text:
GROWTH RESPONSE ON-FARM OF TWO JATROPHA CURCAS (L.) VARIETIES AS AFFECTED BY MYCORRHIZAE AND ORGANIC MANURE IN THE LOCALITY OF DANG-NGAOUNDERE (ADAMAWA-CAMEROON)

In view of contributing to the establishment and diversification of Jatropha curcas cultivation in the Guinea –savannah of the Adamawa region of Cameroon, a study was carried out to see the effect of organic manure, mycorrhizae and chemical fertilizer on the growth performance of Jatropha curcas in the locality of Dang (Ngaoundere). The experimental design was a split plot with two factors, the first being the origin or accession at two levels (Chad and Cameroon), the second being 4 treatments (organic manure, mycorrhizae, positive control=chemical fertilizer (NPK-20 10 10) and negative control) in three replicates. Data was collected on growth parameters as influenced by the different treatments, total biomass yields, carbon sequestration and root mycorrhizal colonization. Results indicate that fertilizers used have a highly significant effect (P <0.001) on the growth, development and dry biomass produced at 180 days after sowing, as well as on the level of carbon sequestration. Accessions of local origin (Cameroon) adapted well in the Ngaoundere environment. Organic manure was the most effective in the improvement of growth and development of plants, followed by chemical fertilizers and mycorrhizae. Chemical fertilizer had a negative effect on root mycorrhizal colonization. Organic fertilizers followed by mycorrhizae are seemingly advantageous for the promotion of Jatropha curcas cultivation in the Ngaoundere locality.

INTRODUCTION
Sustainable development policies, the fight against climate change, the high cost of hydrocarbons and the populations' dependence on fossil fuels have motivated research interest on renewable energy resources which are non-polluting and accessible to all (Antonios, 2002). The use of petroleum products by industries produces more carbon dioxide, which is harmful to the environment and health (Péan et al., 2005). Industries are in search of new sources to substitute fossil fuel whose world reserve can run out by the year 2050 (Chisti, 2007). The use of plant species in the production of biogas is envisaged. This can contribute in lowering high cost of fuel prices, the world's dependence on fossil fuels and especially contribute to the reduction of green-house gas emission (Fall, 2007). Bio-fuels are renewable energy sources with no negative impact on the environment (Sall, 2007). In order to ensure food security, research in the domain of bio-energy should be oriented towards non-edible, oil-producing plant species with potentials for bio-fuel production (Dieye, 2007;Jacquet et al., 2009). It is therefore important to produce bio-fuels without compromising agricultural crops destined for human consumption (Bellefontaine et al., 2001;Nwaga, 2009). Jatropha curcas (pourghère), a shrub belonging to the family Euphorbiaceae, seems a likely alternative to solve the problem of bio-energy without competing with food crops. This plant can be grown in association with other food crops (Dauriat et al., 2001). The toxic end-products (oil and cake) are neither used by humans or livestock, but are appreciated in traditional pharmacopeia (Bellefontaine et al., 2001;Olivier, 2007). The plant adapts to tropical and subtropical climate and the semi-arid regions with annual rainfall of 500 or 700 mm. It withstands long periods of drought (3 years), all soil types and requires very little maintenance. It is used for life-fencing and along livestock corridors (Bellefontaine et al., 2001Olivier, 2007Tchobsala et al, 2008. The plant has a plethora of other virtues: oil extracted from the seeds can serve as biofuels or for traditional soap making, rubbing oil, as well as insecticide or nematocide in crop protection (Hammaoui, 2006). The cake obtained after oil extraction is excellent organic manure for crops and when detoxified, can be used as feed for livestock (Kasuya et al., 2013). The practice of sustainable fertilization with mycorrhizae inoculation and the use of organic manure constitute an alternative for chemical fertilizers which carry serious risks of environmental pollution (Lachance et Rouleau, 2004;Bunch, 2004;Megueni et al., 2011). Organic manures (compost, fowl droppings) and bio-fertilizers (mycorrhizae and rhizobia) permit not only the cleansing of the environment but also increases soil nutrients which are important for the growth of plants (Nwaga, 2000a;2009b;Tremier et al., 2002). The general objective of this work is to contribute to the establishment of J. curcas in the guinea-savannah region of Ngaoundere (Cameroon). Specifically, this work will consist of: 1) evaluating the impact of mycorrhizae on the growth and development of J. curcas on-farm at Dang; 2) compare the impact of organic manure and mineral fertilizers on the development of the plant; 3) compare the adaptation rate of two accessions of J. curcas in the environment of the Adamawa-Cameroon.

MATERIAL AND METHODS
The trial was carried out on-farm, around the University campus at Dang-Ngaoundere, situated at an altitude of 1084 m, latitude 7°41'132''N and longitude 13°52'374''E. This is a locality in the Adamawa region (Cameroon), characterized by a guinea-savannah climate with two seasons: a rainy season (mid-March to mid-September) and a dry season (mid-September to mid-March).
Rainfall varies between 1200 and 2000 mm, with an average of 1479 mm with a coefficient of varies between 22°C and 24°C and the relative humidity is highest (80%) in the months of July to August. The soils are basaltic and granites, favorable for agriculture and animal husbandry. The planting materials are ripe seeds of Jatropha curcas harvested in the locality of Moundou (South of Chad) and from Garoua (North Cameroon). There is no noticeable morphological difference between the seeds from the different origins ( Figure 1). The trial site was cleared and cleaned, tilled and demarcated into 10 by 6 m plots. Three beds separated with a space of 2 m each were made on each plot. 5 holes with 2 m spacing were dug in the middle of each bed. The experimental design was a split plot with factor 1 being the origin of the seeds and factor 2 being 4 levels of fertilization (organic manure, mycorrhizae, chemical fertilizer and control) with three repetitions. J. curcas plants were placed 2 m apart in between lines and holes. Organic manure (OM) consisted of dried cow dung (2.5kg/hole). Chemical fertilizer (CF) applied was NPK (20-10-10) at a dose of 250g/hole). These two inputs were applied two weeks after seed germination. Mycorrhizae (M) inoculum obtained from the Biotechnology Centre in Yaoundé (Cameroon) was a mixture of Glomus clarum, Gigaspora margarita and Scutellespora sp. at a concentration of 20 spores/gram of substrate. 20 g of this substrate mixture was introduced into each hole during planting (Nwaga, 2009). Seeds were pre-treated by soaking in cold water for 12 hours. Three healthy pre-treated seeds were put in each hole at a depth of 3 -5 cm. Thinning was done 14 days after germination to leave one plant per hole. Plots were maintained clean by hand weeding. Data were collected on germination and survival of plants, growth parameters and fresh weight of plants as well as root mycorrhization (nodulation) from a sample of 30 plants. The mean leaf surface area was calculated using the formula by RAUNKIAER (Ngatchou, 1987) as: Leaf Surface = 2/3(L* l) (Where L = length of limb, and l = width of limb) Dry matter (DM) was determined from plant samples dried under ambient temperature (air dried) for two days, followed by the oven at 105°C until constant dry weight was attained. The rate of mycorrhizal colonization was evaluated as per Kormanick and Mc Graw (1992).
The statistical package «Statgraphic plus 5.0» was used for analysis of variance (ANOVA) and for determining interactions between treatments. The Duncan test was used to compare the means.

Germination rate of seeds at two weeks after planting
Seeds of J. curcas germinated between 6 to 14 days after sowing (DAS). Konaté (2006), Sharma (2007) and Nenwôla (2009) also observed that germination in J. curcas was averagely 10 days. There was a significant difference (p < 0.05) in the impact of the different treatments on the germination rate of the seeds originating from Chad ( Figure 2). The germination rate for the seeds originating from Chad were 49.0 ± 7.2%; 54.0 ±16.1% and 48.0 ±13.8 % respectively, on plots that received organic manure, mycorrhizae and control. These values were 83.0±16.6; 96±19.5 and 82±13.5 % respectively for the same treatments with the seeds originating from Cameroon. And for the latter, the difference is highly significant (p < 0.001) between treatments. Seeds originating (accessions) from Cameroon, had a high germination rate as compared to those from Chad. Mycorrhizae stimulated the germination of seeds from both origins; 96.0±19.5 % for Cameroon accessions and 54.0±16.1 % for Chad accessions. This was likely due to water retention at the level of the radicles at the end of germination, a phenomenon commonly produced by fungi mycorrhizae. Earlier studies confirm the beneficial role of mycorrhizae in water and mineral supply/conduction in plants on dry land (Perret et al., 2000;Thuries et al., 2000).  Values on the bars from the same accession followed by the same letter are not significantly different (p < 0.05).

Effect of different fertilizing agents on the growth of Jatropha curcas
The difference was highly significant (P<0:001) between the different treatments. Slow in the beginning, the growth rate (vertical) increased progressively with time ( Figure 4) All the allocated treatments stimulated a vertical growth in all the J. curcas plants. Ouédraogo (2000) and Singh & Rathod (2002)

Effect of the different treatments on the number of leaves of J. curcas
The different treatments had highly significant (P< 0:001) effects on the increase in leaf number at 180 DAS ( Figure 5) Leaf formation was slow between 120 and 180 DAS. This period corresponds to the dry season months of November and December, when plants are stressed as a result of lack of water. The number of leaves on the control treatment from both accessions remained low throughout the trial period. Increase in the number of leaves on the treated plots suggests that fertilization (organic and mycorrhizae) enhances photosynthetic activity which is necessary for effective physiological functioning of the plant. This leaf increase could have an important role in the fight against climate change in the Adamawa region of Cameroon, given their importance in Carbon sequestration through photosynthesis, thus limiting soil exposure to harmful solar radiation and consequently increasing soil relative humidity and reducing erosion (Wiesenhiitter, 2003). Bunch (2004) and Sall (2007) have shown that leaves represent recyclable biomass and organic matter which could produce mineral elements necessary for plant nutrition, with J. curcas having remarkable physiological potentials. Leye et al. (2009) reported that the inoculation of J. curcas with Glomus sp led to a significant increase in leaf number. Organic manure provides nutrients which help in increasing the organic matter, stabilizing the pH, improving the structure and the water retention capacity of the soil; this explains the increase in the development of J. curcas as observed in all the plots amended with organic manure. There was a highly significant correlation between plant height and leaf number in both accession (R=0.839; P< 0.001).

