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Cited by 32Plant tissue culture and regenerationPlant Stress Responses and ToleranceBanana Cultivation and Research

Ex vitro acclimatization of plantain plantlets micropropagated in temporary immersion bioreactor

Open access
Aragón, C., Carvalho, L., González, J., Escalona, M., Amâncio, S.·2010·Biologia plantarum
DOI 10.1007/s10535-010-0042-y
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Abstract

Plantain (Musa ABB CEMSA 3/4) plantlets were micropropagated in temporary immersion bioreactors (TIB) or in gelled medium (GM). After ex vitro transfer ROS accumulation was determined by infiltrating leaves with nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB). Stomatal cells were more stained with NBT and DAB in GM plants than in TIB plants, but the difference disappeared at the end of acclimatization. At the end of the in vitro phase, GM plantlets showed higher activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR), while activities of catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GT) were higher in TIB grown plantlets. At the end of acclimatization GT, SOD, CAT and MDHAR stabilized at low values of activity in plantlets derived from both treatments. Concerning the correspondent genes, GM plantlets showed higher expression of all transcripts with the exception of CuZnSOD. The immunobloting of peroxiredoxins (PRXs) showed that chloroplast-located PRXs were expressed at higher levels in TIB plantlets, some showing polymerization. In conclusion, TIB grown plantlets had an improved anti-oxidative response when compared with GM.

AI snapshot

Written from the paper’s full text.

TL;DR

Plantain plantlets grown in temporary immersion bioreactors (TIB) showed a stronger antioxidative response and less ROS accumulation after ex vitro transfer than plantlets grown on gelled medium (GM). By the end of acclimatization, antioxidant enzyme activities in GM and TIB plantlets converged, but TIB plantlets had higher chloroplast peroxiredoxin expression and appeared better prepared for ex vitro conditions.

Key findings

  • Stomatal guard cells were more stained with NBT and DAB in GM plants than in TIB plants after ex vitro transfer, but the difference disappeared at the end of acclimatization.
  • At the end of the in vitro phase, GM plantlets had higher activities of APX, DHAR, MDHAR and GR, while CAT, SOD and GT activities were higher in TIB-grown plantlets. In Table 2, relative CAT activity was 116% in TIB versus 100% in GM at day 0, while SOD-B was 72% in TIB versus 100% in GM.
  • At the end of acclimatization, GT, SOD, CAT and MDHAR stabilized at low activities in plantlets from both treatments; exceptions were APX, which remained rather high in GM, and DHAR, which remained rather high in TIB.
  • GM plantlets showed higher expression of all monitored genes except CuZnSOD, which increased significantly only on day 21. Immunoblotting showed chloroplast-located PRXs were expressed at higher levels in TIB plantlets, with some polymerization; 2-cys PRX was hardly detected in GM and highest on day 7 in TIB.

Methods

  • Plantain (Musa ABB CEMSA 3/4) plantlets were micropropagated through three 21-d subculture cycles; the last subculture used either GM in Magenta vessels (50 cm³ medium, 5 shoots/vessel) or TIB in containers (200 cm³ medium, 5 shoots/bioreactor, immersed 4 min every 3 h), with PPFD 45 ± 5 µmol m⁻² s⁻¹ and a 16-h photoperiod.
  • After transfer to ex vitro conditions, microcuttings were placed in pots with moistened peat and Perlite (1:1, v/v) in glass chambers at PPFD 200 ± 10 µmol m⁻² s⁻¹, 16-h photoperiod, initial RH 98% decreased to ambient, and 25 ± 2 °C light / 22 ± 1 °C dark. Sampling occurred at day 0, days 1–7, and days 14 and 21.
  • Analyses included ROS detection by NBT and DAB infiltration, stomatal and trichome indices, enzyme activity assays for APX, DHAR, MDHAR, GR, SOD, CAT, GT and PRX, native PAGE activity staining, PRX immunoblotting, and real-time RT-PCR normalized to Act2. Experiments were performed three times with 3 repetitions, and statistics used non-parametric Kruskal-Wallis or Mann-Whitney tests at 5% significance.

Limitations

  • The paper does not state an explicit limitations section.
  • Evident from the text: the study is limited to one plantain cultivar (Musa ABB CEMSA 3/4) and a 21-d ex vitro acclimatization period, so generalization to other cultivars, species, or longer field performance is not established.
  • Evident from the text: conclusions rely on physiological, biochemical, transcript and immunoblot measurements rather than direct agronomic or yield outcomes.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1007/s10535-010-0042-y

Cites

39 of 39 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

2 of 10 cited sources checked · 1 supported · 0 flagged · accuracy not yet measured

  • Interpretationcitation

    The polymerization strategy of Type II PRX-E on day 7 is a response to stress conditions.

