Biological Agriculture & Horticulture

An International Journal for Sustainable Production Systems

ISSN: 0144-8765 (Print) 2165-0616 (Online) Journal homepage: http://www.tandfonline.com/loi/tbah20

Grafting tomato onto tobacco rootstocks is a

practical and feasible application for higher
growth and leafing in different tobaccotomato
unions

Mehmet Haberal, Didem Aksoy Krpe, zlem Darcansoy eri & Feride Iffet
Sahin

To cite this article: Mehmet Haberal, Didem Aksoy Krpe, zlem Darcansoy eri & Feride Iffet
Sahin (2016): Grafting tomato onto tobacco rootstocks is a practical and feasible application
for higher growth and leafing in different tobaccotomato unions, Biological Agriculture &
Horticulture, DOI: 10.1080/01448765.2016.1169218

To link to this article: http://dx.doi.org/10.1080/01448765.2016.1169218

Published online: 07 Apr 2016.

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 Biological Agriculture & Horticulture, 2016
http://dx.doi.org/10.1080/01448765.2016.1169218

Grafting tomato onto tobacco rootstocks is a practical and

feasible application for higher growth and leafing in different
tobaccotomato unions
Mehmet Haberala,b, Didem Aksoy Krpea, zlem Darcansoy eria and Feride Iffet Sahina,c
a
Institute of Transplantation and Gene Sciences, Baskent University, Ankara, Turkey; bFaculty of Medicine, Department
of Surgery, Baskent University, Ankara, Turkey; cFaculty of Medicine, Department of Medical Genetics, Baskent
University, Ankara, Turkey
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ABSTRACT ARTICLE HISTORY

A previously developed grafting strategy was applied to graft a commercial Received 23 October 2014
cultivated variety of tomato (Solanum lycopersicum L. H-2274) onto Accepted 18 March 2016
Nicotiana rustica L. (cv. Hasankeyf ) and Nicotiana tabacum L. (cv. Samsun) KEYWORDS
rootstocks. Higher growth and leafing, and earlier flower onset were found Grafting; Nicotiana rustica;
in grafted than in non-grafted and self-grafted plants. Significant 22.7 and Nicotiana tabacum; Solanum
34.3% increases in fruit yield were obtained with Samsun and Hasankeyf lycopersicum
rootstocks, respectively. Leaves of tobacco-grafted plants had significantly
elevated levels of nicotine, and highest leaf nicotine levels were measured
at 90days after transplantation (DAT). Leaves from upper sampling levels of
plants had significantly less nicotine independent of the sampling DAT. Fruits
of all tobacco-grafted plants had significantly higher amounts of nicotine
than non-grafted scions. Nicotine levels were higher in fruits sampled from
lower parts of the plants for different harvest DAT and rootstocks. Increases
in leaf nicotine concentration of tobacco-grafted plants were higher when
compared with that of fruit. Grafting tomato onto tobacco rootstocks is a
practical and feasible application for different tobaccotomato unions.

Introduction
Grafting of horticultural crops has been widely used for improvement of crop yield, quality and plant
growth, to increase resistance to biotic and abiotic stress, to change varieties or cultivars and to opti-
mise cross-pollination and pollination. Grafting has been used to improve resistance of the scion to
soil-borne diseases (Lee 1994; Trionfetti Nisini et al. 2002), low (Bulder et al. 1990) and high (Rivero
et al. 2003) temperatures, and high salt concentration (Ruiz et al. 2005), and to enhance nutrient
uptake (Ruiz et al. 1997).
Grafting performance depends on compatibility between scion and rootstock. For successful graft-
ing, vascular tissues of rootstock and scion should contact each other, and cambial division with sec-
ondary vascular tissues should occur at the transplant site (Moore 1984). Considering horticultural
crops, various graft combinations of rootstocks and scions of different species have been investigated
for the assessment of grafting efficiency (Kacjan-Marsic & Osvald 2004; Khah et al. 2006; Gisbert
et al. 2011). Incompatibility of rootstocks and scions cause decline in productivity and fruit quality
(Trionfetti Nisini et al. 2002).

