Special Issue "Plant Tissue Culture"

A special issue of Plants (ISSN 2223-7747).

Deadline for manuscript submissions: closed (31 October 2020).

Special Issue Editor

Dr. Iyyakkannu Sivanesan
Website
Guest Editor
Department of Bioresources and Food Science, Institute of Natural Science and Agriculture, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 05029, South Korea
Interests: plant tissue culture; secondary metabolites; hydroponics; environmental protection
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Special Issue Information

Dear Colleagues,

Plant tissue culture has led to breakthroughs in understanding and applying the fundamental knowledge gained towards harnessing more from plants. It is an important technique that involves growing cells, tissues, and organs of plants on artificial media under a controlled environment. Several in vitro culture methods have been used to study and improve our knowledge on basic and advanced areas of plant biology, such as biochemistry, cytology, embryology, molecular biology, and physiology. This Special Issue is aimed at covering various aspects of plant tissue culture, such as germplasm conservation, genetic manipulation, morphogenesis, somatic embryogenesis, nutrition, large-scale clonal propagation, and the production of disease-free plants and useful metabolites.

Dr. Iyyakkannu Sivanesan
Guest Editor

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Keywords

  • plant regeneration
  • shoot proliferation
  • somatic embryogenesis
  • germplasm conservation
  • genetic manipulation
  • clonal propagation
  • bioactive compounds
  • virus-free plants
  • cell suspension culture
  • hairy root culture

Published Papers (35 papers)

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Open AccessArticle
Cytokinin-Facilitated Plant Regeneration of Three Brachystelma Species with Different Conservation Status
Plants 2020, 9(12), 1657; https://doi.org/10.3390/plants9121657 - 26 Nov 2020
Abstract
In Africa and Asia, members of the genus Brachystelma are well-known for their diverse uses, especially their medicinal and nutritional values. However, the use of many Brachystelma species as a valuable resource is generally accompanied by the concern of over-exploitation attributed to their [...] Read more.
In Africa and Asia, members of the genus Brachystelma are well-known for their diverse uses, especially their medicinal and nutritional values. However, the use of many Brachystelma species as a valuable resource is generally accompanied by the concern of over-exploitation attributed to their slow growth and general small size. The aim of the current study was to establish efficient micropropagation protocols for three Brachystelma species, namely Brachystelma ngomense (endangered), Brachystelma pulchellum (vulnerable) and Brachystelma pygmaeum (least concern), as a means of ensuring their conservation and survival. This was achieved using nodal segments (~10 mm in length) as the source of explants in the presence of different concentrations of three cytokinins (CK) namely N6-benzyladenine (BA), isopentenyladenine (iP) and meta-topolin riboside (mTR), over a period of 6 weeks. The highest (25 µM) concentration of cytokinin treatments typically resulted in significantly higher shoot proliferation. However, each species differed in its response to specific CK: the optimal concentrations were 25 µM mTR, 25 µM iP and 25 µM BA for Brachystelma ngomense, Brachystelma pulchellum and Brachystelma pygmaeum, respectively. During the in vitro propagation, both Brachystelma ngomense and Brachystelma pygmaeum rooted poorly while regenerated Brachystelma pulchellum generally lacked roots regardless of the CK treatments. Following pulsing (dipping) treatment of in vitro-regenerated shoots with indole-3-butyric acid (IBA), acclimatization of all three Brachystelma species remained extremely limited due to poor rooting ex vitro. To the best of our knowledge, the current protocols provide the first successful report for these Brachystelma species. However, further research remains essential to enhance the efficiency of the devised protocol. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Advancements in Low-Chill Blueberry Vaccinium corymbosum L. Tissue Culture Practices
Plants 2020, 9(11), 1624; https://doi.org/10.3390/plants9111624 - 23 Nov 2020
Abstract
The demand for blueberry Vaccinium corymbosum L. (and hybrids) plants has significantly increased in the last 30 years due to its market expansion. In vitro propagation of sterile plants are required for commercial purposes but also for research applications such as plant transformation. [...] Read more.
The demand for blueberry Vaccinium corymbosum L. (and hybrids) plants has significantly increased in the last 30 years due to its market expansion. In vitro propagation of sterile plants are required for commercial purposes but also for research applications such as plant transformation. Thus far, tissue culture characteristics of the tropical-adapted blueberry have been scarcely studied. In this study we present the following findings: (i) zeatin, a hormone used to promote plant growth, should be used in the 1–2 mg/L range to promote plant architecture optimal for transformation experiments; (ii) red-blue LED lights induce more production of meristems and biomass than white LED or fluorescent lights; (iii) levels as high as 1000 mg/L of decontamination agents (the antibiotics timentin and cefotaxime) can be used to eliminate Agrobacterium overgrowth without inhibiting plant growth during plant transformation experiments; (iv) kanamycin, paromomycin, and geneticin, which are widely used antibiotics to select transgene-carrying transformants, cannot be efficiently used in this system; (v) glufosinate, a widely used herbicide, shows potential to be used as an effective selectable marker for transformed plants. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Metabolic Profiling of Primary Metabolites and Galantamine Biosynthesis in Wounded Lycoris radiata Callus
Plants 2020, 9(11), 1616; https://doi.org/10.3390/plants9111616 - 20 Nov 2020
Abstract
Plants are continuously exposed to abiotic and biotic factors that lead to wounding stress. Different plants exhibit diverse defense mechanisms through which various important metabolites are synthesized. Humans can exploit these mechanisms to improve the efficacy of existing drugs and to develop new [...] Read more.
Plants are continuously exposed to abiotic and biotic factors that lead to wounding stress. Different plants exhibit diverse defense mechanisms through which various important metabolites are synthesized. Humans can exploit these mechanisms to improve the efficacy of existing drugs and to develop new ones. Most previous studies have focused on the effects of wounding stress on the different plant parts, such as leaves, stems, and roots. To date, however, no study has investigated the accumulation of primary and galantamine content following the exposure of a callus to wounding stress. Therefore, in the present study, we exposed Lycoris radiata calli to wounding stress and assessed the expression levels of several genes involved in metabolic pathways at various time points (0, 3, 6, 12, 24, 48, 72, and 96 h of exposure). Furthermore, we quantify the primary and galantamine content using gas chromatography–time-of-flight mass spectrometry and the high-performance liquid chromatography qRT-PCR analysis of eight galantamine pathway genes (LrPAL-2, LrPAL-3, LrC4H-2, LrC3H, LrTYDC2, LrN4OMT, LrNNR, and LrCYP96T) revealed that seven genes, except LrN4OMT, were significantly expressed following exposure to wounding stress. Galantamine contents of calli after 3, 6, 12, 24, 48, 72, and 96 h of exposure were respectively 2.5, 2.5, 3.5, 3.5, 5.0, 5.0, and 8.5 times higher than that after 0 h of exposure. Furthermore, a total of 48 hydrophilic metabolites were detected in the 0 h exposed callus and 96 h exposed callus using GC-TOFMS. In particular, a strong positive correlation between galantamine and initial precursors, such as phenylalanine and tyrosine, was observed. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Breaking the Dormancy of Snake’s Head Fritillary (Fritillaria meleagris L.) In Vitro Bulbs—Part 2: Effect of GA3 Soaking and Chilling on Sugar Status in Sprouted Bulbs
Plants 2020, 9(11), 1573; https://doi.org/10.3390/plants9111573 - 13 Nov 2020
Abstract
The bulb is the main propagation organ of snake’s head fritillary (Fritillaria meleagris L.), a horticulturally attractive and rare geophyte plant species. In this study, we investigated the effect of soaking bulbs in GA3 solution (1, 2, and 3 mg L [...] Read more.
The bulb is the main propagation organ of snake’s head fritillary (Fritillaria meleagris L.), a horticulturally attractive and rare geophyte plant species. In this study, we investigated the effect of soaking bulbs in GA3 solution (1, 2, and 3 mg L−1) combined with low-temperature treatment (7 °C) on breaking the dormancy of in vitro bulbs. Sugar status (total soluble sugars, glucose, and fructose content) was analyzed in different parts of the sprouted bulbs. The results showed that the soluble sugar concentration was highest in bulbs soaked in GA3. The main sugar in fritillary bulbs was glucose, while fructose content was much lower. Glucose concentration dramatically increased after bulb chilling (7 °C), and its accumulation was predominantly detected in the lower sprout portion during the first weeks of sprouting. Sugar concentration was significantly lower in nonchilled bulbs, which indicates the importance of low temperature in bulb development and sprouting. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Screening of Bioactive Metabolites and Biological Activities of Calli, Shoots, and Seedlings of Mertensia maritima (L.) Gray
Plants 2020, 9(11), 1551; https://doi.org/10.3390/plants9111551 - 12 Nov 2020
Abstract
Mertensia maritima (L.) Gray is threatened with extinction owing to climate change, poor seed germination, and ocean warming. In vitro explant-culture is used for ex situ preservation and plantlet massive production. In vitro cell and organ cultures serve as an alternative plant material [...] Read more.
