Journal of Plant Growth Regulation

https://doi.org/10.1007/s00344-019-09941-w

Smoke-saturated Water and Karrikinolide Modulate Germination,

Growth, Photosynthesis and Nutritional Values of Carrot (Daucus
carota L.)
Arshiya Akeel1 · M. Masroor Akhtar Khan1 · Hassan Jaleel1 · Moin Uddin2

Received: 1 July 2018 / Accepted: 24 January 2019

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Plant-derived smoke is a positive regulator of seed germination and growth with regard to many plant species. Of the several
compounds present in plant-derived smoke, karrikinolide or ­KAR1 (3-methyl-2H-furo[2,3-c]pyran-2-one) is considered to
be the major active growth-promoting compound. To test the efficacy of smoke-saturated water (SSW) and K ­ AR1 on carrot
(Daucus carota L.), two separate pot experiments were simultaneously conducted in the same environmental conditions.
SSW and ­KAR1 treatments were applied to the plants in the form of aqueous solutions of variable concentrations. Prior to
sowing, seeds were soaked in the solutions of SSW (25.8 µg L−1, 51.6 µg L−1,103.2 µg L−1 and 258.0 µg L−1) and ­KAR1
(0.015 µg L−1, 0.150 µg L−1, 1.501 µg L−1 and 15.013 µg L−1). Percent seed germination, vegetative growth, photosynthesis
and nutritional values were the major parameters through which the plant response to the applied treatments was investigated.
The results obtained indicated a significant improvement in all the plant attributes studied. In general, SSW (51.6 µg L−1) and
­KAR1 (1.501 µg L−1) proved optimum treatments for most the parameters. As compared to the control, 51.6 µg L −1 of SSW
and 1.501 µg L−1 of ­KAR1 increased the percent seed germination by 58.0% and 54.4%, respectively. Over the control, the
values of plant height and net photosynthetic rate were enhanced by 33.9% and 40.9%, respectively, due to 51.6 µg L−1 of
SSW, while the values of these parameters were increased by 25.2% and 34.0%, respectively, due to 1.501 µg L−1 of K ­ AR1.
In comparison with the control, treatment 51.6 µg L−1 of SSW increased the contents of β-carotene and ascorbic acid by
32.7% and 37.9%, respectively, while the treatment 1.501 µg L−1 M of K ­ AR1 enhanced these contents by 42.0% and 48.4%,
respectively. This study provides an insight into an affordable and feasible method of crop improvement.

Keywords 3-Methyl-2H-furo[2,3-c]pyran-2-one · Daucus carota L. · Growth promoter · Karrikinolide · Plant-derived

smoke · Photosynthesis · Seed germination

Introduction and aqueous extract of plant-derived smoke. A number of

studies have established the potency of smoke in stimulating
Plant-derived smoke has widely been accepted for its seed germination and improving seedling vigor of a number
stimulatory role in seed germination and seedling growth of plant species including lettuce (Drewes et al. 1995; Ren
(Drewes et al. 1995; Pierce et al. 1995; Thomas and van et al. 2017), red rice (Doherty and Cohn 2000), wild oats
Staden 1995). This dynamic discovery was first documented (Adkins and Peters 2001), bean (van Staden et al. 2006),
by de Lange and Boucher (1990). Since then, substantial okra as well as tomato (Kulkarni et al. 2007a), maize (Soós
research has been conducted to evaluate the effect of aerosol et al. 2009), papaya (Chumpookam et al. 2012), and wheat
(Aslam et al. 2015; Iqbal et al. 2016, 2018). These findings
led to the rejection of earlier thought, which indicated that
* Arshiya Akeel the stimulatory effect of smoke was limited to fire-prone
arsh.aqeel@gmail.com species only.
1
Department of Botany, Aligarh Muslim University, Aligarh, Recent studies affirmed that efficacy of plant-derived
India smoke extends beyond germination (Sparg et al. 2005; van
2
Botany Section, Women’s College, Aligarh Muslim Staden et al. 2006). Plant-derived smoke has, in fact, been
University, Aligarh, India found to modulate somatic embryogenesis (Senaratna et al.

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Vol.:(0123456789)
 Journal of Plant Growth Regulation

1999), flowering (Keeley 1993), photosynthesis (Zhou The present study aimed at exploring the growth-accel-
et al. 2013), rooting (Taylor and van Staden 1996), and eration potential of smoke-saturated water (aqueous smoke
yield (Kulkarni et al. 2008) in plants. These findings led ­ AR1 with regard to germination, growth, pho-
extract) and K
to the expansion of smoke technology to various agricul- tosynthesis and nutritional contents of carrot (Daucus carota
tural fields, including commercially cultivated crops (van L.). Secondly, as karrikins are expensive, the experiment
Staden et al. 2006; Kulkarni et al. 2010), in alleviating the was aimed at answering the question of whether we can use
stress of auxin (Asaf et al. 2014), boron (Khan et al. 2014), smoke-water in place of karrikins to enhance the potential
and salinity (Jamil et al. 2013). of plants regarding seed germination, other physiological
Although the study of compounds present in plant- attributes and yield.
derived smoke extract is elusive and arduous, rigorous
work in this area opens up the way for identification of
highly active compound present in the smoke. Recently, a Materials and Methods
karrikinolide or ­KAR1 (3-methyl-2H-furo[2,3-c]pyran-2-
one), obtained from burned cellulose (Flematti et al. 2004) Plant Material and Treatments
and plant-derived smoke (van Staden et al. 2004; Com-
mander et al. 2008) has been established as the main ger- In connection with the present study, two pot experiments
mination stimulant. It has been established through various were conducted on carrot (D. carota L.), family Apiaceae.
studies that the growth promoting activity of smoke is due Seeds of carrot (var. Pusa Kesar) for this experiment were
to the presence of this compound ­(KAR1). Five analogues purchased from the local seed market (Kashvin Seeds Pvt.
of ­KAR1 ­(KAR2–KAR6), also known as karrikins, have Ltd., Ahmedabad, India).
till date been discovered and confirmed from smoke solu- Smoke-saturated water (SSW) was prepared using a labo-
tion, using a chemical synthesis method (Flematti et al. ratory-made setup (Fig. 1). The smoke, generated by burning
2009). Karrikins have closely related chemical structures, wheat straw, was passed into double distilled water (DDW)
containing a butenolide moiety fused to pyran ring with through continuous bubbling. When the water turned dark
various methyl substitutions. Although ­KAR1 is the most yellowish and no more smoke seemed to be absorbed by the
important stimulant for most species, seed germination can water (that is, when the value of optical density of smoke-
be greatly affected by other analogues too (Nelson et al. water turned constant), it was considered smoke-saturated
2008). ­KAR1 is water soluble, thermostable, long-lasting water. The SSW was filtered through a filter paper (What-
in solution and highly active at low concentrations, down man No. 1) and labeled as stock solution of SSW. Four
to ­10−9 M (Light et al. 2009); also, it has no mutagenic concentrations of SSW, that is, 25.8 µg L−1, 51.6 µg L−1,
and genotoxic effects (Trinh et al. 2010). As a matter of 103.2 µg L−1 and 258.0 µg L−1 were prepared using DDW. A
fact, the stimulating effect of ­KAR1 on seed germination control treatment (DDW) was also employed simultaneously.
has been reported for various plant species (Daws et al. Thus, there were five treatments, whose effect was studied
2007; Flematti et al. 2007; Stevens et al. 2007). Because on carrot (D. carota L.) plants. Another experiment was
­KAR1 is active and potent in stimulating seed germination conducted concurrently in the same environment, wherein
at very low concentrations, it can widen the environmental ­ AR1 (0.015 µg L−1, 0.150 µg L−1,
four concentrations of K
conditions under which the seeds can germinate (Jain et al. 1.501 µg L and 15.013 µg L−1) were used as four kar-
−1

