the control tobacco shoots, including bacteria from 59 families, however, it was largely dominated by Mycobacteriaceae . The antibiotic treatment resulted in a decline of species richness (the number of families was reduced 4.5-fold) and increased domination by the Mycobacteriaceae family. The results imply that the diversity of plant-associated microbiome might represent a significant factor contributing to the efficient propagation of in vitro tissue culture.

In vitro plant tissue cultures face various unfavorable conditions, such as mechanical damage, osmotic shock, and phytohormone imbalance, which can be detrimental to culture viability, growth efficiency, and genetic stability. Recent studies have revealed a presence of diverse endophytic bacteria, suggesting that engineering of the endophytic microbiome of in vitro plant tissues has the potential to improve their acclimatization and growth. Therefore, the aim of this study was to identify cultivated tobacco (Nicotiana tabacum L.) endophytic bacteria isolates that are capable of promoting the biomass accumulation of in vitro tobacco shoots. Forty-five endophytic bacteria isolates were obtained from greenhouse-grown tobacco plant leaves and were assigned to seven Bacillus spp. and one Pseudomonas sp. based on 16S rRNA or genome sequence data. To evaluate the bacterial effect on in vitro plant growth, tobacco shoots were inoculated with 22 isolates selected from distinct taxonomic groups. Four isolates of Bacillus cereus group species B. toyonensis, B. wiedmannii and B. mycoides promoted shoot growth by 11–21%. Furthermore, a contrasting effect on shoot growth was found among several isolates of the same species, suggesting the presence of strain-specific interaction with the plant host. Comparative analysis of genome assemblies was performed on the two closely related B. toyonensis isolates with contrasting plant growth-modulating properties. This revealed distinct structures of the genomic regions, including a putative enzyme cluster involved in the biosynthesis of linear azol(in)e-containing peptides and polysaccharides. However, the function of these clusters and their significance in plant-promoting activity remains elusive, and the observed contrasting effects on shoot growth are more likely to result from genomic sequence variations leading to differences in metabolic or gene expression activity. The Bacillus spp. isolates with shoot-growth-promoting properties have a potential application in improving the growth of plant tissue cultures in vitro.

Plants exposed to drought stress conditions often increase the synthesis of anthocyanins—natural plant pigments and antioxidants. However, water deficit (WD) often causes significant yield loss. The aim of our study was to evaluate the productivity as well as the anthocyanin content and composition of berries from cultivated Fragaria vesca “Rojan” and hybrid No. 17 plants (seedlings) grown under WD. The plants were grown in an unheated greenhouse and fully irrigated (control) or irrigated at 50% and 25%. The number of berries per plant and the berry weight were evaluated every 4 days. The anthocyanin content and composition of berries were evaluated with the same periodicity using HPLC. The effect of WD on the yield parameters of two evaluated F. vesca genotypes differed depending on the harvest time. The cumulative yield of plants under WD was not less than that of the control plants for 20–24 days after the start of the experiment. Additionally, berries accumulated 36–56% (1.5–2.3 times, depending on the harvest time) more anthocyanins compared with fully irrigated plants. Our data show that slight or moderate WD at a stable air temperature of about 20 °C positively affected the biosynthesis of anthocyanins and the yield of F. vesca berries.

The heavy blooming of apple trees results in the inefficient usage of energy and nutritional material, and additional expenditure on fruitlet thinning is required to maintain fruit quality. A possible solution for controlling the fruit load on trees is the development of new cultivars that self-eliminate excess fruitlets, thus controlling yield. The aim of our study was to identify biological differences in apple cultivars in terms of blooming intensity and fruitlet load self-regulation. In total, 19 apple cultivars were studied in the years 2015–2017. The dynamics of fruitlet self-elimination, seed development in fruitlets and fruits, photosynthetic parameters, carbohydrates, and plant hormones were evaluated. We established that apple cultivars self-eliminating a small number of fruitlets need a lower number of well-developed seeds in fruit, and their number of leaves and area per fruit on a bearing branch are larger, compared to cultivars, self-eliminating large numbers of fruitlets. A higher carbohydrate amount in the leaves may be related to smaller fruitlet self-elimination. The amount of auxin and a high indole-3-acetic acid/zeatin ratio between leaves of cultivar groups with heavy blooming were higher than in cultivars with moderate blooming. A lower amount of abscisic acid was found in heavy-blooming cultivars during drought stress. All these parameters may be used as markers for the selection of different apple genotypes that self-eliminate fruitlets.