Mertensia maritima (L.) Gray is threatened with extinction owing to climate change, poor seed germination, and ocean warming. In vitro explant-culture is used for ex situ preservation and plantlet massive production. In vitro cell and organ cultures serve as an alternative plant material source to investigate the biological activities and phytochemical profiles of rare plants. We aimed to develop an efficient callus and shoot production protocol and investigate bioactive metabolites, antioxidants, and enzyme inhibitory potential of M. maritima calli, shoots, and in vivo seedlings. The effects of combinations of different plant growth regulators, 6-BA (N6-benzyladenine), 6-KN (Kinetin), TDZ (Thidiazuron), and NAA (1-Naphthylacetic acid), in MS (Murashige and Skoog) nutrient medium were studied. The highest callus proliferation was obtained after 5-week cultivation over a 16-h photoperiod on growth medium MS enriched with 4 µM each of 6-BA and NAA. The medium with 2 µM 6-BA and 4 µM 6-KN had the best shoot induction rate (91.1%) with a mean of 13.4 shoots. The combination of two cytokinins (6-BA and 6-KN) was found to be effective in M. maritima shoot regeneration. The rooting frequency was 100% in ½ MS with Indole-3-butyric acid (IBA 2 µM). The number of detected compounds and chemical composition in the M. maritima shoots and seedlings extracts were similar. The total amount of phenolics in the shoots was 216.4% and 369.5% higher than in seedlings and calli, respectively. The total amount of flavonoids in the shoots was 241.1% and 429.3% higher than in seedlings and calli, respectively. The best antioxidant activity was obtained in the shoots, followed by seedlings and calli. However, the order was seedlings > calli > shoots regarding metal chelating ability. The strongest acetylcholinesterase inhibition properties were obtained in the calli, followed by seedlings and shoots. However, the tested samples can be ranked as seedlings > shoots > calli in butylcholinestrase inhibition assay. This study is the first report on the enzyme inhibitory effects of M. maritima extracts, providing valuable contributions to the scientific community. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Transformation and Characterization of Δ12-Fatty Acid Acetylenase and Δ12-Oleate Desaturase Potentially Involved in the Polyacetylene Biosynthetic Pathway from Bidens pilosa
Plants 2020, 9(11), 1483; https://doi.org/10.3390/plants9111483 - 03 Nov 2020
Abstract
Bidens pilosa is commonly used as an herbal tea component or traditional medicine for treating several diseases, including diabetes. Polyacetylenes have two or more carbon–carbon triple bonds or alkynyl functional groups and are mainly derived from fatty acid and polyketide precursors. Here, we [...] Read more.
Bidens pilosa is commonly used as an herbal tea component or traditional medicine for treating several diseases, including diabetes. Polyacetylenes have two or more carbon–carbon triple bonds or alkynyl functional groups and are mainly derived from fatty acid and polyketide precursors. Here, we report the cloning of full-length cDNAs that encode Δ12-fatty acid acetylenase (designated BPFAA) and Δ12-oleate desaturase (designated BPOD) from B. pilosa, which we predicted to play a role in the polyacetylene biosynthetic pathway. Subsequently, expression vectors carrying BPFAA or BPOD were constructed and transformed into B. pilosa via the Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of transgenes and selection marker genes in the obtained transgenic lines. The copy numbers of transgenes in transgenic lines were determined by Southern blot analysis. Furthermore, 4–5 FAA genes and 2–3 OD genes were detected in wild-type (WT) plants. Quantitative real time-PCR revealed that some transgenic lines had higher expression levels than WT. Western blot analysis revealed OD protein expression in the selected transformants. High-performance liquid chromatography profiling was used to analyze the seven index polyacetylenic compounds, and fluctuation patterns were found. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Breaking the Dormancy of Snake’s Head Fritillary (Fritillaria meleagris L.) In Vitro Bulbs—Part 1: Effect of GA3, GA Inhibitors and Temperature on Fresh Weight, Sprouting and Sugar Content
Plants 2020, 9(11), 1449; https://doi.org/10.3390/plants9111449 - 27 Oct 2020
Cited by 1
Abstract
Bulbs are the main vegetative reproductive organs of Fritillaria meleagris L. In nature, as well as in vitro, they become dormant and require low temperatures for further growth during the next vegetative period. In the present study, using 10 μM of gibberellic acid [...] Read more.
Bulbs are the main vegetative reproductive organs of Fritillaria meleagris L. In nature, as well as in vitro, they become dormant and require low temperatures for further growth during the next vegetative period. In the present study, using 10 μM of gibberellic acid (GA3), or gibberellin biosynthesis (GA) inhibitors—ancymidol (A) and paclobutrazol (P)—the dynamic changes in soluble sugars, fructose and glucose content, fresh weight and sprouting capacity were investigated. F. meleagris bulbs were cultured on medium with GA3 and GA inhibitors for 1, 2 and 5 weeks at two different temperatures (24 and 7 °C). GA3 improved bulb fresh weight, as well as sprouting percentage at both tested temperatures, compared to the control. The highest fresh weight increase (57.7%) and sprouting rate (29.02%) were achieved when bulbs were grown at 24 °C for 5 weeks. In addition, soluble sugar content was the highest in bulbs grown for 5 weeks on medium supplemented with GA3. The main sugar in fritillary bulbs was glucose, while fructose content was lower. The sensitivity of bulbs to GA inhibitors differed and significantly affected sugar content in bulbs. To our knowledge, this is the first study of the sugar composition in F. meleagris bulbs during breaking of the bulb’s dormancy and its sprouting. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
The Establishment of an Efficient Callus Induction System for Lotus (Nelumbo nucifera)
Plants 2020, 9(11), 1436; https://doi.org/10.3390/plants9111436 - 25 Oct 2020
Abstract
The lotus (Nelumbo nucifera) is one of the most popular aquatic plants in Asia, and has emerged as a novel model for studying flower and rhizome development, and primary and secondary metabolite accumulation. Here, we developed a highly efficient callus induction [...] Read more.
The lotus (Nelumbo nucifera) is one of the most popular aquatic plants in Asia, and has emerged as a novel model for studying flower and rhizome development, and primary and secondary metabolite accumulation. Here, we developed a highly efficient callus induction system for the lotus by optimizing a series of key factors that affect callus formation. The highest efficient callus production was induced on immature cotyledon and embryo explants grown on Murashige and Skoog (MS) basal medium containing an optimized combination of 3 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L 6-benzylaminopurine (6-BA). In addition, lotus callus induction was proven to be influenced by lotus genotypes, light conditions, the developmental stages of explants and the time of explant sampling. Collecting immature cotyledons from seeds of the genotype “Shilihe 1”, at 9 days post pollination, and to culture the explants in darkness, are proposed as the optimum conditions for lotus callus induction. Interestingly, highly efficient callus induction was also observed in explants of immature embryo derived aseptic seedlings; and a small amount of lotus benzylisoquinoline alkaloid (BIA) and obvious expression of BIA biosynthetic genes were detected in lotus callus. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Early Development of Direct Embryos in the Cultured Anthers of Manihot esculenta Crantz
Plants 2020, 9(10), 1315; https://doi.org/10.3390/plants9101315 - 06 Oct 2020
Abstract
Cassava is one of the most important sources of energy. To meet the growing demand, genetic improvement is of utmost importance. Its cross-pollinating nature limits the opportunity of exploitation of hybrid vigor and demands the development of homozygous lines through doubled-haploid technologies. The [...] Read more.
