Journal of Applied Phycology

https://doi.org/10.1007/s10811-018-1703-z

Influence of seaweed extracts and plant growth regulators on in vitro

regeneration of Lycopersicon esculentum from leaf explant
S. Vinoth 1,2 & P. Gurusaravanan 3 & S. Sivakumar 2 & N. Jayabalan 2

Received: 28 July 2018 / Revised and accepted: 4 December 2018

# Springer Nature B.V. 2019

Abstract
An efficient in vitro regeneration protocol was developed for tomato from leaf explant using plant growth regulators, organic
elicitors, polyamines, and seaweed extracts. Initially, excised leaf explant was cultured on medium containing different concen-
trations of auxins viz., 2,4-dichloropheonoxyacetic acid (2,4-D; 0.5–3.0 mg L−1), picloram (Pic; 0.5–3.0 mg L−1), 1-
naphthaleneacetic acid (NAA; 0.5–3.0 mg L−1), and indole-3-acetic acid (IAA; 0.5–3.0 mg L−1). The most effective auxin
was tested in combination with efficient cytokinins such as kinetin (Kin; 0.5–3.0 mg L−1), 6-benzylaminopurine (BAP; 0.5–
3.0 mg L−1), and thidiazuron (TDZ; 0.5–3.0 mg L−1). To achieve the best organogenic capability, different elicitors (casein
hydrolysate, yeast extract, glutamine and adenine sulfate), polyamines (putrescine, spermidine, and spermine), and seaweed
(Gracilaria edulis and Sargassum wightii) extracts were tested at various concentrations. Among the plant growth regulators,
additives, and seaweed extracts tested, the medium supplemented with 1.5 mg L−1 Pic + 1.0 mg L−1 TDZ + 80 mg L−1 casein
hydrolysate induced maximum of 28.6 shoots per organogenic callus. Rapid elongation was achieved in the medium fortified
with isopentenyladenine (1.2 mg L−1) and G. edulis extract (30%) and the seaweed S. wightii extract induced rooting of elongated
shoots. The presence of plant growth regulators in seaweed extracts were further confirmed by FTIR and HPLC analysis. HPLC
analysis of seaweed extracts and standard plant growth regulators revealed the presence of NAA and isopentenyladenine in
S. wightii. The standard chromatogram of NAA correlated with the chromatogram of G. edulis. Hence this regeneration system
provides an additional platform for generating transgenic plants in an efficient manner.

Keywords Organogenesis . Plant growth regulators . Polyamines . Tomato . Seaweeds

Introduction effective genotype-independent regeneration protocol that can

be applied to commercially important cultivars to reduce the
Tomato (Lycopersicon esculentum Mill.) is the second most yield loss. Despite numerous reports on plant regeneration in
consumed vegetable in the world, and it is seventh most im- tomatoes (Padliskikh and Yarmishin 1990; Fari et al. 1992;
portant crop species after maize, rice, wheat, potato, soyabean, Izadpanah and Khosh-Khui 1992; Bhatia et al. 2004; Gubis
and cassava (Bergougnoux 2014). Genetic improvement of et al. 2004; Jabeen et al. 2005; Afroz et al. 2010; Chaudhary
tomato is under serious consideration because of the adverse et al. 2010; Otroshy et al. 2013), an efficient protocol for shoot
environmental conditions that severely limit its yield and pro- regeneration which could be used for recovery of genetically
duction. The ultimate aim of the researchers is to develop an transformed plants is still lacking. Such a protocol depends on
several factors like genotype of the cultivar, type of culture
vessel, type of media, concentrations, and combination of
* S. Vinoth plant growth regulators, additives, method of inoculation,
[email protected] pH of the medium, light, temperature, age, size, and orienta-
tion of explant (El-Farash et al. 1993).
1
Department of Biotechnology, Aarupadai Veedu Institute of Due to inefficient regeneration protocols, the efficiency
Technology, Chennai, Tamil Nadu 603 104, India of genetic transformation procedures is generally low
2
Department of Botany, Bharathidasan University, (Hamza and Chupeau 1993; Frary and Earle 1996), as
Tiruchirappalli, Tamil Nadu 620 024, India currently available regeneration systems do not develop
3
Department of Botany, Bharathiar University, Coimbatore, Tamil into shoots in the selective media (Peres et al. 2001).
Nadu 641 046, India Therefore, researchers are focusing to develop innovative
 J Appl Phycol

