Distinctive development of embryo and endosperm caused by male gametes irradiated with carbon-ion beam.

KEY MESSAGE: In Cyrtanthus mackenii, development of embryo and endosperm were differentially affected by fertilization of male gametes with DNA damage and mutations. Pollen irradiation with ionizing radiations has been applied in plant breeding and genetic research, and haploid plant induction has mainly been performed by male inactivation with high-dose irradiation. However, the fertilization process of irradiated male gametes and the early development of embryo and endosperm have not received much attention. Heavy-ion beams, a type of radiation, have been widely applied as effective mutagens for plants and show a high mutation rate even at low-dose irradiation. In this study, we analyzed the effects of male gametes of Cyrtanthus mackenii irradiated with a carbon-ion beam at low doses on fertilization. In immature seeds derived from the pollination of irradiated pollen grains, two types of embryo sacs were observed: embryo sac with a normally developed embryo and endosperm and embryo sac with an egg cell or an undivided zygote and an endosperm. Abnormalities in chromosome segregation, such as chromosomal bridges, were observed only in the endosperm nuclei, irrespective of the presence or absence of embryogenesis. Therefore, in Cyrtanthus, embryogenesis is strongly affected by DNA damage or mutations in male gametes. Moreover, various DNA contents were detected in the embryo and endosperm nuclei, and endoreduplication may have occurred in the endosperm nuclei. As carbon-ion irradiation causes chromosomal rearrangements even at low doses, pollen irradiation can be an interesting tool for studying double fertilization and mutation heritability.

Characterization of a novel mutant with inhibition of storage root formation in sweet potato.

Sweet potato is a widely cultivated crop with storage roots. Although many studies have been conducted on the mechanism of its storage root formation, the details have not been fully elucidated. We screened mutant lines with inhibition of storage root formation to clarify parts of the mechanism. In this study, the process of storage root formation in one of the mutant lines, C20-8-1, was investigated. The inhibition of storage root formation was observed during the early stages of growth. The roots in C20-8-1 did not show histological differences compared to those in wild type. The transition from fibrous roots to pencil roots, which are the developmental stages prior to mature storage root formation, was delayed or inhibited in C20-8-1. The upregulation of starch biosynthesis-related genes and downregulation of lignin biosynthesis genes with storage root swelling were not confirmed in the root of C20-8-1 during the developmental transition stage, suggesting that most of the roots in C20-8-1 are in the pre-transition state toward the storage root swelling. C20-8-1 showed a mutant phenotype during the critical period of storage root swelling initiation, and further clarification of this mutation is expected to provide new insights into storage root formation.

Effect of Growth Stages on Anthocyanins and Polyphenols in the Root System of Sweet Potato.

The storage roots of purple-fleshed sweet potato contain a variety of anthocyanins and polyphenols. Little is known about changes in the total content and composition of anthocyanins and polyphenols in the early growth stages of the root system. In this study, we investigated the changes in anthocyanins and polyphenols in the root system of purple-fleshed sweet potato cultivars at 15, 30, 45, and 60 days after transplant (DAT). Unexpectedly, the highest percentage of acylated anthocyanins in three purple-fleshed cultivars among all growth stages was at 15 DAT. On the other hand, the total polyphenol content in the early growth stages of the root system increased rapidly toward 45 DAT, just before the beginning of storage root enlargement, and then decreased rapidly as the storage roots began to enlarge. These data indicate that the early growth stage of the root system is a critical time. This timing may present a strategy to maximize the accumulation of polyphenols with high antioxidant activity, as well as acylated anthocyanins, to protect against abiotic and biotic stresses.

Blueberry Stem Extract Prevents Lacrimal Hyposecretion in Non-obese Diabetic Mice via Activation of AMPK.