Cassava is one of the most important sources of energy. To meet the growing demand, genetic improvement is of utmost importance. Its cross-pollinating nature limits the opportunity of exploitation of hybrid vigor and demands the development of homozygous lines through doubled-haploid technologies. The problems in callus-mediated embryogenesis, such as longer processing time and genetically unstable nature, can be overcome by direct embryogenesis. Conditions to produce embryos directly from microspores in cultured anthers were optimized. The optimum stress pretreatment condition was 40 °C for 6 h after culturing the anthers into the induction medium. For proembryo formation, 2% sucrose and 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or 1 mg/l 1-naphthaleneacetic acid were optimum. Globular embryos were formed by subculturing proembryos into the medium with 0.5 mg/l 2,4-D and 5 mg/l 6-benzylaminopurine after two weeks of culturing. Light microscopy of cultured anthers demonstrated the formation of multicellular structures and their further development into proembryos. Microscopic studies showed proembryos emerging through the damaged anther wall. Monoallelic banding in simple sequence repeat (SSR) analysis indicated homozygous or haploid states in some of the originated embryos. The conditions optimized in this study were effective in the early development of direct embryos after two weeks of culture initiation. This is the first report of the formation of direct embryos in cultured anthers of cassava. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Synthetic Seed Technology Development and Production Studies for Storage, Transport, and Industrialization of Bracken Spores
Plants 2020, 9(9), 1079; https://doi.org/10.3390/plants9091079 - 22 Aug 2020
Abstract
Bracken fern (Pteridium aquilinum var. latiusculum (Desv.) Underw. ex A. Heller) has long been grown industrially in South Korea. Conventional propagation methods, including planting rhizomes and in vitro seedling culture, are labor intensive and expensive, and thus not commercially suitable. We aimed [...] Read more.
Bracken fern (Pteridium aquilinum var. latiusculum (Desv.) Underw. ex A. Heller) has long been grown industrially in South Korea. Conventional propagation methods, including planting rhizomes and in vitro seedling culture, are labor intensive and expensive, and thus not commercially suitable. We aimed to develop a system to produce synthetic seeds using fern spores (SFS). Synthetic seeds were prepared by mixing bracken spores and alginate matrix. Spore germination and gametophyte and sporophyte growth and development from SFS proceeded normally. Spore density affected gametophyte and sporophyte numbers. SFS prepared using cold (4 °C) long-term storage spores (even 7-year-old spores) could effectively form sporophytes. The highest germination was observed at 25 °C. Soaking-treated SFS successfully formed sporophytes, even after 30 days of storage at 4 °C; indeed, sporophytes formed even after five days of storage at 25 °C during transport conditions. SFS were sown in plug trays for commercial use. Young sporophytes grown from plug seedlings were greenhouse cultivated, and transplanting within eight weeks was effective for root growth and growing-point formation. Developing synthetic seeds is a feasible solution for facilitating efficient transport and the handling of small-sized fern spores; furthermore, this SFS technology provides the basis for fern seedling culture and fern spore industrialization. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
In Vitro Responses of Plant Growth Factors on Growth, Yield, Phenolics Content and Antioxidant Activities of Clinacanthus nutans (Sabah Snake Grass)
Plants 2020, 9(8), 1030; https://doi.org/10.3390/plants9081030 - 14 Aug 2020
Abstract
Clinacanthus nutans, commonly known as Sabah snake grass, is one of the more important medicinal plants in Malaysia’s herbal industry. C. nutans has gained the attention of medical practitioners due to its wide range of bioactive compounds responsible for various biological activities, [...] Read more.
Clinacanthus nutans, commonly known as Sabah snake grass, is one of the more important medicinal plants in Malaysia’s herbal industry. C. nutans has gained the attention of medical practitioners due to its wide range of bioactive compounds responsible for various biological activities, such as anti-cancer, anti-venom and anti-viral activities. Due to its high pharmacological properties, the species has been overexploited to meet the demands of the pharmaceutical industry. The present study was conducted to establish a suitable in vitro culture procedure for the mass propagation of C. nutans. Murashige and Skoog (MS) basal medium, supplemented with different types of cytokinins, auxins, basal medium strength and sucrose concentrations, were tested. Based on the results, a full-strength MS basal medium supplemented with 12 µM 6-benzylaminopurine (BAP) and 30 g/L sucrose was recorded as the best outcome for all the parameters measured including the regeneration percentage, number of shoots, length of shoots, number of leaves and fresh weight of leaves. In the analysis of the phenolics content and antioxidant activities, tissue-cultured leaf extracts assayed at 100 °C exhibited the highest phenolic content and antioxidant activities. The propagation of C. nutans via a plant tissue culture technique was recorded to be able to produce high phenolic contents as well as exhibit high antioxidant activities. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Efficient Tissue Culture Protocol for Magnolia lucida (Magnoliaceae) and Confirmation of Genetic Stability of the Regenerated Plants
Plants 2020, 9(8), 997; https://doi.org/10.3390/plants9080997 - 05 Aug 2020
Abstract
Magnolia lucida (Magnoliaceae) is classified as an endangered species by the International Union for Conservation of Nature. It has high commercial value owing to its attractive tree shape and flowers. We adopted an excellent genotype of M. lucida as the parent material and [...] Read more.
Magnolia lucida (Magnoliaceae) is classified as an endangered species by the International Union for Conservation of Nature. It has high commercial value owing to its attractive tree shape and flowers. We adopted an excellent genotype of M. lucida as the parent material and established a mini-cut orchard through grafting to provide trunk shoots explants over the long-term. Optimal sterilization was achieved using a combination of 75% ethanol for 30 s, one percent benzalkonium bromide for five minutes, and 0.1% mercuric chloride for five minutes. Modified Murashige and Skoog medium (ML) was the optimal medium for the growth of M. lucida. Addition of one mg/L of 6-benzyl adenine (BA) and 0.05 mg/L of α-naphthaleneacetic acid (NAA) to the medium increased the shoot induction rate to 95.56%, and the ML medium containing 0.4 mg/L BA and 0.04 mg/L NAA achieved the maximum multiplication rate (284.56%). Dark treatment for seven days, followed by continuous light treatment could better resolve the challenge of difficult rooting in M. lucida plants. Using random amplified polymorphic DNA and inter simple sequence repeat markers, we confirmed the genetic uniformity and stability of the regenerated plants. Our protocol should be helpful for the propagation and conservation of this endangered plant. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Regeneration of Genetically Stable Plants from in Vitro Vitrified Leaves of Different Carnation Cultivars
Plants 2020, 9(8), 950; https://doi.org/10.3390/plants9080950 - 28 Jul 2020
Cited by 1
Abstract
This study was conducted to investigate the efficacy of shoot regeneration from different leaf types (normal leaves and vitrified leaves) from three different carnation cultivars ‘Kumbuyl’, ‘Denev’, and ‘Jinju’ using different combinations of 3-indole butyric acid (IBA) and thidiazuron (TDZ) concentrations. The shoot [...] Read more.
This study was conducted to investigate the efficacy of shoot regeneration from different leaf types (normal leaves and vitrified leaves) from three different carnation cultivars ‘Kumbuyl’, ‘Denev’, and ‘Jinju’ using different combinations of 3-indole butyric acid (IBA) and thidiazuron (TDZ) concentrations. The shoot tips cultured on Murashige and Skoog (MS) basal media (Type 1 media) produced normal leaves, while those cultured-on media supplemented with plant growth regulators and/or vitamin (Type 2 media and Type 3 media) produced vitrified leaves for all cultivars. Culture of normal leaf segments on MS medium containing different combinations of IBA and TDZ concentrations induced callus in all treatments; however, the callus was unable to induce shoots and finally became necrotic. In contrast, no callus induction was observed in the control (hormone-free treatment). When vitrified leaf segments underwent the same treatments, shoots were induced from the vitrified leaves (derived from Type 2 media) but were unhealthy and gradually died, whereas those induced from Type 3 media were vitrified and healthy. The optimal combination for the best shoot regeneration and number of shoots per explants varied depending on the genotypes used. The vitrified shoots induced from the leaves of Type 3 media transformed into normal shoots and survived well under greenhouse conditions. According to the results of random amplified polymorphic DNA (RAPD) analysis, the banding patterns of twelve primers that were detected in vitrified leaf-induced normalized shoots were identical to those of normal in vitro grown plants, indicating that no genetic variation had occurred during the procedure. Taken together, this study indicates that vitrified leaves can be used for shoot regeneration of recalcitrant carnation cultivars, regardless of the genotypes and types of vitrified leaves. However, as the number of shoots per explants was still low, further investigation is warranted to obtain a more efficient shoot regeneration protocol for genetic transformation of the cultivars. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Effect of Mesos Components (MgSO4, CaCl2, KH2PO4) on In Vitro Shoot Growth of Blackberry, Blueberry, and Saskatoon
Plants 2020, 9(8), 935; https://doi.org/10.3390/plants9080935 - 24 Jul 2020
Abstract
Berry fruit species are, in many countries, considered biologically and economically valuable and important species of small fruits. The aim of this work was to examine the influence of either decreased or increased mesos concentrations (MgSO4, CaCl2, and KH [...] Read more.