protocols using supplementation of additives to the regen- seaweed samples were confirmed by using FTIR and
eration medium that could enhance the regeneration capa- HPLC analysis.
bility of the shoots (Baskaran and Jayabalan 2010).
Additives (polyamines, casein hydrolysate, amino acids,
yeast extract, adenine sulfate) have a vital role in plant Materials and methods
tissue culture by stimulating the cell division, morphogen-
esis, cell proliferation, secondary metabolism, root forma- Explant preparation
tion, stabilization of membranes, senescence, apoptosis,
chromatin organization, ribosome biogenesis, protein syn- Seeds of tomato (Lycopersicon esculentum) Co-3 cultivar
thesis, and protein-DNA interactions (Hussain et al. 2011; were procured from Tamil Nadu Agricultural University,
Chong-Perez et al. 2012; Satish et al. 2016a). Coimbatore. Seeds were surface sterilized in 70% (v/v) etha-
Seaweeds are considered as marine biostimulants that nol for 15 s and 0.1% mercuric chloride for 2 min. Surface
improve the efficiency of nutrition, tolerance to abiotic sterilized seeds were rinsed with sterile water three times and
stress, and/or quality characteristics of crops, regardless inoculated in the medium fortified with 30% Sargassum
of their nutrient content. Naturally available seaweed ex- wightii extract (Vinoth et al. 2012). About 12 days old, leaf
tracts stimulate plant growth and enhance the germina- explant was excised from the in vitro raised seedlings and
tion rate, root system development, as well as increased used for the organogenesis.
leaf area, number of leaves, tillers, weight of the plant,
fruit quality, and plant vigor (Hong et al. 2007; Rayorath Seaweed sample preparation and culture conditions
et al. 2008; Khan et al. 2009; Sunarpi et al. 2010; Craigie
2011; Babu and Rengasamy, 2012; Vinoth et al. 2012; The seaweeds Gracilaria edulis (Rhodophyta) and Sargassum
Chbani et al. 2013; Vinoth et al. 2014; Arioli et al. wightii (Phaeophyta) were collected from the coastal area of
2015; Layek et al. 2018; Prakash et al. 2018). These Rameswaram (9.28° N 79.3° E) and transported to the testing
extracts are marketed as liquid biostimulants and chemi- laboratory at Bharathidasan University, Tiruchirappalli.
cal analyses have revealed the presence of a wide variety Impurities were eliminated from the seaweeds which were
of plant growth promoting substances such as micro-nu- washed thoroughly with clean water three times and shade-
trients, auxins, cytokinins, and betaines. They also pos- dried. Dried materials were powdered using electric blender;
sess a number of bio-active compounds such as complex about 500 g of seaweeds were added to 1 L distilled water and
minerals, vitamins, macro-nutrients (Ca, K, and P) and boiled in a 2-L Erlenmeyer flask for 50 min. The extracts were
micro-nutrients (Cu, Zn, B, Mn, Co, and Mo), antioxi- initially filtered through muslin cloth and then through
dants, organic acids, and amino acids (Khan et al. 2011; Whatman no. 1 filter paper (Vinoth et al. 2014). The filtrate
Vinoth et al. 2014; Satish et al., 2016b). Gorka and was stored at 4 °C for further experimental studies.
Wieczorek (2017) reported the presence of nine phyto- The modified Murashige and Skoog (mMS) medium was
hormones such as indoleacetic acid (IAA), indolebutyric composed of MS salts (Murashige and Skoog 1962) and B5
acid (IBA), phenyleacetic acid (PA), naphtyleacetic acid vitamins (Gamborg et al. 1968). The medium was fortified
(NAA), trans-zeatin, kinetin (Kin), isopentenyladenine with 3% sucrose, various concentrations of plant growth reg-
(2iP), 6-benzylaminopurine (BAP), and abscisic acid ulators, additives, polyamines, and pH was adjusted to 5.8
(ABA) in seaweeds using RP-HPLC-PDA method. prior to autoclaving at 121 °C for 15 min. The cultured ex-
Recent studies have confirmed the presence of IAA, plants were maintained at 25 ± 2 °C under Philips fluorescent
2iP, ABA, and salicylic acid in Pyropia yezoensis and lamps (80 μmol photons m−2 s−1) and 16 h light/8 h dark
Bangia fuscopurpurea (Mori et al. 2017). Commercially cycle. The explants/cultures were subcultured every 2 weeks.
available seaweed extracts have been tested in crop
plants by application as foliar spray or as soil drench/ Organogenic callus induction
fertigation (Carrasco-Gill et al. 2018). Greenhouse-
tested plants had positive results after the application of The excised 12-day-old leaf explant was cultured on the mMS
seaweeds compared to the control, but the role in plant medium fortified with different concentrations of auxins viz.,
tissue culture is still lacking. Therefore, we aimed to 2,4-dichloropheonoxyacetic acid (2,4-D; 0.5–3.0 mg L−1), pi-
develop an efficient regeneration protocol with suitable cloram (Pic; 0.5–3.0 mg L−1), 1-naphthaleneacetic acid
combinations and concentrations of plant growth regula- (NAA; 0.5–3.0 mg L−1), and indole-3-acetic acid (IAA; 0.5–
tors, additives, polyamines, and seaweed extracts that 3.0 mg L−1). After two subcultures the cultures were exam-
would trigger the growth of tomato plant cells at ined for the organogenic callus formation which was identi-
in vitro. Furthermore, identification of possible functional fied by the presence of green compact nodular texture. The
groups and presence of plant growth hormones in observed non-organogenic calli like green friable callus
J Appl Phycol

(GFC), yellowish green friable callus (GYFC), yellowish fri- of auxins NAA (0.2–1.0 mg L−1), IAA (0.2–1.0 mg L−1),
able callus (YFC), and yellowish brown callus (YBCC) were and IBA (0.2–1.0 mg L −1) in combination with 30%
discarded and were not used for the next round of experiment. S. wightii extract. The rooted plantlets were initially
After identification of suitable concentration of auxins for the washed with water to remove adherent medium and trans-
induction of organogenic callus, it was subcultured 2 times in ferred to a paper cup containing soil rite. The plantlets
the same medium for efficient proliferation. There were ten were covered with transparent plastic bags and hardened
replicates per treatment and each experiment was repeated plantlets were maintained in an environmental plant
three times. growth chamber (Sanyo, Japan). After 2 weeks, plantlets
were transferred to plastic pots containing soil, sand, and
Adventitious shoot proliferation vermiculite in the ratio of 2:1:1 and the survival rate was
recorded.
After 6 weeks of culture, about 200 mg of proliferated
organogenic callus was transferred to the adventitious shoot
initiation medium (SIM). The SIM medium was composed of Fourier-transform infrared spectroscopy and analysis
various concentrations and combinations of cytokinins and of seaweeds
auxin. The best responding auxin concentration was tested
in combination with kinetin (Kin; 0.5–3.0 mg L−1), 6- The finely powered samples of seaweeds, S. wightii and
benzylaminopurine (BAP; 0.5–3.0 mg L−1), and thidiazuron G. edulis, of about 10 mg each were mixed with 100 mg of
(TDZ; 0.5–3.0 mg L−1). During every subculture, non- potassium bromide (KBr) and pellet was prepared as per the
organogenic callus was excised and discarded. Maximum of KBr pellet technique. The pellet was examined spectrophoto-
about 30 calli were tested for each treatment and each exper- metrically in the IR ranges between 450 and 4000 cm−1 and
iment was repeated three times. the spectrum of the samples was recorded.