CONTACT zlem Darcansoy eri odiseri@baskent.edu.tr

2016 Informa UK Limited, trading as Taylor & Francis Group
 2 M. Haberal et al.

In general, plants of the same family are grafted to obtain highly compatible grafts. Grafting tech-
niques are widely used in the Solanaceae family i.e. in tomato and eggplant to improve fruit quality
and yield (Flores et al. 2010; Gisbert et al. 2011; Moncada et al. 2013). Grafting of tobacco scion on
tomato rootstock was also studied to evaluate nicotine accumulation in different tissues (Solt 1957;
Solt & Dawson 1958). Tomato has economic importance in Turkey, with 1012 million tonnes annual
production in 20102013 being the fourth most produced commodities in these years (FAO 2014).
In addition, tobacco production of Turkey constitutes 1.5% of worldwide production placing Turkey
in the tenth rank (FAO 2014) in 2013. Nicotiana tabacum L. and Nicotiana rustica L. are two of the
commercially cultivated variants in Turkey. A novel and promising cultivation technique for the
improvement of tomato plant performance and yield by grafting tomato onto tobacco rootstocks has
previously been defined (Yasinok et al. 2009), and the grafted plants patented as Tomacco (Patent no:
TR-2008-05391-B). Fruit and leaf nicotine increased in grafted plants; however, nicotine levels were
within safe consumption limits for fruits. The salt tolerance and adaptive response of tomato cv. Elazg
grafted onto N. tabacum cv. Samsun and N. rustica cv. Hasankeyf has been assessed and enhancement
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of salt tolerance observed (eri et al. 2015). Two tobacco cultivars have also been demonstrated to
have different adaptive responses against salt stress (eri et al. 2013). For the establishment of the
patent and the technique in routine agricultural applications, and to demonstrate the sustainability
of the technique with different scion-rootstock pairs, the performance of new graft unions using N.
tabacum and N. rustica as rootstocks, and a commercially available tomato cultivar was investigated.
Nicotine is accumulated in response to wounding and is synthesised in roots and then transported
to the aerial organs (Dawson 1942; Baldwin et al. 1997). Nicotine accumulation in leaves and fruits
may vary between the graft unions due to compatibility and wound recovery, plant age and sampling
stem level. Therefore, in this study the question of whether nicotine levels show variation in different
parts of the grafted plants and/or depending on harvest time was also addressed.

Material and methods

Growth conditions of scion and rootstocks
Plants were grown and grafting applied, in the plastic greenhouses of the Institute of Transplantation
and Gene Sciences, Bakent University, Kazan-Ankara, Turkey. Turkish local cultivars N. tabacum cv.
Samsun and N. rustica cv. Hasankeyf, were used as rootstocks in grafting experiments. A Solanum
lycopersicum L. commercial cv. H-2274 (May-Agro Seed Corporation, Bursa, Turkey) was used as scion.
Tobacco and tomato seeds were sown into cells containing seedling substrate (Klasman-Deilmann
GmbH, Geeste, Germany) and grown for 50 and 25days, respectively at 2025C with a relative
humidity of 5060%. Plants were transferred to soil in individual pots containing soil:fertilizer, 2:1,
the fertilizer derived from excreta of cows fed by clover silage in the farm of Bakent University, and
grown for another 2025days at 2025C with a relative humidity of 5560% in a greenhouse.

Grafting
The cleft grafting method was applied when the scion had three or four true leaves and the rootstocks
had six or seven true leaves (Yasinok et al. 2009) during May 2009. For the assessment of the effect
of grafting on plants, tomato plants were also self-grafted. In brief, the rootstock stem was scratched
from the middle at right angles with a sterile razor blade. The base of the scion was cut into a thin,
narrow wedge and inserted into the cut of the rootstock. The graft union was wrapped with Parafilm
to enhance wound healing. Grafted and self-grafted plants were kept for 10days under controlled
conditions (2025C with a relative humidity of 9095%, and a photoperiod of 16:8h; light:dark).
Grafted, self-grafted and non-grafted control tomato plants were kept at 2025 C with a relative
humidity of 5060% for a week, and then transplanted to soil field in greenhouse 60cm apart from
each other. Temperature was controlled during the growing season in the 2025C range during
 Biological Agriculture & Horticulture 3

daytime temperature and night time temperatures were kept above 15C. Irrigation was maintained by
applying water at 23day intervals prior to the first harvest and on alternate days after the first harvest
via a drip irrigation system with drip tubes placed at the base of each plant, each with a flow rate of
2lh1. Soil moisture content was measured (ECH2O-10, Decagon Devices, Pullman, WA, USA). An
animal based fertilizer was applied to the soil 30days before the transplantation.