a. Provenance Cameroun
b. Provenance Tchad Figure 5: Variations in the number of leaves at different points in time as affected by the different treatments.
DAS : Day after sowing ; OM: organic manure; M: Mycorrhizae; CF: Chemical fertilizer; C: control 3.5 Effects of the different treatments on the leaf surface area (cm 2 ) of Jathopha curcas Leaf surface area showed a significant (P< 0.001) increase with the input of the various fertilizing agents and attained maximum from 90 to 120 DAP (Fig. 6). From 120 DAP, leaf surface area reduced greatly in both accessions. This reduction period corresponds to the dry season months of November and December when most plants loose considerable amounts of their leaves in order to withstand the dry season. This work is similar to that of Ouédraogo (2000), which showed that under stress conditions, J. curcas reduced surface leaf area or sheds leaves in order to withstand the drought.
a. Origin Cameroon b. Origin Chad

Effect of the treatments on plant biomass yield
The effect of organic manure, chemical fertilizer and mycorrhizae on the dry weight of Jatropha curcas plants is presented on figure 7. The different treatments significantly (P< 0,001) influenced values of this parameter. Total dry biomass produced by the Cameroon accession was highest in organic manure (239±9.6 g) followed by chemical fertilizer (129.9±3.1 g), then by mycorrhizae (114.7±0.4 g). The lowest dry biomass yield was obtained from control (99.2±1.9 g). These values from the Chad accession were 176.4±3.6, 74.2±12.4, 46.5±9.5 and 43.2±2.8 g respectively for the same treatments. These results are in accordance with those of Tchuenteu (2009) who noted that organic manure increased biomass yields in Ricinus communis. Fertilization has a beneficial effect on dry biomass yields in J. curcas plants (Can et al., 2004;Janaludin et al., 2006).
There is a strong and positive correlation (R=0.854; P< 0.001 for Chad accession and R=0.892; P< 0.001 for Cameroon) between dry weight and height. Values on the same set of bars followed by the same letters are not significantly different.

Effect of treatments on water content of the plants
Results obtained from the Cameroon accession shows high water content (74.1±3.1%) of plants on Mycorrhizae treated plots compared to plants on organic manure (70.7±7.3 %), control (71.7±4.3 %) and chemical fertilizer (68.5±5.3) treated plots. The effect of these treatments on the Chad accession was completely different from that of Cameroon accession (Figure 8). The high water content in plants of the Cameroon accession could be due to a more favorable response to environmental conditions. Mycorrhizae contribute more in water uptake and resistance to drought, which justifies the higher water content in plants in the mycorrhizae inoculated plots (Perrin, 1991;Megueni et al., 2011).
According to Ouédraogo (2000), J. curcas retains three major adaptive characteristics which enhance its resistance to drought: a strong root development, a cuticle protection of aboveground structures and a reduction or shedding of leaves, which highly limits loses through transpiration. Actually, leaf loss (defoliation) was more pronounced on the organic manure treated plots (61%) and on the mycorrhizae treated plots (32%) for the Cameroon accession. Improvement with organic manure and mycorrhizae conferred to the plant potentials for resistance to environmental stress.

Effect of different treatments on the mycorrhization rate of the J. curcas roots
Root colonization by mycorrhizae showed a highly significant difference (P< 0,001) between the seeds from the two origins (Table 4). The roots mycorrhization rate was lowest on chemical fertilizer treated plots compared to the other treatments. This shows that chemical fertilizer pollutes the environment and disturbs the development of soil microflora (Nwaga, 2000 ;Tremier et al., 2002 ;Lachance and Rouleau, 2004). The sustainable management of soils for crop production would require the use of organic and biological fertilizers (Estève, 1996;Fresco, 2003 andMegueni et al., 2011).


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Tissue Printing and Dual Excitation Flow Cytometry for Oxidative Stress—New Tools for Reactive Oxygen Species Research in Seed Biology

The intracellular homeostasis of reactive oxygen species (ROS) and especially of superoxide anion and hydrogen peroxide participate in signaling cascades which dictate developmental processes and reactions to stresses. ROS are also biological molecules that play important roles in seed dormancy and germination. Because of their rapid reactivity, short half-life and low concentration, ROS are difficult to measure directly with high accuracy and precision. In presented work tissue printing method with image analysis and dual excitation flow cytometry (FCM) were developed for rapid detection and localization of O2•− and H2O2 in different part of seed. Tissue printing and FCM detection of ROS showed that germination of wild oat seeds was associated with the accumulation of O2•− and H2O2 in embryo (coleorhiza, radicle and scutellum), aleurone layer and coat. To verify if printing and FCM signals were specified, the detection of O2•− and H2O2 in seeds incubated in presence of O2•− generation inhibitor (DPI) or H2O2 scavenger (CAT) were examined. All results were a high level of agreement among the level of ROS derived from presented procedures with the ones created from spectrophotometric measured data. In view of the data obtained, tissue printing with image analysis and FCM are recommended as a simple and fast methods, which could help researchers to detection and level determination of ROS in the external and inner parts of the seeds.

Introduction
Reactive Oxygen Species (ROS) have long been known to be a damaging compound for biomolecules [1] and also signaling intermediates [2]. ROS are by-product of aerobic metabolism under natural conditions and plants have evolved an array of self-protective defensive tools to counteract loss of redox homeostasis of the cell [3,4]. Detoxification of ROS by antioxidative defense system comprising of both non-enzymatic and enzymatic antioxidants is paramount for the maintenance of physiological level of ROS and function of ROS as a signal molecules in a number of cellular processes.
Several works in the last decade strongly convey the role of ROS at all stages of seed life from embryogenesis to germination [5]. Out of several ROS, superoxide anion (O 2 •− ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (HO•) have been implicated in dormancy releasing and germination [6][7][8]. ROS accumulation occurs both in quiescent dry seeds during after-ripening (AR) and metabolically active seeds [9,10]. ROS participate in dormancy release during AR through the direct oxidation of a subset of biomolecules, such as nucleic acid, especially mRNA [11]. During seed imbibition, their metabolic activity vary dramatically in most of the cell organelles like glyoxysomes, peroxisomes and mitochondria, results in ROS accumulation [12,13]. Likewise, the enzymatic activity of NADPH oxidase, which transfer electrons across the plasma membrane from cytoplasmic NADPH to molecular oxygen, is an important source of O 2 •− in the plasma membrane [14]. Bailly et al. [5] proposed that the Timing of generation, diffusion and degradation of various types of ROS within various cellular compartments leads to that ROS at different concentrations would play distinctly different roles therein, thus eventually leading to different fates of cells. Therefore, it has become very important to determine the location and concentration of ROS. Because ROS arise from various mechanisms, various qualitative and quantitative techniques are currently available. Furthermore, due to instability, short half-life and mutual interference of most oxygen radicals, ROS are not an easy object to analyze. Protocols for ROS detection and determination of its concentration in cell are available, like spectrophotometry [17], histochemical staining for stereomicroscopy and light microscopy [18,19], fluorescence [20,21], chemiluminescence [22], high-performance liquid chromatography (HPLC) [23], fluorescent protein-based redox probe [24], electron spin resonance (ESR) [25] and electrochemical biosensors [26]. However, each technique has its advantages and disadvantages, so it is recommended to use more than one method to test ROS in cells, trying to rule out specific drawbacks of single techniques [27]. Tissue printing onto membranes is a simple and rapid method employed to study the localization of nucleic acid, proteins and metabolites from freshly cut surface of a bisected tissue. Seed tissue imprints on nitrocellulose membranes were described by Reference [28] for localization of extensin in soybean seed coat. The KI/starch-or DAB-mediated tissue printing has been proposed for direct detection of H 2 O 2 production in seedlings [29][30][31] and fruits [32]. A procedure based on the reduction of nitro blue tetrazolium (NBT) by O 2 •− , visualized by the formation of the dark blue formazan and oxidation of 3,3 -diaminobenzidine (DAB) by H 2 O 2 , visualized by the formation of the yellow complex, have recently been used for a localization of ROS in whole seeds or embryos isolated from seeds [33][34][35][36][37][38][39][40]. However, there is no evidence of studies using NBT-or DAB-mediated tissue printing for ROS analysis in seeds. Detection and histochemical localization of ROS provides information about in situ distribution and accumulation of ROS in different part of tissue or organ. An important objective for analysis of stained tissue or tissue imprinting signal is to statistically compare staining intensity for a particular component that has been located in the analyzed object. The stained area can be detected and quantification analyzed with the methods of freeware or commercial applications for image analysis.
The cost effective answer for quantitative analysis is ImageJ, image processing program inspired by NIH (National Institutes of Health) Image (https://imagej.nih.gov/ij/index.html). Image analysis with ImageJ is rapid and simply method, which proceeds as follows: acquisition of an image, preprocessing of the image for facilitating further processing, selection of pixels of interest and extraction of characteristic features. The quantitative analysis of O 2 •− and H 2 O 2 level based on histochemical staining was used for whole leaves [41][42][43]. However, there is no evidence of studies using method of quantification with the help of digital image analysis of histochemical staining and tissue printing results for ROS level estimation in seed. Fluorescence is one of the excellent technique for visualize ROS due to the sensitivity and selectivity offered by fluorescent probe [44]. Detection of ROS by fluorescence involves the oxidation of the fluorescent probe itself but the probe being a stable molecule in the reduced state with fluorescence properties. Two specific dyes are available to measure intracellular ROS using fluorescence microscopy or standard fluorometer. Dihydroethidium (DHE) can detect intracellular

In Situ Localization of O2 •− and H2O2 by Histochemical Staining
To verify the tissue printing results, the detection and localization of O2 •− and H2O2 were performed by histochemical NBT or DAB staining in the seeds imbibed in water, DPI or CAT. The experiment was first carried out using whole seeds but the staining was not completed; ROS were accumulating on the surface of coleorhiza of seeds when the coat was ruptured ( Figure S2a-d). However, stained longitudinally bisected half caryopses showed accumulation of O2 •-and H2O2 in the coleorhiza, radicle, scutellum, aleurone layer and coat ( Figure S3). The obtained data confirmed the results of tissue printing experiment ( Figure 1). ROS were produced both on the outer and inner side of the cover. Furthermore, the signal intensity of stained longitudinally bisected half seeds was stronger than printing signal of half seeds. The tissue printing results shown, that ROS signal, not related with mechanical tissue damage, can be registered only when the tissue printing was done in less than 2 min after the cut was done. Probably, if the histochemical staining from NBT (last 10 min) or DAB (last up to 90 min) has been used to detect ROS in half seeds, resulted with very strong signal which might be related to mechanical damage. Therefore, the embryos were isolated from dry and imbibed seeds before NBT or DAB staining and it was found that embryos were stained ( Figure 2).