BACKGROUND/AIM: Tears secreted from the lacrimal gland are essential for preserving the ocular surface. Thus, dysfunction of the lacrimal gland in Sjögren's syndrome (SS) can lead to dry eye, resulting in a reduced quality of life. We previously reported that blueberry 'leaf' water extract prevents lacrimal hyposecretion in male non-obese diabetic (NOD) mice in a SS-like model. In this study, we investigated the effect of blueberry 'stem' water extract (BStEx) on lacrimal hyposecretion in NOD mice. MATERIALS AND METHODS: Male NOD mice were fed 1% BStEx or control (AIN-93G) for 2, 4, or 6 weeks from 4 weeks of age. Pilocarpine-induced tear secretion was measured using a phenol red-impregnated thread. The lacrimal glands were histologically evaluated by HE staining. Inflammatory cytokine levels in the lacrimal glands were measured using ELISA. Immunostaining was performed to examine aquaporin 5 (AQP5) localization. The expression levels of autophagy-related proteins, AQP5, and phosphorylated AMPK were measured using western blotting. RESULTS: After feeding BStEx to mice for 4 or 6 weeks, tear volume was observed to have increased in the BStEx group compared with that in the control group. There were no significant differences in inflammatory cell infiltration, autophagy-related protein expression, or the localization and expression of AQP5 in the lacrimal glands between the two groups. In contrast, AMPK phosphorylation increased in the BStEx group. CONCLUSION: BStEx prevented lacrimal hyposecretion in the SS-like model of male NOD mice, probably by opening tight junctions via the activation of AMPK in lacrimal acinar cells.

Comparison of Proanthocyanidin Content in Rabbiteye Blueberry (Vaccinium virgatum Aiton) Leaves and the Promotion of Apoptosis against HL-60 Promyelocytic Leukemia Cells Using 'Kunisato 35 Gou' Leaf Extract.

Polyphenol-rich rabbiteye blueberry (Vaccinium virgatum Aiton) leaves have attracted attention as a food material. In this study, we compared the total polyphenols, total proanthocyanidin content, and antioxidant activity of the leaves of 18 blueberry varieties and investigated the seasonal variation in polyphenols. We also evaluated the anti-cancer cell proliferation properties of the rabbiteye blueberry leaf specific cultivar 'Kunisato 35 Gou'. Rabbiteye blueberry leaves had significantly higher total polyphenol and total proanthocyanidin values than northern highbush blueberry and southern highbush blueberry leaves. The antioxidant activity of blueberry leaves was highly positively correlated with both the total polyphenol and total proanthocyanidin content. Variations were observed in the total polyphenol and total proanthocyanidin content of rabbiteye blueberry leaves harvested at different points in the growing season; leaves collected in fall to winter contained more epicatechin in addition to proanthocyanidins. In the evaluation of anti-cancer cell proliferation properties against HL-60 promyelocytic leukemia cells, the September-harvested extracts of rabbiteye blueberry 'Kunisato 35 Gou' showed strong properties, and the use of an FITC Annexin V apoptosis detection kit with propidium iodide confirmed that this HL-60 cell death occurred via apoptosis. Limiting the harvest time would make rabbiteye blueberry leaves a more functional food ingredient.

Blueberry stem extract and stem active components prevent blue light-emitting diode light-induced retinal photoreceptor cell damage in vitro.

Blue light causes retinal damage that can lead to ocular diseases such as age-related macular degeneration. In this study, we determined the protective effect of blueberry stem extract (BStEx) and active components on blue light-emitting diode (LED) light-induced retinal photoreceptor cell damage in vitro. Photoreceptor cells cultured in the presence of BStEx or components were exposed to blue light to induce cell damage. BStEx, fractions of BStEx containing proanthocyanidins, chlorogenic acid, catechin, and epicatechin prevented the cell damage and/or inhibited the generation of reactive oxygen species (ROS). Furthermore, BStEx reduced apoptosis and cell death, and inhibited the phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase leading to cellular apoptosis induced by blue light exposure. These findings suggest that BStEx and components exert a protective effect against blue light-induced photoreceptor cell damage through the inhibition of MAPK phosphorylation and ROS production.

Blueberry Leaf Extract Prevents Lacrimal Hyposecretion in Sjögren's Syndrome-like Model of Non-obese Diabetic Mice.

