Microtuber Production Research Articles

The Impact of the Growth Regulators and Cultivation Conditions of Temporary Immersion Systems (TISs) on the Morphological Characteristics of Potato Explants and Microtubers

Potatoes (Solanum tuberosum L.) constitute one of the most economically important annual crops. In terms of tissue culture, potato microtubers (MTs) have a number of advantages over conventional plants. These advantages include their small size, which greatly facilitates storage, transport, and germplasm exchange compared to in vitro plants. One effective solution for the production and mass propagation of healthy MTs is the use of temporary immersion systems (TISs). In this study, in a SETISTM system containing kinetin/gibberellic acid (GA)/indole-3-butyric acid (IBA) hormones, we investigated the effects of different nutrient media on the morphological characteristics of potato explants and MTs. We determined the optimal cycling duration (3 h) with an immersion frequency of 2 min. The results revealed that the optimal nutrient medium for culturing single-node potato explants in a SETISTM bioreactor was the M7 medium containing kinetin (2 mg/L), GA (0.5 mg/L), and IBA (0.5 mg/L). The optimal nutrient medium for obtaining potato MTs was the M1 medium (hormone-free) with a high concentration of sucrose (9%) at 18 °C under dark growing conditions. Thus, a universal nutrient medium, employed in a bioreactor, was selected for the mass propagation of potato MTs for both domestic and foreign potato varieties.

β-Estradiol Improves Response of Potato Genotypes to Salt Stress and Enhances Microtuber Production Under In Vitro Conditions

β-Estradiol Improves Response of Potato Genotypes to Salt Stress and Enhances Microtuber Production Under In Vitro Conditions

Standardization of protocol for in vitro tuberization in potato (Solanum tuberosum) cultivar Kufri Sindhuri

Potato (Solanum tuberosum L.) is an economically important dicotyledonous and tuber crop which is ranked as the fourth most cultivated food crop after wheat, rice and maize. Asexual propagation of potato is done through tubers which are prone to large number of fungal and viral diseases. Microtubers produced through tissue culture serve as an essential component for production of disease-free quality potato seed. The present study was carried out during 2018 and 2019 at School of Biotechnology, Sher-e-Kashmir University of Agriculture Science and Technology of Jammu, Jammu to standardize in vitro microtuber production protocol in potato variety Kufri Sindhuri using different explants. Nodal segments were the most suitable explants for culture establishment which resulted in maximum survival with least contamination and mortality. Murashige and Skoog medium supplemented with BAP (1.5 mg/litre) and NAA (0.1 mg/litre) resulted in 100% shoot regeneration with 3.75 shoots per explant. Vigorous shoot proliferation was achieved by fortification of calcium pentothenate (2 mg/litre) and gibberellic acid (0.25 mg/litre) in establishment medium. Pre-tuberization was done by incubating cultures for 28 days in liquid multiplication medium supplemented with NAA (0.5 mg/litre). Maximum microtubers (24) per culture flask were obtained in 10 days when tuberization medium was fortified with 80 g/litre of sucrose while maximum diameter of 0.9 cm was recorded in the presence of growth retardant chlorocholine chloride (500 mg/litre). Complete darkness was an essential factor for microtuber induction. The harvested microtubers (G0) were stored at 4°C after treating them with fungicides.

Technology for producing healthy seed potatoes from botanical seeds

The presented material on the improvement of seed potatoes through botanical seeds will improve the varietal and sowing qualities of potatoes. The selected varieties are capable of forming berries on plants in the conditions of the Lower Volga region, and the selected seeds have high germination capacity. The technological process allows us to work out agricultural techniques that ensure the production of microtubers at every stage, from sowing seeds and growing high-quality seedlings.

Expressing the Saccharomyces cerevisiae SUC2 gene encoding invertase apoplastic localization in the transgenic potato (Solanum tuberosum L.) does not contribute to microtubers production of identical physiological age

Study was aimed at assessing the role of apoplastic invertase in synchronization of the microtuber formation in potato plants in vitro. A non-transgenic potato (Solanum tuberosum L.) plants (hereinafter nonTPs) and a line of potato plants, which expressed the SUC2 gene of Saccharomyces cerevisiae encoding invertase apoplastic localization under the control of the B33 promoter of the patatin gene (hereinafter TPs), was used to achieve this purpose. Cell divisions in the axillary meristems of single-node cuttings were synchronized by exposing to 7°C for 24 h in a MS medium (without agar). After the low-temperature, the synchronization (LTS) of proportion of simultaneously dividing cells in meristems of nonTPs was four-fold greater than without chilling. The LTS did not change the number of dividing cells in the axillary meristems of TPs. The LTS contributed to an increase in the mass and size of microtubers in both potato lines. All microtubers of nonTPs were of physiological maturity. At the same time, microtubers of TPs were physiologically immature, with high glucose content. During the tuber formation, the activity of acidic invertases in microtubers of TPs was higher than that of nonTPs. Microtubers of TPs were larger than the nonTPs, more hydrated with low starch amount. Propably glucose accumulated in the microtubers of the TPs may act as a signal to trigger cell division. It indicates the regulatory function of apoplastic invertase and sugars. The high activity of apoplastic invertase plays a negative role in the production of potato microtubers of identical physiological age.