Berry fruit species are, in many countries, considered biologically and economically valuable and important species of small fruits. The aim of this work was to examine the influence of either decreased or increased mesos concentrations (MgSO4, CaCl2, and KH2PO4) on shoot multiplication of five cultivars of three small fruit species (Amelanchier alnifolia var. cusickii, Rubus fruticosus ‘Black Satin’ and ‘Loch Ness’, and Vaccinium corymbosum ‘Brigitta Blue’ and ‘Toro’). Mesos nutrients were manipulated from half to four times their base concentration. The results indicate that mesos manipulation significantly influences the number and length of shoots in most of the studied cultivars. The greatest multiplication rate for A. alnifolia was achieved with tripled mesos, whereas ‘Black Satin’ and ‘Loch Ness’ reacted positively to a lower (1–2x) concentration of mesos. Decreasing the concentration of mesos to half led to worse quality in both blackberry and Saskatoon shoots. ‘Brigitta Blue’ was more sensitive to greater mesos concentrations compared to ‘Toro’. Optimizing the mineral nutrition of plants cultivated in vitro enhances their multiplication rate and contributes to a higher production of good quality plantlets. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Phloroglucinol Mediated Plant Regeneration of Ornithogalum dubium as the Sole “Hormone-Like Supplement” in Plant Tissue Culture Long-Term Experiments
Plants 2020, 9(8), 929; https://doi.org/10.3390/plants9080929 - 23 Jul 2020
Abstract
Tissue culture is an essential requirement in plant science to preserve genetic resources and to expand naturally occurring germplasm. A variety of naturally occurring and synthetic hormones are available to induce the processes of dedifferentiation and redifferentiation. Not all plant material is susceptible [...] Read more.
Tissue culture is an essential requirement in plant science to preserve genetic resources and to expand naturally occurring germplasm. A variety of naturally occurring and synthetic hormones are available to induce the processes of dedifferentiation and redifferentiation. Not all plant material is susceptible to tissue culture, and often complex media and hormone requirements are needed to achieve successful plant propagations. The availability of new hormones or chemicals acting as hormones are critical to the expansion of tissue culture potentials. Phloroglucinol has been shown to have certain hormone-like properties in a variety of studies. Ornithogalum dubium, an important geophyte species, was used to characterise the potential of phloroglucinol as the sole plant-like hormone in a tissue culture experiment. Tissue culture, plant regeneration, total phenolic and genetic variability were established by applying a variety of methods throughout long-term experiments. Phloroglucinol did induce callus formation and plant regeneration when used as the sole supplement in the media at a rate of 37%, thus demonstrating auxin/cytokines-like properties. Callus formation was of 3 types, friable and cellular, hard and compact, and a mixture of the two. The important finding was that direct somatogenesis did occur albeit more frequently on younger tissue, whereby rates of induction were up to 52%. It is concluded that phloroglucinol acts as a “hormone-like” molecule and can trigger direct embryogenesis without callus formation. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Phytochemical Analysis and Establishment of Embryogenic Cell Suspension and Agrobacterium-mediated Transformation for Farmer Preferred Cultivars of West African Plantain (Musa spp.)
Plants 2020, 9(6), 789; https://doi.org/10.3390/plants9060789 - 24 Jun 2020
Cited by 1
Abstract
Banana and plantain are among the foremost staple food crops providing food and livelihood to over 500 million people in tropical countries. Despite the importance, their production is hampered due to several biotic and abiotic stresses. Plant tissue culture techniques such as somatic [...] Read more.
Banana and plantain are among the foremost staple food crops providing food and livelihood to over 500 million people in tropical countries. Despite the importance, their production is hampered due to several biotic and abiotic stresses. Plant tissue culture techniques such as somatic embryogenesis and genetic transformation offer a valuable tool for genetic improvement. Identification and quantification of phytochemicals found in banana and plantain are essential in optimizing in vitro activities for crop improvement. Total antioxidants, phenolics, flavonoids, and tannins were quantified in various explants obtained from the field, as well as in vitro plants of banana and plantain cultivars. The result showed genotypic variation in the phytochemicals of selected cultivars. The embryogenic cell suspensions were developed for three farmer-preferred plantain cultivars, Agbagba, Obino l’Ewai, and Orishele, using different MS and B5-based culture media. Both culture media supported the development of friable embryogenic calli (FEC), while MS culture media supported the proliferation of fine cell suspension in liquid culture media. The percentage of FEC generated for Agbagba, Obino l’Ewai, and Orishele were 22 ± 24%, 13 ± 28%, and 9 ± 16%, respectively. Cell suspensions produced from FECs were successfully transformed by Agrobacterium-mediated transformation with reporter gene constructs and regenerated into whole plants. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Adventitious Shoot Regeneration from In Vitro Leaf Explants of the Peach Rootstock Hansen 536
Plants 2020, 9(6), 755; https://doi.org/10.3390/plants9060755 - 16 Jun 2020
Abstract
In the present study, an efficient system for the in vitro regeneration of adventitious shoots from the peach rootstock Hansen 536 leaves has been established. Twenty regeneration media containing McCown Woody Plant Medium (WPM) as a basal salt supplemented with different concentrations and [...] Read more.
In the present study, an efficient system for the in vitro regeneration of adventitious shoots from the peach rootstock Hansen 536 leaves has been established. Twenty regeneration media containing McCown Woody Plant Medium (WPM) as a basal salt supplemented with different concentrations and combinations of plant growth regulators (PGRs) were tested. Expanded leaves along with their petiole from 3-week-old elongated in vitro shoot cultures were used as starting explants. The highest regeneration rate (up to 53%) was obtained on WPM basal medium enriched with 15.5 μM N6-benzylaminopurine (BAP). The influences on leaf regeneration of the ethylene inhibitor silver thiosulphate (STS) and of different combinations of antibiotics added to the optimized regeneration medium were also investigated. The use of 10 μM STS or carbenicillin (238 μM) combined with cefotaxime (210 μM) significantly increased the average number of regenerating shoots per leaf compared to the control. In vitro shoots were finally elongated, rooted and successfully acclimatized in the greenhouse. The results achieved in this study advances the knowledge on factors affecting leaf organogenesis in Prunus spp., and the regeneration protocol described looks promising for the optimization of new genetic transformation procedures in Hansen 536 and other peach rootstocks and cultivars. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Micropropagation and Production of Health Promoting Lignans in Linum usitatissimum
Plants 2020, 9(6), 728; https://doi.org/10.3390/plants9060728 - 09 Jun 2020
Abstract
Linum usitatissimum commonly known as flax or linseed is an important medicinal plant, produces medicinally potent lignans, used in the treatment of several human diseases. Lignans limited production in the natural plants does not meet the increasing market demand. This study was conducted [...] Read more.