BACKGROUND/AIM: This study evaluated the effect of blueberry leaf hot water extract (BLEx) on Sjögren's syndrome (SS)-like lacrimal hyposecretion in male non-obese diabetic (NOD) mice. MATERIALS AND METHODS: NOD or BALB/c mice were fed 1% BLEx or control (AIN-93G) for 2 weeks from the age of 4 to 6 weeks. Pilocarpine-induced tear volume was measured using a phenol red-impregnated thread. The lacrimal glands were evaluated histologically by H&E staining. The IL-1ß and TNF-α levels in the lacrimal gland tissue were measured by ELISA. The mRNA expression levels of secretion-related proteins were measured by real-time PCR. LC3 I/II and arginase 1 expression levels were measured by western blot. RESULTS: After feeding with BLEx, pilocarpine-induced tear secretion in NOD mice was increased. In contrast, the mRNA expression levels of the cholinergic muscarinic M3 receptor, aquaporin 5, and ion channels related to lacrimal secretion were not changed by BLEx administration. In addition, the protein expression of arginase 1, which was recently reported to be involved in tear hyposecretion in NOD mice, was also not improved by BLEx administration. Although infiltration in the lacrimal gland of NOD mice was not decreased, the levels of TNF-α and the autophagy-related protein LC3 were significantly suppressed by BLEx treatment. CONCLUSION: BLEx treatment may ameliorate lacrimal hyposecretion in NOD mice by delaying the progression of autoimmune disease by suppressing autophagy in lacrimal glands.

Comprehensive effects of heavy-ion beam irradiation on sweet potato (Ipomoea batatas [L.] Lam.).

Sweet potato is a major root crop with nutritious tuberous roots. The mechanism of tuberous root development has not yet been adequately elucidated. Genetic resources are required to develop the molecular understanding of sweet potato. Heavy-ion beams were applied to hexaploid sweet potato for an increase in genetic variation, after which the comprehensive effects of heavy-ion beam irradiation were investigated. In vitro cultured shoots with an axillary bud of 'Beniharuka' were irradiated with Ar-ions at a dose of 1-5 Gy and C-ions at a dose of 5-20 Gy, and three irradiated lines were separated from each irradiated shoot. The shoot regeneration was inhibited at high doses of each ion irradiation. Ar-ion irradiation had an especially high biological effect on shoot regeneration. A total of 335 lines were obtained, consisting of 104 and 231 lines derived from Ar- and C-ion irradiation, respectively. The change in the DNA content of the lines was analyzed by flow cytometry to evaluate the irradiation-induced damage to the DNA. The two lines demonstrated significant differences in the DNA content and changes at the chromosome level. The screening for the morphological mutants was conducted in the field. Some irradiated lines showed inhibited or no tuberous root phenotype as mutant candidates. Additionally, the high-yield mutant candidates were dominated by Ar-ion irradiation. It was indicated that heavy-ion beam mutagenesis is effective in broadening the range of the phenotypes corresponding to tuberous root formation in hexaploid sweet potato.

Highly polymerized proanthocyanidins (PAC) components from blueberry leaf and stem significantly inhibit SARS-CoV-2 infection via inhibition of ACE2 and viral 3CLpro enzymes.

With the current worldwide pandemic of COVID-19, there is an urgent need to develop effective treatment and prevention methods against SARS-CoV-2 infection. We have previously reported that the proanthocyanidin (PAC) fraction in blueberry (BB) leaves has strong antiviral activity against hepatitis C virus (HCV) and human T-lymphocytic leukemia virus type 1 (HTLV-1). In this study, we used Kunisato 35 Gou (K35) derived from the rabbit eye blueberry (Vaccinium virgatum Aiton), which has a high PAC content in the leaves and stems. The mean of polymerization (mDP) of PAC in K35 was the highest of 7.88 in Fraction 8 (Fr8) from the stems and 12.28 of Fraction 7 (Fr7) in the leaves. The composition of BB-PAC in K35 is that most are B-type bonds with a small number of A-type bonds and cinchonain I as extension units. A strong antiviral effect was observed in Fr7, with a high polymerized PAC content in both the leaves and stems. Furthermore, when we examined the difference in the action of BB-PAC before and after SARS-CoV-2 infection, we found a stronger inhibitory effect in the pre-infection period. Moreover, BB-PAC Fr7 inhibited the activity of angiotensin II converting enzyme (ACE2), although no effect was observed in a neutralization test of pseudotyped SARS-CoV-2. The viral chymotrypsin-like cysteine protease (3CLpro) of SARS-CoV-2 was also inhibited by BB-PAC Fr7 in leaves and stems. These results indicate that BB-PAC has at least two different inhibitory effects, and that it is effective in suppressing SARS-CoV-2 infection regardless of the time of infection.