    “The polymerization strategy of Type II PRX-E on day 7 is a response to stress conditions (Konig et al. 2002).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Konig et al. 2002 source

      In “Chloroplast 2-Cys Prx Catalyzes Trx-Dependent Peroxide Reduction” (page unavailable), the paper reports that salt stress caused oligomers of barley 2-Cys Prx to appear and suggests this may help detoxify peroxides. It does not establish the claim for Type II PRX-E or for day 7; Figure 7 instead describes two days of salt stress. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Findingown data

    The adaxial side showed the highest stomatal index in in vitro GM propagated plantlets.

    “The adaxial side showed the highest stomatal index in in vitro GM propagated plantlets”
    Quote in the paperNot a substantial claim
  • Findingown data

    CuZnSOD expression only increased significantly on day 21.

    “CuZnSOD which only increased significantly on day 21.”
    Quote in the paperNot a substantial claim
  • Findingown data

    At the end of the in vitro phase, TIB plantlets showed higher activities of CAT, SOD and GT than GM plantlets.

    “while activities of catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GT) were higher in TIB grown plantlets.”
    • Also stated in: § Results · plain
      “TIB plantlets evidenced higher SOD, CAT, PRX and GT activities (Fig. 3).”
    Quote in the paperSubstantial claim
  • Findingown data

    At the end of the in vitro phase, GM plantlets showed higher activities of APX, DHAR, MDHAR and GR than TIB plantlets.

    “At the end of the in vitro phase, GM plantlets showed higher activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR), while activities of catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GT) were higher in TIB grown plantlets.”
    • Also stated in: § Results · plain
      “The activities of the ascorbate-glutathione cycle enzymes APX, DHAR, MDHAR and GR were higher in GM plantlets on day 0 while TIB plantlets evidenced higher SOD, CAT, PRX and GT activities (Fig. 3).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 3
  • May combine two claims. 12

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The transition of plantlets to ex vitro conditions under high irradiance can lead to production of reactive oxygen species (ROS).

    “Moreover, the transition of the plantlets to an ex vitro conditions under high irradiance can lead to the production of reactive oxygen species (ROS; for review see Baťková et al. 2008).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Baťková et al. 2008 source

  • May combine two claims.

    Findingown data

    CAT and MnSOD showed peaks of expression on day 3 while the expression of APX was constantly high.

    “CAT and MnSOD showed peaks of expression on day 3 while the expression of APX was constantly high.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    2-cys PRX was hardly detected in GM grown plantlets and showed the highest quantity on day 7 in TIB.

    “2-cys PRX was hardly detected in GM grown plantlets and showed the highest quantity on day 7 in TIB.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The cytosol PRX II-C was present in TIB plantlets from day 0 while in GM it was only detected after some days after ex vitro transfer.

    “The cytosol PRX II-C was present in TIB plantlets from day 0 while in GM it was only detected after some days after ex vitro transfer.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both SOD A and SOD B were present at higher levels in GM than in TIB on day 0, decreasing slightly with time.

    “Both were present at higher levels in GM than in TIB on day 0, decreasing slightly with time.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The staining pattern was mostly similar in GM and TIB propagated plantlets with the exception of high staining percentage in guard cells on the abaxial side of in vitro GM plantlets.

    “The staining pattern was mostly similar in GM and TIB propagated plantlets with the exception of high staining percentage in guard cell on the abaxial side of in vitro GM plantlets.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    With the exception of GR and PRX, all enzymes studied reached their maximum activity between days 3 and 5 in GM propagated plantlets, decreasing to minimum values on day 7.

    “With the exception of GR and PRX, all the enzymes studied reached their maximum activity between days 3 and 5 in GM propagated plantlets, decreasing to minimum values on day 7.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    CAT showed one isoenzyme with higher activity in TIB, and it was unaltered with time.

    “CAT showed one isoenzyme with higher activity in TIB, and it was unaltered with time.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Two isoenzymes of APX were detected; APX-A activity decreased with time while APX-B activity increased slightly in both treatments.

    “It was possible to detect two isoenzymes of APX; APX-A, whose activity decreased with time while the activity of APX-B increased slightly in both treatments.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    From three PRX in chloroplasts, Type II E was the most abundant in both treatments with maximum on day 7.

    “From three PRX in chloroplasts Type II E was the most abundant in both treatments with maximum on day 7.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Cytosol located type II PRX C was hardly detected during acclimatization; in GM grown plantlets it was not detected on day 0.

    “Cytosol located type II PRX C was hardly detected during acclimatization.In fact, in GM grown plantlets this PRX was not even detected on day 0.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Evidence of oxidative stress during micropropagation has often been reported.

    “Evidence of oxidative stress during micropropagation has often been reported (Van Huylenbroeck et al. 2000, Carvalho et al. 2001).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Van Huylenbroeck et al. 2000 source
    • cites Carvalho et al. 2001 source

  • May combine two claims.