Effect of additives and polyamines on multiple shoot

proliferation High-performance liquid chromatography analysis
of seaweed extracts
To attain high frequency shoot proliferation, the best
responded concentration of auxin and cytokinin was tested About 200 g of each seaweed sample (S. wightii and
in combination with additives, elicitors, and polyamines. G. edulis) were submerged in 200 mL of methanol.
Various concentrations of casein hydrolysate (CH; 20– After 15 days, the extract was centrifuged at 10,000 rpm
100 mg L−1), yeast extract (YE; 5–25 mg L−1), glutamine for 10 min and the supernatants were transferred to a fresh
(10–50 mg L−1) and adenine sulfate (Ads; 5–25 mg L−1) were vial and dried in a Petri plate. The dried material was
examined for the multiple shoot proliferation. Further, com- redissolved in methanol and filtered through 0.1 μm sy-
petence of polyamines was tested for the multiple shoot pro- ringe filter. Standards of NAA and 2iP were purchased
liferation, with the mMS supplemented with various concen- from Sigma-Aldrich. The HPLC analysis was performed
trations of polyamines viz., spermine (5–25 mg L −1 ), on a Shimadzu LC-10 AT VP instrument equipped with
spermidine (5–25 mg L−1), and putrescine (5–25 mg L−1). SPD-10 AV vp UV/VIS detector, FCV-10 AL low-
Well-proliferated shoots of about greater than 2 cm were trans- pressure gradient unit, GT-104 degasser, and C18 column.
ferred to shoot elongation medium. The mobile phase consisted of 70% acetonitrile and 30%
water was used at a flow rate of 1 mL min−1. About
Effect of seaweed extracts and plant growth 15 μL of each sample and standard was injected.
regulators on shoot elongation and rooting Identification was by comparison of retention times of
sample with standard PGRs (Vinoth et al. 2014).
Mini shoots were primarily excised from the clump and
was transferred to shoot elongation medium supplemented
with PGRs and seaweed extract. The shoot elongation Statistical analysis
medium was composed of N6-(2-isopentyl) adenine
(2iP; 0.4–2.0 mg L −1 ), gibberellic acid (GA 3 0.4– The statistical significance of the data obtained (percentage of
2.0 mg L−1), 20% (v/v) G. edulis extract and 1.5% (w/v) response for callus induction, mean number of shoots per
sucrose and solidified with 0.8% (w/v) agar. After 3 weeks callus culture, mean shoot, and root length) was determined
of culture, the elongated shoots were lifted from the cul- by one-way analysis of variance (ANOVA) (SPSS v. 17 for
ture bottle and the base of the shoots of about 0.25 cm Windows 7). The mean values were compared by using
was excised and transferred to rooting medium composed Duncan’s multiple range test (P < 0.05).
 J Appl Phycol

Results callus was subcultured on the same medium for callus prolif-
eration (Fig. 1b). The medium supplemented with 2,4-D and
Induction of organogenic callus NAA-induced non-organogenic calli like yellowish friable
callus (YFC), yellowish brown friable callus (YBFC), and
Induction and selection of organogenic callus culture is a key yellowish green friable callus (YGFC) were discarded. The
preliminary part of the indirect organogenesis. The leaf ex- medium fortified with 0.5 mg L−1 of 2,4-D recorded much
plants were inoculated in the mMS medium containing 2,4- less organogenic callus forming efficiency (57.8%) compared
D (0.5–3.0 mg L−1), Pic (0.5–3.0 mg L−1), NAA (0.5– to the other hormones tested.
3.0 mg L−1), and IAA (0.5–3.0 mg L−1). After 2 weeks of
culture, the efficiency of different auxins in induction of Proliferation of shoots
organogenic callus was observed (Fig. 1a). The medium for-
tified with 1.5 mg L−1 Pic induced organogenic callus with To examine the effects of cytokinins in shoot induction,
highest frequency of about 86.5% (Table 1). The organogenic organogenic calli were transferred to mMS medium

Fig. 1 Effect of PGRs and

seaweed extracts on indirect
organogenesis of tomato using
leaf explant. a Initiation of callus
from mature leaf explant (× 1); b
Callus proliferation and initiation
of shoots from callus (× 1.2); c
Multiple shoot induction (× 0.5);
d Mini shoots cultured on mMS
medium containing 30% G. edulis
(× 0.7); e Elongated shoots
cultured in the mMS medium
supplemented with 20% S. wightii
(× 0.7); f Rooting of in vitro
elongated shoots on mMS
medium supplemented with 20%
of S. wightii (× 0.7); g Hardened
in vitro derived plant in artificial
growth chamber and maintained
in it for 7 days (× 0.5); h
Acclimatized plant outside the
artificial growth chamber (× 0.4)
J Appl Phycol