Improved Production of Mashua (Tropaeolum tuberosum) Microtubers MAC-3 Morphotype in Liquid Medium Using Temporary Immersion System (TIS-RITA®)

Essential molecules are embedded within the millenary crop Tropaeolum tuberosum (mashua); these compounds are critical for the Andean people’s traditional diet and extensively utilized by the pharmaceutical industry in Peru. In the Andean region, conventional cropping techniques generate microtubers susceptible to a viral infection, which substantially endangers mashua’s production. Therefore, we developed an innovative in vitro technique condition for enhancing the agriculture process for micro tubers production. The temporary immersion system (TIS) permits the production of high-quality microtubers in a reduced space, a lower amount of time, and in large quantities compared with tubers grown under traditional conditions. To obtain T. tuberosum’s microtubers via TIS, we propagated seedlings, utilizing TIS-RITA® vessels. A set of immersion frequency times were evaluated. Interestingly, results showed that immersion at 2 min every 3 h was more beneficial compared with 2 min every 5 h based on microtubers produced after 10 weeks from the treatments, revealing an efficient frequency setting which outputted improved microtubers quality and production.

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies.

Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L−1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen (LN, cryotherapy) treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20–100%). The most effective result (70–100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in “Dunluce” free of PVS having higher numbers of microtubers and in “V500’ free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.

Synthesis of biocompatible Fe3O4 and MnO2 nanoparticles for enhanced tuberization in potato (Solanum tuberosum L.)

Iron oxide (Fe3O4) and manganese dioxide (MnO2) Nanoparticles (NPs) were synthesized via green synthesis approach using beetroot (Beta vulgaris) leaf extract and evaluated as nanofertilizer for studies of In-vitro microtuberization of potato. Successfully biogenesis of NPs were demonstrated through UV–visible spectroscopy, FTIR, XRD, SEM-EDX, and TEM analysis. In-vitro micortuberization analysis, single nodal explants of potato were placed on media (Murashige-Skoog plant growth medium devoid of original Fe and Mn salt) added with different concentrations of metal oxide-NPs, and physiological, biochemical and molecular changes were observed via using standard methods. The interaction of the NPs with the nodal explants significantly induced early tuber induction and tuber growth upon application of Fe3O4NPs (4.0 mg L-1) and MnO2NPs (1.0 mg L-1) in comparison to untreated potato tissues. Molecular analysis of potato tissues revealed enhanced expression of primary tuber inducing genes viz. Calcium-Dependent Protein Kinases (StCDPK), Calmodulin (StCaM1), and Lipoxygenase (StLOX) enzyme activity show a positive correlation of tuber induction with added NPs. Further elemental analysis via EDX exhibited that the addition of biocompatible metal oxide NPs in the growth media induced the cytosolic Ca+2 burst leading to enhanced expression of major tuber induction pathway genes resulting in early and enhanced potato tuberization. Absorption of metal-oxide NPs in microtubers was evaluated by FTIR and EDX mapping. This study is the first report on the molecular mechanism involved in regulating NPs induced the potato tuberization under In-vitro conditions. The study also indicated that application of the metal-oxide NPs as nano-nutrient to enhanced potato microtuber production.

Microtubers production by using Temporary Immersion System (TIS) bioreactor to potato varieties

As the national potatoes growth has become lower from year to year, it is now known that the problems came from the lack of certified potato seed varieties and the minimum access to sophisticated technology to make a good potato variety. The solution that can be made is to utilize microtubers as an efficient factor. The purpose of this research is to find the most effective method in micro tubers cultivation in vitro by comparing the conventional tissue culture method and the Temporary Immersion System (TIS) bioreactor method to four different potato varieties (Granola L., Dayang Sumbi, Atlantic Malang, Maglia). This research uses a split-plot design with a completely randomized design by using two factorial. The result of this study shows that the microtubers in the multiplication and production step using the TIS bioreactor method has a higher average compared to the conventional tissue culture method. As the various details, Dayang Sumbi has the highest parameter such as most sprouts, primer roots, diameter, wet weights, and fastest time growth. Granola L. excel in planlet height and most tubers. Atlantic Malang in the most multiplication and nodus. Meanwhile Maglia excel in most leaves.