In Situ Localization of O 2 •− and H 2 O 2 by Histochemical Staining
To verify the tissue printing results, the detection and localization of O 2 •− and H 2 O 2 were performed by histochemical NBT or DAB staining in the seeds imbibed in water, DPI or CAT. The experiment was first carried out using whole seeds but the staining was not completed; ROS were accumulating on the surface of coleorhiza of seeds when the coat was ruptured ( Figure S2a-d).
However, stained longitudinally bisected half caryopses showed accumulation of O 2 •and H 2 O 2 in the coleorhiza, radicle, scutellum, aleurone layer and coat ( Figure S3). The obtained data confirmed the results of tissue printing experiment ( Figure 1). ROS were produced both on the outer and inner side of the cover. Furthermore, the signal intensity of stained longitudinally bisected half seeds was stronger than printing signal of half seeds. The tissue printing results shown, that ROS signal, not related with mechanical tissue damage, can be registered only when the tissue printing was done in less than 2 min after the cut was done. Probably, if the histochemical staining from NBT (last 10 min) or DAB (last up to 90 min) has been used to detect ROS in half seeds, resulted with very strong signal which might be related to mechanical damage. Therefore, the embryos were isolated from dry and imbibed seeds before NBT or DAB staining and it was found that embryos were stained ( Figure 2). In the embryo isolated from dry seeds accumulation of ROS was not detected ( Figure 2). When seeds were incubated in presence of water for 8 h, embryo showed a significant accumulation of O 2 •− and H 2 O 2 in coleorhiza and scutellum or in coleorhiza, respectively, as compared to the same parts of the dry embryo. The intensity of the signal increased during imbibition time and was more evident after 16 h. However, when the seeds were incubated in presence of DPI or CAT, intensity of staining was fully decreased. In the embryo isolated from dry seeds accumulation of ROS was not detected ( Figure 2). When seeds were incubated in presence of water for 8 h, embryo showed a significant accumulation of O2 •− and H2O2 in coleorhiza and scutellum or in coleorhiza, respectively, as compared to the same parts of the dry embryo. The intensity of the signal increased during imbibition time and was more evident after 16 h. However, when the seeds were incubated in presence of DPI or CAT, intensity of staining was fully decreased.

O2 •− and H2O2 Quantification by ImageJ
Digital image analysis package ImageJ was applied for quantification of the intensity of stained area in tissue printing from seeds and histochemical stained embryo. The quantification by the measure of staining intensity of printing signal showed that after incubation of seeds in presence of water for 8 h, the content of O2 •− was increased in embryo (

O 2 •− and H 2 O 2 Quantification by ImageJ
Digital image analysis package ImageJ was applied for quantification of the intensity of stained area in tissue printing from seeds and histochemical stained embryo. The quantification by the measure of staining intensity of printing signal showed that after incubation of seeds in presence of water for was about 1.5-fold higher. Prolongation of incubation for up to 16 h progressively increased the intensity of the signal; the signal was ca. 1.8-fold higher in embryo, coleorhiza, radicle and aleurone layer from 16 h-imbibed seeds than signal from the dry seeds. Furthermore, in this time point, intensity of the staining in scutellum was only 1.3-fold higher than in scutellum from dry seeds ( Figure 4d). The signal of H2O2 was not detected when the seeds were incubated in presence of CAT for 8 and 16 h.  representing shades of gray. Vertical bars indicate ± SD. One-way ANOVA with the Duncan's post hoc test was used to determine the significance of differences. Mean values with different letters (a-c) are significantly different (p < 0.05, n = 5). The NBT histostains demonstrate that during incubation of seeds in presence of water for 8 h, content of O2 •− was increased in coleorhiza and scutellum 1.5 times in comparison to the same parts of embryo from dry seeds (Figure 5a,b). During further incubation up to 16 h, O2 •− showed about 2.5 and 3 times higher level in coleorhiza and scutellum, respectively, than in coleorhiza and scutellum from dry embryo. The signal of O2 •− was fully suppressed when the seeds were incubated in presence The NBT histostains demonstrate that during incubation of seeds in presence of water for 8 h, content of O 2 •− was increased in coleorhiza and scutellum 1.5 times in comparison to the same parts of embryo from dry seeds (Figure 5a  of DPI for 8 and 16 h. After 8 h of imbibition in water, the amount of H2O2 was ca. 1.7 times higher in coleorhiza than in coleorhiza from dry embryo and remained constant up to 16 h (Figure 5c). The signal of H2O2 was fully suppressed when the seeds were incubated in presence of CAT for 8 and 16 h.    Fluorescence-based signal for H 2 O 2 was also detected in different part of seed by flow cytometry (FCM), when seeds were stained using 6-carboxy-2 ,7 -dichlorodihydrofluorescein diacetate di(acetoxymethyl ester) (CDCDHFDA-AM). The fluorescence signal was more strongly in embryo (Figure 7a

O2 •− and H2O2 Quantification by Spectrophotometric Method
To test the precision of tissue printing method with ImageJ and FCM to ROS estimation, intensity of tissue printing signal and stained embryo signal to results obtained using the UV-VIS method were compared. In agreement with higher accumulation of O2 •− estimated by tissue printing (Figures 1 and  2) and FCM (Figure 6

O 2 •− and H 2 O 2 Quantification by Spectrophotometric Method
To test the precision of tissue printing method with ImageJ and FCM to ROS estimation, intensity of tissue printing signal and stained embryo signal to results obtained using the UV-VIS method were compared. In agreement with higher accumulation of O 2 •− estimated by tissue printing (Figures 1 and 2) and FCM (Figure 6  content of H2O2 was about 1.6-fold higher than content of H2O2 in the same parts of dry seeds. Prolongation of incubation up to 16 h increased the content of H2O2; in this time point, H2O2 showed about 1.7-2.2 times higher level than in dry seeds. Furthermore, the level of H2O2 in scutellum was not significantly changed throughout the whole period of imbibition (Figure 9d). The content of H2O2 was decreased when the seeds were incubated in presence of CAT.

Discussion
Seed germination is a critical developmental step regulated by multiple plant endogenous signals, such as phytohormones, reactive oxygen species (ROS) or reactive nitrogen species (RNS) [49][50][51]. ROS are efficiently interlinked with the environmental condition and major hormonal regulators, such as gibberellin, abscisic acid and ethylene, which are associated with seed dormancy and germination [52][53][54]. A number of studies have shown that ROS promote the germination of several seeds, both dormant and non-dormant [6][7][8], including Avena fatua [36,55].
Endogenously produced ROS acts as a cell signaling molecules and as a redox potential regulators. However, despite their positive role, their accumulation in the cells leads to oxidative stress. Therefore, understanding the role of ROS as cellular messenger and response for the stress, requires its precise localization, quantification and global dynamics of ROS in different parts of the seed. Due to short half-life and high reactivity, the detection of ROS in the cell has always been very

Discussion
Seed germination is a critical developmental step regulated by multiple plant endogenous signals, such as phytohormones, reactive oxygen species (ROS) or reactive nitrogen species (RNS) [49][50][51]. ROS are efficiently interlinked with the environmental condition and major hormonal regulators, such as gibberellin, abscisic acid and ethylene, which are associated with seed dormancy and germination [52][53][54]. A number of studies have shown that ROS promote the germination of several seeds, both dormant and non-dormant [6][7][8], including Avena fatua [36,55].
Endogenously produced ROS acts as a cell signaling molecules and as a redox potential regulators. However, despite their positive role, their accumulation in the cells leads to oxidative stress. Therefore, understanding the role of ROS as cellular messenger and response for the stress, requires its precise localization, quantification and global dynamics of ROS in different parts of the seed. Due to short half-life and high reactivity, the detection of ROS in the cell has always been very challenging.
During past decades, a variety of methods for detection and quantification of ROS, including its reactive intermediates, have been applied.
Tissue printing method has become an important tool for visualization and localization of different molecules in plant tissue [56]. In this study, it has been used to detect and determine the localization of ROS in non-dormant Avena fatua L. seeds during imbibition. One of known techniques of detection of O 2 •− is histochemical staining based on the reduction of nitro blue tetrazolium by O 2 • , visualized by the formation of the dark blue formazan [18]. Employment of this reaction together with the tissue printing method, made the detection and localization of O 2 •− in plant tissue possible. According to our knowledge, the detection and localization of O 2 •− using the NBT-mediated tissue printing in plant material is unavailable. In our protocol, O 2 •− was localized in embryo, coleorhiza, radicle, scutellum, aleurone layer and coat during seeds imbibition up to 16 h (Figures 1 and 2). However, when the seeds were incubated in presence of DPI, intensity of staining was completely decreased. Until now, the tissue printing for H 2 O 2 detection and localization was used only for seedlings of Glycine max, Pisum sativum, Phaseolus vulgaris, Helianthus annuus, Cucumis sativus and Solanum tuberosum [29]. It is worth noting, that aforementioned procedure was based on the oxidation of KI to I 2 by H 2 O 2 . Used nitrocellulose membranes were presoaked with starch and KI, reagent mixture for the histochemical assay of H 2 O 2 [57]. The DAB-mediated tissue printing, has been proposed for direct detection of H 2 O 2 production also for seedlings of Zea mays [30] and Lycopersicon esculentum [31], as well as fruits of L. esculentum [32]. Usage of this method allowed for identification of H 2 O 2 signals in coleorhiza and radicle of embryo, aleurone layer and coat during seeds imbibition up to 16 h (Figures 1 and 4). Nonetheless, when the seeds were incubated in presence of CAT, intensity of staining was entirely decreased. ROS accumulation in coleorhiza and radicle can be associated with elongation growth and cell wall loosening. Previously, ROS have been demonstrated to play a role in radicle elongation in Lepidium sativum [58] and Lactuca sativa [33]. Taken as a whole, presented results show that NBT-or DAB-mediated tissue printing for O 2