Table 1 Effect of auxins on organogenic callus induction from leaf Table 2 Effect of Pic in combination with different cytokinins
explant of tomato concentration for shoot bud differentiation from leaf derived callus of
L. esculentum (cv. CO-3)
Concentration of plant Percentage of Type and nature of
growth regulators organogenic callus Concentration of plant Percentage Mean number
(mg L−1) callus formation growth regulators (mg L−1) of shoot initiation of shoots per callus

2,4,D Kin + Pic

0.5 57.8 ± 0.13e YFC 0.5 + 1.5 80.2 ± 0.52c 15.7 ± 0.16c
a
1.0 62.4 ± 0.17 YFC 1.0 + 1.5 82.3 ± 0.76a 17.5 ± 0.39a
1.5 61.7 ± 0.13b YBC 1.5 + 1.5 80.8 ± 0.58b 15.6 ± 0.17b
2.0 59.5 ± 0.30c YBCC 2.0 + 1.5 77.2 ± 0.36d 14.3 ± 0.10d
2.5 58.2 ± 0.25d YBCC 2.5 + 1.5 73.4 ± 0.38e 12.5 ± 0.50e
3.0 56.0 ± 0.18f YBCC 3.0 + 1.5 69.9 ± 0.25f 11.2 ± 0.31f
Pic BAP + Pic
0.5 82.8 ± 0.30c YFC 0.5 + 1.5 61.5 ± 0.18f 09.7 ± 0.36f
1.0 84.2 ± 0.24b YBCC 1.0 + 1.5 72.6 ± 0.13d 12.3 ± 0.12d
1.5 86.5 ± 0.52a GCC 1.5 + 1.5 73.9 ± 0.24c 14.9 ± 0.34c
2.0 78.1 ± 0.33d GCC 2.0 + 1.5 80.1 ± 0.57a 16.8 ± 0.16a
2.5 75.3 ± 0.29e YGCC 2.5 + 1.5 76.7 ± 0.53b 15.4 ± 0.19b
3.0 69.1 ± 0.15f YGCC 3.0 + 1.5 68.5 ± 0.23e 12.1 ± 0.15e
NAA TDZ + Pic
0.5 70.8 ± 0.57f YGC 0.5 + 1.5 80.3 ± 0.73c 16.2 ± 0.17c
1.0 74.6 ± 0.32d YGC 1.0 + 1.5 85.3 ± 0.38a 19.8 ± 0.34a
1.5 76.9 ± 0.38b YGCC 1.5 + 1.5 81.6 ± 0.56b 17.6 ± 0.12b
2.0 78.7 ± 0.18a YGCC 2.0 + 1.5 78.1 ± 0.45d 15.5 ± 0.15d
2.5 75.1 ± 0.23c YGFC 2.5 + 1.5 76.6 ± 0.38e 14.9 ± 0.10e
3.0 73.5 ± 0.50e YGFC 3.0 + 1.5 70.5 ± 0.32f 12.8 ± 0.12f
IAA
The control medium was fortified with 1.5 mg L−1 of Pic. Data were
0.5 67.6 ± 0.43e YGFC
recorded after 3 weeks of culture. Leaf-derived organogenic callus were
1.0 70.3 ± 0.32c YGFC cultured on mMS medium fortified with different concentrations of Kin,
1.5 73.2 ± 0.30a YGCC BAP and TDZ in combination with Pic. Values represent the mean SE.
2.0 71.6 ± 0.17b YGCC Means followed by the same letter in each column are not significantly
different according to Duncan’s multiple range test (P < 0.05)
2.5 69.9 ± 0.13d GFC
3.0 59.0 ± 0.16f GFC
medium supplemented with 1.0 mg L−1 Kin and 1.5 mg L−1
GFC, green friable callus; YGFC, yellowish green friable callus; YFC, Pic. Of the three cytokinin combinations tested, 1.0 mg L−1
yellowish friable callus; YBFC, yellowish brown friable callus; GCC, BAP in combination with 1.5 mg L−1 Pic induced only 9.7
green compact callus; YGCC, yellowish green compact callus; YGFC, shoots per 200 mg of leaf-derived callus culture and its fre-
yellowish green friable callus; YBCC, yellowish brown compact callus
quency was about 61.5%.
Data were recorded after 4 weeks of culture. Leaf explant were cultured
on mMS medium fortified with different concentrations of 2,4-D, Pic,
NAA, and IAA. Values represent the mean SE. Means followed by the
same letter in each column are not significantly different according to
Influence of additives on multiple shoot induction
Duncan’s multiple range test (P < 0.05)
Leaf-derived callus cultures were transferred to medium con-
taining different concentrations of organic elicitors (CH, YE,
supplemented with combination of 1.5 mg L−1 Pic and cyto- glutamine, and Ads). The organogenic callus responded well
kinins (Kin, BAP, and TDZ) at various concentration ranging in the medium supplemented with CH (20–100 mg L−1), YE
from 0.5 to 3.0 mg L−1. Shoot initiation was observed on the (5–25 mg L−1), glutamine (10–50 mg L−1), and Ads (5–
addition of cytokinins to the Pic fortified medium. The medi- 25 mg L−1). Among the different additives tested in this study,
um consisting of 1.0 mg L−1 TDZ and 1.5 mg L−1 Pic-induced the highest frequency of multiple shoot induction was record-
the highest percentage of shoot induction from leaf derived ed on the medium supplemented with CH which exhibited
callus culture of about 85.3% and it produced a maximum of maximum 91% of response with a mean 21.3 shoots per
19.8 shoots (Table 2). The maximum of 17.5 shoots was pro- leaf-derived callus. YE, glutamine and Ads-supplemented
duced from the leaf derived callus culture on the mMS medium produced a maximum response of about 89.3%,
 J Appl Phycol