Influence of the Size of Potato Microtubers (<i>Solanum tuberosum</i> L.) on the Yield of Plants under Semi-Axenic Conditions

The potato (Solanum tuberosum L.) is a vegetable that ranks fifth in the world for human consumption. Its importance is growing more and more in the Senegalese diet. However, the potato production in Senegal does not meet the needs of the market, which maintains dependence on the outside for the supply of quality seeds. In addition, these imported seeds do not often have phytosanitary qualities required for local production in the Sahelian zone. The in vitro production of microtubers, used as seed, has been shown to be one of the most efficient means for propagation of basic material. To overcome the constraints linked to the supply and availability of potato seeds, with high germination capacity, the impact of the microtuber size on the yield of the plants under semi-controlled conditions was examined. The pre-germinated microtubers were produced in vitro from vitroplants of 3 different varieties (Aida, Atlas, Odessa) adapted to the edaphic-climatic conditions of Senegal. The effects of the seed sizes of microtubers, greater than 4 mm, sown under semi-controlled conditions, on the yield of the plants, result in an increase in the ratio, in the vegetative development of the plants, but also in the number and size of the minitubers harvested. The yield of the plants also depends on the variety. It can therefore be envisaged to produce local potato seeds from microtubers and minitubers.

Composable microfluidic spinning platforms for facile production of biomimetic perfusable hydrogel microtubes.

Microtissues with specific structures and integrated vessels play a key role in maintaining organ functions. To recapitulate the in vivo environment for tissue engineering and organ-on-a-chip purposes, it is essential to develop perfusable biomimetic microscaffolds. We developed facile all-aqueous microfluidic approaches for producing perfusable hydrogel microtubes with diverse biomimetic sizes and shapes. Here, we provide a detailed protocol describing the construction of the microtube spinning platforms, the assembly of microfluidic devices, and the fabrication and characterization of various perfusable hydrogel microtubes. The hydrogel microtubes can be continuously generated from microfluidic devices due to the crosslinking of alginate by calcium in the coaxial flows and collecting bath. Owing to the mild all-aqueous spinning process, cells can be loaded into the alginate prepolymer for microtube spinning, which enables the direct production of cell-laden hydrogel microtubes. By manipulating the fluid dynamics at the microscale, the composable microfluidic devices and platforms can be used for the facile generation of six types of biomimetic perfusable microtubes. The microfluidic platforms and devices can be set up within 3 h from commonly available and inexpensive materials. After 10-20 min required to adjust the platform and fluids, perfusable hydrogel microtubes can be generated continuously. We describe how to characterize the microtubes using scanning electron or confocal microscopy. As an example application, we describe how the microtubes can be used for the preparation of a vascular lumen and how to perform barrier permeability tests of the vascular lumen.

Obtaining potato microtubers in a liquid nutrient medium

Obtaining virus-free potato seed material in Kazakhstan is mainly carried out using the method of isolation of apical meristems with the subsequent production of test-tube plants from them on an agar nutrient medium and the further formation of minitubers in greenhouse. Obtaining microtubers from meristem plants in production of potato seed material in vitro is not carried out, although this method is one of the most successful methods for increasing the potato material in vitro . Based on the above issue, we’ve set a goal to optimize the MS liquid nutrient medium for obtaining potato microtubers in vitro from meristem plants with the prospect of introducing this procedure into the process of obtaining seed material at seed farms. The paper presents new data applicable at the initial stages of potato seed production, for production of the first tuberous generation of healthy material, in particular, for replication of plants in vitro and production of microtubers. The results are presented on analysis of morphometric data of plant development in vitro obtained at cultivation of single-bud potato cuttings on ten variants of the Murashige-Skuga liquid medium with various combinations of sucrose concentrations (20 and 30 g/l), kinetin (0,2 and 4 mg/l), gibberellic acid (0, 0,5 and 1 mg/l) in 220 ml glass vessels. The perfect liquid nutrient medium for obtaining developed potato plants in vitro for further production of microtubers has been defined. Efficiency of liquid nutrient medium for obtaining potato microtubers has been determined when growing plants in vitro USING media with various combinations of concentrations of sucrose (60 and 90 g/l), kinetin (0 and 2 mg/l) and 6-benzylaminopurine (0 and 5 mg/l). The results of the study can form the basis for cultivation of plants in a bioreactor with the purpose of mass production of virus-free potato microtubers.