Linum usitatissimum commonly known as flax or linseed is an important medicinal plant, produces medicinally potent lignans, used in the treatment of several human diseases. Lignans limited production in the natural plants does not meet the increasing market demand. This study was conducted to establish an easy and rapid method for the in vitro micropropagation and production of potent lignans and antioxidant secondary metabolites in linseed. The results indicated that hypocotyl explants under the effects of thidiazuron (TDZ: 0.5 mg/L) + kinetin (Kn: 0.5 mg/L) in the basal growth media, resulted in the optimal shoot organogenesis parameters (shoot induction frequency: 86.87%, number of shoots: 6.3 ± 0.36 and shoots length: 6.5 ± 0.54 cm), in 4 weeks. Further, TDZ supplementation in the culture media efficiently activated the antioxidant system in the in vitro raised shoots, wherein maximum production of total phenolic content, TPC (34.33 ± 0.20 mg of GAE/g DW); total flavonoid content, TFC (8.99 ± 0.02 mg of QE/g DW); DPPH free radical scavenging activity (92.7 ± 1.32%); phenylalanine ammonia-lyase activity, PAL (8.99 ± 0.02 U/g FW); and superoxide dismutase expression, SOD (3.62 ± 0.01 nM/min/mg FW) were observed in the shoot cultures raised in presence of TDZ: 0.5 mg/L + Kn: 0.5 mg/L. Nonetheless, considerable levels of pharmacologically active lignans such as secoisolariciresinol (SECO: 23.13–37.10 mg/g DW), secoisolariciresinol diglucoside (SDG: 3.32–3.86 mg/g DW) and anhydrosecoisolariciresinol diglucoside (ANHSECO: 5.15–7.94 mg/g DW) were accumulated in the regenerated shoots. This protocol can be scaled up for the commercial production of linseed to meet the market demands for lignans. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
In Vitro Propagation of an Endangered Helianthus verticillatus by Axillary Bud Proliferation
Plants 2020, 9(6), 712; https://doi.org/10.3390/plants9060712 - 03 Jun 2020
Abstract
Helianthus verticillatus (Asteraceae), whorled sunflower, is a perennial species restricted to a few locations in the Southeastern United States. Habitat loss has caused H. verticillatus to become rare, and since 2014, it has been federally listed as an endangered species. As a part [...] Read more.
Helianthus verticillatus (Asteraceae), whorled sunflower, is a perennial species restricted to a few locations in the Southeastern United States. Habitat loss has caused H. verticillatus to become rare, and since 2014, it has been federally listed as an endangered species. As a part of the recovery plan for the restoration and protection of H. verticillatus, an efficient micropropagation protocol based on axillary shoot proliferation was developed. Various concentrations of 6-benzylaminopurine (BAP; 0 to 4.44 µM) were examined for their morphogenetic potential in the regeneration of six genotypes of H. verticillatus from the nodal explants derived from greenhouse-grown plants. Both the BAP concentration and genotype had significant effects on the regeneration capacity of H. verticillatus. Although the induced buds were observed on ½-strength Murashige and Skoog medium without plant growth regulators, a higher rate of induction and bud development were achieved on media with either 0.88 or 2.22 µM BAP, regardless of the genotype. Successful rooting of the induced shoots was achieved within four weeks after the transfer from the induction medium to the fresh ½-strength MS medium, but the rooting efficiency was dependent on the plant’s genetic background. Regenerated plantlets, with well-developed shoots and roots, were acclimatized successfully to greenhouse conditions with a 97% survival rate. Simple sequence repeats (SSRs) markers were employed to assess the genetic uniformity of the micropropagated plants of H. verticillatus. No extraneous bands were detected between regenerants and their respective donor plants, confirming the genetic fidelity and stability of regenerated plants. To our knowledge, the protocol developed in this study is the first such report for this endangered species. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Production of Flavonoids in Callus Cultures of Sophora flavescens Aiton
Plants 2020, 9(6), 688; https://doi.org/10.3390/plants9060688 - 28 May 2020
Abstract
Flavonoids, including maackiain (Maac) from Sophora flavescens Aiton roots, have many pharmacological properties, such as antitumor, antimicrobial, and antifungal activities. This research aimed to develop an in vitro plant and callus culture system for S. flavescens for the purpose of generating an alternative [...] Read more.
Flavonoids, including maackiain (Maac) from Sophora flavescens Aiton roots, have many pharmacological properties, such as antitumor, antimicrobial, and antifungal activities. This research aimed to develop an in vitro plant and callus culture system for S. flavescens for the purpose of generating an alternative production system for enhancing Maac production, as Maac is usually present in very small amounts in S. flavescens’ roots. We arranged the optimal conditions of different tissues of S. flavescens and supplemented the medium with various plant growth regulators (PGRs). The highest induction and proliferation rates of callus was shown in combination treatments of all concentrations of thidiazuron (TDZ) and picloram. In addition, calli induced with leaf explants cultured on 2.0 mg/L picloram and 0.5 mg/L 6-benzyladenine (BA) in Murashige and Skoog (MS) medium had the highest accumulation of the active metabolite Maac. In vitro shoots were regenerated on medium containing combinations of TDZ and α-Naphthalene acetic acid (NAA). A reliable protocol for the mass production of secondary metabolites using a callus culture of S. flavescens was successfully established. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Micropropagation, Genetic Fidelity and Phenolic Compound Production of Rheum rhabarbarum L.
Plants 2020, 9(5), 656; https://doi.org/10.3390/plants9050656 - 22 May 2020
Abstract
An efficient micropropagation protocol for Rheum rhabarbarum L. was developed in this study. The in vitro rhubarb plants obtained in the multiplication stage (proliferation rate: 5.0 ± 0.5) were rooted in vitro (96% rooting percentage) and acclimatized ex vitro in floating perlite, with [...] Read more.
An efficient micropropagation protocol for Rheum rhabarbarum L. was developed in this study. The in vitro rhubarb plants obtained in the multiplication stage (proliferation rate: 5.0 ± 0.5) were rooted in vitro (96% rooting percentage) and acclimatized ex vitro in floating perlite, with 90% acclimatization percentage. To assess the genetic fidelity between the mother plant and in vitro propagated plants, sequence-related amplified polymorphism (SRAP) markers were used. All banding profiles from the micropropagated plants were monomorphic and similar to those of the mother plant indicating 100% similarity. Regarding the polyphenolic profile, gallic, protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic, caffeic, syringic, p-coumaric and ferulic acid were present in different amounts (2.3–2690.3 μg g−1 dry plant), according to the extracted matrix. Aglicons and glycosides of different classes of flavonoids were also identified. The rhizome extracts (both from in vitro and field grown plants) contained resveratrol, a stilbene compound with high antioxidant properties, ranging between 229.4 to 371.7 μg g−1 plant. Our results suggest that in vitro propagation of Rheum rhabarbarum L. represents a reliable alternative to obtain a large number of true-to-type planting material with high bioactive compound content of this valuable nutritional and medicinal species. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Developing a Sufficient Protocol for the Enhancement of α-Glucosidase Inhibitory Activity by Urena lobata L. Aeroponic Hairy Roots Using Exogenous Factors, a Precursor, and an Elicitor
Plants 2020, 9(4), 548; https://doi.org/10.3390/plants9040548 - 23 Apr 2020
Abstract
Aeroponics is considered as a potential method for the culture of herbal plants due to the high growth rate, quantity and quality enhancement of secondary metabolites, and substantial environmental progress associated with this method. The aim of this study was to develop a [...] Read more.
Aeroponics is considered as a potential method for the culture of herbal plants due to the high growth rate, quantity and quality enhancement of secondary metabolites, and substantial environmental progress associated with this method. The aim of this study was to develop a sufficient protocol for successful Urena lobata hairy root induction by Agrobacterium rhizogenes ATCC 15834, using a precursor and elicitor to enhance α-glucosidase inhibitory activity (GIA) of aeroponic hairy roots (AHRs) in greenhouse conditions. In this study, we found that the optimized procedure (10 min, Woody plant medium (WPM), 1/25 salt strength) had an outstanding effect with a reduction in the rooting time (RT), promotion of the rooting rate (RR), and increase in the fresh weight (FW) and dry weight (DW) compared with the original procedure (30 min, Murashige and Skoog (MS) medium, 1/25 salt strength) after 30 days of culture. The highest DW, GIA, flavonoid (FLA) and phenolic (PHEL) contents were observed for individual addition of 10 mM phenylalanine (PA) or 50 mM chitosan (CS) in the late exponential phase (eighth week) with 15 days of elicitation compared to the control AHRs. However, individual treatment was less effective than the combination of the two. Positive correlations among the GIA, FLA and PHEL indicate that AHRs accumulated phenolic compounds, leading to an increase in the GIA by a synergistic effect. In conclusion, the culture of Urena lobata AHRs with PA and CS is an efficient procedure to produce GIA material in greenhouse conditions. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
In Vitro Propagation of Gastrochilus matsuran (Makino) Schltr., an Endangered Epiphytic Orchid
Plants 2020, 9(4), 524; https://doi.org/10.3390/plants9040524 - 18 Apr 2020
Abstract
Gastrochilus matsuran (Makino) Schltr. (Orchidaceae) populations are declining quickly because of overexploitation, climatic changes, and deforestation; therefore, mass-production protocols are required for this orchid. Natural propagation of this species is often hampered by meager seed germination and slow growth. Thus, our aim was [...] Read more.