Horticultural crops of the Ribes genus are valued for their anthocyanin-rich fruits, but until now, there were no data about the genes and regulation of their flavonoid pathway. In this study, the coding sequences of flavonoid pathway enzymes and their putative regulators MYB10, bHLH3 and WD40 were isolated, and their expression analyzed in fruits with varying anthocyanin levels from different cultivars of four species belonging to the Ribes genus. Transcription levels of anthocyanin synthesis enzymes and the regulatory gene RrMYB10 correlated with fruit coloration and anthocyanin quantities of different Ribes cultivars. Regulatory genes were tested for the ability to modulate anthocyanin biosynthesis during transient expression in the leaves of two Nicotiana species and to activate Prunus avium promoters of late anthocyanin biosynthesis genes in N. tabacum. Functional tests showed a strong capability of RrMyb10 to induce anthocyanin synthesis in a heterologous system, even without the concurrent expression of any heterologous bHLH, whereas RrbHLH3 enhanced MYB-induced anthocyanin synthesis. Data obtained in this work facilitate further analysis of the anthocyanin synthesis pathway in key Ribes species, and potent anthocyanin inducer RrMyb10 can be used to manipulate anthocyanin expression in heterologous systems.

Blackcurrant (Ribes nigrum L.) is commercially important species in Northern Europe and New Zealand. The blackcurrant is rich in phenolic compounds, mainly anthocyanins, which are responsible for antioxidant activity of blackcurrant berries. Effect of interspecific hybridization on anthocyanin amount and composition in berries of Ribes hybrids is unknown. Our aim was to evaluate the possibility of interspecific hybridisation in Ribes genus in order to increase amount of anthocyanins and to improve their composition, and also to establish cross combinations for breeding blackcurrant cultivars with higher amount of stable anthocyanins and high antioxidant activity. Interspecific Ribes hybrids with similar biological traits to blackcurrant were selected from a pool of 30-60 plants, anthocyanin amount and composition in berries was established. Intersectional cross combinations of R. nigrum and R. sanguineum (Calobotrya section) or R. aureum (Symphlocalyx section) enable creating hybrids with larger total anthocyanin amount and larger proportion of cyanidins compared to blackcurrant. Breeding of blackcurrant with redcurrant (from Rybesia section) allows increasing total anthocyanin amount in berries and does not alter anthocyanin composition.

Interactions between host plants and endophytic microorganisms play an important role in plant responses to pathogens and environmental stresses and have potential applications for plant stress management under in vitro conditions. We assessed the effect of endophytic bacteria on the growth and proliferation of domestic apple cv. Gala shoots in vitro. Further, a model apple cell suspension system was used to examine molecular events and protein expression patterns at an early stage of plant-endophyte interaction. Among the seven strains used in the study, Bacillus spp. strains Da_1, Da_4, and Da_5 and the Pseudomonas fluorescens strain Ga_1 promoted shoot growth and auxiliary shoot proliferation. In contrast, Bacillus sp. strain Oa_4, P. fluorescens strain Ga_3 and P. orientalis strain G_12 inhibited shoot development. In the cell suspension, the effects of the association between endophytic bacteria and plant cells were specific to each strain. Modulation of the cellular redox balance was monitored in the apple cells using a 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) probe, and strain-specific effects were observed that correlated with the in vitro shoot development results. Proteomic analysis revealed differences in protein expressions in apple cells co-cultivated with different Bacillus spp. strains that had contrasting effects on cellular redox balance and shoot development. The Bacillus sp. strain Da_4, which enhanced shoot development and oxidation of H2DCFDA, induced differential expression of proteins that are mainly involved in the defense response and regulation of oxidative stress. Meanwhile, treatment with Bacillus sp. strain Oa_4 led to strong upregulation of PLAT1, HSC70-1 and several other proteins involved in protein metabolism and cell development. Taken together, the results suggest that different cell signaling and response events at the early stage of the plant-endophyte interaction may be important for strain-dependent regulation of cellular redox balance and development of shoot phenotype.

Botrytis cinerea Pers.: Fr is an important strawberry disease-causing pathogen with a broad host range. Classical B. cinerea identification is complicated due to the lack of morphological polymorphism between species. The use of molecular tools helps to identify pathogens fast and accurately. This study aimed to determine Botrytis spp. isolates and evaluate the genetic diversity of grey mould population in Lithuania. During June-August of 2012-2014, 273 isolates were sampled from 12 different areas of Lithuania. All samples were isolated from infected fruits, and single-spore isolates were extracted. B. cinerea isolates were identified using B. cinerea species-specific primers Bc108(+)/Bc563(-). The polymerase chain reaction (PCR) showed two bands characteristic of two specific DNA fragments of B. cinerea - upper and lower band of 360 and 480 bp, respectively. These two bands reflect pathogen genotype differentiation and could be used for cryptic species detection. The cryptic species analysis revealed that resistant group I accounted for 16.95% and sensitive group II for 83.05% of the Lithuanian collection of B. cinerea isolates. The precise identification of the B. cinerea cryptic species is important for the species-specific fungicide resistance and aggressiveness. Four microsatellite markers used in this study revealed genetic diversity of B. cinerea. The 158 isolates were identified as B. cinerea. The most polymorphic microsatellite marker was BC6 (0.88) and the least polymorphic - BC7 (0.79). The isolates clustered into three genetic groups. The first group consisted of 45 strains, the second group of 15 and the third group of 4 isolates. Our data show genetic diversity within the Lithuanian population of B. cinerea. One of the management tools is recognition and identification of the pathogen which leads to optimal and efficient disease management.