2006). Additionally, it has been proven that it can play rikinolide treatments. Simultaneously, a control treatment
positive role in enhancing seedling growth of weeds (Daws (DDW) was also employed. Thus, there were five treatments
et al. 2007) and medicinal herbs (MousaviNik et al. 2016). in this experiment too, whose effect was studied on carrot
Thus, this relatively new technology of smoke application plants. ­KAR1 was purchased from Toronto Research Chemi-
offers great potential with regard to both conventional and cals, Canada. The amount of smoke present per mL of SSW
organic agriculture. stock was calculated during the extraction process given in
Carrot is an important horticultural crop, widely culti- the next section.
vated and consumed around the world. It was chosen for this
study because of its marvelous nutritional value. Carrot has Smoke‑Saturated Water Extraction and Analysis
been ranked tenth in terms of its nutritional value; among
38 other fruits and vegetables, it is seventh for its contribu- The original method of Ren et al. (2017) of smoke-water
tion to human nutrition (Alasalvar et al. 2005). Carrot is rich extraction was slightly modified for this purpose. The SSW
in beta-carotene and ascorbic acid; also, it is an excellent stock-solution was prepared using wheat straw. After extrac-
source of iron, calcium, phosphorus, folic acid and vitamin tion, the smoke-water was filtered using a filter paper (What-
B. Carrot is also high in fiber, carotenoids, vitamins C and E man No. 1). Thereafter, the neutral SSW, thus obtained, was
and phenolic compounds (Alasalvar et al. 2001). filtered again and dried in vacuum. This dried neutral extract

13
Journal of Plant Growth Regulation

Fig. 1  Apparatus to generate

smoke-saturated water
Iron stands

Rubber tubing

Inverted
funnel

Petri dish containing

wheat straw

Hot plate Glass tube

immersed in
dis­lled water
Heat Regulator Vacuum flask

AC Adapter Smoke water

(220 volt 5A
to 12 volt 2A)
Speed
regulator
Vacuum
pump

of SSW was weighed to calculate the amount of smoke pre- of a homogeneous mixture of field-soil and organic manure.
sent per mL of SSW stock solution and based on these data, Before transferring into pots, the soil–manure mixture was
different concentrations of SSW (µg L−1) were prepared. sterilized for 30 min, using an autoclave running at 121 °C
The concentrated, neutral and water-free smoke-extract, temperature and 15 Psi pressure. Soil samples were ana-
thus attained, was analyzed by liquid chromatography mass lyzed in the Central Laboratory for Soil and Plant Analysis,
spectrometry (LC–ESI–MS) at the Sophisticated Analytic Indian Agricultural Research Institute, New Delhi. Physi-
Instrument Facility, Central Drug Research Institute (CDRI), cal and chemical characteristics of the soil were as follows:
Lucknow, India. texture: sandy-loam, pH 8.07, E.C.: 0.36 m mhos cm−1, and
available N, P, and K: 96.42, 6.72 and 143.0 mg kg−1 soil,
Experimental Procedure respectively. Thirty seeds per pot were sown equidistantly.
After recording germination percentage, extra seedlings
Healthy carrot seeds were surface sterilized by soaking in were clipped out, maintaining three plants per pot. Each
0.5% NaOCl solution containing 1% Tween-20 surfactant for treatment was replicated four times.
10 min and then rinsed with sterilized water for 3 min. As for
presoaking seed treatment, the sterilized seeds were soaked Measurements
in scheduled concentrations of SSW and ­K AR 1, using
DDW as control. After 12 h of presoaking seed-treatment Germination Percentage
with SSW solutions, seeds were rinsed with sterile water
to remove inhibitory compounds (if any) present in SSW. Number of germinated seeds was counted on per pot basis.
Prior to sowing, the treated seeds were air-dried. Seeds were considered germinated when the radical pro-
The pot-experiments were conducted with completely truded by about 2 mm in length. Percent germination was
randomized design in the natural conditions of a net-house calculated according to the formula:
in the Department of Botany, AMU, Aligarh. Seeds were
Total number of seeds germinated
sown in plastic pots (25 cm diameter × 25 cm height) as per Percent germination = × 100
Total number of seeds sown
treatments schedule. Each experimental pot carried 8.5 kg

