Recent Articles

Below are the titles and abstracts from the most recent articles published in In Vitro Cellular and Developmental Biology – Plant. Click on the title to view the full article at Springerlink.


  • A methodology for large-scale Athyrium sheareri gametophyte proliferation and sporophyte production using tissue culture
    on June 10, 2019 at 12:00 am

    Abstract Tissue culture methods using gametophytes are considered the easiest ways to mass-produce fern sporophytes. The aim of this study was to develop a practical propagation method for the ornamental fern, Athyrium sheareri. The gametophytes obtained from in vitro spore germination were used as experimental materials. We used the chopping method to investigate the culturing conditions for proliferating gametophytes and the blending method for evaluating the mass production of sporophytes in mixed soil. Gametophyte proliferation was determined via Knop medium, various concentrations of Murashige and Skoog (MS) basal medium (1, 1/2, 1/4), and media components (sucrose, nitrogen source, and activated charcoal). The fresh weight of the gametophytes increased by more than 24-fold in 1/2 MS medium. In addition, 1 g of gametophyte could produce a maximum of 255.3 sporophytes in a mixed soil of 7.5 cm2 area. Treating gametophytes with exogenous plant growth […]

  • Development of a new culture medium and efficient protocol for in vitro micropropagation of Ceratonia siliqua L.
    on June 10, 2019 at 12:00 am

    Abstract A new basal culture medium was developed and tested using a rapid and efficient protocol of in vitro axillary shoot bud proliferation of Ceratonia siliqua L., an important Mediterranean Fabaceae plant species. In a first experiment, the new formulated ‘LA’ mineral composition significantly improved shoot growth and proliferation as compared with Murashige and Skoog medium (MS, 1962) in both solid and liquid culture media. However, the liquid culture system proved to be the most suitable for shoot induction, shoot length (about fourfold higher), and multiplication rate (about two-fold higher), the difference being significant. The measured growth and proliferation parameters were further improved when LA mineral composition was optimized, in a second experiment. The highest multiplication rate (6.3) was achieved during the second subculture using the optimized ‘LAC’ medium. Noticeably, hyperhydricity and shoot-tip necrosis symptoms […]

  • Plant tissue culture media and practices: an overview
    on June 1, 2019 at 12:00 am

    Abstract This review presents an overview of the culture media and practices used in plant tissue culture and developmental biology. The compositions of the most commonly used basal media, especially Murashige and Skoog (MS) and modified MS (MMS), Gamborg’s B5 medium and B5 modifications, Woody Plant Medium (WPM), and Driver and Kuniyuki Woody plant medium (DKW) are discussed, along with typical basal medium manipulations to elicit and support various developmental responses. The most commonly used plant growth regulators and their applications to promote various developmental responses are examined, along with a presentation of the classical phytohormone developmental models for organogenesis and somatic embryogenesis. Elaborated developmental models for both organogenesis and somatic embryogenesis, with emphasis on discrete developmental steps, occasional need for multiple manipulations in culture to achieve a single developmental step, and identification […]

  • The in vitro propagation system of Citrus × latifolia (Yu. Tanaka) Yu. Tanaka (Rutaceae) affects the growth and depletion of nutriments
    on June 1, 2019 at 12:00 am

    Abstract Among citrus fruits, Citrus × latifolia (Yu. Tanaka) Yu. Tanaka stands out given its diverse uses, and there is a great demand for plants of high phytosanitary and genetic quality. In the present study, the in vitro micropropagation process of C. latifolia was studied in RITA®-type bioreactor, semisolid medium, and partial immersion with liquid culture medium. Explants that were 2 mo of age were used and apices of 0.5 cm were extracted and placed in each culture system. In all the systems, MS (Murashige and Skoog) culture medium was used, supplemented with 6-benzylaminopurine (BAP) and kinetin (KIN). After 30 d, the number of shoots, the size of the shoots, the number of leaves, and the depletion of nutrients from the culture medium were analyzed. With the data obtained, ANOVA and Tukey’s mean comparison tests were performed. The RITA® bioreactors, with the addition of 40 mL of medium per explant in a 4-h-interval […]

  • Bixa orellana L. (achiote) tissue culture: a review
    on June 1, 2019 at 12:00 am