•−
and H 2 O 2 , respectively, are a very rapid and specific methods which can be used for detection and visualization of ROS in seeds. Rapidity of this protocol (within 10 s, see Materials and Methods for details) can completely avoid the interference of wound-induced ROS. Usage of image processing software for further analysis of received results, is fundamental and allow to extract very useful information from images. Results analysis can be done using open source image analysis tools, such as CellProfiler [59], Fiji/ImageJ [60] or Icy [61] can be used. ImageJ, a Java-based application, currently very popular in human science, thanks to its versatility can also be used as a tool for the quantification of histological results [62]. In the field of plant and seed science, ImageJ provides a way to measure many parameters, such as length, width, area or shape [63] and has become necessary tool for plant physiology, such as leaf disease or leaf color changes [64].
There are several example studies where the image processing method was used for the quantitative estimate of ROS accumulation in leaves. ImageJ has been used for determination of O 2 •− and H 2 O 2 level in leaves [41][42][43]65] and roots [66]. According to available data, O 2 •− and H 2 O 2 in seeds are visualized by staining with NBT or DAB, respectively. The data interpretation was based on subjective visual estimation and provided only qualitative results. Stained tissue can be digitalized and opened in Fiji/ImageJ for quantification of blue (NBT for O 2 •− ) or yellow (DAB for H 2 O 2 ) color intensity (in the negative, corresponding to the lighter tones of gray). In this work, aforementioned method has been applied for NBT-or DAB-mediated tissue printing of whole seeds and histochemical stained embryo, as well as used to detect the spatial distribution of the NBT or DAB intensity at different parts of seeds. As a result, an image with NBT or DAB only staining is generated and the average intensity of its pixels can be quantified after the selection of specific ROI (Region of Interest). In digital image analysis, the pixel intensity values for color range from 0 to 255, wherein, 0 represents the darkest shade of the color and 255 represent the lightest shade of the color as a standard. Combining tissue printing and image processing with Fiji/ImageJ software, it was possible to obtain a relative ROS concentration based on the pixel intensity resulting from the NBT or DAB signal (Figures 1-5). Obtained results demonstrate that the Fiji/ImageJ is a promising tool for the quantification of ROS in different part of seeds. Oxidative stress can be studied using a large variety of methodological approaches, including fluorescence: microscopy or classical fluorimeter. Several fluorescent dyes have been tested so far. They allowed to obtain information on the localization of ROS and thus the dynamics of oxidative stress in plants exposed to unfavorable environmental conditions. In present study, small-molecule fluorescent ROS dyes (DHE and CDCDHFDA-AM) and dual excitation flow cytometry, which can provide information at the single cell level, have been used for ROS quantification in different parts of seeds. The fluorescence signal showed that during imbibition of seeds up to 16 h, the content of O 2  (Figure 7f). To confirm that FCM measures the ROS level, caryopses were treated with DPI or CAT. The application of DPI or CAT, fully reduced of fluorescence signal intensity.
In plant physiology, the fluorescence probes DHE and CDCDHFDA-AM are perhaps the most frequently used when studying ROS signaling and oxidative stress in plant cells and green algae using fluorescence microscopy and fluorometry [67]. To our knowledge, the present study is the first application of dual excitation FCM with DHE and CDCDHFDA-AM to determination of O 2 •and H 2 O 2 level in seeds. The results obtained demonstrate that FCM is a promising tool for the precise localization and quantification of ROS in different part of seeds.
To validate results obtained using tissue printing with image analysis and FCM methods for ROS analysis in seed material, UV-VIS method was used to measure O 2 •− and H 2 O 2 levels. In this study, it was proved a high correlation between mean gray value in tissue printing signal, FCM signal and the biochemically-quantified amount of O 2 •− and H 2 O 2 (Figures 8 and 9). In agreement with higher accumulation of O 2 •− estimated by tissue printing (Figures 1-3) and FCM (Figures 6 and 7),

Seed Material
Avena fatua (wild oat) spikelets, which contained 2-3 florets covered with glumes, were collected in July 2010 near Szczecin (Poland). After collection, the florets (a single caryopsis, covered by the lemma and palea) were dried at room temperature (RT) for 7 days to a constant moisture content (ca. 12%). To obtain non-dormant caryopses (seeds), dormant florets were stored dry under ambient relative humidity for up to 4 months in darkness at 25 • C.
In all the experiments, seeds (25 in each of 5 replicates) were incubated at 20 • C in the dark, in Petri dishes (ø 6 cm) on one layer of filter paper (Whatman No. 1) moistened with 1.5 mL of distilled water or with solutions: diphenyleneiodonium (DPI) (10 −4 M) (Merck, Darmstadt, Germany) or catalase (CAT) (1000 U) (Merck, Darmstadt, Germany) for 8 or 16 h. After treatment whole seeds and embryo, coleorhiza, radicle, coat (testa + pericarp) and aleurone layer isolated from caryopses were used for analysis.

Localization of O 2
•− and H 2 O 2 using tissue printing was determined according to Liu et al. [31] with some modification. After incubation whole seeds were placed on Petri dish and cut using razor blade. To obtain a high-quality cut surface without damaging tissue, each edge of the razor blade was used several times. Longitudinally bisected half seed was immediately used for tissue printing. Nitrocellulose blotting membrane (Amersham™ Protran™ 0.2 µm NC, Amersham, UK) was soaked in 6 mM NBT (Merck, Darmstadt, Germany) (in 10 mM Tris-HCl, pH 7.4) for O 2 •− or 2 mg mL −1 DAB-HCl solution (pH 3.8) (Merck, Darmstadt, Germany) for H 2 O 2 localization and then air-dried at 25 • C for 30 min in darkness. The soaked membrane was placed on three layers of filter paper (Whatman No 1) (Merck, Darmstadt, Germany). The half seed was placed with its cut surface down on membrane, covered with parafilm and pressed for 10 s. Then, the half seed or embryo was carefully removed with the forceps. After 5 min at RT, when reaction between ROS and NBT or DAB was completed, membranes were photographed (Canon EOS 500, Tokyo, Japan). All the images were saved as TIFF files, with 3072 × 2304 pixel resolution and 24 bits RGB color depth (pixel transformation factor = 1, no scaling of result images). The image analysis was conducted using the ImageJ (version 1.51s/Java 1.6, LOCI, University of Wisconsin, U.S.). NBT or DAB stained membranes were converted to gray scale for analysis. Staining can be assessed by setting a 'threshold' using the thresholding tool. However, to limit the ROS determination to the defined parts of the seeds or embryos, specific ROI (Region of Interest; using one of the drawing tools) was analyzed. Minimum and maximum threshold values were established to remove background staining and a mean gray value for the plaques was then calculated. In digital image analysis, the pixel intensity values for color range from 0 to 255, wherein, 0 represents the darkest shade of the color and 255 represent the lightest shade of the color as a standard. The data are presented as a mean gray value of the total plaque area to give a relative staining density for each sample [68]. The dried membranes can be kept at room temperature without losing signals or the original resolution of printed images for several months. To verify whether there is a production of wound-induced ROS under experimental conditions, tissue printing was performed at 0, 1, 2 and 4 min after cutting.

In Situ Localization and Level of O 2 •− and H 2 O 2 and Content Determination by ImageJ
Generation of ROS in situ was detected by monitoring the reduction of nitro blue tetrazolium (NBT) as described by Beyer and Fridovich [18] or polymerization of 3,3 -diaminobenzidine (DAB) according to Thordal-Christensen et al. [19]. After incubation embryos were isolated from seeds and stained for 10 min in darkness in 6 mM NBT (in 10 mM Tris-HCl, pH 7.4) or 90 min in darkness in 1 mg ml-1 DAB containing 0.05% (v/v) Tween-20 and 10 mM Na 2  The stained samples were transferred to 2 mL of the isolation buffer, pH 7.4, containing 45 mM MgCl 2 , 30 mM sodium citrate, 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) and 0.1% (v/v) Triton X-100 [69] and using a razor blade, were chopped for 2 min. Subsequently, the suspension was passed through a 50 µm nylon mesh. The labelled cells were analyzed using flow cytometer (Partec, Germany) with an air-cooled 20 mV argon-ion laser and HBO mercury arc lamp. In the flow chamber, each cell crosses a region of fluorescence excitation. There, the green-or red-fluorescence emissions are excited consecutively with blue light at 488 nm and UV and recorded by photomultiplier tubes. The flow cytometer was equipped with dichroic mirror with the edge at 420 nm (TK420), a full mirror (FM) and a long pass filter with the edge at 515 nm (OG515). The final gated cell populations contained 20,000 cells and signals were recorded on a histogram by logarithmic amplifiers. The histograms present the fluorescence intensity (log; Geo Mean) on the x-axis and cell count on the y-axis in gated population of cells. The relative O 2 •− and H 2 O 2 level was expressed as the mean fluorescence intensity (percentage of the control).

Determination of O 2 •− and H 2 O 2 Content by Spectrophotometric Method
Extracellular O 2 •− production was estimated using the method developed by Misra and Fridovich [17]. After incubation, the embryos, coleorhiza, radicle, scutellum, coat (testa + pericarp) and aleurone layer were isolated from seeds. Sample were ground and homogenized by pestle and mortar in cold 50 mM Tris-HCl pH 7.5 with 2% (w/v) PVPP (fresh weight: buffer, 1:10, w/v). The homogenate was centrifuged for 20 min at 14,000× g at 4 • C and then the supernatant was immediately used. The reaction mixture was composed of 0.05 mL of 50 mM Tris-HCl pH 7.5, 0.05 mL of 60 mM epinephrine (in 0.5 M HCl) and 0.05 mL of supernatant. The oxidation of epinephrine to adrenochrome was measured in reaction mixture at 480 nm for 2 min. In each test, oxidation of adrenaline was carried out in the reaction mixture (without extract). The epinephrine extinction coefficient was ε = 4.02 mM −1 cm −1 . The results were expressed as relative unit corresponds to the rate of epinephrine oxidation in extracts of sample from dry caryopses calculated as µmol min −1 g −1 FW). Extracellular H 2 O 2 content was determined according to Velikova et al. [70]. After incubation, the embryos, coleorhiza, radicle, scutellum, coat (testa + pericarp) and aleurone layer were isolated from seeds. Sample were ground and homogenized by pestle and mortar in cold 1% (w/v) trichloroacetic acid (TCA) (fresh weight:TCA, 1:10, w/v). After 20 min of centrifugation at 14,000× g at 4 • C, the resulting supernatant was immediately used for spectrophotometric analysis. The content of H 2 O 2 was measured in reaction mixture (0.5 mL of 10 mM potassium phosphate buffer pH 7.0, 1 mL of 1 M KI in 10 mM potassium phosphate buffer, pH 7.0 and 0.5 mL of the supernatant) at 390 nm after incubation at 25 • C for 60 min. A standard curve was prepared by using the H 2 O 2 standard. The results were expressed as nmol H 2 O 2 g −1 FW.

Data Analysis
All the experiments were carried out in five biological replicates and the results are expressed as mean ± SD. The means were analyzed for significance using one-way analysis of variance, ANOVA (Statistica for Windows version 13.0, Stat-Soft Inc., Tulsa, OK, USA). Data were checked for normality and homogeneity of variance and met these criteria. Duncan's multiple range test was used to test for significance of differences (p ≤ 0.05). Every experiment was repeated three times and the results presented correspond to a representative single experiment.