13
 Journal of Plant Growth Regulation

All observations and calculations were carried out in (OD) of the pigment extract was recorded with the help of
accordance with ISTA rules (2010). a spectrophotometer (Shimadzu UV-1700, Tokyo, Japan) at
662, 645 and 470 nm to determine the content of chloro-
Growth Attributes phyll a, chlorophyll b and total carotenoids, respectively.
Total chlorophyll content was estimated by adding up the
Growth attributes, like plant height, leaf area and number contents of chlorophyll a and chlorophyll b. The content of
of leaves per plant, were measured at 60 and 120 days after each photosynthetic pigment was finally expressed as mg g−1
sowing (DAS). Length, diameter, fresh and dry weights of leaf FW.
roots were determined as per standard procedures. Plants
were uprooted and washed under running tap water to SEM Study of Leaf Stomata
remove adhering foreign particles. The clean plants were
surface-dried using blotting paper. Plant height and root Stomatal frequency and pore size were determined with
length were measured using a meter scale. Root diameter the help of a scanning electron microscope (SEM) (JEOL,
was measured using Vernier caliper. After measuring fresh JSM-6510LV, Japan) at the Ultra Sophisticated Instrumen-
weight, the plants were dried at 80 °C for 24 h using a hot- tation Facility Centre, AMU, Aligarh. Fresh leaf samples
air oven; plant dry weight was recorded thereafter. Leaf area were employed for SEM. Prior to scanning, the samples
was estimated using a leaf area meter (LA211, Systronics, were coated with gold. Stomatal frequency was calculated
Hyderabad, India). by counting the number of stomata in the microscope field of
view. The pore size of stomata was determined in microm-
Photosynthetic Attributes eters (µm) using scale bars in SEM. Three leaves were ana-
lyzed from each replicate, and each treatment was replicated
PS II Activity four times for the stomatal study.

Chlorophyll fluorescence (Fv/Fm) was measured on the Nutritional Value

adaxial surface of fully expanded leaves using a portable
chlorophyll fluorometer (PAM-2000, Walz, Effeltrich, Ger- Fresh tissue of carrot was used to assess its nutritional value
many). The test for maximum quantum efficiency (Fv/Fm) of at 120 DAS. The procedure is given below:
PSII photochemistry was carried out by pre-darkening (dark
adaptation) of the leaf using a PPFD of 3000 µmol m−2 s−1 β‑Carotene Assay
as saturating flash for the duration of 1 s.
Extraction for the β-carotene analysis was carried out
Leaf Gas Exchange Parameters according to the Association of Official Analytical Chem-
ists (AOAC 1980). Fifty mL of 95% ethanol was added to
Net photosynthetic rate (PN), stomatal conductance (gs) 10 g of the macerated sample in a conical flask; this con-
and intercellular C­ O2 concentration (Ci) were recorded on a coction was maintained at 70–80 °C for 20 min in a water
sunny day on fully expanded penultimate leaves at light-sat- bath, shaking it periodically. The supernatant was decanted;
urating intensity of plants between 11.00 and 12.00 O’clock then, it was allowed to cool at room temperature. The etha-
(noon), using portable photosynthetic system (LICOR-6400, nol concentration of the supernatant was brought to 85%
Lincoln, NE, USA). The atmospheric conditions at the time by adding 15 mL of distilled water; it was further cooled in
of measurement were: photosynthetically active radiation an ice-water container for about 5 min. The cooled super-
(PAR): 682 µmol m−2 s−1; air temperature: 25 °C; and rela- natant was transferred into a separating funnel, followed
tive humidity: 66%. by addition of 25 mL of petroleum ether (pet-ether). The
PS II activity and leaf gas exchange parameters were stud- separating funnel was swirled gently to obtain a homog-
ied at 60 DAS only because these parameters are greatly enous mixture, and it was later allowed to stand until two
affected by the age of plant (Bielczynski et al. 2017). separate layers were obtained. The bottom layer, contain-
ing ethanol and other pigments, was run off into a beaker,
Photosynthetic Pigments while the top layer, containing carotenoids, was collected
into a 250-mL conical flask. The bottom layer was trans-
Contents (concentrations) of chlorophyll and carotenoids ferred into the separating funnel; it was re-extracted with
were estimated in the fresh leaves according to the method of 10 mL of pet-ether for 5–6 times until the extract turned
Lichtenthaler and Buschmann (2001). Fresh tissue (100 mg) fairly yellow. The entire pet-ether was collected into a 250-
from the interveinal area of the leaf was ground with 10 mL mL conical flask and transferred into a separating funnel for
of 100% acetone using mortar and pestle. The optical density re-extraction with 50 mL of 80% ethanol. After extraction,

13
Journal of Plant Growth Regulation

the petroleum-ether-carotenoid phase was made up to the Results

volume of 50 mL. The final extract was poured into sample
bottles for further analysis. LC–ESI–MS Analysis

Spectrophotometer Reading and Calculation LC–ESI–MS analysis revealed the presence of several
compounds present in SSW. The mass spectrum (Fig. 2)
The content of β-carotene in the petroleum-ether extract was confirmed the presence of ­KAR1 (+ 3H+). Peaks related to
determined spectrophotometrically (UV-1700, Shimadzu, karrikin-related compounds have also been identified like
Tokyo, Japan), recording the absorbance (OD) at 450 nm. strigyl acetate and xylose derivatives.
The concentration of carotenes, expressed as β-carotene
(mg/100 g), was calculated according to Khalil et al. (2015) Effect of SSW and KAR1 on D. carota L.
using the response factors as follows:
A× d × V The effect of different dilutions of SSW and various con-
𝛽-carotene = centrations of K­ AR1 was significant on the performance of
A1% 1 cm × w
carrot plants with regard to the parameters studied. In gen-
where A—absorbance, d—dilution, V—volume (mL), eral, 51.6 µg L−1 of SSW and 1.501 µg L−1 of K ­ AR1 proved
A1%1 cm—coefficient of absorbance (2592 for petroleum- effective for the parameters studied, whereas the control gave
ether), w—weight of the sample (g). the lowest value invariably.