Table 3 Effect of different concentrations of organic elicitors on the most effective in induction of shoots from the leaf-derived
multiple shoot proliferation from organogenic callus cultures of tomato
organogenic callus. Spermidine-supplemented medium in-
Organic elicitors Percentage Mean number duced maximum of 28.6 shoots with the highest percentage
and additives (mg L−1) of shoot proliferation of shoots per callus of response of about 94.3% (Fig. 1c). Of the various concen-
trations of spermine tested, maximum percentage of response
CH
was found in the medium fortified with 10 mg L−1 and it
20 79.1 ± 0.72e 15.5 ± 0.52e
produced maximum of 26.9 shoots per callus mass.
40 84.5 ± 0.35d 16.7 ± 0.15d
Putrescine-supplemented medium induced a maximum of
60 88.4 ± 0.61b 19.6 ± 0.52b
22.5 shoots per organogeneic callus which was lower than
80 91.0 ± 0.30a 21.3 ± 0.46a
the other polyamines tested (Table 4). We found that increas-
100 86.2 ± 0.15c 17.2 ± 0.24c
ing the concentration of polyamines leads to browning of
Yeast extract tissues.
5 79.3 ± 0.64e 16.0 ± 0.18e
10 85.8 ± 0.55c 17.3 ± 0.15c Effect of seaweed extracts and PGRs on shoot
15 87.1 ± 0.49b 19.3 ± 0.37b elongation and rooting
20 89.3 ± 0.51a 19.7 ± 0.53a
25 81.7 ± 0.23d 16.9 ± 0.18d Regenerated shoots longer than 3 cm were excised and cul-
Glutamine tured in shoot-elongation medium consisting of PGRs and
10 76.3 ± 0.13e 14.5 ± 0.33e seaweed extract. As we found that seaweed extracts played a
20 80.3 ± 0.37c 15.7 ± 0.27c vital role in shoot elongation and rooting, the combined effect
30 84.8 ± 0.24b 16.1 ± 0.35b of plant growth regulators with extracts were analyzed. The
40 86.5 ± 0.18a 17.0 ± 0.20a elongation medium was supplemented with 2iP (0.4–
50 77.9 ± 0.32d 14.4 ± 0.15d 2.0 mg L−1), GA3 (0.4–2.0 mg L−1), and 30% G. edulis ex-
Ads tract. We observed that 30% G. edulis extract was an optimal
5 71.7 ± 0.52d 11.2 ± 0.10d concentration in shoot elongation. Therefore we directly test-
10 76.5 ± 0.56c 13.8 ± 0.13c ed 30% G. edulis with plant growth regulators (2iP and GA3).
15 81.3 ± 0.74a 15.6 ± 0.13a The best response of 95.8% and shoot length of 14.7 cm was
20 79.1 ± 0.32b 14.1 ± 0.12b obtained in medium containing 1.2 mg L−1 2iP and 30%
25 69.8 ± 0.56e 09.5 ± 0.10e G. edulis extract. The GA3 (1.6 mg L−1) and G. edulis
(30%) extract-supplemented medium induced maximum
CH, casein hydrolysate; Ads, adenine sulfate
shoot length of 12.5 cm with 92.7% response (Table 5; Fig.
The medium supplemented with 1 mg L−1 TDZ and 1.5 mg L−1 Pic 1d).
served as control. Data were recorded after 3 weeks of culture. Leaf-
derived organogenic callus were cultured on MS medium fortified with Well-elongated shoots were transferred to rooting medium
different concentrations of organic elictors for multiple shoot prolifera- supplemented with NAA (0.2–1.0 mg L−1), IAA (0.2–
tion. Values represent the mean SE. Means followed by the same letter in 1.0 mg L−1), IBA (0.2–1.0 mg L−1), and 20% S. wightii extract
each column are not significantly different, according to Duncan’s mul- (Fig. 1e). Of the auxins evaluated, 0.2 mg L−1 IBA and 20%
tiple range test (P < 0.05)
S. wightii extract-containing medium shown the best response
producing a maximum root length of 23.7 cm with 18.9 mean
86.5%, and 81.3% and induced 19.7, 17.1, and 15.6 shoots per
roots per elongated shoots (Table 6; Fig. 1f and g). The rooted
leaf-derived organogenic callus, respectively (Table 3). YE
plantlets were carefully taken from the culture bottle and
and yeast maltose broth have high amino acid content and
washed and hardened in a plastic cups containing soil-rite
carbon, protein, vitamins, fermentable carbohydrate, and other
and placed in a plant growth chamber for 2 weeks before
saccharides as energy sources. The shoot forming capability
being transferred to greenhouse conditions (Fig. 1h). The
was least in the medium supplemented with 25 mg L−1 Ads
plantlets transferred from plant growth chamber to greenhouse
which promoted the lowest number of shoots (15.6) among
conditions showed a survival rate of 90%.
the additives tested.
FTIR analysis of seaweeds
Influence of polyamines on multiple shoot induction
Infrared absorption and functional groups present in sea-
To enhance the multiple shoot proliferation, different concen- weeds, S. wightii and G. edulis, were evaluated using FTIR.
trations of PAs (spermine, spermidine, and putrescine) be- About 10 bands were observed in the analysis of S. wightii
tween 5 and 25 mg L−1 were tested. Of the three polyamines between 3436.80 and 768.13 cm−1. The functional groups
tested, medium supplemented with 10 mg L−1 spermidine was present in the seaweed were amide N–H stretch, alkyl C–H
J Appl Phycol