Effects of Growth Regulators, Media and Explant Types on Microtuberization of Potato

Microtubers (MT) are an innovative approach for nuclear seed production in potato (Solanum tuberosum L.). They are produced under in vitro conditions using different protocols. However, content and type of growth media as well as explant types significantly affect the MT production efficiency. This study was conducted to develop an efficient protocol for MT production by evaluating different growth regulators at different concentrations, types of growth media and explants in two separate experiments. In Experiment 1, the effects of six different growth regulators, and their three application rates on MT formation were compared. The effects of two growth media and three explant types on MT production were compared in Experiment 2. As a result, our studies indicated that cotton based liquid MS medium containing 0.1 mg L−1 Thidiazuron, and using whole plants as explant can be used for efficient MT production in potato.

Production of potato (Solanum tuberosum, L.) microtubers using plastic culture bags.

We have developed a system using plastic culture bags with forced aeration system for both liquid medium and gaseous phase to produce microtubers of potato (Solanum tuberosum L.). The production of microtubers under sterile conditions is a good way to produce disease-free materials for crop production, and bioreactors have been used for this purpose. However, bioreactors are expensive and difficult to handle. The plastic culture bags are relatively inexpensive and are easy to store and sterilize because they can be flattened. Microtuber production involves two stages: plant proliferation in one medium, followed by microtuber production in a different medium. Both steps are carried out using the same culture bag. Using this system, we produced 100 to 300 microtubers per 8 l culture bag, depending on the potato cultivar. We varied the nutrient concentrations in the media and found that a lower sucrose concentration in the plant proliferation medium and lower nitrogen concentration in the microtuber production medium both increased the total numbers of microtubers per bag. Notably, a higher concentration of potassium phosphate increased the numbers of larger microtubers. This is beneficial because larger microtubers are much more tolerant to field conditions. We produced about 250,000 microtubers per year in a 66 m2 tissue culture room using the culture bag system. These microtubers have been planted directly in the field and utilized for seed potato production.

Manipulating the Structural Transformation of Fullerene Microtubes to Fullerene Microhorns Having Microscopic Recognition Properties.

We report the production of fullerene microtubes (FMTs), having solid cores bisecting their tubular cavities, from solutions of mixtures of fullerene C60 and C70 and have demonstrated the structural transformation of FMTs to fullerene microhorns (FMHs) upon their exposure to alcohol/mesitylene mixtures at 25 °C. The conically shaped microhorns have hollow interiors and exhibit preferential recognition of silica particles over fullerene C70, polystyrene (PS) latex, PS hydroxylate, or PS carboxylate particles of similar dimensions due to strong electrostatic interactions between negatively charged FMHs and positively charged silica particles.

Intercalated Intermetallic Compounds AlTi3 and Fe2Ti in Microrods and Microtubes Obtained by Invariant Reaction of Mechanically Milled System Al43Ti36Fe21.

This paper reports the production of intermetallic microrods and microtubes from the decomposition of an intermetallic compound in an AlTiFe system. The intermetallic compound was obtained by mechanosynthesis of elemental powders of Al, Ti and Fe over 300 h at 400 rpm, sintering from compacted powder particles at 300 MPa per minute and at 900 °C for 3600 s in an argon atmosphere. The milled and sintered samples were analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM). The intermetallic AlTi3 and Fe3Al phases were obtained during the milling process. After sintering, a decomposition of these intermetallic phases was found—Al3Ti0.75Fe0.25, Al3Ti, FeTi, AlTi3, Ti9Al23, Fe2Ti, Al86Fe14 and Al0.4Fe0.6. As a result of the decomposition, we observed the formation of hexagonal rods with intercalated phases of AlTi3 and Fe2Ti.

Efficient synthesis of carbon microtubes–gold nanoparticles composite: optical and micro-analytical study

This article reports a new synthesis method for the production of carbon microtubes–AuNPs composite (CMT–AuNPs) based on sucrose. The CMT showed the ability to functionalize gold nanoparticles. The UV–Vis spectroscopy was used to determine the surface plasmon resonance (SPR) of the nanoparticles and the absorbance of the CMT. The results showed two absorption bands centered at 287 nm and 527 nm, associated to carbon and gold structures, respectively. The Raman spectra revealed two bands, D and G, located at 1345 cm−1 and 1570 cm−1. Transmission electron microscope (TEM) and scanning electron microscope (SEM) were used to obtain structural properties of the CMT, the results show an approximate diameter size of 0.8–1.5 µm. Metallic gold NPs were observed between 15 and 30 nm adhered to the CMT surface. The NPs were analyzed with high-resolution imaging HRTEM and chemical analysis through energy dispersive X-ray spectroscopy EDS. This is the first report that uses sucrose as a precursor to obtain carbon microtubes with gold nanoparticles and opens up new ways to fabricate this material.