Vitamin C Determination
Effect of SSW and KAR1 on Germination Percentage
Ascorbic acid (vitamin C) was determined by using the
2,6-dichlorophenol indophenol titrimetric method (AOAC Of the treatments applied, 51.6 µg L −1 of SSW and
2000). 1.501 µg L−1 of ­KAR1 significantly enhanced the percent
germination by 58.1 and 54.4%, respectively, as compared
to the control (Fig. 3).
Extraction

Fresh and dry carrot mass was macerated with 20% met- Effect of SSW and KAR1 on Growth Attributes
aphosphoric acid. Ten milliliters of extracting solution/g
of dry test portion was used for this purpose. The mixture The growth of plants, raised from the seeds soaked in dif-
was homogenized in a high-speed blender at 18,000×g ferent concentrations of SSW and ­KAR1, was stimulated
(in ice and darkness) for 1 min and then centrifuged at in respect with height of plants, number of leaves per plant
9000×g (refrigerated at 4 °C) for 20 min. This procedure and leaf area per plant. The SSW and K­ AR1 treatments also
was repeated twice, and the two resulting supernatants were significantly enhanced the length, diameter fresh weight and
mixed together. dry weight of roots per plant as compared with the control.
Of the various seed-soaking treatments, 51.6 µg L−1 of SSW
Titration and 1.501 µg L−1 of K ­ AR1 proved optimum, increasing the
plant height by 38.6 and 25.2%, respectively, over the control
Fifty milliliters of extracted solution was mixed with 25 mL at 60 DAS, while at 120 DAS, the percent increase in plant
of 20% metaphosphoric acid. Ten milliliters of this solution height was 29.7 and 25.4%, respectively, as a result of the
was dispensed in a small flask followed by adding 2.5 mL above-mentioned treatments (Tables 1, 2). The application of
of acetone. It was then titrated using indophenol solution as 51.6 µg L−1 of SSW increased the number of leaves and leaf
indicator until a faint pink color persists for 15 s. area per plant by 40.4 and 62.5% at 60 DAS, whereas at 120
DAS, this increment was to the extent of 65.5 and 48.1%,
respectively. At 60 DAS, 1.501 µg L−1 of ­KAR1 exhibited
Statistical Analysis
maximum increase in the values for these growth attrib-
utes (36.4 and 52.3%, respectively). At 120 DAS, 57.8 and
Data of the present study were analyzed using one-way
42.0% increase in leaf number and leaf area, respectively,
analysis of variance (ANOVA) using SPSS 21 statisti-
were recorded in comparison with the control (Tables 1, 2).
cal software (IBM Corp., Armonk, NY, USA). Treatment
Application of 51.6 µg L−1 of SSW resulted in maxi-
means were separated using Duncan’s multiple range test
mum increase in the length (37.2%), diameter (33.5%),
at p < 0.05. The results are presented as mean values ± S.E.
fresh weight (41.8%) and dry weight (43.0%) of carrot
(standard errors) in tables and figures.

13
 Journal of Plant Growth Regulation

Fig. 2  Mass spectrum of SSW showing K

­ AR1

Fig. 3  Effect of various concentrations of SSW (Control- seed-germination of carrot (D. carota L.). Means within a column
DDW, T1-25.8 µg L−1, T2-51.6 µg L−1, T3-103.2 µg L−1, and followed by the same letter(s) are not significantly different (p < 0.05).
T4-258.0 µg L−1) and ­ KAR1 (Control-DDW, T1-0.015 µg L−1, Error bars (%) show ± S.E
T2-0.150 µg L−1, T3-1.501 µg L−1 and T4-15.013 µg L−1) on percent

root, compared to the control. Similarly, the treatment Effect of SSW and KAR1 on Photosynthetic
1.501 µg L−1 of K
­ AR1 maximally enhanced the values for Attributes
length (42.2%), diameter (41.3%), fresh weight (53.6%)
and dry weight (65.8%) of carrot root over the control Priming of carrot seeds with 1.501 µg L−1 of K
­ AR1 resulted
(Table 3). in positive effects on all the photosynthetic parameters,

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Journal of Plant Growth Regulation

Table 1  Effect of presoaking seed treatment with smoke-saturated water (SSW) on plant height, number of leaves, leaf area, and total content of
leaf chlorophyll and carotenoids of Daucus carota L. recorded at 60 and 120 DAS (days after sowing)
Parameters DAS Smoke-saturated water concentrations
Control (DDW) 25.8 µg L−1 51.6 µg L−1 103.2 µg L−1 258.0 µg L−1

Plant height (cm) 60 32.50 ± 0.62e 40.70 ± 0.55b 43.50 ± 0.53a 38.20 ± 0.59c 36.10 ± 0.31d
120 47.20 ± 0.85d 54.20 ± 0.80bc 61.20 ± 0.67a 56.30 ± 0.29ab 51.10 ± 1.10cd
Number of leaves ­plant−1 60 7.33 ± 0.34d 8.66 ± 0.41bc 10.33 ± 0.34a 9.66 ± 0.28ab 8.33 ± 0.37cd
120 8.66 ± 0.24d 13.00 ± 0.58ab 14.33 ± 0.34a 11.66 ± 0.33bc 10.33 ± 0.88c
Leaf area plant (­ cm2) 60 642 ± 9.64e 937 ± 14.93b 1043 ± 12.66a 865 ± 10.79c 748 ± 10.12d
120 1038 ± 8.02e 1465 ± 11.0ab 1537 ± 9.54a 1371 ± 10.12c 1163 ± 9.07d
Total chlorophyll content (mg g−1 FW) 60 2.63 ± 0.05d 3.17 ± 0.02b 3.34 ± 0.04a 3.06 ± 0.03b 2.87 ± 0.06c
120 2.47 ± 0.02d 2.87 ± 0.04b 3.11 ± 0.06a 2.77 ± 0.03b 2.64 ± 0.04c
Total carotenoids content (mg g−1 FW) 60 0.280 ± 0.002d 0.329 ± 0.002ab 0.340 ± 0.006a 0.321 ± 0.002b 0.297 ± 0.005c
120 0.164 ± 0.002d 0.193 ± 0.003ab 0.197 ± 0.002a 0.186 ± 0.003bc 0.178 ± 0.004c

Each value represents the mean of four replicates written with ± S.E. Means within a row followed by the same letter(s) are not significantly dif-
ferent (p < 0.05)
DDW double distilled water

Table 2  Effect of presoaking seed treatment with karrikinolide ­(KAR1) on plant height, number of leaves, leaf area, total chlorophyll content,
total carotenoids content of Daucus carota L. recorded at 60 and 120 DAS (days after sowing)
Parameters DAS KAR1 concentrations
Control (DDW) 0.015 µg L−1 0.150 µg L−1 1.501 µg L−1 15.013 µg L−1