Conclusions
ROS are hot topics in seed biology because they function both as agents of damage and mediators of cellular signals. This small molecules are now known to play an important role in seed dormancy and germination, stress responses and environmental interactions. Understanding of their functions needs knowledge about the level of ROS, especially superoxide anion and hydrogen peroxide. Hence, it is necessary to use sensitive and specific methods in order to understand the contribution of each signaling molecule to various biological processes.
The quantitative method for ROS determination based on tissue printing with image analysis and flow cytometry are simple and fast. This methods can be used for determination of ROS accumulation in the external and inner parts of the seeds. Finally, all steps of tissue printing protocol was done within 10 s which avoided interference of ROS resulting of tissue damage. Image analysis applied to NBT-or DAB-mediated tissue printing and FCM provides a very efficient means of quantifies ROS level in seed sample. It has been practically proven, by comparison of received results with the UV-VIS results, that developed methods provide repeatable, accurate and reliable results. Furthermore, the use of image analysis methods avoids the effects of human subjectivity. These methods have the potential to significantly enhance both precision and reproducibility and making quantitative methods more readily accessible to most plant laboratories.

Acknowledgments:
The author would like to thank Jan Kępczyński for critical remarks on some parts of the manuscript.

Conflicts of Interest:
The author declares no conflict of interest.


extfieldsofstudy:
Materials Science
Medicine

text:
Freeze-Drying of Blueberries: Effects of Carbon Dioxide (CO2) Laser Perforation as Skin Pretreatment to Improve Mass Transfer, Primary Drying Time, and Quality

Freeze-dried berry fruits are generally consumed as they are, whole and without peeling or cutting, as the conservation of their original shape and appearance is often desired for the final product. However, usually, berries are naturally wrapped by an outer skin that imparts a barrier to vapor flow during freeze-drying, causing berry busting. Photo-sequence, experimental, and theoretical methodologies were applied to evaluate the application of CO2 laser microperforations to blueberry skin. Under the same set of freeze-drying conditions, blueberries with and without perforations were processed. The results showed that the primary drying time was significantly reduced from 17 ± 0.9 h for nontreated berries to 13 ± 2.0 h when nine microperforations per berry fruit were made. Concomitantly, the quality was also significantly improved, as the percentage of nonbusted blueberries at the end of the process increased from an average of 47% to 86%. From a phenomenological perspective, the analysis of the mass transfer resistance of nontreated fruits, in agreement with reported studies, showed a Type II curvature, with a sharp decrease at low time, followed by a linear increase. In contrast, blueberries with nine perforations depicted a Type III regime, with a saturation curvature toward the time axis. It was demonstrated that CO2-laser microperforation has high potential as a skin pretreatment for the freeze-drying of blueberries.

Introduction
Freeze-drying (FD) or lyophilization is a dehydration method that has been proven to produce high-quality fruit and vegetable products, as compared with other drying processes. Since it is carried out at a low temperature (typically a maximum of 38 to 54 • C shelf temperature), product damage is minimized. Thus, it is used for delicate, high-sensitive, and high-value products to maintain their color, flavor, shape, and nutritional characteristics [1][2][3]. Examples of these kinds of foods are berry fruits such as blueberries, strawberries, maquiberries, and cranberries, among others. Freeze-dried berry fruits are generally consumed as they are, whole and without peeling or cutting, as conservation of their original shape and appearance is often desired for the final product [4]. However, most of these berries are naturally wrapped by a waxy outer skin that imparts barrier to water vapor flow during FD, as happens transfer needs to be estimated, the Manometric Temperature Measurement (MTM) methodology has been proven to be an effective tool. Moreover, Pikal et al. [21], concluded that a high resistance to mass transfer at the beginning of the sublimation process is due to the presence of a surface barrier resulting from a structure different from that of the dried layer, a conclusion that has been supported by scanning electron microscopy. This observation may be applied to berry FD, but it needs to be tested.
Thus, the objectives of this study were as follows: (1) to describe the busting process of blueberry freeze-drying through experimental and phenomenological approaches, and (2) to evaluate the use of CO2 laser microperforation technology to reduce the processing time as well as product explosion and its associated consequences.

The Freeze-Drying Processes
Similar to other dehydration processes, FD includes simultaneous heat and mass transfer. A simple but conceptual explanation of the phenomenological processes taking place during a typical like-berry-fruit FD operation is described in Figure 1. A product, initially completely frozen, is placed into a freeze dryer chamber. Then, the chamber pressure is lowered under the water triple point, and heat is supplied by mean of a heated shelf and/or the surrounding air temperature (primary drying or sublimation). As heat flows through the dried layer through heat transfer, sublimation latent heat is delivered, and a sublimation interface recedes leaving, resulting in a thicker porous layer of dried material, which acts as a resistance to heat transfer, as well as water vapor flow, toward the product surface through mass transfer, and finally, to the condenser trap, where water is separated before gases are expelled by the vacuum pump. Both heat and the mass transfer rate are the essential causes that make the FD process slow down. In FD, if heat supplied to the sublimation interface is equal to the latent heat associated with the sublimation rate of ice (m × ΔHS = q), the saturated pressure of sublimation (Pi) and its corresponding temperature (Ti) become stable, and the sublimation proceeds normally. However, if the heat supplied to the sublimation interface is not enough, the sublimation rate will drop; conversely, if the total resistance to vapor diffusion is too large (RT), then Pi and Ti may rise. As a result, frozen water will melt and collapse, and/or bust may take place [20,21]. During berry-fruit FD processing, in addition to the drylayer resistance (Rdl), the skin operates as an important barrier (Rsk) that further extends the A product, initially completely frozen, is placed into a freeze dryer chamber. Then, the chamber pressure is lowered under the water triple point, and heat is supplied by mean of a heated shelf and/or the surrounding air temperature (primary drying or sublimation). As heat flows through the dried layer through heat transfer, sublimation latent heat is delivered, and a sublimation interface recedes leaving, resulting in a thicker porous layer of dried material, which acts as a resistance to heat transfer, as well as water vapor flow, toward the product surface through mass transfer, and finally, to the condenser trap, where water is separated before gases are expelled by the vacuum pump. Both heat and the mass transfer rate are the essential causes that make the FD process slow down. In FD, if heat supplied to the sublimation interface is equal to the latent heat associated with the sublimation rate of ice (m × ∆H S = q), the saturated pressure of sublimation (P i ) and its corresponding temperature (T i ) become stable, and the sublimation proceeds normally. However, if the heat supplied to the sublimation interface is not enough, the sublimation rate will drop; conversely, if the total resistance to vapor diffusion is too large (R T ), then P i and T i may rise. As a result, frozen water will melt and Foods 2020, 9, 211 4 of 18 collapse, and/or bust may take place [20,21]. During berry-fruit FD processing, in addition to the dry-layer resistance (R dl ), the skin operates as an important barrier (R sk ) that further extends the processing times. Additionally, the low skin permeability can generate explosion or bust problems, as vapor cannot escape at the needed rate so as not to provoke a pressure lift just under the skin [5].

Mass Transfer Resistance
The estimation of R T through experimental and theoretical approaches would explain and describe the phenomenological process associated with the berry busting process. The interaction effects between the blueberry fruit characteristics, such as mass transfer area (A) the product mass transfer resistance (R T ), and the freeze-drying processing conditions, have been studied by using the semiempirical MTM procedure, which was originally developed to assess the temperature of the sublimation interface within the product and the dried-layer mass transfer resistance. The method has a large body of literature information [22][23][24][25][26][27]. The principle of MTM is based on the flow of water vapor from the product chamber to the condenser being momentarily interrupted during the primary drying time. The MTM is based on a rapid increase in the chamber pressure by closing the freeze-dryer butterfly valve ( Figure 2). During this perturbation process, the chamber pressure will rapidly increase due to the continued sublimation of ice. Since the composition of the vapor phase in the chamber is nearly all water vapor, sublimation will stop when the chamber pressure reaches the vapor pressure of ice at the sublimation interface (diving force ∆P = 0); consequently, the pressure rise will cease. The dynamics of this pressure rise process can be theoretically described by equation (1), which is derived from the phenomenological heat and mass balance over a system, defined as the void volume of the product chamber [28]: where P(t) is the system pressure (void volume of the freeze-drying chamber), P i is the interphase pressure of the product, P 0 is the initial pressure of the system, N is the number of fruit units, M H2O is the water molecular weight; V is the void volume of the freeze drier system, R is the universal gas constant, T S is the shelf temperature, A is the mass transfer area per fruit unit, R T is the total area normalized product resistance to mass transfer, ∆T is the temperature gradient across the frozen layer-normally fixed at 1 K, L' is the geometric product characteristic-ice thickness, and X is the linear term in equation (1) associated with external air infiltration. Tang et al. [28] generated simulations of pressure rise curves to analyze the relative contribution to the chamber pressure rise (P(t)), of the many terms in Equation (1), concluding that a resistance-dominated period, which takes place in the first 5 to 10 s of the pressure rise, is mainly expressed by the first exponential term, so the others terms are nearly neglected as part of the summation.

Materials
The experiments were carried out with fresh blueberries of the Huertos de Chile brand (variety Vaccinium corymbosum), which were bought at a local market. Blueberries with a diameter of 14-15 mm were selected.

Blueberry Characterization and Freezing
The blueberry fruits used as raw material to carry out a set of experimental treatments were characterized to ensure a homogeneous quality of said material. Total Soluble Solids (TSS), or • Brix, was selected for this purpose as it is one of the main quality indicators of the blueberry fruit [29]. To determine • Brix, blueberry fruits were weighed using a RADWAG balance (AS 220/C/2, Radom, Poland) and then crushed and homogenized for 5 min with 10 mL of distilled water until a homogeneous juice was obtained. The resulting juice was filtered, and aliquots were taken for • Brix analysis with a HI 96,680 digital refractometer (Hanna instruments, Rothe Island, USA).
Blueberries to be used in the set of experimental treatments were first frozen in a convection freezer (MT05, Alaska, Minas Gerias city, Brasil) at −40 • C for 2 h to obtain a product temperature equal to or less than −35 • C.
A series of experiments, divided into two steps, was carried out in order to achieve the research objectives: (i) (Blueberries were freeze-dried without any treatment, where the busting process was observed through photographic monitoring. The fraction of busted blueberries was computed throughout time. (ii) The same freeze-drying process was applied to blueberries under different treatments: Whole, without any treatment. b.
Cut in half. c.
The Pressure Increase Test (PIT) was performed to estimate the treatment effect on primary drying time, and the MTM method was applied to evaluate the mass transfer phenomena, as affected by blueberry characteristics (skin, A, R T ) and the busting process. The final fraction of busted blueberries was also evaluated. Table 1 depicts a summary of the experimental setup, showing its two steps-visual observation/evaluation of the busting process of whole blueberries and a total of 6 treatments to evaluate the effect of CO 2 -laser microperforations on primary drying time and quality.