    Abstract Bixa orellana L. (achiote) is a commercially important plant grown for its natural dye annatto, which is derived from the arils of seeds. Annatto, which contains the antioxidant bixin, is used in food, cosmetic, and textile industries as a natural colorant. Even though B. orellana can be propagated by seed, established tissue culture and in vitro propagation protocols exist for this plant. Organogeneses, both axillary and adventitious, as well as somatic embryogenesis, have been successfully induced from different explant sources, including cotyledons, hypocotyls, roots, stems, and leaves, and effective acclimatization protocols exist for regenerated plantlets. This present mini-review highlights the achievements in the tissue culture of achiote. In addition to using in vitro techniques for the clonal propagation of elite plants, knowledge from these protocols could be used to establish mass production systems, such as bioreactors, to facilitate year-round […]

  • A new temporary immersion system for commercial micropropagation of banana ( Musa AAA cv. Grand Naine)
    on June 1, 2019 at 12:00 am

    Abstract Temporary immersion systems (TIS) are among the best options for commercial micropropagation. However, although successes have been reported with TIS, it is necessary to establish the most suitable system. The aim of this research was to compare the efficiency of different banana (Musa AAA cv. Grand Naine) micropropagation systems. In addition, a liquid medium with partial immersion and a semi-solid culture medium were evaluated. At 28 d of culture, the number and length of shoots, number of leaves per shoot, chlorophyll content, stomatal index, and the percentage of closed stomata were recorded. Additionally, the survival percentage and ex vitro development of the plantlets were evaluated during acclimatization. Results showed that TIS have a higher multiplication rate with respect to partial immersion and semi-solid medium. The chlorophyll content, stomatal index, and stomata functioning were affected by the different culture systems. The different […]

  • Evaluation of canavanine as an allelochemical in etiolated seedlings of Vicia villosa Roth: protoplast co-culture method with digital image analysis
    on June 1, 2019 at 12:00 am

    Abstract Strong allelopathic activities of etiolated seedlings of a leguminous plant, Vicia villosa Roth (hairy vetch), were confirmed by a protoplast co-culture method. The combination of 1% Cellulase RS and 1% Driselase 20 in 0.4 M mannitol solution was used for protoplast isolation. The inhibitory effects of V. villosa protoplasts on the growth of recipient Lactuca sativa L. (lettuce) protoplasts were determined using 96-well culture plates with 50 μL of liquid medium consisting of Murashige and Skoog’s basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM 6-benzyladenine, 3% (w/v) sucrose, and 0.4 M mannitol in each well. The allelopathic activity of V. villosa epicotyl protoplasts (2 × 102 to 105 mL−1), on cell division of lettuce protoplasts (6 × 103 to 5 × 104 mL−1), was stronger than that of roots and was stronger than on […]

  • Wounding and medium formulation affect de novo shoot organogenic responses in hypocotyl-derived explants of annatto ( Bixa orellana L.)
    on June 1, 2019 at 12:00 am

    Abstract Bixa orellana L. (annatto) is the unique exploited source for the production of bixin and norbixin natural dyes, which are widely used in the food, pharmaceutical, cosmetic, and textile industries. In tissue culture, adequate culture conditions are essential for the development of the plant. Among them, the saline composition of the culture medium and the type of explants are limiting factors. In this study, the effect of MS (Murashige and Skoog), Correia and colleagues (JADS) medium, Woody Plant Medium (WPM), and Driver Kuniyuki Walnut (DKW) culture media and wounding of hypocotyl explants on de novo shoot organogenesis (DNSO) of annatto were evaluated. It was demonstrated that JADS medium improved the nutritional status of shoots, which promoted the best development and quality of the plants generated after acclimatization, when compared to MS medium. The use of JADS medium for annatto DNSO during 45 d of culture was sufficient to regenerate and […]

  • Goji berry ( Lycium barbarum L.) in vitro multiplication improved by light-emitting diodes (LEDs) and 6-benzylaminopurine
    on June 1, 2019 at 12:00 am

    Abstract In Brazil, Lycium barbarum L. (goji berry) is an agronomically valuable, imported, commercialized plant. The present study evaluated the effects of cytokinins and light sources on optimization of in vitro multiplication of goji berry by axillary bud proliferation. Nodal explants were excised from plants 90 d after in vitro germination and cultured on Murashige and Skoog (MS) medium containing 30 g L−1 sucrose; 7 g L−1 agar-agar; and 0, 2, 4, 8, or 16 μM 6-benzylaminopurine (BAP), 6-furfurylaminopurine (KIN), or thidiazuron (TDZ). After 60 days, regenerated lateral buds and shoot lengths were evaluated. The regenerated shoots were inoculated in MS supplemented with 0.0, 2.5, 5.0, 10.0, or 20.0 μM BAP under white fluorescent lamps or mixed treatment of red/blue (RB) light-emitting diode (LED) lamps. After 60 days in vitro cultivation, the number of formed shoots, leaf number, and shoot length […]