Table 4 Effect of different

concentrations of polyamines on Polyamines (mg L−1) Percentage of shoot proliferation Mean number of shoots per callus
multiple shoot prolifereation from
organogenic callus cultures of Spermine
L. esculentum (cv. CO-3) 5 90.5 ± 0.72b 25.3 ± 0.13b
10 93.2 ± 0.92a 26.9 ± 0.33a
15 88.7 ± 0.76c 23.8 ± 0.24c
20 82.3 ± 0.91d 17.6 ± 0.12d
25 76.4 ± 0.52e 13.5 ± 0.16e
Spermidine
5 87.6 ± 0.56c 23.2 ± 0.52c
10 94.3 ± 0.96a 28.6 ± 0.32a
15 89.8 ± 0.36b 26.7 ± 0.30b
20 83.5 ± 0.32d 18.5 ± 0.18d
25 79.7 ± 0.50e 16.9 ± 0.52e
Putrescine
5 75.8 ± 0.53e 14.7 ± 0.15e
10 85.6 ± 0.38b 20.3 ± 0.18b
15 87.1 ± 0.33a 22.5 ± 0.36a
20 80.3 ± 0.17c 17.8 ± 0.12c
25 76.1 ± 0.24d 15.2 ± 0.10d

The medium supplemented with 1 mg L−1 TDZ, 1.5 mg L−1 Pic, and 80 mg L−1 CH served as control. Data were
recorded after 3 weeks of culture. Leaf-derived organogenic callus were cultured on MS medium fortified with
different concentrations of polyamines for multiple shoot proliferation. Values represent the mean SE. Means
followed by the same letter in each column are not significantly different, according to Duncan’s multiple range
test (P < 0.05)

stretch, carboxylic acid O–H stretch, alkynl C=C stretch, aro- gave 11 bands ranges from 3435.14 to 776.80 cm−1. The
matic C=C bending, sulfonyl chlorides S=O stretch, sulfites functional groups present in G. edulis were amide, alkyl, al-
S=O stretch, sulfoic acids S=O stretch, and aromatic C–H kenes, alcohols, phenols, carboxylic acids, and sulfoides
bending (Table 7; Fig. 2). The spectral analysis of G. edulis (Table 8; Fig. 3).

Table 5 Effect of 2iP, GA3, and

G. edulis on shoot elongation Concentration of plant growth Percentage of response Mean shoot length (cm)
from mini shoots of L. esculentum regulators and extract
(cv. CO-3)
Control 63.7 ± 0.18f 6.8 ± 0.30f
−1
2iP (mg L ) + GE (%)
0.4 + 30 87.9 ± 0.77e 8.1 ± 0.13e
0.8 + 30 91.4 ± 0.95c 8.6 ± 0.17d
1.2 + 30 95.8 ± 0.92a 14.7 ± 0.12a
1.6 + 30 93.1 ± 0.96b 12.5 ± 0.13b
2.0 + 30 89.4 ± 0.72d 9.5 ± 0.10c
GA3 (mg L−1) GE (%)
0.4 + 30 80.4 ± 0.53e 7.1 ± 0.12d
0.8 + 30 86.2 ± 0.52d 8.3 ± 0.21c
1.2 + 30 91.1 ± 0.90b 11.7 ± 0.30b
1.6 + 30 92.7 ± 0.58a 12.5 ± 0.36a
2.0 + 30 90.3 ± 0.91c 11.4 ± 0.13b

GE G. edulis
MS Medium without supplementation of seaweed extract and PGRs served as control. Shoots of about 2 cm were
cultured on MS medium fortified with different concentrations of 2iP and GA3 in combination with 30%
G. edulis. Values represent the mean SE. Means followed by the same letter in each column are not significantly
different according to Duncan’s multiple range test (P < 0.05)
 J Appl Phycol

Table 6 Effect of auxins and

S. wightii on rooting of elongated Concentration of plant growth Percentage of root Average no. of Average no. of root
shoots of L. esculentum (cv. CO- regulators and extract induction roots/shoot length (cm)
3)
Control 90.0 ± 0.10cd 10.6 ± 0.17de 09.4 ± 0.15f
−1
NAA (mg L ) + SW (%)
0.2 + 20 84.9 ± 0.18e 12.6 ± 0.13e 12.3 ± 0.13d
0.4 + 20 90.1 ± 0.11d 13.1 ± 0.13d 13.6 ± 0.17c
0.6 + 20 91.5 ± 0.28c 14.2 ± 0.15c 14.9 ± 0.15b
0.8 + 20 95.6 ± 0.17a 15.9 ± 0.17a 16.9 ± 0.10a
1.0 + 20 93.2 ± 0.30b 15.1 ± 0.17b 13.2 ± 0.13c
IAA (mg L−1) + SW (%)
0.2 + 20 95.8 ± 0.25a 16.1 ± 0.16a 17.4 ± 0.13a
0.4 + 20 91.3 ± 0.17b 14.8 ± 0.13b 15.8 ± 0.17b
0.6 + 20 88.1 ± 0.12c 13.9 ± 0.17c 13.8 ± 0.13c
0.8 + 20 85.5 ± 0.13d 11.2 ± 0.13d 11.6 ± 0.13d
1.0 + 20 84.2 ± 0.16e 9.9 ± 0.10e 10.2 ± 0.15e
IBA (mg/L) + SW (%)
0.2 + 20 95.9 ± 0.10b 16.8 ± 0.16c 19.2 ± 0.17b
0.4 + 20 97.3 ± 0.31a 18.9 ± 0.19a 23.7 ± 0.13a
0.6 + 20 95.2 ± 0.56c 16.2 ± 0.10b 17.9 ± 0.16c
0.8 + 20 93.5 ± 0.17d 15.6 ± 0.15d 15.2 ± 0.00d
1.0 + 20 90.7 ± 0.16e 13.2 ± 0.13e 13.5 ± 0.15e