Gastrochilus matsuran (Makino) Schltr. (Orchidaceae) populations are declining quickly because of overexploitation, climatic changes, and deforestation; therefore, mass-production protocols are required for this orchid. Natural propagation of this species is often hampered by meager seed germination and slow growth. Thus, our aim was to establish an effective protocol for the in vitro propagation of G. matsuran and reduce the risk of its extinction. We investigated the impacts of culture media, coconut water (CW), and plant hormones (gibberellic acid (GA3), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA), and thidiazuron (TDZ)) on asymbiotic germination, multiplication and conversion of protocorms, and plantlet development. Maximal seed germination (93.3%) was achieved on ½ MS medium without vitamins plus 5% CW, 1 µM NAA, and 1.5 µM GA3. Secondary protocorm formation was best achieved on ½ MS medium without vitamins plus 2 µM TDZ. The conversion of protocorms into seedlings was maximized by supplementation with 2 µM IBA or 1 µM NAA. Acclimatized plantlets that exhibited exuberant growth on sphagnum moss were reintroduced to tree trunks in a natural habitat, with a 67% survival rate. This in vitro propagation procedure would be helpful for the mass production and conservation of this rare epiphytic orchid. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Effect of Gibberellic Acid on Production of Biomass, Polyphenolics and Steviol Glycosides in Adventitious Root Cultures of Stevia rebaudiana (Bert.)
Plants 2020, 9(4), 420; https://doi.org/10.3390/plants9040420 - 30 Mar 2020
Abstract
In current study, the effect of gibberellic acid was tested for production of biomass, polyphenolics and Steviol glycosides in adventitious root cultures of Stevia rebaudiana. Adventitious cultures were induced from the roots of in vitro grown plantlets on Murashige and Skoog (MS) [...] Read more.
In current study, the effect of gibberellic acid was tested for production of biomass, polyphenolics and Steviol glycosides in adventitious root cultures of Stevia rebaudiana. Adventitious cultures were induced from the roots of in vitro grown plantlets on Murashige and Skoog (MS) medium containing combination of gibberellic acid (GA3; 0.5, 1.0, 1.5 and 2.0 mg/L) and naphthalene acetic acid (NAA; 0.5 mg/L). Initially, a known mass of inoculum roots were shifted into suspension media augmented with various GA3 concentrations. The growth behavior of adventitious roots was recorded every 3 days for a period of 30 days. Maximum biomass biosynthesis (13.12 g/flask) was noticed in exponential phase on 27th day in the suspension containing 2.0 mg/L of GA3. Other GA3 concentrations also displayed optimum patterns of biomass accumulation as compared to the control. Adventitious roots were investigated for total phenolic content (TPC) and production (TPP), total flavonoid content (TFC) and production (TFP), and 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-based antioxidant potential. Maximum phenolics (TPC 9.84 mg gallic acid equivalent (GAE)/g-dry weight (DW)) and TPP (147.6 mg/L), TFC (5.12 mg Quercitin equivalent (QE)/g-DW) and TFP (76.91 mg/L) were observed in 2.0 mg/L GA3 treated cultures. The same concentration of gibberellic acid enhanced antioxidant activity (77.2%). Furthermore, maximum stevioside (7.13 mg/g-DW), rebaudioside-A (0.27 mg/g-DW) and dulcoside-A (0.001 mg/g-DW) were observed in roots exposed to 2.0 mg/L GA3. This is the first report on the application of GA3 on biomass accumulation and secondary metabolite production in S. rebaudiana. The current study will be helpful to scale up the adventitious root cultures in bioreactors for the production of biomass and pharmaceutically important secondary metabolites. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Feasible Production of Lignans and Neolignans in Root-Derived In Vitro Cultures of Flax (Linum usitatissimum L.)
Plants 2020, 9(4), 409; https://doi.org/10.3390/plants9040409 - 25 Mar 2020
Abstract
Flax lignans and neolignans impart health benefits, particularly in treating different types of cancers, due to their strong phytoestrogenic and antioxidant properties. The present study enhances the comprehension on the biosynthesis of antioxidant lignans and neolignans in root-derived in vitro cultures of flax [...] Read more.
Flax lignans and neolignans impart health benefits, particularly in treating different types of cancers, due to their strong phytoestrogenic and antioxidant properties. The present study enhances the comprehension on the biosynthesis of antioxidant lignans and neolignans in root-derived in vitro cultures of flax (both callus and adventitious root). The results presented here clearly showed that the adventitious root culture efficiently produced a higher amount of lignans (at day 40) and neolignans (at day 30) than callus culture of flax. High performance liquid chromatography (HPLC) analysis revealed that the accumulations of secoisolariciresinol diglucoside (SDG, 5.5 mg g−1 DW (dry weight)) and dehydrodiconiferyl alcohol glucoside (DCG, 21.6 mg/g DW) were 2-fold higher, while guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG, 4.9 mg/g DW) and lariciresinol glucoside (LDG, 11.9 mg/g DW) contents were 1.5-fold higher in adventitious root culture than in callus culture. Furthermore, the highest level of total phenolic production (119.01 mg/L), with an antioxidant free radical scavenging activity of 91.01%, was found in adventitious root culture at day 40, while the maximum level of total flavonoid production (45.51 mg/L) was observed in callus culture at day 30 of growth dynamics. These results suggest that adventitious root culture can be a good candidate for scaling up to industrial level to commercially produce these pharmacologically and nutritionally valuable metabolites. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Somatic Embryogenesis and Plantlet Regeneration in the Carica papaya L. cv. Eksotika
Plants 2020, 9(3), 360; https://doi.org/10.3390/plants9030360 - 12 Mar 2020
Abstract
A highly efficient protocol for regeneration of Carica papaya L. cv. Eksotika somatic embryos from immature zygotic embryos was developed. This study was designed to overcome the obstacles in regeneration of somatic embryos from immature zygotic embryos of “Eksotika”, especially problems associated with [...] Read more.
A highly efficient protocol for regeneration of Carica papaya L. cv. Eksotika somatic embryos from immature zygotic embryos was developed. This study was designed to overcome the obstacles in regeneration of somatic embryos from immature zygotic embryos of “Eksotika”, especially problems associated with formation of better root quality and callus formation at the base of somatic embryos. Somatic embryos were generated by incubation of immature zygotic embryos in half-strength salt Murashige and Skoog (MS) medium with full-strength vitamins supplemented with 7.5 mg L−1 2,4-D, 100 mg L−1 L-glutamine, 50 mg L−1 myo-inositol, 45 mg L−1 adenine sulphate, 0.33% gelrite, and 6% sucrose, followed by transfer to maturation medium consisting of ½ MS medium supplemented with 5 mg L−1 phloroglucinol, 100 mg L−1 L-glutamine, 100 mg L−1 myo-inositol, 68 mg L−1 adenine sulphate, 0.38% gelrite, and 3% sucrose. After that, well-formed somatic embryos were transferred to MS medium containing 3% sucrose and 0.8% agar for shoot production. The embryos were elongated in MS medium supplemented with 1 mg L−1 gibberellic acid, 0.5 mg L−1 indole-3-butyric acid, 100 mg L−1 myo-inositol, and 3.76 mg L−1 riboflavin. Root regeneration was achieved on MS medium containing 7.9 mg L−1 phloroglucinol and supported with vermiculite after 4 days of cultivation on ½ MS medium with 2 mg L−1 indole-3-butyric acid. After the rooting phase, in vitro plantlets were acclimatized in peat moss soil. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Synergistic Effect of NaCl Pretreatment and PVP on Browning Suppression and Callus Induction from Petal Explants of Paeonia Lactiflora Pall. ‘Festival Maxima’
Plants 2020, 9(3), 346; https://doi.org/10.3390/plants9030346 - 09 Mar 2020
Abstract
Browning is prevalent in tissue cultures of Paeonia lactiflora Pall. (herbaceous peony), and severely affects and restricts the growth and differentiation of the explants. In this study, dipping excised explants in a sodium chloride (NaCl) solution as a pretreatment, adding polyvinyl pyrrolidone (PVP) [...] Read more.