Growth monitoring and yield assessment

Measurements of height and leaves were performed between 30 and 140days after transplanting (DAT),
and the onset of flowering (FO) was evaluated. Ripe fruits were collected and weighed for 200days.
Fruit yield per plant and fruit weight were calculated.

Relative water content and pigment concentration of leaves

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At each sampling, height of the plants was measured. Accordingly, each leaf sampling was performed
from lower, medium and upper part of each plant. In order to determine relative water content (RWC)
of leaves, leaf samples from lower, medium and higher levels of plants at 90, 110, and 140 DAT were
sampled, and fresh (FW), dry (DW), and turgor weights (TW) were measured. Samples were washed
with distiled water and FW were measured. Then samples were floated in distiled water for 24h and
TW was measured. Samples were dried at 60C for 2days and DW was measured. RWC was also
calculated according to the formula (Celikkol Akcay et al. 2010):
RWC (%) = [(FW) (DW) (TW) (DW)1 ] 100
For the determination of total chlorophyll (a+b) of leaves from lower, medium and higher levels
at 90, 110, and 140 DAT were sampled. A sample of fresh leaves was put in pure acetone (Merck,
Darmstadt, Germany), and incubated at 4 C for 4 days. Absorbance was measured at 644.8 and
661.6nm, and concentration of chlorophyll a+b in leaves was calculated according to Lichtenthaler
and Buschmann (2001). Anthocyanin concentration of leaf samples were determined by crushing
fresh leaf samples in 1ml of 79% (v/v) methyl alcohol (Merck) and 1% HCl (Merck) (v/v) solution.
Samples were incubated at 4C for 4days, and absorbance of the sample solution was measured at
530 and 657nm (=34,300M1cm1) (Mancinelli 1990; Giusti & Wrolstad 2001).

Nicotine analysis
Two grams of leaves and 5g of fruit samples were homogenised in 1.5 and 1ml of distiled water, respec-
tively. A Dispomix homogenizer (Zug, Switzerland) was used at 4004000g for 40s in clockwise
and counter-clockwise directions. Nicotine was extracted from samples by toluene (Merck) (Siegmund
et al. 1999). Diphenylamine (Sigma-Aldrich, St. Louis, MO, USA) was used as an internal standard to
spike samples. Nicotine in samples was measured by Agilent 6890 Series Gas Chromatograph (Santa
Clara, CA, USA) equipped with a flame ionisation detector at the Anadolu University Medicinal Plants,
Drugs and Scientific Research Center (Eskiehir, Turkey). An HP-1 (250.32mm, 0.17m) column
was used. Nitrogen was used as the carrier gas. An injection volume of 1l and an injector temper-
ature of 200C were employed. The temperature programme was 40C for 5min; then increased to
a final temperature of 220C (with 15Cmin1), which was held constant for 10min. The detector
temperature was 250C. Nicotine standards (Dr. Ehrenstorfer GmbH, Augsburg, Germany) were
injected for quantitative content analysis.

Statistical analysis
Statistical analyses were performed using SPSS 11.5 software (SPSS Inc., Chicago, IL, USA). All data
are expressed as mean standard error of the means (SEM). Mean differences between grafted,
 4 M. Haberal et al.

non-grafted, and self-grafted plants were statistically evaluated by one-way analysis of variance, and
post hoc Tukey analyses were carried out to find groups whose mean differences were significant at
p<0.05. Relative water, total chlorophyll and anthocyanin contents of leaves were evaluated from six
biological replicates within the same sampling level. Nicotine contents from different plants, measured
from two biological and two technical replicates, were compared within the same sampling level and
between harvest levels of the same plant.

