Effect of activated charcoal and ascorbic acid on in vitro morphogenesis and o-dihydroxyphenols content in Paphiopedilum insigne

Abstract

Phenolic compounds limit micropropagation of many orchids in vitro. The aim of the study was to estimate the effect of activated charcoal (AC);1, 2 or 4 g/L) or ascorbic acid (AA; 10, 20 or 30 mg/L) added to the half strength MS medium on the growth and o-dihydroxyphenols content in Paphiopedilum insigne in vitro. A positive effect of AC on the shoot and root formation has been found. The highest multiplication rate (5.6 shoots/explant) and rooting frequency were obtained on medium containing 2 g/L of AC. However, AC reduced the leaf number as compared to the control. The lowest content of o-dihydroxyphenols was marked in Paphiopedilum insigne leaves when the shoots were grown on medium with 10 mg/L AA, followed by AC at 1 or 2 g/L.

Full text

48
Short Communication Horticultural Science (Prague), 49, 2022 (1): 48–51
https://doi.org/10.17221/68/2020-HORTSCI
Paphiopedilum insigne, (Orchideaceae) is a pot plant
with unique owers shaped like slippers. It is also used
as a cut ower (Ng, Saleh 2011).
In vitro techniques increase the availability of plants,
but there are many diculties like tissue or media
browning due to phenolic compounds (Ndakidemi et
al. 2014), what may lead to poor regeneration (Skrzy-
pek et al. 2007).
To limit phenolic production, compounds like acti-
vated charcoal (AC) or ascorbic acid (AA) are used.
Nongdam and Chongtham (2011) report that AC ad-
sorb phenols. Its’ positive eect was noted in Phalae-
nopsis cornu-cervi (Rittirat et al. 2012), Cymbidium
aloifolium (Nongdam, Chongtham 2011).
Ascorbic acid catches free radicals from cutting
tissues or phenols oxidation (Wojcieszyńska, Wil-
czek 2006).
Its’ positive influence wasnoted inBra-
chylaena huillensis (Ndakidemi et al. 2014) and Ty-
lophora indica (Faisal et al. 2007).
e aim of the study was to estimate the effect
ofAC and AA on regeneration and phenolic com-
pounds content inPaphiopedilum insigne cultures.
e explants were rosettes, 7–10 mm high and
12–15 mm wide with 3 leaves and 1–2roots, taken
from stable cultures started from asymbiotic seeds
germination and passaged 3times on regulators free
1/2 Murashige and Skoog (MS) medium (Murashige,
Skoog 1962). Explants were placed on MS medium
with macronutrients reduced by1/2, with 0.05 mg/L
thiamine, 0.25mg/L pyridoxine, 0.25mg/L nicotinic
acid, 1mg/L glycine, 50mg/L myo-inositol, 15g/L
sucrose, 5mg/L kinetin (KIN), and 1mg/L
benzylad-
Effect of activated charcoal and ascorbic acid on in vitro
morphogenesis and o-dihydroxyphenols content
in Paphiopedilum insigne
M P, M P*, P S
Institute of Horticultural Production, Faculty of Horticulture and Landscape Architecture,
University of Life Sciences in Lublin, Lublin, Poland
*Corresponding author: marzena.parzymies@up.lublin.pl
Citation: Poniewozik M., Parzymies M., Szot P. (2022): Effect of activated charcoal and ascorbic acid on in vitro morpho-
genesis and o-dihydroxyphenols content in Paphiopedilum insignae. Hort. Sci. (Prague), 49: 48–51.
Abstract: Phenolic compounds limit micropropagation ofmany orchids invitro. Theaim ofthe study wasto esti-
mate theeffect ofactivated charcoal (AC); 1, 2 or 4g/L) or ascorbic acid (AA; 10, 20 or 30mg/L) added tothe half
strength MS medium on thegrowth and o-dihydroxyphenols content inPaphiopedilum insigne invitro. A positive
effect ofAC on theshoot and root formation hasbeen found. Thehighest multiplication rate (5.6shoots/explant)
and rooting frequency were obtained on medium containing 2 g/L ofAC. However, AC reduced theleaf number
ascompared tothe control. Thelowest content ofo-dihydroxyphenols wasmarked inPaphiopedilum insigne leaves
when theshoots were grown on medium with 10mg/L AA, followed byAC at1 or 2g/L.
Keywords: lady slipper; orchid; micropropagation; phenolic compounds; tissue culture
Supported by the University of Life Sciences in Lublin, Poland.