Plant height (cm) 60 32.50 ± 0.62d 36.20 ± 0.55c 38.40 ± 0.72b 40.70 ± 0.55a 34.60 ± 0.36c
120 47.20 ± 0.85e 54.10 ± 0.45c 57.30 ± 0.21b 59.20 ± 0.53a 50.60 ± 0.67d
Number of leaves ­plant−1 60 7.33 ± 0.34c 8.66 ± 0.60abc 9.33 ± 0.65ab 10.00 ± 0.58a 7.66 ± 0.33bc
120 8.66 ± 0.24d 10.33 ± 0.34c 12.66 ± 0.36b 13.66 ± 0.33a 9.66 ± 0.25c
Leaf area p­ lant−1 ­(cm2) 60 642 ± 9.64d 714 ± 10.21c 838 ± 9.87b 978 ± 9.00a 697 ± 11.59c
120 1038 ± 8.02d 1208 ± 8.0c 1321 ± 9.85b 1474 ± 8.39a 1186 ± 9.07c
Total chlorophyll content (mg g−1 FW) 60 2.63 ± 0.06d 3.08 ± 0.02bc 3.16 ± 0.03ab 3.23 ± 0.04a 2.97 ± 0.05c
120 2.47 ± 0.02e 2.71 ± 0.05c 2.83 ± 0.02b 2.98 ± 0.03a 2.58 ± 0.04d
Total carotenoids content (mg g−1 FW) 60 0.280 ± 0.002d 0.324 ± 0.002bc 0.331 ± 0.004ab 0.334 ± 0.003a 0.315 ± 0.003c
120 0.164 ± 0.002c 0.185 ± 0.002b 0.189 ± 0.003ab 0.195 ± 0.004a 0.182 ± 0.003b

Each value represents the mean of four replicates written with ± S.E. Means within a row followed by the same letter(s) are not significantly dif-
ferent (p < 0.05)
DDW double distilled water

enhancing Fv/Fm, gs, Ci, and PN by 11.1, 28.3, 17.1 and 20.1% at 60 and 120 DAS, respectively. Compared to the
33.9%, respectively, over the control (Figs. 4, 5, 6, 7). The control, 1.501 µg L−1 of ­KAR1 gave maximum content of
seed priming effect of 51.6 µg L−1 of SSW (the optimum chlorophyll (22.8%) and carotenoids (19.2%) at 60 DAS,
SSW concentration) regarding photosynthetic parameters whereas at 120 DAS, the increment in chlorophyll content
was evidenced by the significant increase in Fv/Fm (11.9%), and carotenoids content was recorded at 20.7 and 18.9%,
gs (35.9%), Ci (22.6%), and PN (40.9%), in comparison with respectively, as a result of this ­KAR1 treatment (Tables 1, 2).
the control (Figs. 4, 5, 6, 7).
The contents of photosynthetic pigments, that is, total Effect of SSW and KAR1 on SEM Analysis of Carrot
chlorophyll and carotenoids, were affected positively by Leaves
SSW and ­KAR1 treatments. As compared with the control,
51.6 µg L−1 of SSW enhanced the total chlorophyll content According to SEM analysis, SSW and K ­ AR1 treatments
by 27.0 and 25.9% at 60 and 120 DAS, respectively, whereas resulted in significant enhancement (stomatal frequency
this treatment enhanced the carotenoids content by 21.4 and 251.28 ± 2.60 mm−2, pore diameter 1.2 µm and pore length

13
 Journal of Plant Growth Regulation

Table 3  Effect of presoaking Treatments Parameters

seed treatment with smoke-
saturated water (SSW) and Root length (cm) Root diameter (cm) Root fresh weight Root dry
karrikinolide ­(KAR1) on root (g plant−1) weight
length, root diameter, root (g plant−1)
fresh and dry weight of Daucus
carota L. recorded at 120 DAS Smoke-saturated water concentrations
(days after sowing) Control (DDW) 12.10 ± 0.15e 2.81 ± 0.03e 34.28 ± 1.21d 8.57 ± 0.06d
0.250 µg L−1 12.80 ± 0.21d 3.23 ± 0.02d 39.34 ± 1.10c 9.66 ± 0.03c
0.625 µg L− 1 13.50 ± 0.10c 3.35 ± 0.01c 52.65 ± 1.04a 9.78 ± 0.05c
−1
1.250 µg L 16.60 ± 0.12a 3.75 ± 0.01a 45.78 ± 1.26b 12.26 ± 0.02a
−1
2.250 µg L 14.40 ± 0.15b 3.53 ± 0.02ab 44.63 ± 1.11ab 12.05 ± 0.06b
KAR1 concentrations
0.015 µg L−1 15.70 ± 0.12c 3.65 ± 0.02c 42.60 ± 1.14bc 10.65 ± 0.04c
−1
0.150 µg L 16.60 ± 0.10b 3.72 ± 0.01b 45.78 ± 1.26b 10.99 ± 0.05b
−1
1.501 µg L 17.20 ± 0.15a 3.97 ± 0.03a 52.65 ± 1.04a 14.21 ± 0.03a
−1
15.013 µg L 13.20 ± 0.26d 3.42 ± 0.02d 39.34 ± 1.10c 10.23 ± 0.02d

Each value represents the mean of five replicates written with ± S.E. Means within a row followed by the
same letter(s) are not significantly different (p < 0.05)

Fig. 4  Effect of various concentrations of SSW (Control- phyll fluorescence of carrot (D. carota L.) leaves. Means followed by
DDW, T1-25.8 µg L−1, T2-51.6 µg L−1, T3-103.2 µg L−1 and the same letter(s) are not significantly different (p < 0.05). Error bars
T4-258.0 µg L−1) and ­ KAR1 (Control-DDW, T1-0.015 µg L−1, (%) show ± S.E
T2-0.150 µg L−1, T3-1.501 µg L−1 and T4-15.013 µg L−1) on chloro-