Results
Growth monitoring and yield assessment
Grafting success is one of the important parameters for the assessment of feasibility in routine agricul-
tural applications Grafting success was 96 and 91% for Hasankeyf and Samsun rootstocks, respectively.
Since sufficient survival rates were obtained with both rootstocks, tests were carried out to assess
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whether similar survival rates could be attained with more cost effective optimizations for larger
production capacity. Relative humidity is an important parameter for grafting success. Plants were
incubated at a relative humidity of 9095% for 10days after grafting. Graft performance of tobacco
unions was also tested at a relative humidity of 8085%. However, graft performance decreased to 77
and 80% for Hasankeyf and Samsun rootstocks, respectively, so the incubation humidity was set at
9095% for better graft performance in all experiments.
The height of non-grafted, self-grafted and tobacco-grafted plants did not differ significantly at
140 DAT (Table 1). At 140 DAT foliation of the H2274/H union was significantly higher than the
other groups. Tobacco grafted plants had significantly earlier flower onset than non-grafted plants. In
addition, FO of H2274/H plants were significantly less than self-grafted and H2274/S plants.
H2274/H grafted plants had greater fruit yield than non-grafted and self-grafted plants (Table 1).
Statistically significant 22.7 and 34.3% increases in fruit yield of tomato cv. H-2274 compared with
non-grafted plants were obtained by utilizing cv. Samsun and cv. Hasankeyf tobacco grafting, respec-
tively . In addition, a 16.7% of fruit yield increase of self-grafted plants in comparison with non-grafted
plants was obtained. Average fruit weights obtained from grafted, non-grafted, and self-grafted plants
did not differ significantly.

RWC and pigment concentration of leaves

RWC is a quantitative indication of water status of plants, and specific tissues. RWC was lower in
tobacco-grafted and self-grafted plants than in non-grafted plants at 90 DAT, was unaffected by grafting
at 110 DAT, and was again lower in self-grafted, but not tobacco-grafted, than the non-grafted at 140
DAT in all sampling levels (Table 2).
Decrease in total chlorophyll content causes decrease in rate of photosynthesis. Effectively there
was no difference in chlorophyll concentrations at any of the leaf levels. Self or tobacco grafted plants
never differed significantly in chlorophyll concentration from non-grafted plants. In only, H2274/H

Table 1.Effects of grafting on height and leaf number 140 days after transplanting (DAT), onset of flowering (FO), yield and fruit
weight.

H2274 SG H2274/H H2274/S

Height (cm) (140 DAT) 150.611.4 a 145.86.8 a 154.88.8 a 158.17.4 a
Leaves (140 DAT) 392 a 344 a 483 b 413 a
FO (DAT) 342 a 321 a, b 263 c 301 b
Fruit yield per plant (gplant1) 2100.495.2 c 2452.3110.1b 2820.4113.3 a 2577.2114.1 b
Fruit weight (g) 108.28.3 a 112.44.5 a 114.35.2 a 110.24.4 a
Notes: All data are expressed as meanstandard error. Means of the grafted (H2274/H and H2274/S), non-grafted (H2274), and
self-grafted (SG) plants followed by the same letter are not statistically significant according to post hoc Tukey analyses (p>0.05).
 Biological Agriculture & Horticulture 5

Table 2.Stress assessment parameters (relative water content (RWC), chlorophyll (Chl a+b) and anthocyanin concentrations) of
non-grafted (H2274), self-grafted (SG), and grafted (H2274/H and H2274/S) plants on different days after transplanting (DAT).

DAT H2274 SG H2274/H H2274/S

90 RWC (%) Low 91.142.45 a 81.812.00 b 84.883.61 b 80.062.98 b
Medium 85.352.69 a 79.933.40 b 73.874.45 b 74.894.99 b
High 87.952.77 a 78.071.52 b 75.118.05 b 72.343.82 b
Chl a+b (mggFW1) Low 1.520.24 a 1.540.30 a 1.810.08 a 2.200.30 a
Medium 1.420.27 a 1.480.08 a 1.830.15 a 1.920.19 a
High 2.020.04 a, b 1.420.11 b 2.310.19 a 2.010.24 a, b
Anthocyanin (molgFW1) Low 0.150.02 a 0.890.10 b 0.140.01 a 0.130.01 a
Medium 0.140.01 a 0.890.05 b 0.140.01 a 0.130.01 a
High 0.200.09 a 0.880.08 b 0.150.01 a 0.130.01 a
110 RWC (%) Low 97.070.14 a 93.073.72 a 96.522.04 a 90.021.68 a
Medium 95.402.34 a 93.951.93 a 91.572.93 a 91.313.63 a
High 92.770.40 a 89.651.77 a 90.493.61 a 86.143.03 a
Chl a+b (mggFW1) Low 1.210.09 a 1.350.17 a 1.020.07 a 1.140.13 a
Medium 1.420.18 a 1.320.09 a 1.020.08 a 1.230.22 a
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High 1.430.29 a 1.400.19 a 0.950.02 a 1.100.08 a