Browning is prevalent in tissue cultures of Paeonia lactiflora Pall. (herbaceous peony), and severely affects and restricts the growth and differentiation of the explants. In this study, dipping excised explants in a sodium chloride (NaCl) solution as a pretreatment, adding polyvinyl pyrrolidone (PVP) to the culture medium, storing planted explants at 4 °C for 24 h, and transferring planted explants to a new medium after 24 h were considered as browning-suppression methods in tissue cultures of herbaceous peony ‘Festival Maxima’. The treated petal explants were cultured in a culture room with a 16-hour photoperiod, 25 °C temperature, and 80% relative humidity in darkness for 4 to 8 weeks. The results demonstrated that dipping excised explants in a 0.5 g·L−1 NaCl solution, adding 0.5 g·L−1 PVP to the medium, storing planted explants at 4 °C for 24 h, and transferring planted explants to the same fresh medium after 24 h could effectively inhibit browning. Adding PVP to the medium led to the greatest browning suppression percentage of 95%. Storing planted explants at 4 °C for 24 h reduced the effectiveness of other treatments in suppressing browning. After 8 weeks, dipping excised explants in a NaCl solution resulted in the highest callus induction percentage of 75%, while storing explants at 4 °C for 24 h suppressed callus formation. It was observed in all treatments that decreases in browning was accompanied with higher levels of phenols and lower activities of phenylalanine ammonia-lyase (PAL) and polyphenoloxidase (PPO). Overall, the results suggest that dipping in a NaCl solution was effective in alleviating the browning issues of herbaceous peony tissue cultures, and had positive synergistic effects with PVP on browning suppression and callus induction. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
In Vitro Regeneration Potential of White Lupin (Lupinus albus) from Cotyledonary Nodes
Plants 2020, 9(3), 318; https://doi.org/10.3390/plants9030318 - 03 Mar 2020
Abstract
The tissue culture regeneration system of Lupinus albus has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful [...] Read more.
The tissue culture regeneration system of Lupinus albus has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful application of genetic engineering. This aim of this study was to develop standardized, efficient protocol for successful shoot induction from cotyledonary node of white lupin. In this study, 5 day old aseptically cultured seedlings were used to prepare three explants (half cotyledonary node, HCN; whole cotyledonary node, WCN; and traditional cotyledonary node, TCN), cultured on four concentrations of M519 medium (M519, ½ M519, 1/3 M519, and ¼ M519), containing four carbohydrate sources (sucrose, fructose, maltose, and glucose), and stimulated with various combinations of KT (kinetin), and NAA (naphthalene acetic acid) for direct shoot regeneration. High frequency of 80% shoot regeneration was obtained on ½ M519 medium (KT 4.0 mg L−1 + NAA 0.1 mg L−1) by using HCN as an explant. Interestingly, combinations of (KT 4.0 mg L−1 + NAA 0.1 mg L−1 + BAP 1.67 mg L−1), and (KT 2.0 mg L−1 + NAA 0.1 mg L−1) showed similar shoot regeneration frequency of 60%. Augmentation of 0.25 g L−1 activated charcoal (AC) not only reduced browning effect but also improved shoot elongation. Among the all carbohydrate sources, sucrose showed the highest regeneration frequency with HCN. Additionally, 80% rooting frequency was recorded on ½ M519 containing IAA 1.0 mg L−1 + KT 0.1 mg L−1 (indole acetic acid) after 28 days of culturing. The present study describes establishment of an efficient and successful protocol for direct plant regeneration of white lupin from different cotyledonary nodes. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Comparative Analysis of In Vitro Responses and Regeneration between Diverse Bioenergy Sorghum Genotypes
Plants 2020, 9(2), 248; https://doi.org/10.3390/plants9020248 - 14 Feb 2020
Abstract
Sorghum has been considered a recalcitrant plant in vitro and suffers from a lack of regeneration protocols that function broadly and efficiently across a range of genotypes. This study was initiated to identify differential genotype-in vitro protocol responses across a range of bioenergy [...] Read more.
Sorghum has been considered a recalcitrant plant in vitro and suffers from a lack of regeneration protocols that function broadly and efficiently across a range of genotypes. This study was initiated to identify differential genotype-in vitro protocol responses across a range of bioenergy sorghum parental lines and the common grain sorghum genotype Tx430 in order to characterize response profiles for use in future genetic studies. Two different in vitro protocols, LG and WU, were used for comparisons. Distinct genotype-protocol responses were observed, and the WU protocol performed significantly better for plantlet regeneration. Most bioenergy genotypes performed as well, if not better than Tx430, with Rio and PI329311 as the top regenerating lines. Genotypes displayed protocol-dependent, differential phenolic exudation responses, as indicated by medium browning. During the callus induction phase, genotypes prone to medium browning exhibited a response on WU medium which was either equal or greater than on LG medium. Genotype- and protocol-dependent albino plantlet regeneration was also noted, with three of the bioenergy genotypes showing albino plantlet regeneration. Grassl, Rio and Pink Kafir were susceptible to albino plantlet regeneration, with the response strongly associated with the WU protocol. These bioenergy parental genotypes, and their differential responses under two in vitro protocols, provide tools to further explore and assess the role of genetic loci, candidate genes, and allelic variants in the regulation of in vitro responsiveness in sorghum. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of Viola serpens Wall. ex Ging
Plants 2020, 9(2), 246; https://doi.org/10.3390/plants9020246 - 14 Feb 2020
Abstract
An efficient protocol of plant regeneration through indirect organogenesis in Viola serpens was developed in the present study. Culture of leaf explants on MS (Murashige and Skoog) medium supplemented with 2.0 mg/L 6-benzyladenine and 0.13 mg/L 2,4-dichloro phenoxy acetic acid. Adventitious shoot formation [...] Read more.
An efficient protocol of plant regeneration through indirect organogenesis in Viola serpens was developed in the present study. Culture of leaf explants on MS (Murashige and Skoog) medium supplemented with 2.0 mg/L 6-benzyladenine and 0.13 mg/L 2,4-dichloro phenoxy acetic acid. Adventitious shoot formation was observed when calli were transferred on to MS medium containing 0.5 mg/L α-naphthalene acetic acid and 2.25 mg/L kinetin, which showed the maximum 86% shoot regeneration frequency. The highest root frequency (80.92%) with the 5.6 roots per explant and 1.87 cm root length was observed on MS medium supplemented with 2 mg/L indole-3-butyric acid. The plantlets were transferred to the mixture of sand, coffee husk and soil in the ratio of 1:2:1 in a pot, and placed under 80% shade net for one month. It was then transferred to 30% shade net for another one month, prior to transplantation in the field. These plantlets successfully acclimatized under field conditions. A Sequence Characterized Amplified Region (SCAR) marker was also developed using a 1135 bp amplicon that was obtained from RAPD (Random Amplification of Polymorphic DNA) analysis of six accessions of V. serpens. Testing of several market samples of V. serpens using the SCAR marker revealed successful identification of the genuine samples of V. serpens. This study, therefore, provides a proficient in vitro propagation protocol of V. serpens using leaf explants and a SCAR marker for the authentic identification of V. serpens. This study will be helpful for conservation of authentic V. serpens. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
An Efficient Method for In Vitro Shoot-Tip Culture and Sporophyte Production Using Selaginella martensii Spring Sporophyte
Plants 2020, 9(2), 235; https://doi.org/10.3390/plants9020235 - 12 Feb 2020
Cited by 1
Abstract
Selaginella martensii, an evergreen perennial fern that is native to South America and New Zealand, is named “frosty fern” because of its beautiful white-colored leaves and it is used as an ornamental plant. Efficient propagation methods for this species have not been [...] Read more.
Selaginella martensii, an evergreen perennial fern that is native to South America and New Zealand, is named “frosty fern” because of its beautiful white-colored leaves and it is used as an ornamental plant. Efficient propagation methods for this species have not been developed. We aimed to develop an efficient propagation method for S. martensii through in vitro culture. We investigated culture conditions that are suitable for shoot-tip proliferation and growth. The optimum shoot-tip culture conditions were determined while using Murashige and Skoog (MS) medium (quarter, half, full, or double strength) and macronutrients (sucrose and two nitrogen sources) at various concentrations. In MS medium, the shoot tips formed a maximum of 6.77 nodes per explant, and each node formed two new shoot tips (i.e., 26 or 64 shoot tips). When using branching segments containing an angle meristem, the shoot-to-rhizophore formation ratio could be controlled by medium supplementation with plant-growth regulators. Sporophytes that were grown from shoot tips in vitro were acclimated in ex vitro soil conditions and successfully survived in the greenhouse. Numerous shoot tips could be obtained from in vitro-grown sporophytes and be proliferated ex vitro to produce a large number of plants. This method provides a way of shortening the time that is required for producing a large stock of S. martensii planting material. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Enhanced Somatic Embryo Induction of a Tree Peony, Paeonia ostii ‘Fengdan’, by a Combination of 6-benzylaminopurine (BA) and 1-naphthylacetic Acid (NAA)
Plants 2020, 9(1), 3; https://doi.org/10.3390/plants9010003 - 18 Dec 2019
Cited by 2
Abstract
Somatic embryogenesis is a preferred method for vegetative propagation due to its high propagation efficiency. In this study, zygotic embryos, cotyledons, and hypocotyls of Paeonia ostii ‘Fengdan’ were used as the explant to induce somatic embryogenesis. The results showed that a combination of [...] Read more.