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Horticultural Science (Prague), 49, 2022 (1): 48–51 Short Communication
https://doi.org/10.17221/68/2020-HORTSCI
Cultures were kept at 28oC ± 2 oC and 16-hour
photoperiod with thelight intensity of30mol/m2/s.
e data wasanalyzed with Statistica 13 (StatSoft),
according toone – way ANOVA. e significance
ofdifferences was estimated with theTukey’s con-
fidence intervals atthe level ofsignificance α=0.05.
Activated charcoal and ascorbic acid had no effect
on the number of regenerated P. insigne plants, but
AC significantly enhanced the shoot multiplication
(Table1). ehighest shoot number (5.6) was noted
inthe presence of 2g/L of AC. Similar results were
previously obtained by Nongdam and Chongtham
(2011) in Cymbidium aloifolium and by Fasal et al.
(2007) in Tylophora indica. We observed that the
shoot ofP. insigne grown inthe presence of AC pro-
duced less leaves as compared to control, but only
AC at 1 g/L influenced formation of bigger leaves
(Table2). epositive effect ofAC on rosettes height
wasnoted inCattleya walkeriana (de Faria et al. 2002)
or Miltonia flavescens (Morales et al. 2003). Rittiratet
al. (2012) reported thatin presence ofAC Phalaenop-
sis cornu-cervi leaves were longer and wider.
Aswasshown inTable2, theaddition ofAC tothe
medium resulted inan increase of the rooting rate
by 20%, whereas AA slighty reduced this process
ascompared tothe control. Activated charcoal had
also positive effect on theroot length and weight.
e positive effect of AC on rooting in vitro
wasnoted bymany authors. Yan et al. (2006) proved
thatAC darkened media promoting roots develop-
ment. A higher number ofroots inpresence ofAC
was observed in Phalaenopsis cornu-cervi (Ritti-
rat et al. 2012), Miltonia flavescens and Oncidium
trulliferum (Morales et al. 2003)
Table 1. e influence of ascorbic acid (AA) and activated
charcoal (AC) on the number of regenerated plants
and multiplication rate of Paphiopedilum insigne
Antioxidants Mean No. of regenerated
plants/treatment
Multiplication
rate
Control 7.83a*1.19b
AA 10 (mg/L)7.83a1.87b
AA 20 (mg/L)8.33a1.49b
AA 30 (mg/L)8.67a1.27b
AC 1 (g/L)8.33a1.98b
AC 2 (g/L)9.50a5.60a
AC 4 (g/L)9.33a2.60b
*Means followed by the same letter do not differ signifi-
cantly at P=0.05
Table 2. Influence of ascorbic acid (AA) and activated charcoal (AC) on morphological features of Paphiopedilum
insigne in tissue culture
Antioxidants
Leaves Weight of
rosettes
(mg)
Roots
Number/
plant
Length
(mm)
Width
(mm)
Presence
(%)
Number/
plant
Length
(mm)
Weight
(mg)
Control 3.76a* 12.04 5.03 80.03b75ab 1.89b3.61c11.98b
AA 10 (mg/L) 2.31b9.69 3.97 89.57b74ab 2.26b3.61c19.05b
AA 20 (mg/L) 3.23a10.78b4.45 79.78b72b1.81b3.94c17.02b
AA 30 (mg/L) 3.75a12.41 4.69 89.89b77ab 2.45b4.97c27.21b
AC 1 (g/L) 2.55b12.46 4.82 187.55a90ab 4.27a10.86a153.12a
AC 2 (g/L) 2.35b7.33 3.04 219.14a95a5.02a8.86b140.15a
AC 4 (g/L) 2.56b11.44b4.27 173.23a95a3.96a8.48b113.98a
*Means followed by the same letter do not differ significantly at P=0.05
enine (BA). Media were supplemented with AC (1, 2
or 4g/L) or AA (10, 20 or 30mg/L). emedium with-
out additions wasthe control. e
pH wasadjusted
to5.7. Media were gelled with 6.75g/L agar. ere
were 6 flasks with 10 plants per treatment, intwo
repetitions. e study lasted 16weeks. A content
ofo-dihydroxyphenols inP. insigne leaves wasdone
with Folin-Ciocalteu reagent according toSingel-
ton and Rossi method (1965). Briefly, the0.5 mL
ofthe diluted sample wasreacted with 2ml ofFo-
lin-Ciocalteu reagent and 10ml water for 3min,
and then 10ml 10% ofsaturated sodium carbon-
ate solution. ereadings were done after 30min
at760nm with the use of Cary100Varian spec-
trophotometer. Caffeic acid wasused asa reference
standard, and theresults were expressed ascaffeic
acid equivalent.