Anthocyanin (molgFW1) Low 0.650.01 a 0.460.02 b 0.230.05 c 0.160.04 c
Medium 0.470.07 a 0.470.02 a 0.240.05 b 0.220.04 b
High 0.440.01 a 0.500.02 a 0.270.04 b 0.210.06 b
140 RWC (%) Low 92.591.40 a 74.491.96 b 93.375.09 a 91.140.71 a
Medium 91.991.99 a 68.172.29 b 91.442.22 a 91.170.53 a
High 90.790.30 a 65.512.15 b 88.862.00 a 89.380.73 a
Chl a+b (mggFW1) Low 1.110.01 a 0.940.16 a 1.170.06 a 1.130.05 a
Medium 1.010.09 a 0.990.08 a 1.140.04 a 1.040.03 a
High 1.100.09 a 1.020.02 a 1.250.04 a 1.250.04 a
Anthocyanin (molgFW1) Low 0.390.10 a 0.650.02 b 0.370.04 a 0.310.04 a
Medium 0.460.10 a 0.810.03 b 0.340.02 a, c 0.290.04 c
High 0.550.02 a 0.870.04 b 0.380.05 a, c 0.290.07 c
Notes: All data are expressed as meanstandard error. Means of the grafted, non-grafted, and self-grafted plants of the same harvest
level followed by the same letter are not statistically significant according to post hoc Tukey analyses (p>0.05).

high leaves at 90 DAT chlorophyll concentration in grafted plants significantly exceeded self-grafted
(Table 2).
Regarding anthocyanin, self-grafted concentrations were significantly higher than non-grafted
throughout, except medium and high leaves at 110 DAT; whereas anthocyanin accumulations in self-
grafted plant leaves were significantly higher than in other groups except medium and high leaves
at 110 DAT (Table 2). H2274/H had lower anthocyanin concentration than non-grafted only at 110
DAT. H2274/S had lower anthocycanin concentration than non-grafted at 110 DAT (all leaf levels)
and 140 DAT (medium and high leaves only).

Effect of grafting on nicotine concentration

Leaf samples collected from tobacco-grafted plants had significantly elevated levels of nicotine (Table
3). Highest nicotine levels were measured at 90 DAT collected from low and medium levels of tobacco
grafted plants. Samples collected from high levels of tobacco-grafted plants had significantly less nico-
tine than other levels independent of sampling time. H2274/H leaf nicotine concentration seemed to
be affected by plant age since leaf nicotine concentration were lower 120 and 160 DAT in all sampling
levels, except for the sample collected at 90 DAT from high level. Leaf nicotine concentration of the
scion was 8.831.05mgkgFW1 and of rootstocks was 104.520.38 and 294.5114.93mgkgFW1
for cvs. Samsun and Hasankeyf, respectively, prior to grafting. Interestingly, cv. Hasankeyf had higher
leaf nicotine than cv. Samsun, and the levels were lower than the ones obtained after grafting.
Fruits of tobacco-grafted plants had significantly higher concentrations of nicotine than those of
non-grafted scions independent of harvest time and level (Table 4). Nicotine concentration of fruits
harvested 120160 DAT from medium level of self-grafted plants was 1.010.02gkgFW1 and did
 6 M. Haberal et al.

Table 3.Nicotine concentrations of leaves sampled from different levels of the non-grafted (H2274), self-grafted (SG), and tobac-
co-grafted (H2274/H and H2274/S) plants on different days after transplanting (DAT).

Leaf nicotineSEM (mgkgFW1)

DAT Sampling level H2274 H2274/H H2274/S SG
90 Low 9.120.94 a 1129.877.85 b 2020.34114.31 c
Medium 14.541.69 a 1043.858.66 b 1961.543.67 c
High 9.420.05 a 333.2312.29 d 662.5728.68 e
120 Low 9.790.07 a 327.121.75 b 1054.3015.3 8c
Medium 12.631.36 a 294.6314.77 b 934.802.17 c 12.900.28 a
High 18.820.88 d 273.5915.74 e 412.340.35 f
160 Low 15.061.52 a 454.553.09 b 1961.3967.69 c
Medium 18.530.07 a 400.904.92 e 1929.8794.58 c
High 20.131.29 d 305.504.57 f 443.419.82 g
Notes: All data are expressed as meanstandard error. Means of the non-grafted and tobacco-grafted plants are compared for
sampling levels only, and the means of the same DAT followed by the same letter are not statistically significant according to post
hoc Tukey analyses (p>0.05).
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Table 4.Nicotine concentrations of fruits harvested from different levels of the non-grafted (H2274), self-grafted (SG), and tobac-
co-grafted (H2274/H and H2274/S) plants on different periods.