Prediction of the Adult T-Cell Leukemia Inhibitory Activity of Blueberry Leaves/Stems Using Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics.

Although Vaccinium virgatum Aiton leaves and stems inhibit adult T-cell leukemia (ATL) cells, leaves and stems can differ between individual plants and by time and location. In this study, leaf and stem components were profiled in the same individual plant using direct-injection electron ionization-mass spectrometry (DI-EI-MS) metabolomics, with the aims of analyzing the anti-ATL activity, and quantifying proanthocyanidins (PACs). Leaves, stems, and leaf/stem mixtures showed distinct and characteristic spectra. Anti-ATL activity was stronger in stems than leaves, and the PAC content was higher in stems than leaves. These data were subjected to bivariate analysis to identify the factor (m/z) responsible for the inhibitory effect of ATL based on the highest coefficient of determination (R2). The results of this DI-EI-MS metabolomics analysis suggest that among PACs contained in V. virgatum stems and leaves, the fragment ion at m/z 149 contributes significantly to anti-ATL activity.

Effective Utilization of Vaccinium virgatum Aiton Stems as Functional Materials: Major Constituent Analysis and Bioactivity Evaluation.

We previously reported that rabbit-eye blueberry (Vaccinium virgatum Aiton) leaves exhibit multiple functions. In this study, we evaluated whether V. virgatum stems can also be used as functional materials similar to leaves and clarified the major constituents and their biological activity (antioxidant activity and anti-adult T cell leukemia (ATL) activity). Water extracts of V. virgatum stems were separated into 19 fractions using a Diaion HP-20 open column. Sugars and organic acids were detected in the highly water-soluble fractions. Polyphenols and proanthocyanidin were detected in the hydrous methanol-soluble fractions. In biological activity evaluations, a difference in antioxidant activity was observed in the water-containing methanol-eluted fractions, and fractions exhibiting anti-ATL activity differed depending on cell type. These results suggest that blueberry stems, like leaves, are rich in polyphenols and exhibit antioxidant activity and inhibit ATL cell growth. In the future, aerial parts of blueberries, including stems and leaves, could be used as functional materials and/or medicinal resources.

Inhibition of adult T-cell leukemia cell proliferation by polymerized proanthocyanidin from blueberry leaves through JAK proteolysis.

We have previously reported that the proanthocyanidin (PAC) fraction of blueberry leaf extract (BB-PAC) inhibits the proliferation of HTLV-1-infected adult T-cell leukemia (ATL) by inducing apoptosis. In the present study, we further analyzed the structure of BB-PAC and elucidated the molecular mechanism underlying the inhibitory function of HTLV-1-infected and ATL cells. After hot water extraction with fractionation with methanol-acetone, BB-PAC was found to be concentrated in fractions 4 to 7 (Fr7). The strongest inhibition of ATL cell growth was observed with Fr7, which contained the highest BB-PAC polymerization degree of 14. The basic structure of BB-PAC is mainly B-type bonds, with A-type bonds (7.1%) and cinchonain I units as the terminal unit (6.1%). The molecular mechanism of cytotoxicity observed around Fr7 against ATL cells was the degradation of JAK1 to 3 and the dephosphorylation of STAT3/5, which occurs by proteasome-dependent proteolysis, confirming that PAC directly binds to heat shock protein 90 (HSP90). JAK degradation was caused by proteasome-dependent proteolysis, and we identified the direct binding of PAC to HSP90. In addition, the binding of cochaperone ATPase homolog 1 (AHA1) to HSP90, which is required for activation of the cofactor HSP90, was inhibited by BB-PAC treatment. Therefore, BB-PAC inhibited the formation of the HSP90/AHA1 complex and promoted the degradation of JAK protein due to HSP90 dysfunction. These results suggest that the highly polymerized PAC component from blueberry leaves has great potential as a preventive and therapeutic agent against HTLV-1-infected and ATL cells.