Freeze-Drying Process
All experiments were carried out in a Martin Christ freeze-dryer, model Alpha 2-4 LSCplus (Martin Christ Gefriertrocknungsanlagen, Osterode, Germany), which can operate at a total vacuum pressure of up to 0.001 mbar, provided with an MKS Baratron 622 capacitance manometer (MKS Instruments) and a condenser that can operate at temperatures down to −85 • C (see Figure 2). It has three shelves of 0.021 m 2 each that can be temperature controlled with a wireless temperature monitoring system, which, in turn, allows for sample temperature monitoring through pt100 port sensors. The samples or blueberries were placed onto the three shelves, considering a total load of 60 units. In order to assess the product temperature, one blueberry fruit per shelf was inserted with a miniature pt100 sensor (PT 100 Mini-Epsilon LSC Plus, Martin Chris, ostero de am Harz, Germany). A standard freeze-drying processing condition was fixed at 0.13 mbar (13 Pa) with a condenser temperature of −85 • C and a shelf temperature of 20 • C, while the air temperature was also 20 • C. The freeze-drier system includes dedicated software (SCADA Software V1 LPC plus Martin Chris, ostero de am Harz, Germany) to set, control, and monitor the freeze-drying process at the selected processing condition. It samples data every 5 s. A butterfly valve with an approximate closing time of 0.5 s, mounted in the cylindrical duct connecting the freeze-drying chamber and the condenser chamber, can be managed by the software to perform Pressure Increase Tests (PIT) (Figure 2). The SCADA Software LPC plus software allows graphic visualization and recording of several process variables: tray temperature, blueberry temperature, condenser temperature, chamber absolute pressure (capacitive sensor), and PIT data.
Foods 2020, 9, x FOR PEER REVIEW 6 of 19 freeze-drier system includes dedicated software (SCADA Software V1 LPC plus Martin Chris, ostero de am Harz, Germany) to set, control, and monitor the freeze-drying process at the selected processing condition. It samples data every 5 s. A butterfly valve with an approximate closing time of 0.5 s, mounted in the cylindrical duct connecting the freeze-drying chamber and the condenser chamber, can be managed by the software to perform Pressure Increase Tests (PIT) (Figure 2). The SCADA Software LPC plus software allows graphic visualization and recording of several process variables: tray temperature, blueberry temperature, condenser temperature, chamber absolute pressure (capacitive sensor), and PIT data.

Determination of Primary Drying Time
After freezing, the FD process proceeds to primary-drying or sublimation, and finally to secondary drying or water desorption. The primary drying stage-conventionally, the most timeconsuming part of the process-was investigated in the present study. In order to determine the end of the primary drying time or the transition from the primary drying stage to the final secondarydrying, which can be estimated with the automatic Pressure Increase Test (PIT), the freeze-drier software system was implemented. It works by temporarily closing the butterfly valve between the product chamber and the ice condenser (see Figure 2), allowing a pressure increase in the product chamber. If the pressure increase remains below a set limit, usually 10% whilst the valve is closed, the software program assumes that there is no further sublimation water left in the product, and the primary drying phase can be considered to be finished, and this time is recorded. Another way to estimate the end of the primary drying stage is to observe the difference between the shelf and assessed product temperature; when they equalize, no more sublimation heat is absorbed, meaning the sublimation is over.

Visual Registry of the Blueberry FD Process.
A photo-sequence methodology was used to capture and visually analyze the dynamics of the busting process while blueberries are being freeze-dried. Images of the top-shelf blueberries were taken by a photographic camera (Flea ® 3 FL3-GE-03S2C-C Color GigE Camera, FLIR Systems, Wilson Ville, OR, USA) located at the upper part of the freeze drier system ( Figure 2

Determination of Primary Drying Time
After freezing, the FD process proceeds to primary-drying or sublimation, and finally to secondary drying or water desorption. The primary drying stage-conventionally, the most time-consuming part of the process-was investigated in the present study. In order to determine the end of the primary drying time or the transition from the primary drying stage to the final secondary-drying, which can be estimated with the automatic Pressure Increase Test (PIT), the freeze-drier software system was implemented. It works by temporarily closing the butterfly valve between the product chamber and the ice condenser (see Figure 2), allowing a pressure increase in the product chamber. If the pressure increase remains below a set limit, usually 10% whilst the valve is closed, the software program assumes that there is no further sublimation water left in the product, and the primary drying phase can be considered to be finished, and this time is recorded. Another way to estimate the end of the primary drying stage is to observe the difference between the shelf and assessed product temperature; when they equalize, no more sublimation heat is absorbed, meaning the sublimation is over.

Visual Registry of the Blueberry FD Process
A photo-sequence methodology was used to capture and visually analyze the dynamics of the busting process while blueberries are being freeze-dried. Images of the top-shelf blueberries were taken by a photographic camera (Flea ® 3 FL3-GE-03S2C-C Color GigE Camera, FLIR Systems, Wilson Ville, OR, USA) located at the upper part of the freeze drier system ( Figure 2). A dataset of images taken at a rate of 12 photographs per min was then visually inspected to calculate the fraction of busted blueberries over time.

Estimation of the Dry-Layer Mass Transfer Resistance (R T )
The MTM method was used to evaluate the influence of product characteristics in the FD process. The needed chamber pressure-rise as a function of time can be experimentally monitored, and the acquired data can then be regressed through Equation (1), where unknown parameters such as P i , X, A T (where A T = N × A unitary ), and R T can be estimated. The minimum conditions on the experimental procedure have been reported in order to obtain reliable results. The data collection time could not be longer than 30 s, since a longer time would allow a considerable product temperature increase because of the closed system, and within that time, there needed to be sufficient data collection to observe the complete development of the exponential part of Equation (1), which accounts for the pressure rise controlled by the product resistance. During pressure increase runs, the dynamic pressure increase was monitored with a cDAQ module/9215 data acquisition system and LabVIEW software, manufactured by National Instruments (11500 N Mopac Expwy, Austin, Texas, United States), allowing a sampling period of as low as 10 ms. To successfully apply the MTM method, the product of the geometric characteristics and the particular void volume of the chamber system needed to be adjusted. It has been demonstrated that the computed value of the exponential expression Q (without considering t) should be equal to or higher than 0.2 (1/h) to ensure complete depiction of the exponential phase of the pressure rise in the product chamber [28]: For a given value of R T , Q allows the minimum number of units (N, number of blueberry fruits) that must be loaded in a particular freeze-drier system (characterized by its void volume V) to be estimated to obtain a value equal to or higher than 0.2 (1/h). A total resistance value (R T ) of approximately 3 (torr h cm 2 /g) has been reported to be acceptable to carry out this assessment.

CO 2 Laser Microperforations and Laser System Settings
As mentioned in the introduction section, CO 2 laser microperforation is a convenient pretreatment as it is a noncontact technology that significantly mitigates the chance of physical and microbiological contamination of materials that are typically associated with traditional cutting or contact devices or fluids. In this respect, the most-used pretreatment technology is to machine-cut the blueberry fruits into halves, which effectively avoids the busting process and reduces the primary drying time. Then, both pretreatment technologies were evaluated: CO 2 laser perforation at four levels of perforation-1, 3, 6, and 9 perforations per blueberry fruit-and FD blueberry cut into halves. Finally, the results were compared with each other and with those of FD whole blueberries.
A 100 W CO2-laser system (Firestar t100, Synrad Inc., Mukilteo, Wash., U.S.A.) was used to carry out the perforations of blueberries. The system was equipped with a 125 mm focusing lens (FH series Flyer, Synrad Inc.) and a computer interface with laser marking software (WinMark Pro, Synrad Inc.).
The system was operated at a continuous wavelength of 10.6 µm and a frequency of 10 kHz. Perforations were made in a square grid pattern with a density of 2.0 × 2.0 mm.
The CO2 laser was set at 120 pulses, duration of 1 millisecond, linear speed of 100 cm per second, distance of 128 mm between the laser and the surface of the blueberry, and a percentage of power that was determined experimentally by observing in a microscope (Helmut Hund GmbH, H600/12) the depth of the perforation, until finding the configuration of the laser that allowed us to perforate until 1/3, varying the power of the laser (whose maximum was 100 watts).
A unit of frozen (−35 • C) blueberry was loaded onto an aluminum tray (20 cm × 20 cm) to perform microperforations; then, blueberry samples were refrozen at −35 • C and kept until FD experiments.

Blueberry Characteristics
Blueberry size was selected within the diameter range of 14 to 15 mm. Assuming a spherical shape for the blueberry fruit, and, given a diameter of 14.5 mm (1.45 cm), its surface area (A = 4πr 2 ) results in a unit area of 6.6 cm 2 .
Total Soluble Solids of the blueberries used as raw material for experimental procedures showed a characteristic quality indicator within the narrow enough range of 9.4 ± 0.7 • Brix.

Busting Process of Nontreated Whole Blueberries
The whole blueberry FD process was set as described in the methodology section. A slide dataset that consists of images extracted from the photo sequence every 20 min was used to visually quantify the fraction of busted blueberries over time; this evolution is depicted in Figure 3. The busted blueberries are identified by red circles, reflecting the progression of the busting process, which starts with zero busted at 20 min and describes a saturation curve, achieving a maximum of 26 busted blueberries at 180 min.

Blueberry Characteristics
Blueberry size was selected within the diameter range of 14 to 15 mm. Assuming a spherical shape for the blueberry fruit, and, given a diameter of 14.5 mm (1.45 cm), its surface area ( = 4 ) results in a unit area of 6.6 cm 2 .
Total Soluble Solids of the blueberries used as raw material for experimental procedures showed a characteristic quality indicator within the narrow enough range of 9.4 ± 0.7 °Brix.