  • Increased freezing stress tolerance of Nicotiana tabacum L. cv. Bright Yellow-2 cell cultures with the medium addition of Ascophyllum nodosum (L.) Le Jolis extract
    on June 1, 2019 at 12:00 am

    Abstract The study of bioactive components of the brown alga Ascophyllum nodosum (L.) Le Jolis has shown that they promote growth and increase productivity of plants. However, a standardized model system providing consistent responses to such bioactive components has yet to be established. Given that freezing stress, especially in northern climates, reduces plant growth and productivity, a technique was developed to protect plant cells under freezing stress using the natural products made from the abundant A. nodosum as a cell culture supplement. In this study, a homogenous cell culture system of Nicotiana tabacum L. cultivar Bright Yellow-2 (BY-2) suspension cells was used to investigate the bioactivity and protection level of this alga extract under freezing temperatures, and BY-2 cell growth, physiology, and molecular changes were measured in the presence or absence of the extract. The results indicated that A. nodosum extract significantly improved BY-2 cell […]

  • Micropropagation of Nelumbo nucifera ‘Weishan Hong’ through germfree mature embryos
    on June 1, 2019 at 12:00 am

    Abstract Asian lotus (Nelumbo nucifera Gaertn.) is an economically important aquatic plant that is widely cultivated in Asia. It is mainly propagated by dividing rhizomes, which maintains genotype stability but is also laborious and time-consuming. An efficient in vitro method that ensures genetic stability is highly desirable. An optimized method was developed to produce intact and sterile mature embryos by soaking fruits in sterile water containing 2% (v/v) plant preservative mixture. Using mature embryos as explants, an improved micropropagation procedure was established in N. nucifera ‘Weishan Hong.’ The growth patterns of explants differed with the types and concentrations of cytokinins. Media containing 0.5 to 2.0 mg L−1 kinetin were suitable for elongation of unexpanded stems, while media with 1.0 to 2.0 mg L−1 6-benzylaminopurine (BAP) the stems were shorter. Higher concentrations of about […]

  • In vitro cloning via direct somatic embryogenesis and genetic stability assessment of Paphiopedilum niveum (Rchb.f.) Stein: the endangered Venus’s slipper orchid
    on June 1, 2019 at 12:00 am

    Abstract Wild populations of Paphiopedilum niveum (Rchb.f.) Stein, an endangered orchid species, have been threatened with extinction due to climate change and overcollection. Conventional methods of propagation are unable to meet market demands. The current study reports the production of genetically stable, protocorm-derived protocorm-like bodies (PLBs) of P. niveum that can be used for commercial production. These PLBs are referred to hereafter as somatic embryos (SEs). Factors affecting SE induction and SE proliferation were examined. The highest percentage of SE formation (68.33 ± 11.77%) and the maximum number of SEs per explant (5.19 ± 0.67) were obtained on modified Vacin and Went (MVW) medium containing 0.1 mg L−1 1-naphthaleneacetic acid (NAA). The highest increase in fresh weight (FW) of proliferated SEs (183.33 ± 28.93 mg per 100 mg initial FW) was gained on […]

  • Physiological and morphological analyses of Thymus vulgaris L. in vitro cultures under polyethylene glycol (PEG)-induced osmotic stress
    on June 1, 2019 at 12:00 am

    Abstract Morphological and physiological parameters of polyethylene glycol (PEG)-induced drought-stressed Thymus vulgaris L. (thyme) seedlings were examined. To start in vitro selection for drought stress tolerance and to provide a more efficient method for the production of pharmaceutical substances, callus induction and production of essential oils were analyzed. Thyme seeds were germinated on medium containing 0, 2, 4, 6, and 8% (w/v) PEG; and, for callus induction, stem explants were cultured on medium supplemented with 1 mg L−1 2,4-dichlorophenoxyacetic acid with different concentrations of PEG. Four weeks after PEG-induced water stress, a significant decrease in seedling growth, chlorophyll and carotenoid contents, and callus weight and induction was observed. However, no significant difference was found in the germination rate of seeds. Organic osmolytes, e.g., proline and carbohydrates, were influenced by PEG-induced osmotic stress. Reducing […]