Vaccinium virgatum Aiton Leaves Extract Suppressed Lipid Accumulation and Uric Acid Production in 3T3-L1 Adipocytes.

Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.

Double Mutant Analysis with the Large Flower Mutant, ohbana1, to Explore the Regulatory Network Controlling the Flower and Seed Sizes in Arabidopsis thaliana.

Two growth processes, cell proliferation and expansion, determine plant species-specific organ sizes. A large flower mutant in Arabidopsis thaliana, ohbana1 (ohb1), was isolated from a mutant library. In the ohb1 flowers, post-mitotic cell expansion and endoreduplication of nuclear DNA were promoted. The whole-genome resequencing and genetic analysis results showed that the loss of function in MEDIATOR16 (MED16), a mediator complex subunit, was responsible for the large flower phenotypes exhibited by ohb1. A phenotypic analysis of the mutant alleles in MED16 and the double mutants created by crossing ohb1 with representative large flower mutants revealed that MED16 and MED25 share part of the negative petal size regulatory pathways. Furthermore, the double mutant analyses suggested that there were genetically independent pathways leading to cell size restrictions in the floral organs which were not related to the MED complex. Several double mutants also formed larger and heavier seeds than the wild type and single mutant plants, which indicated that MED16 was involved in seed size regulation. This study has revealed part of the size-regulatory network in flowers and seeds through analysis of the ohb1 mutant, and that the size-regulation pathways are partially different between floral organs and seeds.

Effects of Dietary Blueberry (Vaccinium ashei Reade) Leaves on Mildly Postprandial Hypertriglyceridemia.

Prevention of postprandial hypertriglyceridemia is an important consideration for reducing the risk of developing cardiovascular disease. While blueberry fruits have been reported to ameliorate lipid metabolism in humans, there are only few research reports on the effects of blueberry leaves (BL). Here, we investigated the efficacy of BL on postprandial hyperlipidemia in subjects with high fasting triacylglycerol (TG) concentrations. Randomized, double-blind, cross-over design study was conducted. The subjects consumed a BL containing beverage or a placebo beverage before a fat-enriched test meal. Blood samples were collected prior to and 1, 2, 3, 4, and 5 hours after consuming the test beverage. The postprandial serum TG and remnant-like particle cholesterol (RLP-C) concentrations were significantly lower in the BL beverage compared with those in the placebo beverage. Additionally, BL was more effective in subjects with high fasting ghrelin with gastric emptying function. In current study, fasting ghrelin correlated with the increase in postprandial serum TG, suggesting that BL ameliorates hypertriglyceridemia through delayed gastric emptying. In conclusion, this pilot study suggests that BL may be useful as an early dietary therapy for treating postprandial hyperlipidemia.

Blueberry Leaf Polyphenols Prevent Body Fat Accumulation in Mice Fed High-fat, High-sucrose Diet.

Blueberry leaf is currently a popular dietary supplement. Effects of dietary blueberry leaf and its active components on body fat accumulation were examined. C57BL/6J mice were fed high-fat, high-sucrose diet with or without 3% blueberry leaf extract (BLEx) or 3% concentrated-polyphenolic BLEx (CP BLEx) for 8 weeks. Compared to mice fed a high-fat, high-sucrose diet without blueberry leaf, BLEx and CP BLEx significantly reduced body weight and adipose tissue weight gain. Adipocytes were also smaller and and liver lipid accumulatioin was significantly inhibited in mice fed either BLEx or CP BLEx. These effects tended to be more pronounced in mice fed CP BLEx compared to in mice fed BLEx. Together, results suggest that blueberry leaf inhibits body fat accumulation typically observed in mice fed a high-fat, high-sucrose diet, and that inhibition is attributable to polyphenolic components in leaf extracts.

Subchronic toxicity evaluation of leaves from rabbiteye blueberry (Vaccinium virgatum Aiton) in rats.