Busting Process of Nontreated Whole Blueberries
The whole blueberry FD process was set as described in the methodology section. A slide dataset that consists of images extracted from the photo sequence every 20 min was used to visually quantify the fraction of busted blueberries over time; this evolution is depicted in Figure  3. The busted blueberries are identified by red circles, reflecting the progression of the busting process, which starts with zero busted at 20 min and describes a saturation curve, achieving a maximum of 26 busted blueberries at 180 min.  Note that the dynamic of the busted blueberry percentage increased by up to 43% after 3 h of freeze-drying, and then it stayed constant (Figure 4). This means that the resistance to the mass flow of sublimated water is high at the beginning, and then it decreases as the number of busted blueberries increases. Then, a normal FD process should continue with increasing resistance as the dried layer gets thicker. The main process parameters and measurements of the whole blueberries FD, treatment 1 in Table 1, are depicted in Figure 5.
As expected, the percentage pressure increase (PIT) was low at the beginning of the process until around 2.5 h, in agreement with that shown in Figure 4. Then, it stayed approximately constant for 2.5 h, before decreasing at a moderate rate until 13 h, and finally, a very low decrease continued to get down to values under 10% after around 17 h. The product temperature also achieved the shelf temperature at approximately 15 h. Two important outcomes can be extracted from this result. First, normally, the resistance to vapor flow from the FD product is low at the beginning of the process, as no dried layer exists; however, in this case, resistance seems to have been high at the process initiation due to the high skin resistance. Then, as the busting process proceeds, the resistance goes down, before finally increasing again as the dried layer develops. Secondly, the results indicate that the primary drying time is in the range of 15 to 20 h. From the experiments carried out in triplicate, the end of the     Note that the dynamic of the busted blueberry percentage increased by up to 43% after 3 h of freeze-drying, and then it stayed constant (Figure 4). This means that the resistance to the mass flow of sublimated water is high at the beginning, and then it decreases as the number of busted blueberries increases. Then, a normal FD process should continue with increasing resistance as the dried layer gets thicker. The main process parameters and measurements of the whole blueberries FD, treatment 1 in Table 1, are depicted in Figure 5. As expected, the percentage pressure increase (PIT) was low at the beginning of the process until around 2.5 h, in agreement with that shown in Figure 4. Then, it stayed approximately constant for 2.5 h, before decreasing at a moderate rate until 13 h, and finally, a very low decrease continued to get down to values under 10% after around 17 h. The product temperature also achieved the shelf temperature at approximately 15 h. Two important outcomes can be extracted from this result. First, normally, the resistance to vapor flow from the FD product is low at the beginning of the process, as In order to quantify the effect of the blueberry skin on mass transfer, the MTM method was applied. It has been demonstrated that the computed value of the exponential expression of Equation (1) (Q; without considering t) should be equal to, or higher than, 0.2 (1/h) (Equation (2)) to ensure complete depiction of the exponential phase of the pressure rise in the product chamber. This phase reflects the influence of the resistance (R T ) on the mass transfer phenomena. For a given value of R T , Equation (2) allows us to estimate the minimum number of fruit units N (number of blueberry fruits) that must be loaded in a particular freeze-drier system (characterized by its void volume V). A value of R T of approximately 3 (torr h cm 2 /g) has been reported to be acceptable to carry out this assessment [28]. To satisfy the above condition, the following set of values were considered: R/M H2O = 3.461 (torr L/g-K); N ≥ 60 units; A = 6.6 (cm 2 /unit); V = 32.5 (L); T S = 293 K; R T = 3.0 (torr h cm 2 /g). These data were input into Equation (2), resulting in a Q value of 4.1 > 0.2.
All published studies about the application of the MTM method have used a cylindrical shape (vials in the pharmaceutical industry), where the mass transfer area (A) does not change as the FD proceeds (one-dimensional axial mass transfer). However, assuming a spherical geometry to represent the blueberry shape, area A does change over time. For this reason, in this study, the quotient (A T /R T ), was taken as a regression variable, together with P i and X (see Equation (1)). Experimental data of the pressure rise vs. time over 30 s were generated every 20 min. The pressure rise data were taken at a rate of 100 data/s and then averaged to generate a data set of 5 data points per second. These data were then regressed against Equation (1) to obtain (A T /R T ). In order to show the evolution of the process, Figure 6 depicts the first 10 s of some of the pressure rise vs. time experimental data and their respective regression curves. As expected, the regressions showed neglected values for variable X (in the range of 0.001 to 0.0001), indicating a good seaming of the FD system (no air infiltration). Also, the change of P i values was minor in comparison with the changes of (R T /A T ), meaning that the latter is the main agent of chamber pressure rise [28]. (1) (Q; without considering t) should be equal to, or higher than, 0.2 (1/h) (Equation (2)) to ensure complete depiction of the exponential phase of the pressure rise in the product chamber. This phase reflects the influence of the resistance (RT) on the mass transfer phenomena. For a given value of RT, Equation (2) allows us to estimate the minimum number of fruit units N (number of blueberry fruits) that must be loaded in a particular freeze-drier system (characterized by its void volume V). A value of RT of approximately 3 (torr h cm 2 /g) has been reported to be acceptable to carry out this assessment [28]. To satisfy the above condition, the following set of values were considered: R/MH2O = 3.461 (torr L/g-K); N ≥ 60 units; A = 6.6 (cm 2 /unit); V = 32.5 (L); TS = 293 K; RT = 3.0 (torr h cm 2 /g). These data were input into Equation (2), resulting in a Q value of 4.1 > 0.2.
All published studies about the application of the MTM method have used a cylindrical shape (vials in the pharmaceutical industry), where the mass transfer area (A) does not change as the FD proceeds (one-dimensional axial mass transfer). However, assuming a spherical geometry to represent the blueberry shape, area A does change over time. For this reason, in this study, the quotient (AT/RT), was taken as a regression variable, together with Pi and X (see Equation (1)). Experimental data of the pressure rise vs. time over 30 s were generated every 20 min. The pressure rise data were taken at a rate of 100 data/s and then averaged to generate a data set of 5 data points per second. These data were then regressed against Equation (1) to obtain (AT/RT). In order to show the evolution of the process, Figure 6 depicts the first 10 s of some of the pressure rise vs. time experimental data and their respective regression curves. As expected, the regressions showed neglected values for variable X (in the range of 0.001 to 0.0001), indicating a good seaming of the FD system (no air infiltration). Also, the change of Pi values was minor in comparison with the changes of (RT/AT), meaning that the latter is the main agent of chamber pressure rise [28].  It can be seen that at the beginning (0 h 40 min), the pressure rise was low, and it kept increasing up to 2 h 20 min. Then, it started to decrease, approximating the pressure rise observed at the process initiation (10 h 00 min). The (A T /R T ) values obtained by the regression analysis at different processing times, together with a tendency line, are shown in Figure 7. It can be seen that at the beginning (0 h 40 min), the pressure rise was low, and it kept increasing up to 2 h 20 min. Then, it started to decrease, approximating the pressure rise observed at the process initiation (10 h 00 min). The (AT/RT) values obtained by the regression analysis at different processing times, together with a tendency line, are shown in Figure 7. This value started at a low value, which means that a high RT value has an important effect in the quotient (AT/RT), and it increased rapidly over time up to 4 h, as some of the blueberry fruits busted and RT decreased, in spite of the AT value that should have been decreasing during this period. This reveals that the main effect is that given by the important reduction of RT (busting process). If an average AT over time is assumed (for example, AT = 31.4 cm 2 ), the resistance RT as a function of time can be approximated, and its result is shown in Figure 8, including a tendency line, where, contrary to a normal FD process, RT started at a high value and then decreased sharply before finally increasing again.
This observation reflects the presence of a surface barrier, which appears to be the blueberry skin, which, after being exposed to increasing vapor pressure for some time, cracks, and the effective resistance decreases. This event does not occur at the same time for all FD blueberry fruits, resulting in the sharp, but not instantaneous, RT reduction. This is in agreement with what was observed by Lu and Pikal [30], Pikal et al. [21], and Pikal [23], where some experiments using water solutions with different solutes showed initial values of RT significantly larger than zero, which suggests a surface barrier resulting from a different structure for the dried product near the surface, which was attributed to the formation of a high-resistance "skin" during the freezing step of the FD process, a conclusion that was supported by scanning electron microscopy. These authors, after performing various experiments with different compositions, suggested four types of resistance curves: Type I, with a linear dependence of RT on the dried layer thickness (or concomitant elapsed time); Type II. with a sharp decrease in RT after a short period of time, followed by a linear increase in RT with increasing time; and Type III and IV, with a saturation curvature toward the time axis, with Type III being less severe than Type IV. The bust process described in this study is clearly a Type II resistance curve. This value started at a low value, which means that a high R T value has an important effect in the quotient (A T /R T ), and it increased rapidly over time up to 4 h, as some of the blueberry fruits busted and R T decreased, in spite of the A T value that should have been decreasing during this period. This reveals that the main effect is that given by the important reduction of R T (busting process). If an average A T over time is assumed (for example, A T = 31.4 cm 2 ), the resistance R T as a function of time can be approximated, and its result is shown in Figure 8, including a tendency line, where, contrary to a normal FD process, R T started at a high value and then decreased sharply before finally increasing again.
This observation reflects the presence of a surface barrier, which appears to be the blueberry skin, which, after being exposed to increasing vapor pressure for some time, cracks, and the effective resistance decreases. This event does not occur at the same time for all FD blueberry fruits, resulting in the sharp, but not instantaneous, R T reduction. This is in agreement with what was observed by Lu and Pikal [30], Pikal et al. [21], and Pikal [23], where some experiments using water solutions with different solutes showed initial values of R T significantly larger than zero, which suggests a surface barrier resulting from a different structure for the dried product near the surface, which was attributed to the formation of a high-resistance "skin" during the freezing step of the FD process, a conclusion that was supported by scanning electron microscopy. These authors, after performing various experiments with different compositions, suggested four types of resistance curves: Type I, with a linear dependence of R T on the dried layer thickness (or concomitant elapsed time); Type II. with a sharp decrease in R T after a short period of time, followed by a linear increase in R T with increasing time; and Type III and IV, with a saturation curvature toward the time axis, with Type III being less severe than Type IV. The bust process described in this study is clearly a Type II resistance curve. These results indicate that the reduction of the initial value of RT, considering a pretreatment to the blueberry skin, would enhance the water vapor flow at the beginning of the process, and consequently would reduce the primary drying time and/or increase the quality yield by bringing down the fraction of busted blueberry units at the end of the process.