7.6 µm) in the stomatal frequency and pore size. SSW dilution, Effect of SSW and KAR1 on Nutritional Value
that is, 51.6 µg L−1, increased the frequency of leaf stomata by of Carrot
28.4% as compared to control, whereas diameter and length of
stomatal pores were approximately 3.0 and 10.5 µm, respec- Nutritional value of carrot tissue was assayed to evalu-
tively. Likewise, ­KAR1, applied at 1.501 µg L−1, resulted in ate the extent of modulation of β-carotene content due
25.0% increase in stomatal frequency, over the control, meas- to the application of presoaking seed treatments by SSW
uring 1.3 µm of pore diameter and 11.7 µm of pore length and ­KAR1. SSW, applied at 51.6 µg L−1, proved the best
(Fig. 8). for tissue β-carotene content, in comparison with the

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Journal of Plant Growth Regulation

Fig. 5  Effect of various concentrations of SSW (Control- lular ­CO2 concentration (µmol mol−1) of carrot (D. carota L.) leaves.
DDW, T1-25.8 µg L−1, T2-51.6 µg L−1, T3-103.2 µg L−1, and Means followed by the same letter(s) are not significantly different
T4-258.0 µg L−1) and ­ KAR1 (Control-DDW, T1-0.015 µg L−1, (p < 0.05). Error bars (%) show ± S.E
T2-0.150 µg L−1, T3-1.501 µg L−1 and T4-15.013 µg L−1) on intercel-

Fig. 6  Effect of various concentrations of with SSW (Control- tal conductance (mol m−2 s−1) of carrot (D. carota L.) leaves. Means
DDW, T1-25.8 µg L−1, T2-51.6 µg L−1, T3-103.2µg L−1, and followed by the same letter(s) are not significantly different (p < 0.05).
T4-258.0 µg L−1) and ­ KAR1 (Control-DDW, T1-0.015 µg L−1, Error bars (%) show ± S.E
T2-0.150 µg ­L , T3-1.501 µg L−1 and T4-15.013 µg L−1) on stoma-
−1

control. Over the control, this treatment enhanced the Discussion

tissue β-carotene content by 37.9%. On the other hand,
1.501 µg L−1 of KAR1 resulted in 48.4% increase in tissue Modulating plant growth, stature and yield has been a
β-carotene content, over the control (Fig. 9). farming goal in professional agronomy and horticulture. In
In the case of leaf ascorbic acid content, a progressive this regard, usage of sustainable agricultural practices and
increase was manifested as a result of SSW treatments. application of modern technologies are feasible options
Treatment with 51.6 µg L−1 of SSW enhanced the ascor- to increase the agricultural productivity. Use of organic
bic acid content by 32.2%, over the control. Similarly, fertilizers and plant-derived pesticides/herbicides for seed
1.501 µg L−1of ­KAR1 resulted in 42.0% increase in ascor- or crop treatment are workable examples of efforts carried
bic acid content, in comparison with the control (Fig. 10).

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 Journal of Plant Growth Regulation

Fig. 7  Effect of various concentrations of seed presoaking with SSW tosynthetic rate (µmol m−2 s−1) of carrot (D. carota L.) leaves. Means
(Control-DDW, T1-25.8 µg L−1, T2-51.6 µg L−1, T3-103.2 µg L−1, followed by the same letter(s) are not significantly different (p < 0.05).
and T4-258.0 µg L−1) and ­KAR1 (Control-DDW, T1-0.015 µg L−1, Error bars (%) show ± S.E
T2-0.150 µg L−1, T3-1.501 µg L−1 and T4-15.013 µg L−1) on net pho-

Fig. 8  Leaf stomatal response of carrot (D. carota L.) observed under scanning electron microscope due to seed presoaking with a Control, b
SSW (T2–51.6 µg L−1), c ­KAR1 (T3-1.501 µg L−1)

out in the direction of sustainable agriculture. In fact, horticultural industries to improve plant growth (Harris
there has been an increase in demand for such naturally et al. 1999; Khalil et al. 2001; Rashid et al. 2006) and
derived agrochemicals to achieve the sustainable produc- the vitamin content and nutritional value (Janeczko et al.
tion of crops. The results of the present study provide new 2015) of various crops. Germination-promoting com-
insights concerning the effects of SSW and ­K AR 1 with pounds (such as karrikinolide) or other growth-stimulat-
regard to growth and development of carrot. ing compounds can be safely incorporated into the seed
The seed-priming technique employed in this inves- through the process of imbibition to achieve such goals.
tigation is a widespread technique in agricultural and The results, obtained in this regard, are briefly discussed
below.

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Journal of Plant Growth Regulation

Fig. 9  Effect of various concentrations of seed presoaking with SSW carotene content of carrot (D. carota L.). Means followed by the
(Control-DDW, T1-25.8 µg L−1, T2-51.6 µg L−1, T3-103.2 µg L−1, same letter(s) are not significantly different (p < 0.05). Error bars (%)
and T4-258.0 µg L−1) and ­KAR1 (Control-DDW, T1-0.015 µg L−1, show ± S.E
T2-0.150 µg L−1, T3-1.501 µg L−1 and T4-15.013 µg L−1) on beta

Fig. 10  Effect of various concentrations of with (Control- bic acid content of carrot (D. carota L.). Means followed by the
DDW, T1-25.8 µg L−1, T2-51.6 µg L−1, T3-103.2 µg L−1, and same letter(s) are not significantly different (p < 0.05). Error bars (%)
T4-258.0 µg L−1) and ­ KAR1 (Control-DDW, T1-0.015 µg L−1, show ± S.E
T2-0.150 µg L−1, T3-1.501 µg L−1 and T4-15.013 µg L−1) on ascor-

Effect of SSW and KAR1 on Percent Seed-germination response of plants to smoke-water/smoke-