Blueberry leaf may contain multiple compounds with beneficial effects. We conducted a 90-day toxicity study in rats to evaluate the safety of consuming the leaves of rabbiteye blueberry (Vaccinium virgatum Aiton; RB species). Powdered leaves were administered daily by oral gavage at doses of 500, 1000, and 2500 mg/kg body weight to male and female Sprague-Dawley rats for 90 days. Treatment did not result in death or changes in the behavior and external appearance of the animals. No alterations were observed in hematological and serum chemical parameters, urinalysis, food consumption, body weight gain, or absolute and relative organ weights at the end of the treatment period, with the exception of some leukocyte percentages in male rats treated with 500 and 1000 mg/kg blueberry leaf powder. The findings indicate that rabbiteye blueberry leaf is safe for consumption and should be investigated as a candidate functional food.

Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method for Analyzing Blueberry Leaf Metabolites That Inhibit Adult T-cell Leukemia Proliferation.

Metabolic profiling is often used to identify possible correlations between a compound's metabolic profile and biological activity. Direct-injection electron ionization-mass spectrometry "fingerprinting" is useful for characterizing biological materials. We demonstrate the utility of direct-injection electron ionization-mass spectrometry for metabolic profiling using 100 different extracts of leaves from 20 blueberry cultivars collected at 5 time points from April to December 2008. A qualitative direct-injection electron ionization-mass spectrometry method was used to profile the major and/or minor constituents in the blueberry leaf extracts. Blueberry leaf extracts could be distinguished by principal component analysis based on the absolute intensity of characteristic fragment ions. Twenty cultivars were categorized into four species, and the most appropriate discriminative marker m/z value for identifying each cultivar was selected statistically. Correlated m/z values indicating the collection month were determined in the same analysis, and air temperature variance factors were extracted from score plots by principal component analysis. We previously reported that blueberry extracts inhibit the proliferation of adult T-cell leukemia cells. Leaves of Vaccinium virgatum collected in December of 2008 exhibited significantly greater inhibition of adult T-cell leukemia cell proliferation than other species. Highly bioactive cultivars or species were identified by direct-injection electron ionization-mass spectrometry metabolomics analysis of blueberry leaf extracts. The components extracted based on our direct-injection electron ionization-mass spectrometry analyses could be used to construct a model to predict anti-adult T-cell leukemia bioactivity. This is the first study to report a relationship between seasonal variation and bioactivity of natural products using a direct-injection electron ionization-mass spectrometry metabolomics method.

Cryopreservation of blueberry shoot tips derived from in vitro and current shoots using D cryo-plate technique.

Cryopreservation is an important tool for long-term storage of plant germplasm that is currently used for plant germplasm storage at many institutes worldwide. Recently, novel cryogenic procedures (V and D cryo-plate methods) have been developed. In this study, the most suitable conditions for preserving blueberry shoot tips derived from in vitro and current shoots using the D cryo-plate method were investigated. The D cryo-plate method has advantages such as higher regrowth after cryopreservation and a more user-friendly process compared with conventional cryogenic methods. The optimum duration of desiccation for regrowth of shoot tips from each shoot type was 1 h. To induce dehydration tolerance for the shoot tips, the effects of two cryoprotection treatments (sucrose preculture and loading solution [LS] treatment) on shoot regrowth after cryopreservation were investigated. The combined effect of both treatments significantly increased percentage regrowth (approximately 90%). No regrowth of shoot tips was attained without the two treatments. Thus, preculture and LS treatment were effective to induce dehydration tolerance for cryopreservation of blueberry shoot tips. The optimized conditions for blueberry shoot tips using the D cryo-plate technique were: preculture with 0.3 M sucrose for 1 day, LS treatment (2 M glycerol +0.4-1.0 M sucrose) for 30 min, and air dehydration for 1 h. This optimized procedure was applied to additional blueberry cultivars shoot tips derived from in vitro shoots (regrowth 46.7-100%) and current shoots (regrowth 17.2-62.7%). Furthermore, in vitro shoot tips were suitable material for the D cryo-plate method in blueberry.