Fruit nicotineSEM (gkgFW1)

DAT Harvest level H2274 H2274/H H2274/S SG
0120 Low 0.980.06 a 2.800.01 b 4.120.03 c
Medium 1.000.01 a 1.700.02 d 2.600.01 e
120160 Medium 0.980.05 a 1.730.01 b 5.580.01 c 1.010.02 a
High 1.040.01 a 1.690.01 b 4.090.02 d
Notes: All data are expressed as meanstandard error. Means of the non-grafted and grafted plants of the same days after trans-
planting (DAT) followed by the same letter are not statistically significant according to post hoc Tukey analyses (p>0.05).

not statistically differ from the nicotine concentration of fruits of non-grafted plants harvested from
same level and time. Increases in leaf nicotine concentration of tobacco-grafted plants were higher
when compared to that of fruit levels. Nicotine concentration of fruits harvested from cv. Samsun
grafted scions was higher than those of cv. Hasankeyf. Similar to leaf nicotine concentrations, higher
nicotine was found in fruits harvested from lower level than from higher levels.

Discussion
Using the cleft grafting method, successful graft unions of H-2274 commercial cultivar with tobacco
rootstocks were obtained. Furthermore, the results obtained with tobacco unions were better than
the success of self-grafted plants (65%) in concordance with previous findings (Yasinok et al. 2009).
Better graft performance of the tobacco unions may be associated with high water content of tobacco
stem (Yasinok et al. 2009), thick stem diameter of tobacco or strength of the tobacco rootstock, which
are important factors affecting graft success in cleft grafting. Better graft performance can be obtained
with higher humidity.
Since the root system of plants affects vegetative growth, growth performance of grafted and non-
grafted plants may vary. Incompatibility may occur at early stages of grafting since vascular connections
between rootstock and scion are not exactly completed (Martnez-Ballesta et al. 2010). At 140 DAT
foliation of the H2274/H union was significantly higher than the other groups. Increased foliation of
grafted plants may have been due to increased water and nutrient uptake, and may be correlated with
higher graft performance (Lee 1994; Mohammed et al. 2009).
A positive effect of grafting on flower performance of tobacco grafted plants with different scions
has previously been reported (Yasinok et al. 2009). In the present study earlier flower onset of tobacco
grafted plants in comparison with non-grafted plants is also reported (Table 2). Earlier FO of tobacco
 Biological Agriculture & Horticulture 7