Somatic embryogenesis is a preferred method for vegetative propagation due to its high propagation efficiency. In this study, zygotic embryos, cotyledons, and hypocotyls of Paeonia ostii ‘Fengdan’ were used as the explant to induce somatic embryogenesis. The results showed that a combination of 0.5 mg·L−1 thidiazuron (TDZ) and 0.5 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D) was effective in inducing somatic embryos from the zygotic embryo and cotyledon explants. Hypocotyls only formed somatic embryos on Murashige and Skoog (MS) medium supplemented with both 0.5 mg·L−1 TDZ and 0.5 mg·L−1 1-naphthylacetic acid (NAA). Moreover, the compact callus was effectively produced from zygotic embryo, cotyledon, and hypocotyl explants in medium supplemented with a combination of 3.0 mg·L−1 6-benzylaminopurine (BA) and 1.0 mg·L−1 NAA, and then converted into somatic embryos in the same medium, and the ratio of the explants with embryo induction and number of embryos induced per explant were much higher than those induced by 0.5 mg·L−1 TDZ and either 0.5 mg·L−1 2,4-D or 0.5 mg·L−1 NAA. The MS medium was better than the woody plant medium (WPM) for inducing somatic embryos from zygotic embryo and hypocotyl explants, whereas the WPM was better than the MS medium for somatic embryogenesis induction from cotyledon explants. All of the somatic embryos developed well into mature embryos on their respective media supplemented with both 3.0 mg·L−1 BA and 1.0 mg·L−1 NAA. Overall, the protocols for indirect somatic embryogenesis from zygotic embryo, cotyledon, and hypocotyl of P. ostii ‘Fengdan’ were successfully established, which can greatly facilitate their propagation and breeding processes. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessArticle
Investigating the In Vitro Regeneration Potential of Commercial Cultivars of Brassica
Plants 2019, 8(12), 558; https://doi.org/10.3390/plants8120558 - 29 Nov 2019
Cited by 2
Abstract
In vitro regeneration is a pre-requisite for developing transgenic plants through tissue culture-based genetic engineering approaches. Huge variations among different genotypes of the genus Brassica necessitate the identification of a set of regeneration conditions for a genotype, which can be reliably used in [...] Read more.
In vitro regeneration is a pre-requisite for developing transgenic plants through tissue culture-based genetic engineering approaches. Huge variations among different genotypes of the genus Brassica necessitate the identification of a set of regeneration conditions for a genotype, which can be reliably used in transformation experiments. In this study, we evaluated the morphogenesis potential of four commercial cultivars (Faisal canola, Punjab canola, Aari canola, Nifa Gold) and one model, Westar, from four different explants namely cotyledons, hypocotyls, petioles and roots on three different Brassica regeneration protocols, BRP-I, -II and -III. The regeneration efficiency was observed in the range of 6–73%, 4–79.3%, 0–50.6%, and 0–42.6% from cotyledons, petioles, hypocotyls and roots, respectively, whereas, the regeneration response in terms of average shoots per explant was found to be 0.76–10.9, 0.2–3.2, 0–3.4 and 0–2.7 from these explants. Of the commercial varieties tested, almost all varieties showed poorer regeneration than Westar except Aari canola. In comparison to Westar, its regeneration frequency from cotyledons was up to 7.5-fold higher on BRP-I, while it produced up to 21.9-fold more shoots per explant. Our data show that the explant has strong influence on the regeneration response, ranging from 24% to 92%. While the growth of commercial cultivars was least affected by the regeneration conditions provided, the effect on Westar was twice that of the commercial cultivars. After determining the optimal explant type and regeneration conditions, we also determined the minimum kanamycin concentration levels required to selectively inhibit the growth of untransformed cells for these cultivars. Regenerated shoots of Aari canola could be successfully grown to maturity within 16–18 weeks, with no altered phenotype noted and normal seed yields obtained. Therefore, the commercial variety, Aari canola, could be a good candidate for future genetic transformation studies. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Review

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Open AccessReview
In Vitro Propagation, Phytochemical and Neuropharmacological Profiles of Bacopa monnieri (L.) Wettst.: A Review
Plants 2020, 9(4), 411; https://doi.org/10.3390/plants9040411 - 26 Mar 2020
Cited by 1
Abstract
Bacopa monnieri has been used as a reputed drug in the Indian traditional ayurvedic system for centuries. This medicinal herb with important phytopharmaceuticals has been popularly known as “Brahmi”. In recent years, B. monnieri has been extensively studied for its bioactive constituents, constituents [...] Read more.
Bacopa monnieri has been used as a reputed drug in the Indian traditional ayurvedic system for centuries. This medicinal herb with important phytopharmaceuticals has been popularly known as “Brahmi”. In recent years, B. monnieri has been extensively studied for its bioactive constituents, constituents responsible for memory enhancing effect, and also its diverse other useful effects. It possesses many pharmacological activities such as antioxidant, gastrointestinal, endocrine, antimicrobial, anti-inflammatory etc. The plant has been also used for the treatment of neurological and neuropsychiatric diseases. Due to its multipurpose therapeutic potential, micropropagation using axillary meristems and de novo organogenesis has been extensively studied in the species and is being reviewed. High frequency direct shoot organogenesis can be induced in excised leaf and internode explants in the absence of exogenous phytohormones and the rate of induction is enhanced in the presence of exogenous cytokinins, supplements, growth regulators, etc. Using explants from tissue culture raised plants, direct shoot regeneration leading to production of more than 100 rooted plants/explant within 8–12 weeks period with 85%–100% survival in the field after acclimatization can be expected following optimized protocols. Bioreactor based micropropagation was found to increase the multiplication rate of shoot cultures for the commercial propagation of B. monnieri plants. The maximum content of bacosides has been recorded in shoot biomass using an airlift bioreactor system. Further studies for the biosynthesis of bacosides and other secondary metabolites need to be conducted in the species utilizing untransformed shoot cultures in bioreactors. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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Open AccessReview
Endemic Plant Species Conservation: Biotechnological Approaches
Plants 2020, 9(3), 345; https://doi.org/10.3390/plants9030345 - 09 Mar 2020
Cited by 7
Abstract
Endemic plant species are usually more vulnerable to anthropogenic threats and natural changes and, therefore, hold a higher extinction risk. The preservation of these species is a major concern on a worldwide context and in situ protection alone will not guarantee their conservation. [...] Read more.
Endemic plant species are usually more vulnerable to anthropogenic threats and natural changes and, therefore, hold a higher extinction risk. The preservation of these species is a major concern on a worldwide context and in situ protection alone will not guarantee their conservation. Ex situ conservation measures must be undertaken to support the conservation of these species, and seed banking is the more efficient and cost-effective method. However, when seed banking is not an option, alternative approaches should be considered. Biotechnological tools provide new and complementary options for plant conservation including short-, medium-, and long-term strategies, and their application for plant species conservation has increased considerably in the last years. This review provides information about the status of the use biotechnology-based techniques for the conservation of endemic plant species. Particular attention is given to cryopreservation, since is the only long-term ex situ conservation strategy that can complement and support the other conservation measures. The cryopreservation of plant genetic resources is, however, more focused on crop or economically important species and few studies are available for endemic plant species. The plant material used, the cryopreservation methods employed, and the assessment of cryogenic effects are reviewed. The reasons to explain the difficulties in cryopreserving these species are discussed and new strategies are proposed to facilitate and increase the interest on this matter. We expect that further studies on the conservation of endemic plant species will increase in a near future, thus contributing to maintain these valuable genetic resources. Full article
(This article belongs to the Special Issue Plant Tissue Culture)
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