    Premisecitation

    TIB is an efficient propagation technique that improves plantlet morphology and physiology and represents a low cost option for massive production of plantlets for tropical agriculture.

    “TIB is an efficient propagation technique, that improves plantlet morphology and physiology and it represents a low cost option for the massive production of plantlets for the tropical agricultural industry (e.g.sugarcane, plantain, pineapple) (Lorenzo et al. 2001, Escalona et al. 2003, Aragón et al. 2005).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Lorenzo et al. 2001 source
    • cites Escalona et al. 2003 source
    • cites Aragón et al. 2005 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Photosynthesis can be down regulated either due to a lack of CO2 or ethylene production in closed vessels or due to feedback inhibition of Calvin cycle enzymes by sucrose in the medium.

    “Photosynthesis can be down regulated either due to a lack of CO 2 or ethylene production in closed vessels or due to the feedback inhibition of Calvin cycle enzymes by the sucrose in the medium (Jackson et al. 1991, Tanaka et al. 2005).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Jackson et al. 1991 source
    • cites Tanaka et al. 2005 source
14 of 14

  • The paper relies on this without support.

    Act2 is expressed at a relatively high and constant level in plantain leaves under the experimental conditions.

    “C T values were normalized to the C T value of Act2, a housekeeping gene expressed at a relatively high and constant level.”

  • The paper relies on this without support.

    The polyclonal antibodies against the five peroxiredoxins specifically recognize the intended PRX proteins in plantain.

    “Polyclonal antibodies against cytosolic type II Prx C, chloroplast-located type II Prx E, chloroplast-located 2-cys Prx, chloroplast-located Prx Q and mitochondria-located type II Prx F, kindly given by Prof. K.-J.Dietz (Bielefeld University, GE, USA).”

  • The paper relies on this without support.

    The measured enzyme activities in leaf extracts reflect the in vivo anti-oxidative capacity of the plantlets.

    “The extracts for measuring enzyme activities were prepared at 4 ºC from 0.5 g of frozen leaf tissue ground with 50 % (m/m) polyvinylpolypyrrolidone (PVPP).”

  • The paper relies on this without support.

    The expression levels of the studied genes correspond to the activities or amounts of the corresponding enzymes.

    “The expression of key genes of the anti-oxidative defence system was monitored using real time RT-PCR (Fig. 5).”

  • The paper relies on this without support.

    The differences in enzyme activities and gene expression between GM and TIB are due to the propagation system rather than other uncontrolled factors.

    “Two different techniques, GM (gelled medium) and TIB (temporary immersion bioreactors) were applied for the last subculture (21 d).”
5 of 5

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Plantain genotype differs between abstract and methods

    Minor

    The abstract identifies the plantlets as Musa ABB, while the materials and methods identify them as Musa AAB.

  • Figure captions report a test excluded by the statistical methods

    Minor

    The statistical methods say all situations were analyzed with Kruskall-Wallis or Mann-Whitney tests, but the Figure 2 caption reports a C-Dunnett multiple range test. Figure 5 also reports C-Dunnett.

  • CuZnSOD result cites an unmeasured day 21

    Minor

    The results place a significant CuZnSOD increase on day 21, but the gene-expression figure caption lists measurements only through day 14.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. 10.1093/jxb/53.372.1249 · DOI

Cited by

Sorted by citations

Related

Authors
Aragón, C., Carvalho, L., González, J., Escalona, M., & Amâncio, S.
Affiliations
  • Universidad de Ciego de Ávila (CU)
  • Universidade Técnica de Lisboa (PT)
ORCID
Venue
Biologia plantarum
Publisher
Institute of Experimental Botany
Published
2010-06-01
Volume
54
Issue
2
Pages
237-244
ISSN
  • 0006-3134 (ISSN-L)
  • 1573-8264
DOI
10.1007/s10535-010-0042-y
OpenAlex
W2109579546
Cited by
32
References
39
Citations by year
2024: 1 · 2023: 2 · 2022: 3 · 2021: 1 · 2020: 4 · 2019: 1 · 2018: 2 · 2017: 2 · 2016: 5 · 2015: 2 · 2013: 3 · 2012: 3
FWCI
1.06
Type
journal article
Language
en
Open access
Locations
  • Biologia Plantarum (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
Funders
Fundação para a Ciência e a Tecnologia (SFRH/BPD/5707/2001)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant tissue culture and regeneration
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Stress Responses and Tolerance
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Banana Cultivation and Research
Keywords
plantain, ex vitro acclimatization, temporary immersion bioreactor, micropropagation, reactive oxygen species, antioxidant enzymes, ascorbate-glutathione cycle, peroxiredoxin, nitroblue tetrazolium staining, gene expression

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Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-28.

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