grafted plants may be correlated with wound healing response of tobacco graft unions. In fact, wound
healing of the tomatotobacco graft union is a more difficult process than that of a tomato-tomato
union since it requires establishment of vascular connections between different rootstock and scion.
On the other hand, greater reduction in FO of H2274/H plants than of H2274/S plants may be related
to differences in root systems of the two tobacco rootstocks. Interestingly, Habermann and Wallace
(1958) reported a reduction in FO time of sunflowers grafted onto older host plants. FO difference
between H2274/H and self-grafted plants may be the combined consequence of both root system and
age of the rootstock effects.
In concordance with a reduction in FO, grafted plants had greater fruit yield than non-grafted, and
self-grafted plants (Table 2). Since average fruit weights obtained from grafted, non-grafted, and self-
grafted plants did not differ significantly, altered fruit yield does not seem to be correlated with weight
of individual fruits. In a previous report, total fruit yield increase of 5.0 and 30.1% for cvs. Sweet and
Elazig tomato scions, respectively, grafted on cv. Samsun were obtained (Yasinok et al. 2009). However,
in that study self-grafting had a negative impact on fruit yield of cvs. Sweet and Elazig. Previous
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studies reported rootstock dependent variations in yield of scion cultivars (Romano & Paratore 2001;
Kacjan-Marsic & Osvald 2004; Khah et al. 2006). A recent report on the effectiveness of grafting of
different tomato genotypes onto distinctly different tomato rootstocks demonstrated that increments
in both fruit yield and fruit quality traits of the scion were induced by tomato rootstocks (Flores et
al. 2010). Moreover, Khah et al. (2006), demonstrated that tomato grafting on suitable rootstocks
had positive effects on the cultivation performance, especially in greenhouse conditions. Previously,
negative, neutral (Khah et al. 2006), and positive (Romano & Paratore 2001) impacts of self-grafting
of tomato cultivars on yield have also been demonstrated by other groups. Tomato-tomato grafting
is considered as a well established method for tomato production. The yield increments reported
here with tomatotobacco grafting are compatible to tomato-tomato grafting yields reported in other
studies i.e. 36.9% (Kacjan-Marsic & Osvald 2004) and 11.032.5% with different tomato rootstocks
(Khah et al. 2006).
Enhancement of tomato performance and yield may be related to both the nature of the rootstock
and graft induced changes. In fact, differences in tomato yield of H2274/H and H2274/S are more
probably related to rootstock effect on scion. Increase of the self-grafted tomato yield, compared with
non-grafted plants, on the other hand, seems to be related to graft induced effects. Vigour of the root
system, and altered water and mineral uptake are important factors affecting performance of grafted
plants (Ioannou et al. 2002; Kacjan-Marsic & Osvald 2004).
Graft incompatibility can lead to decreased water and nutrient flow through the graft union and
cause wilting of the plant (Davis et al. 2008). Reduction in leaf RWC (Table 2) may be correlated with
graft induced stress on plants and diminished water transport. Since at 140 DAT, self-grafted plants
had significantly less leaf RWC at all sampling levels, aging had probably more severe effects on self-
grafted plants, which were eliminated by the root system of tobacco grafted plants. Negative impacts
of grafting are more apparent at early stages, when vascular connections are forming, but can also
appear at later stages of growth when the plant has a high demand for water and nutrients (Martnez-
Ballesta et al. 2010).
Chlorophyll concentration of leaves decreases in response to environmental factors through inhi-
bition of the synthesis or through an acceleration of its degradation (Reddy & Vora 1986). Decrease
in total chlorophyll causes decrease in rate of photosynthesis. The chlorophyll content is related to
tissue water content (Defline et al. 1998). In a previous report, early negative effects of grafting on
chlorophyll concentrations 30 and 50days after grafting for cv. Sweet and cv. Elazig, respectively, were
reported (Yasinok et al. 2009). In the present study, evaluation in later developmental periods revealed
that tobaccotomato grafting had no significant effect on total chlorophyll concentration of the scion.
Accumulation of water soluble solutes in plant tissues helps plants to maintain their turgor and
enhances water uptake. Anthocyanin biosynthesis may be induced by environmental factors, and
production and localisation tissues may develop resistance to abiotic stresses. Moreover, nutrient defi-
ciencies, such as phosphorus, magnesium, nitrogen, sulphur and zinc, have long been associated with
 8 M. Haberal et al.

foliar anthocyanin accumulation of different species (reviewed in Chalker-Scott 1999). Anthocyanin