SW S. wightii
MS Medium without supplementation of seaweed extract and PGRs served as control. Elongated shoots were
cultured on MS medium fortified with different concentrations of NAA, IAA, and IBA in combination with 20%
S. wightii. Values represent the mean SE. Means followed by the same letter in each column are not significantly
different according to Duncan’s multiple range test (P < 0.05)

HPLC analysis of seaweed extracts 2.167, respectively (Fig. 4b). To confirm the presence of
PGRs, standard NAA and 2iP were analyzed and compared
HPLC analysis was performed to confirm the presence of with the seaweed chromatograms. The highest peak area of
PGRs. The chromatogram of S. wightii showed five peaks 11379.52 (2.437 RT) and 8769.95 (2.563 RT) was obtained
(2.010, 2.150, 2.407, 2.533, 2.803), in which the highest peak for NAA and 2iP respectively (Fig. 5a and b). HPLC analysis
area of 58.03 mV·s was obtained in the second peak (Fig. 4a). of seaweed extracts and standard plant growth regulators re-
The chromatogram of G. edulis had four peaks at different vealed the presence of NAA and 2iP in S. wightii. The stan-
retention times (RT). The highest peak areas of about dard chromatogram of NAA was in correlation with the chro-
683.240 and 184.469 were obtained at RT of 2.460 and matogram of G. edulis. Hence, this analysis proved the pres-
ence of plant growth regulators in the seaweed and the activity
in shoot elongation and rooting of elongated shoots was due to
Table 7 Infrared absorption and functional groups present in S. wightii
the presence of PGRs.
Frequency range (cm−1) Bond and functional group

3436.80 Amide N–H stretch

2920.18 Alkyl C–H stretch Discussion
2807.51 Carboxylic acid O–H stretch
2723.00 Carboxylic acid O–H stretch Tomato is the major vegetable crop cultivated all over the
2018.77 Alkynl C=C stretch world and it is frequently affected by biotic and abiotic stress-
1593.89 Aromatic C=C bending es that limit its yield and productivity. Plant biotechnology-
1383.73 Sulfonyl chlorides S=O stretch based methods help in the improvement of this crop for sus-
1351.45 Sulfites S=O stretch tainable agriculture. A basic and powerful tool used for crop
1098.94 Sulfoic acids S=O stretch improvement of economic importance is plant tissue culture.
768.13 Aromatic C–H bending Several factors influence the development of genotype
independent and reproducible protocol for tomato. The type
J Appl Phycol

61.5
Fig. 2 FTIR spectrum of
S. wightii
1
2723.00 2018.77
50 1098.94

2807.51
2920.18 768.13
40

1383.73
%T 30
1351.45

20
3436.80

10
1593.89
4.4
4000.0 3000 2000 1500 1000 450.0
cm-1

of explants used not only determines the proportion of in the presence of light induced the best callus proliferation
explants, but also the number of shoots produced per explant (Nitnaware et al. 2011). Therefore, to enhance the shoot-
during organogenesis. Duzyaman et al. (1994) found that the forming efficiency, the most effective cytokinins (Kin, BAP
degree of shoot regeneration was in the order of leaves ≥ cot- and TDZ) were tested in combination with 1.5 mg L−1 Pic.
yledons ≥ hypocotyls, and all cultivars responded similarly. Manipulation of composition and ratio of PGRs is the pri-
Most tissues of tomato seem to have high totipotency; how- mary empirical approach used for optimization of in vitro
ever, the choice of right explant may vary with the genotype propagation methods (Shukla et al. 2012). The highest fre-
used for the study. Organogenic callus was initiated from the quency of callus formation and shoot proliferation was obtain-
leaf explant on medium supplemented with 1.5 mg L−1 Pic ed on the medium supplemented with 1 mg L−1 of TDZ and it
and it enhanced the regeneration ability of the determined induced maximum of 19.8 of shoots per callus. Similarly,
cells. Picloram is a very potent growth regulator capable of Nitnaware et al. (2011) reported that medium supplemented
inducing somatic embryogenesis in wheat (Collins et al. 1978; with TDZ exhibited higher shoot regeneration of 32.4 shoots
He and Lazzeri 2001; Mendoza and Kaeppler 2002), barley per culture. The most effective combination (1.0 mg L−1 Kin
(Kachhwaha and Kothari 1994), finger millet (Eapen and and 1.5 mg L−1 Pic) produced an average of 15.6 shoots per
George 1990; George and Eapen 1990), and fox-tail millet leaf explant with shoot regeneration frequency of 82.3%.
(Vishnoi and Kothari, 1996). In supporting to our result, Shoot regeneration frequency was comparably high when ex-
Kaur and Kothari (2004) reported that picloram was more posed to TDZ supplemented medium than the BAP and Kin
effective over a range of BA concentrations produced both concentrations tested. TDZ is a synthetic phenylurea deriva-
embryogenic and organogenic callus. However, 2,4-D failed tive with cytokinin activity, known to show higher activity and
to develop organogenic callus (Denchev and Conger 1994). to promote better shoot organogenesis in comparison to
On media supplemented with Pic (6.9 mg L−1) and incubated adenine-type cytokinins such as BA and kinetin (D’Onofrio
and Morini 2005; Khurana-Kaul et al. 2010).
The promotional effects of organic elicitors on shoot regen-
Table 8 Infrared absorption and functional groups present in G. edulis
eration have been reported in several plant species
Frequency range (cm−1) Bond and functional group (Komalavalli and Rao 2000; Jain and Babbar 2003;
Baskaran and Jayabalan 2007). Among the additives tested,
3435.14 Amide N–H stretch CH at 80 mg L−1 induced maximum 21.3 shoots after 6 weeks
2910.79 Alkyl C–H stretch of culture. Similarly, Baskaran and Jayabalan (2007) reported
2723.00 Alkyl C–H stretch that combinations of PGRs and CH were more effective in
2028.16 Alkenes C=C asymmetric stretch shoot regeneration and 100 mg L−1of CH significantly in-
1593.82 Aromatic C=C bending creased shoots (32.6 shoots per explant) with lengths of
1458.43 Alkenes CH2 and CH3 4.2 cm after 8 weeks of Psoralea corylifolia culture. CH as
1379.06 Alcohols and phenols O–H bending (in plane) an organic nitrogen source is reported to have positive effects
1352.05 Alcohols and phenols O–H bending (in plane) on somatic embryo development and multiple shoot regener-
1253.01 Carboxylic acids and derivatives O–C ation (Ramakrishnan et al. 2013). The number of shoots pro-
1042.92 Sulfoides S=O stretch duced in the YE supplemented medium was significantly
776.80 Aromatic C–H bending higher than the glutamine and Ads fortified medium. YE has
 J Appl Phycol