Seed‑Germination and Growth Attributes derived compounds has already been studied and promising
results have been obtained by van Staden et al. (2006) for
Seed germination is considered as the vital stage in seed- agricultural crops, and by Kulkarni et al. (2007b, 2008) for
ling establishment, which subsequently determines the plant horticultural crops. Jain and van Staden (2006) reported sig-
growth and successful crop production. Results obtained in nificant increase in percent germination of tomato seeds due
the present study revealed a remarkable increase in per- to the application of 1:500 (v/v) smoke dilution (equivalent
cent seed-germination as a result of SSW and ­KAR1 treat- to 51.6 µg L−1 according to our experimental methodology).
ments. The present results are in agreement with previous Considering various reports, not only the smoke-extract, but
studies regarding different crops (Adkins and Peters 2001; also smoke-derived compounds, have proven potentially
Flematti et al. 2004; van Staden et al. 2004; Jain and van effective in this regard. Seedlings of bean, okra and tomato
Staden 2006; Chiwocha et al. 2009; Tavşanoğlu et al. 2015). treated with butenolide, showed a greater percentage of seed

13
 Journal of Plant Growth Regulation

germination, in addition to improved seedling vigor indices resulting in release of seed-dormancy and promotion of
than the control (van Staden et al. 2006). In this regard, Jain seed-germination in several plant species (van Staden
and van Staden (2006) indicated that butenolide might be et al. 2000; Light et al. 2009). Nelson et al. (2008) com-
instrumental in mobilizing seed reserves into developing pared the influence of gibberellic acid (­ GA3) with those of
tomato seedlings. smoke components. The results showed that smoke-water
The present study also portrayed the substantial potential and butenolide-solutions, in most cases, were more effec-
of smoke-water and karrikinolide to improve physiological tive than ­GA3 in promoting seed-germination and seedling
attributes, as revealed by the positive effects of SSW and vigor of Arabidopsis plants. Daws et al. (2007) reported that
­KAR1 on all the growth parameters studied in the present butenolide-solution was significantly effective in enhancing
study. Our results confirmed that the effects of SSW and the percent seed-germination, rate of seedling growth, and
­KAR1 on various physiological parameters were signifi- seedling biomass with respect to many arable weed species.
cantly positive, as the values of physiological parameters In the case of tomato plant, Kulkarni et al. (2008)
were enhanced considerably over the respective controls reported that the smoke treatments significantly increased
(Tables 1, 2, 3). In this connection, our findings are consist- the number of leaves in tomato plants compared to the
ent with the those of Kulkarni et al. (2008), who reported control. According to results, the smoke extract (1:500)
that smoke-water treatment (1:500 v/v) resulted in maximum resulted in an increment of about three leaves as compared
plant height (14.2 ± 0.4 cm) in the case of tomato. Simi- to the control (seven leaves) as recorded in 57–58 days old
larly, root and shoot lengths, and seedling weight of tropical plants. In addition, the application of smoke extract (1:500)
soda apple were also stimulated due to the application of resulted in thicker stems compared to any other treatment
smoke-water and butenolide solution in a study conducted evaluated. These attributes, as affected by smoke-water treat-
by Kandari et al. (2011). Aremu et al. (2012a) analyzed the ments, contributed to seedling vigor. As a matter of fact, the
positive effects of plant-derived smoke on dry weight of plants, treated with smoke-water and butenolide-solution,
root, root/shoot ratio and leaf area in a tissue-culture study sustained increased growth until the fruiting stage (Kulkarni
conducted on Williams banana. Further, Ghebrehiwot et al. et al. 2008). In a similar study, maize plants, which were
(2013) reported an increase in dry biomass of Eragostis tef drenched with smoke-water, attained more than six leaves,
grass due to plant-derived smoke treatments. whereas the control plants had just more than four leaves as
It has been revealed by several studies that smoke treat- recorded in 30-day-old plants (van Staden et al. 2006). These
ment not only enhanced percent seed-germination, but also results indicate that smoke-water has the ability to improve
resulted in activation and improvement of seedling vigor the aboveground biomass over time. The same is indicated
(Rokich et al. 2002; Brown et al. 2003; Sparg et al. 2005). by the present study.
A significant increase in the seedling-vigor index of crop van Staden et al. (2000) also explained that butenolide,
plants, such as that of tomato, bean, okra and maize, has present in plant derived smoke, might positively affect the
been reported by van Staden et al. (2006), using both smoke- physiological parameters by interacting with endogenous
water and butenolide treatments; as per results, treated plants plant growth regulators. It has also been observed previously
had invariably higher numbers of fruits than controls. In that aqueous solutions of plant-derived smoke might exert
addition, smoke-water treatment significantly increased a hormone like effect on different species, interacting with
the total number of marketable fruits in the case of tomato auxins, cytokinins, gibberellins, ethylene and ABA present
(Kulkarni et al. 2008). Seeds primed with smoke-water dilu- in the plants (Daws et al. 2007). In fact, low concentrations
tion of 1:500 (v/v) resulted in the best growth performance, of SSW and ­KAR1 have been reported to show auxin and
with maximum elongation of shoot and root (Kulkarni cytokinins like activities in vitro in respect with leguminous
et al. 2007b). These findings provide support to the results plants (Jain et al. 2008).
obtained in the present study. According to our findings regarding carrot plants,
Daws et al. (2007) reported that seeds treated with smoke- 51.6 µg L−1 of SSW proved to be the best treatment for plant
water or butenolide-solution germinated faster, showing height, number of leaves and leaf area, whereas 1.501 µg L−1
increased vigor and fresh mass. Seedling vigor determines of ­KAR1 gave optimum values regarding length, diameter,
the potential of plant establishment in pots as well as in and fresh and dry weight of root (Table 3).
the field; it influences growth of plant and economic yield.
Therefore, it can be hypothesized that an increase in seedling Effect of SSW and KAR1 on Photosynthetic
vigor by SSW and K ­ AR1, as observed in the present study, Attributes, Stomatal Response and Nutritional Value
might be due to the growth-promoting effect of SSW and
­KAR1 applied in this study. In the present study, progressive improvement in the pho-
It is reported that smoke-water or butenolide-solution tosynthetic attributes was witnessed as a result of priming
might act like, or interact with other plant hormones, of seeds before sowing, using SSW and ­KAR1 solutions