  • In vitro storage of micropropagated grapevine rootstocks at low temperature
    on June 1, 2019 at 12:00 am

    Abstract A long-term in vitro conservation protocol for five micropropagated grapevine rootstocks (3309 Couderc, 110 Richter, Vitis riparia Gloire de Montpellier, 101-14 Millardet et De Grasset, and Teleki 5C) was developed and optimized by testing the effect of culture containers (polyethylene bags versus glass test tubes), explant types (rooted versus unrooted cuttings), low-temperature regimes (1.6, 4.0, and 7.2°C), and light intensity (low versus dark). Optimal storage conditions were achieved at 4.0°C and low light intensity (3 μmol m−2 s−1), with rooted plantlets established on woody plant medium (WPM) supplemented with 15 g L−1 sucrose and 37 mg L−1 cysteine in polyethylene bags. After 12 and 24 mo of optimal cold storage, the plant condition ratings (3.5 and 2.5 on a scale of 4) and survival rates (75% and 52%) were high, regardless of the rootstock genotype, as determined by visual […]

  • Use of antibiotics to control endophytic bacterial growth migration onto culture medium in Eucalyptus cloeziana F.Muell.: a micropropagation approach
    on May 23, 2019 at 12:00 am

    Abstract The natural endophytic bacterial growth migration onto a culture medium is commonly associated with unnecessary microplant discards. In micropropagation procedures, some bacteria can exude from the internal tissues of plants to colonize the culture medium and compete with the plants for nutrients, which may lead to a reduction in plant development. To find an efficient antibiotic protocol to control this bacterial growth migration onto the culture medium without affecting plant development, Eucalyptus cloeziana F.Muell. microstumps were subjected to four antibiotic treatments for 30 d. They were treated with gentamicin, ciprofloxacin, rifampicin, or Timentin®, in addition to the control treatment (antibiotic free). The effects of the antibiotics were monitored weekly, and the endophytic bacterial community structures were evaluated in two periods of plant development (15 d and 30 d).The denaturing gradient gel electrophoresis (DGGE) […]

  • Iron nanoparticles and potassium silicate interaction effect on salt-stressed grape cuttings under in vitro conditions: a morphophysiological and biochemical evaluation
    on May 20, 2019 at 12:00 am

    Abstract As two newly important components for plant tissue culture, the impacts of iron nanoparticle and potassium silicate were studied on the regeneration and growth of grape cuttings var. Khoshnaw under salinity condition. The treatments consisted of salinity stress (0, 50, and 100 mM NaCl), iron nanoparticles (0.0, 0.08, and 0.8 ppm) and potassium silicate (0, 1, and 2 mM) under an in vitro environment. The overall results indicated that salinity significantly (p ≤ 0.05) increased soluble carbohydrates and carotenoid contents. On one hand, it reduced all studied morphological and physiological traits including shoot number, shoot and root length, shoot and root fresh weight, root volume, and leaf area, along with relative water content (RWC) and chlorophylls’ content. On the other hand, the application of iron nanoparticles and potassium silicate, alone or in combination, could significantly compensate the deleterious effects […]

  • Factors inducing regeneration response in oat ( Avena sativa L.) anther culture
    on May 20, 2019 at 12:00 am

    Abstract The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base […]

  • In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis
    on May 20, 2019 at 12:00 am

    Abstract Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic […]

  • Morphogenesis and in vitro production of caffeoylquinic and caffeic acids in Baccharis conferta Kunth
    on April 23, 2019 at 12:00 am

    Abstract We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and […]

  • Histo-morphological analysis of rice callus cultures reveals differential regeneration response with varying media combinations
    on April 12, 2019 at 12:00 am

    Abstract Genetic transformation of most indica rice (Oryza sativa) cultivars is hampered by poor in vitro culture performance and low regeneration potential. Histological study of primary calli can provide substantial information on their regeneration potential and can be used for early grading of calli expected to develop plantlets on regeneration media. The study was aimed to undertake histological analysis of primary calli derived from mature seeds of five indica rice cultivars viz. KSK-133, KS-282, Shaheen Basmati, Super Basmati, and DilRosh in order to assess their regeneration potential on different media combinations supplemented with various hormone concentrations (N6 + 2 mg/L 2,4-Dichlorophenoxyacetic acid; N6 + 2 mg/L 2–4 D + 2 mg/L Benzylaminopurine and MS + 2 mg/L 2,4-D). Calli with regeneration capability were subjected to histological assays by examining toulidine blue […]

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