Optimized in vitro micro-tuber production for colchicine biosynthesis in Gloriosa superba L. and its anti-microbial activity against Candida albicans

Gloriosa superba L. tubers are a rich source of commercially important colchicine and due to overexploitation, the species has become vulnerable. In the present investigation, in vitro tuber productions were carried out for its propagation and conservation. The in vitro and field-grown tubers were assessed for their colchicine content and antimicrobial activities. Maximum callusing was obtained when the medium was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (kinetin). Among different auxins tested for in vitro tuberous root production, IAA (1.5 mg/L) induced 78.2% tuberous root per callus. In vitro micro-tuber raised in media supplemented with 1.5 mg/L thidiazuron (TDZ) recorded the highest response (72.9%) with 28.4 tubers per explant. Sucrose (6%) with TDZ (1.5 mg/L) produced significantly more micro-tubers per callus. Elicitor treatment with AlCl3 at 125 µM and 150 µM resulted in a significant increase in the micro-tuber and tuberous root production respectively. N6-(2-Isopentenyl) adenine (2ip) (1.0 mg/L) induced the highest frequency of in vitro micro-tuber sprouting and tuber formation compared to 6-benzylaminopurine (BAP) and 1-naphthalene acetic acid (NAA). The elicitor-treatments with AlCl3 significantly increased the colchicine content of in vitro tuberous root and tubers than that of the field grown tubers. The anti-microbial activity of in vitro raised tubers, tuberous roots and AlCl3 treated samples were significantly higher compared to the field grown samples. An optimized tissue culture system for mass propagation of G. superba with conservation aspects and the production of high-value colchicine is presented here, which can be used in various medicinal systems. Manipulation of PGR’S significantly enhanced in vitro tuberization from noncorm bud explants. Elicitor treatment with AlCl3 enhanced the production of colchicine. HPLC analysis revealed significantly higher colchicine content in in vitro raised plants compared to field grown tubers. The study will help in mass propagation, conservation, and commercialization of Gloriosa superba L. for large scale production of colchicine.

A new approach for production of anode microtubes as solid oxide fuel cell support

In this study, a new inexpensive and simple technique has been developed to produce the anode microtubes used as a cell-support in the tubular SOFC system. NiO-GDC (nickel oxide–gadolinium doped ceria) rods with different binder contents were fabricated via thermoplastic extrusion. The rods were transformed into the tubular shape by wicking out the binder during the controlled debinding processing. This was achieved via processing at 600 °C and sintering at 1250 °C or 1400 °C respectively. The wall thickness of microtubes was determined by the content of the binder and the ceramic powders. The mean wall thickness values of fabricated microtubes were 219 µm, 272 µm, and 314 µm. The three-point bending test results show that the best mechanical strength with a bending stress of 164.52 MPa was achieved with the microtubes prepared from the batch 1 containing 20 wt% binder+surfactant and sintered at 1400 °C for 5 h.

Physiological Effect of Potato Genotypes and Salicylic Acid on Plantlets Growth and Microtuber Production under Salt Stress

This study was conducted to investigate the effectiveness of SA (10 and 100 μmol) for induction of salinity tolerance of two potato genotypes “Proventa” and line “97-980” during vegetative and micro-tuberization stages. Results showed that decreasing of plantlet weight and length as well as microtuber weight under saline conditions differed according to genotypes and SA concentration. Although line “97-980” had low percentage of decreasing in weight and length of plantlets compared to “Proventa” under saline condition, Proventa cv. Gave high microtubers weight. Line 97-980 recorded high reducing sugars, phenolics, proline, amino acids and proteins as well as high activity of peroxidase (POD). On contrary, “Proventa” had high concentration of total chlorophylls and high activity of superoxide dismutase(SOD) and catalase (CAT).The decrease in plantlets growth was lower in line 97-980 compared to” Proventa” under low concentration of SA. Microtubers weight in “Proventa” was higher with addition of 10 μmol through increment in most investigated biochemical compounds and high antioxidants enzymes activity. It may be concluded that high content of reducing sugars, phenolics, proline, amino acids, proteins as well as high activity of POD could work as selectable markers for in vitro potato tolerance to salinity. Addition of low concentration of SA had beneficial effect on physiological