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Short Communication Horticultural Science (Prague), 49, 2022 (1): 48–51
https://doi.org/10.17221/68/2020-HORTSCI
e biochemical analyses showed that the con-
tent ofo-dihydroxyphnols inP. insigne leaves invi-
tro was the lowest in the presence of 10 mg/L AA
(0.95%)followed by AC at 1 or 2 g/L AC (1.10 and
1.06% respectively).
Similar results were obtained in Cymbidium (da
Silva 2013). epositive effect of 100 mg/L AA on
the reduction of phenolic compounds production
was observed by Ngomuo et al. (2014) in Musa
spp. in vitro. Ndakidemi et al. (2014) confirmed
that200mg/L ofAA decreased phenols inBrachy-
laena huillensis and Ko et al. (2009) in Cavendish
banana cv. Formosana cultures. AC has a perfect
adsorption ability towards phenolic compounds, but
it depends on interactions between thecompounds
or other effects (Dąbrowski et al. 2005). Inthe pre-
sented research AC at1–2 g/L reduced theamount
of o-dihydroxyphenols, while AC at 4 g/L had no
effect on this process, as compared to the control.
Different effects were noted by Abdelwahd et al.
(2008) inVicia faba, asAC 10 g/L significantly re-
duced browning ofexplants and had a positive effect
on regeneration. da Silva (2013) reported that on
1g/LAC media browning wasnot observed inCym-
bidium tissue culture .
CONCLUSION
Activated charcoal and ascorbic acid affect mi-
cropropagation ofPaphiopedilum insigne. Multipli-
cation rate wasthe highest inthe presence of2g/L
AC, therefore such medium is advised for plants
cultivation in vitro. e rosettes with the highest
number ofleaves were noted on thecontrol media
and inpresence of20 or 30mg/L ofAA, while AC
at1g/L increased thenumber and length of roots.
Figure 2. e influence of ascor-
bic acid and activated charcoal on
morphological features of Paphi-
opedilum insigne in tissue culture
Control
AA 10 mg/L AA 20 mg/L AA 30 mg/L
AC 1 g/L AC 2 g/L AC 4 g/L
0 1 2 cm
Figure 1. e influence of ascorbic acid and activated charcoal on o-dihydroxyphenols content in Paphiopedilum
insigne leaves in vitro
*Means followed by the same letter do not differ significantly at P=0.05
1.44
0.95
1.37 1.34
1.1 1.06
1.46
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control AA 10 mg/L AA 20 mg/L AA 30 mg/L AC 1 g/L AC 2 g/L AC 4 g/L
Content of o-dihydroxyphenols calculated on caffeic acid (%)
a*
d
bb
a
cc
(%

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Horticultural Science (Prague), 49, 2022 (1): 48–51 Short Communication
https://doi.org/10.17221/68/2020-HORTSCI
eamount ofo-dihydroxyphenols inleaves wasthe
lowest on the media with 10 mg/L AA. e addi-
tion ofantioxidants tothe media, except the high-
est amount ofAC, decreased thecontent ofo-dihy-
droxyphenols inleaves.
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Received: April 29, 2020
Accepted: July 2, 2021
Published online: January 5, 2022