Fig. 3 FTIR spectrum of 64.8

G. edulis 60
2723.00 2028.16
55 1253.01 1042.92
1458.43
50 776.80
2910.79
45
1379.06
40 1352.05

%T 35

30

25

20 1593.82
3435.14
15
10.8
4000.0 3000 2000 1500 1000 450.0
cm-1

been reported to promote plant growth due to its high amino concentration of Ads gradually decreased the shoot-forming
acid content (George et al. 2008). Contrary to our results, efficiency and it produced least number of shoots compared to
Baskaran et al. (2011) reported that addition of Ads in combi- the other additives and organic elicitors tested.
nations increased the rapid adventitious shoot regeneration in Polyamines (PAs) are low molecular weight, aliphatic,
P. corylifolia whereas in our study, increase in the polycationic compounds carrying positive charges on nitrogen

Fig. 4 HPLC chromatogram of (a) S. wightii

seaweed extracts. a S. wightii, b
G. edulis

(b) G. edulis
J Appl Phycol

(a) Naphthalene acetic acid

(b) N6-(2-Isopentyl) adenine

Fig. 5 HPLC chromatogram of standard plant growth regulators. a Naphthalene acetic acid b N6-(2-isopentyl) adenine

atoms, a property which facilitates electrostatic interactions spermidine (20 mg L−1) induced 46.4 shoots obtained from
with macromolecules such as DNA, RNA, phospholipids, cell nodal explant and they were elongated in the same medium in
wall components, and proteins (Wallace et al. 2003). In plants, culture duration of 6 weeks. However, in the current investi-
PAs are implicated in a variety of growth and developmental gation, it was observed that the polyamines have poor re-
processes, in addition to abiotic and biotic stress responses sponse towards the shoot elongation.
(Baron and Stasolla 2008). PAs such as putrescine, Seaweeds are biostimulants that enhance seed germination,
spermidine, and spermine are known to play important role simulate growth, increase the concentrations of photosynthetic
in various cellular processes (Bais and Ravishankar 2002). pigments and improve biotic and abiotic stress tolerance
Among the polyamines examined, rapid multiple shoot for- (Battacharyya et al. 2015; Mansori et al. 2016). The medium
mation was observed in the medium fortified with spermidine fortified with seaweed extracts of G. edulis and S. wightii at 30
15 mg L−1. Increase in the concentration of polyamines causes and 20% significantly increased the shooting and rooting of
browning of tissues and it inhibited the regeneration capability plants. Lower concentration (20 and 40%) of the seaweed liq-
of cells. Sivanandhan et al. (2011) reported that Murashige uid extract (SLE) gave a better response in the bioassays, and
and Skoog medium (MS) supplemented with 1.5 mg L−1 of beyond 60% of SLEs showed a significant harmful outcome on
BA, 0.3 mg L−1 of IAA and with the addition of polyamine, the germination of seeds and growth of shoots and roots in all
 J Appl Phycol

four genotypes of finger millet tested (Satish et al. 2016b). This Babu S, Rengasamy R (2012) Effect of Kappaphycus alvarezii SLF treat-
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beneficial effect was due to the presence of PGRs in the sea-
some crop plants. J Acad Indust Res 1:186–195
weed extract. Earlier reports confirmed the presence of auxin Bais PH, Ravishankar GA (2002) Role of polyamines in the ontogeny of
IAA in Ecklonia maxima and Ascophyllum nodosum extracts plants and their biotechnological applications. Plant Cell Tissue Org
(Carrasco-Gill et al. 2018). There are limited numbers of re- Cult 69:1–34
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of NAA and 2ip in S. wightii and NAA in G. edulis. Cytokinins plemented medium. J Hortic Sci Biotechnol 82:908–913
Baskaran P, Jayabalan N (2010) Direct organogenesis from hypocotyls
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