Evaluation of CO2 Laser Microperforation and Blueberries Cut in Half
Experimental results were obtained for all five pretreated blueberries: 1, 3, 6, and 9 perforations and cut in half, treatments 1 to 6 in Table 1. Microperforations were made as described in the methodology, obtaining perforations in a square grid of 2.0 mm × 2.0 mm density, 0.5 mm diameter, and 1/3 of the blueberry diameter in depth, by utilizing 19% power (19 W). The FD system was set at the same conditions used for whole nontreated blueberries, and the same number of blueberry units were loaded in each experiment. The final moisture at the end of primary drying was also controlled and was within the range of 13% (95% CI (10%, 17%)) to 15% (95% CI (12%, 18%)), for all treatments. Figure 9 shows a blueberry with nine and three CO2 laser microperforations in a square arrangement with 2.5 mm spacing. It demonstrates that the CO2 laser microperforation process is minimally invasive with a negligible impact on quality characteristics. These results indicate that the reduction of the initial value of R T , considering a pretreatment to the blueberry skin, would enhance the water vapor flow at the beginning of the process, and consequently would reduce the primary drying time and/or increase the quality yield by bringing down the fraction of busted blueberry units at the end of the process.

Evaluation of CO 2 Laser Microperforation and Blueberries Cut in Half
Experimental results were obtained for all five pretreated blueberries: 1, 3, 6, and 9 perforations and cut in half, treatments 1 to 6 in Table 1. Microperforations were made as described in the methodology, obtaining perforations in a square grid of 2.0 mm × 2.0 mm density, 0.5 mm diameter, and 1/3 of the blueberry diameter in depth, by utilizing 19% power (19 W). The FD system was set at the same conditions used for whole nontreated blueberries, and the same number of blueberry units were loaded in each experiment. The final moisture at the end of primary drying was also controlled and was within the range of 13% (95% CI (10%, 17%)) to 15% (95% CI (12%, 18%)), for all treatments. Figure 9 shows a blueberry with nine and three CO 2 laser microperforations in a square arrangement with 2.5 mm spacing. It demonstrates that the CO 2 laser microperforation process is minimally invasive with a negligible impact on quality characteristics. The PIT and product temperature response were used to evaluate the effect of pretreatment on the primary drying time. Each test was conducted in triplicate to determine the average and standard deviation of the primary drying time. Figure 10 depicts the PIT for the blueberries that were whole and nontreated, cut in half, and microperforated nine times. It is interesting to note that for both cutin-half and nine-times-perforated blueberries, the initial PIT value started higher than that of the whole nontreated fruit and decreased over time. This is due to the weakened pretreated skin or the The PIT and product temperature response were used to evaluate the effect of pretreatment on the primary drying time. Each test was conducted in triplicate to determine the average and standard deviation of the primary drying time. Figure 10 depicts the PIT for the blueberries that were whole and nontreated, cut in half, and microperforated nine times. It is interesting to note that for both cut-in-half and nine-times-perforated blueberries, the initial PIT value started higher than that of the whole nontreated fruit and decreased over time. This is due to the weakened pretreated skin or the area without skin being available to sublimation. The PIT and product temperature response were used to evaluate the effect of pretreatment on the primary drying time. Each test was conducted in triplicate to determine the average and standard deviation of the primary drying time. Figure 10 depicts the PIT for the blueberries that were whole and nontreated, cut in half, and microperforated nine times. It is interesting to note that for both cutin-half and nine-times-perforated blueberries, the initial PIT value started higher than that of the whole nontreated fruit and decreased over time. This is due to the weakened pretreated skin or the area without skin being available to sublimation. If the pressure increase was equal to or less than 10%, then the primary drying time was considered to be over. Table 2 shows the results of the primary drying time for the most significant treatments (treatments 1, 6, and 2 in Table 1). Table 2. Primary drying time of blueberry freeze-drying as affected by CO 2 laser microperforations and cut-in-half treatments.

Microperforations Time [hours]
Without microperforations 17.0 ± 0.9 Nine microperforations 13.0 ± 2.0 Cut in half 6.7 ± 1.2 It can be seen that there was an important primary drying time reduction of 60.6% when blueberries were cut in half, as compared with that of whole nontreated blueberries. However, this option is associated with the loss of the important quality characteristic of maintaining the fruits' original shape. On the other hand, and not less importantly, it can be observed that primary drying time reduced by approximately 23.5% when the blueberry skin was pretreated with nine microperforations, as compared with that of whole nontreated blueberries. This result is explained by the fact that the nine-microperforation pretreatment avoids the initial high resistance (R T ) at the beginning of the process, mitigating the busting process. This means that, in addition to primary time reduction, CO 2 laser microperforation pretreatment can improve the final product quality. It can be seen from Figure 11 that the percentage of nonbusted blueberries significantly increased as the number of perforations per fruit increased, which is statistically sustained by a positive value of the covariance (covariance = +0.634). In addition, the average value of the percentage ofnonbusted blueberries with nine microperforations was 86%, significantly higher than 47% for those with no microperforations. This is supported by their corresponding 95% CIs: Zero microperforations: average 47%; 95% CI (33%, 61%); Nine microperforations: average 86%; 95% CI (73%, 99%).
Nine microperforations 13.0 ± 2.0 Cut in half 6.7 ± 1.2 It can be seen that there was an important primary drying time reduction of 60.6% when blueberries were cut in half, as compared with that of whole nontreated blueberries. However, this option is associated with the loss of the important quality characteristic of maintaining the fruits' original shape. On the other hand, and not less importantly, it can be observed that primary drying time reduced by approximately 23.5% when the blueberry skin was pretreated with nine microperforations, as compared with that of whole nontreated blueberries. This result is explained by the fact that the nine-microperforation pretreatment avoids the initial high resistance (RT) at the beginning of the process, mitigating the busting process. This means that, in addition to primary time reduction, CO2 laser microperforation pretreatment can improve the final product quality. It can be seen from Figure 11 that the percentage of nonbusted blueberries significantly increased as the number of perforations per fruit increased, which is statistically sustained by a positive value of the covariance (covariance = +0.634). In addition, the average value of the percentage ofnonbusted blueberries with nine microperforations was 86%, significantly higher than 47% for those with no microperforations. This is supported by their corresponding 95% CIs: Zero microperforations: average 47%; 95% CI (33%, 61%); Nine microperforations: average 86%; 95% CI (73%, 99%).  Figure 12 also shows that pretreated blueberries are significantly better than those that can be purchased in a local market.
Similar to the analysis made to the whole blueberry process, pressure rise data were regressed against Equation (1) to obtain values of (A T /R T ) to finally assess the effect of two pretreatments (nine perforations and cut in half) on R T behavior during the FD process and compare them with that of the whole nontreated blueberry process. Figures 13 and 14 depict the resistance behavior of cut-in-half and nine-perforation treatments over time, respectively. In both cases, it can be observed that there is a sort of lag period for the R T value, which can be attributed to the fact that the skin resistance (R sk ) is still significantly higher than that of the dried layer, with this lag period being longer for the nine-perforation pretreatment than that of the cut-in-half treatment (about 3 h difference). After the lag period, both of them behave like the normal FD process.  Figure 12 also shows that pretreated blueberries are significantly better than those that can be purchased in a local market. Figure 12. Differences between freeze-dried blueberries as affected by pretreatment and comparison with those that can be found in local markets.
Similar to the analysis made to the whole blueberry process, pressure rise data were regressed against Equation (1) to obtain values of (AT/RT) to finally assess the effect of two pretreatments (nine perforations and cut in half) on RT behavior during the FD process and compare them with that of the whole nontreated blueberry process. Figures 13 and 14 depict the resistance behavior of cut-inhalf and nine-perforation treatments over time, respectively. In both cases, it can be observed that there is a sort of lag period for the RT value, which can be attributed to the fact that the skin resistance (Rsk) is still significantly higher than that of the dried layer, with this lag period being longer for the nine-perforation pretreatment than that of the cut-in-half treatment (about 3 h difference). After the lag period, both of them behave like the normal FD process.      In order to compare all three treatments-whole nontreated, nine-perforation, and cut-in-half blueberries-the tendency lines were represented in the same graph ( Figure 15). This figure summarizes the phenomenological effect of the skin and pretreatments on the blueberry freeze-drying, an effect that is reflected by the R T behavior during FD processing. According to the classification made by Pikal et al. [21], the whole nontreated blueberry FD process, as mentioned, would follow a Type II resistance curve; the cut-in-half blueberries would follow a Type IV process with a severe curvature toward the time axis; and finally, the nine-perforation blueberries would exhibit Type III behavior, with a less severe saturation curvature toward the time axis than that of Type IV. These outcomes phenomenologically describe the effect of the skin and pretreatments on the mass transfer process, which is mainly reflected by the evolution of R T throughout the FD processing time. In order to compare all three treatments-whole nontreated, nine-perforation, and cut-in-half blueberries-the tendency lines were represented in the same graph ( Figure 15). This figure summarizes the phenomenological effect of the skin and pretreatments on the blueberry freezedrying, an effect that is reflected by the RT behavior during FD processing. According to the classification made by Pikal et al. [21], the whole nontreated blueberry FD process, as mentioned, would follow a Type II resistance curve; the cut-in-half blueberries would follow a Type IV process with a severe curvature toward the time axis; and finally, the nine-perforation blueberries would exhibit Type III behavior, with a less severe saturation curvature toward the time axis than that of Type IV. These outcomes phenomenologically describe the effect of the skin and pretreatments on the mass transfer process, which is mainly reflected by the evolution of RT throughout the FD processing time.

Conclusions
The outcomes from the present study demonstrate that CO2 laser microperforation has high potential to be applied as a skin pretreatment for the freeze-drying of blueberries, which offers significant improvement on the process efficiency and final product quality.

Conclusions
The outcomes from the present study demonstrate that CO 2 laser microperforation has high potential to be applied as a skin pretreatment for the freeze-drying of blueberries, which offers significant improvement on the process efficiency and final product quality.
The CO 2 laser treatment allowed the arrangement of microholes that minimally affect the quality and appearance of the fruit, serving as pathways for the flow of water vapor from the sublimating interface through the weakened mass transfer resistance of the berry skin, alleviating the pressure development under it, eventually avoiding the fruit bust, and enhancing the final product quality with a reduced processing time. This makes this novel technological approach an attractive alternative over traditional techniques.
Further research will be required to study other perforation arrangements and perforation depths in blueberries as well as other berry fruits during freeze-drying. The impact on energy consumption and cost-effectiveness should also be evaluated.
Author Contributions: S.A., P.M., J.U. and M.P. conceived, designed the study and wrote the manuscript; R.S., C.R., P.V., Contributed significantly to the design of the paper, edited and wrote some portion of the paper; All authors have read and agreed to the published version of the manuscript.