accumulation in self-grafted plant leaves 90 and 140 DAT (Table 2) may be an osmoprotective response
of leaf tissue associated with low RWC. Less anthocyanin accumulations of H2274/H than self-grafted
plants 110 and 140 DAT, and H2274/S leaves than non-grafted, except for the lower leaf level, and
self-grafted plants 110 and 140 DAT seem to be related to enhanced uptake of water, which is one of
the advantages of use of grafting in cultivation of vegetables (Lee 1994). Rootstock has an influence
on the uptake and translocation of mineral nutrients, and mineral content of aerial plant parts; and
the impact is related to physical characteristics of the root system (Martnez-Ballesta et al. 2010).
Increased rate of mineral transport to leaves, and the ability of the root to take up water and minerals
may be associated with lower leaf anthocyanin level in tobacco grafted plants.
Nicotine is principally synthesised in the roots and transported through the xylem to the shoot.
Systemic and local nicotine accumulation is a response to wounding (Baldwin et al. 1997). In a graft-
ing study of Solt and Dawson (1958), the sites of highest nicotine accumulation were found to be the
graft unions in tobacco grafted on tomato rootstock plants, and some of the nicotine was found to be
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translocated upward to be deposited in leaf and stem. They also reported that bottom leaves of tobacco
stem grafted on tomato rootstock had more nicotine than top leaves. In this study, the leaf samples
collected from tobacco-grafted plants had significantly elevated levels of nicotine (Table 3). Higher
nicotine concentration of leaves sampled from low and medium levels of scion may be correlated to
graft induced local accumulation of nicotine which is dependent on the tobacco rootstock, and its
relatively limited transport through the xylem. Though cv. Hasankeyf had higher leaf nicotine than cv.
Samsun prior to grafting, the leaf nicotine levels in cv. Hasankeyf rootstock grafted plants were lower
than those obtained with grafting onto cv. Samsun rootstocks. It is likely that high levels of nicotine
accumulate in response to tobacco grafting, and the effects of grafting on nicotine accumulation is
dependent on the rootstock and its union compatibility. H2274/H leaf nicotine concentration decreased
with increasing plant age i.e. leaf nicotine concentrations were lower 120 and 160 DAT in all sampling
levels, except for the sample collected at 90 DAT from high level. Decrease of nicotine levels 120 and
160 DAT in H2274/H may be correlated with recovery of the graft union.
Fruits of tobacco-grafted plants had significantly higher concentration of nicotine than those of
non-grafted scions independent of harvest time and level (Table 4). Increases in nicotine concentra-
tions of tobacco-grafted plants were greater in leaves than in fruit. Previously, Dawson (1942) reported
that when tomato scions were grown upon tobacco stocks, nicotine was found in small quantities in
the tomato stems and fruits, and large quantities accumulated in the leaves. Rootstock contributes
to nicotine concentration of whole plant as demonstrated for nicotine accumulation in tobacco (N.
tabacum L.) leaves (Dawson 1942; Lee et al. 1982). Similar to leaf nicotine concentrations, higher nic-
otine was found in fruits harvested from lower than from higher levels. Previously considerably higher
fruit nicotine levels of two tomato graft unions (cv. Sweet and cv. Elazg) obtained by grafting onto
cv. Samsun has been reported (Yasinok et al. 2009). Fruit nicotine levels may vary depending on the
tomato cultivar as well as the growth conditions such as nitrogen supply and its bioavailability to plant
tissues. Therefore, because of its lower nicotine concentration, cv. Samsun and cv. Hasankeyf grafted
H2274 tomato fruits were considered to be safe for consumption as were the previously defined graft
unions (Yasinok et al. 2009). Acceptable dietary intake (ADI) level is 0.0008mgkgbody1 day1 for
nicotine as determined by European Food Safety Authority (2016). Accordingly, for a 60kg individual
ADI for nicotine in 48gday1. If 0.5kg (three or four tomatoes) of H2274/S fruits, which have the
highest nicotine concentration with 5.6gkgFW1, were consumed, the nicotine uptake would be
2.8g, which is below ADI. In addition, it has been reported by other researchers that only 3040%
of orally consumed nicotine reaches the systemic circulation (Andersson et al. 2003).

Conclusions
The effects of grafting reported here are related to the differences in the root system between rootstocks
as well as the grafted and the non-grafted plants. Efficiency of water and nutrient uptake by the roots,
 Biological Agriculture & Horticulture 9

their transport and availability to tissues, and the distribution of growth regulators are possible factors
that affect graft compatibility to scion. Grafting performance depends on compatibility between scion
and rootstock variants, and should be assessed individually for each scion-rootstock pair individually.
The Tomacco plant and the higher nicotine bearing tomato fruits were novel in a previous report by
the authors. The present report showed the sustainability of the novel technique with different root-
stock tobacco species and tomato cultivars. In this report, the fact that nicotine accumulation varied
in different parts of the grafted plants or and with harvest time was demonstrated for the first time.
Previously, improved salt tolerance and adaptive response of cv. Elazg grafted onto cvs. Samsun and
Hasankeyf has also been demonstrated (eri et al. 2015). In conclusion, grafting of tomato on tobacco
is a feasible cultural practice leading to higher tomato yield and performance. Due to the low nicotine
concentration, grafted tomato fruits are considered to be safe for consumption.

Disclosure statement
Downloaded by [Orta Dogu Teknik Universitesi] at 23:58 12 April 2016

No potential conflict of interest was reported by the authors.

Funding
This study was approved by Baskent University Institutional Review Board [Project number DA09/37], and supported
by Baskent University Research Fund.

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