13
Journal of Plant Growth Regulation

(Figs. 4, 5, 6, 7). In this context, our results corroborate compounds in the smoke as suggested by Chiwocha et al.
those of Aremu et al. (2012b), who found an increase in (2009). Overwhelmingly, the results of the present study
the content of leaf-chlorophyll (a/b) in banana (variety Wil- provide new insights into the effects of SSW and ­KAR1 in
liams) plants, when subjected to plant-derived smoke treat- the growth and development of carrot.
ments. Similarly, Ghebrehiwot et al. (2013) also recorded
profound increase in total leaf-chlorophyll content in a cereal
crop (Eragrostis tef) due to plant-derived aerosol smoke. Our Possible Mechanism of Action of SSW/KAR1
findings are also in agreement with the results obtained by
Jamil et al. (2013), who reported that smoke-primed plants The underlying mechanisms of action with regard to plant-
of rice gave greater leaf-chlorophyll content than the plants derived smoke and its derivative compounds have not yet
treated with hydro-priming (seed priming with water). been clearly understood. At present, various scientific groups
Significant improvement in photosynthetic parame- in different laboratories are actively engaged in studying the
ters (Fv/Fm, gs, Ci and PN), recorded in the present study mode of action of SSW/KAR1. Although there are various
(Figs. 4, 5, 6, 7) is in agreement with the findings of Zhou reports confirming the interaction of SSW/KAR1 with phy-
et al. (2013), who reported similar effects of smoke-water tohormones associated with plant development processes.
treatments on photosynthetic activity of Isatis indigotica. An Several investigation reports led to the conclusion that SSW/
increase in PN of carrot plants due to the SSW and K ­ AR1 KAR1 might have effects similar to G ­ A3 in stimulating ger-
treatments, employed in the present investigation, suggests mination and substituting the light requirement in germina-
that these treatments might have enhanced the stomatal tion of lettuce seeds (Drewes et al. 1995; van Staden et al.
opening as manifested by the SEM analyses of leaves that 1995) and in some members of Asteraceae family (Mer-
revealed the positive role of SSW and K ­ AR1 in regulating ritt et al. 2006). Other reports on cognate characteristics
the stomatal response, which might be the reason behind of ­GA3 and SSW/KAR1 include the study of Schwachtje
the increase in photosynthetic attributes like gs, Ci and PN. and Baldwin (2004) on Nicotiana attenuata. Also, Gard-
Carotenoids are lipophilic secondary metabolites derived ner et al. (2001) reported an increase in endogenous G ­ A3 in
from the isoprenoid pathway, which are accumulated in the SSW treated lettuce seeds. According to Meng et al. (2016),
tissue of most plant organs (Howitt and Pogson 2006). The other phytohormones (ABA and IAA) might play pivotal
characteristic bright orange color of carrot is related to its roles in seed germination and seedling growth as well. They
β-carotene content, which is converted by our body into vita- quantified the phytohormones in germinating soybean seed-
min A (Bao and Chang 1994). Ascorbic acid (vitamin C) lings and found that karrikins were responsible for altering
is the most abundant antioxidant found in plants. In view the GA/ABA ratio. IAA is another class of phytohormones,
of the nutritional value of carrot, our study also included whose action might be modified by SSW/ ­KAR1 (Nelson
the tissue contents of β-carotene and ascorbic acid, which et al. 2011). So, the interaction of phytohormones with
were markedly enhanced by seed presoaking treatment with SSW/KAR1 could be contemplated as a possible mode of
SSW and K ­ AR1. This indicated the stimulation of second- action in the SSW/KAR1-related growth response system.
ary plant products in plants treated with SSW and ­KAR1. In
this context, our findings can be supported by the work of
Zhou et al. (2011), who reported significant enhancement in
indigo (bioactive compound) content of the Chinese medic- Conclusion
inal plant (Isatis indigotica) with SSW (1:500 dilution).
Indeed, there are findings, which might confirm the role The technology related to plant-derived smoke and to the
of SSW and K ­ AR1 in enhancing the phenolic content and smoke-derived butenolide hold promise for use in agricul-
other secondary metabolites in plants (Aremu et al. 2012b, ture and horticulture for crop improvement in terms of seed
2014). The results of the present investigation suggested germination, seedling vigor and overall yield. The technique
the stimulating potential of SSW and K ­ AR1 regarding plant is relatively simple and affordable that may prove as a feasi-
growth as well as secondary metabolites in plants. The effi- ble option for the resource-limited farmers to improve their
cacy of smoke extract and its isolated compounds have been crop production. It may be concluded that plant-derived
extensively studied regarding seed-germination; however, smoke exposure exerts positive and stimulatory effect on
there are comparatively fewer reports on plant growth, pho- morphological, physiological, photosynthetic and nutritional
tochemical yield and nutritional attributes of plants, which attributes of carrot plants. It may be suggested that inexpen-
have been investigated in this study in carrot. Most of our sive plant-derived smoke may be used as stimulating source
studies show positive influences of SSW and ­KAR1 on the to enhance the seed germination, growth, photosynthesis and
growth and physiological parameters of carrot, providing nutritional value of crops instead of achieving the same goal
evidence of the presence of some plant-growth promoter by expensive karrikinolide (smoke component).

13
 Journal of Plant Growth Regulation

Acknowledgements We gratefully acknowledge the SAIF, CDRI, Luc- Daws MI, Davies J, Pritchard HW, Brown NA, van Staden J (2007)
know (India), where the LC–ESI–MS analyses of SSW were carried Butenolide from plant-derived smoke enhances germination and
out. Acknowledgement is also due to Prof. Javed Iqbal, Department of seedling growth of arable weed species. Plant Growth Regul
Chemistry, AMU, India, and Mr. Mohammad Shavez, Department of 51:73–82
Microbiology, AMU, India, for critically analyzing the mass spectra de Lange JH, Boucher C (1990) Autecological studies on Audouinia
regarding this study. capitata (Bruniaceae). I. Plant-derived smoke as a seed germi-
nation cue. S Af J Bot 56:700–703
Doherty LC, Cohn MA (2000) Seed dormancy in red rice (Oryza
Compliance with Ethical Standards sativa). XI. Commercial liquid smoke elicits germination. Seed
Sci Res 10:415–421
Conflict of interest The authors declare that they have no conflict of Drewes FE, Smith MT, van Staden J (1995) The effect of a plant-
interests to disclose. derived smoke extract on the germination of light-sensitive let-
tuce seed. Plant Growth Regul 16:205–209
Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD (2004) A
compound from smoke that promotes seed germination. Sci-
ence 305:977–977
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