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EFFECTS OF LEMON JUICE ON PLANT GROWTH FOR DIFFERENT
VARIETIES OF POTATO (SOLANUM TUBEROSUML.) IN VITRO
AL-ZUBAIDI ALI HASSEN ALI1, 2
LIBING JIN1
JIANFEI XU1
1, 2Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China, Ministry of Higher
Education and Scientific Research, Foundation of Technical Education, Iraq
1Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
1Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
ABSTRACT
Lemonjuice concentration (0, 1, 2and 3 ml/L) are used in this study with three
varieties of potatoes (SolanumtuberosumL) to determine the optimal environmental
conditions and their effect on the growth of vegetative for potatoes in vitro. The results
showed that the variety (Zhongshu20)surpassed on the other varieties and the highest rate for
variety in qualities(plant length, root's length, number of leafs, node number, wet weight)
amounted (10.06cm,11.36cm,12.62,10.68and125.02mg) respectively, also surpassed
superiority in 2ml/L concentration on a number of roots and leaf size as shown in the results.
The main aim for using lemon juice in our study; is to use a suitable alternative regulator for
industrial growth regulators with low cost also the difficulty to obtain industrial growth
regulators in some of countries.
KEYWORDS: Lemonjuice, potato’s varieties, Growth regulators, SolanumtuberosumL.,
Tissue culture.
1. INTRODUCTION
Potato (Solanum tuberosum L.) belongs to the solanaceae family which grown
worldwide, most important food for the largest number of peoples in the world. Potato is
usually propagated asexually by tubers. However, regeneration of potato in vitro is easily
done from different explants on Murashige and Skoog MS media (Murashige and Skoog
1962) supplemented with different aux in and cytokine in for diseases free good-quality seeds
and pathogen Virus-free planting materials (Hossain. 1994; Parmar et al 2012, Devi, M.,
Dhaliwal et al. 2008). Both callus induction and plant regeneration from explants need
acceptable mixtures and concentrations of plant growth regulators within the culture MS
media (Ehsanpour and Jones 2000). The growth of plants tissue and organ culture systems
used by forest species make use of natural or artificial plant growth regulators (A. Ghaffoor,
G.B. Shah and K. Waseem 2003).. Without added hormones, most tissues do not remain
viable; and much less grow after the manner. Unfortunately, few studies have dealt with the
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mechanism of hormone action and effective hormones and their concentration have been
derived empirically. A wide range of hormones has been applied to a number of species with
sometimes conflicting results (Rabbani, Askari et al. 2001). The tissues and organs are in
sterile conditions supported by an appropriate culture media which normally contain a
mixture of major and minor salts, vitamins, sugars as a carbon source and plant growth
regulators used formulations to enhance maintenance of plant growth quality of in vitro.
While, the propagation process in vitro is facing a lot of difficulties including high prices of
growth regulators, low efficiency and changes in high temperatures sterilization process, and
the valuable materials from natural plant are not discovered. A method for culturing plant
cells and tissues should provide new means for the commercial processing of even rare plants
and the chemicals they produce (Murashige and Skoog 1962). These technologies are
extended and enhance the usefulness of plants as renewable resources of valuable chemicals.
Biologically active plant-derived chemicals can be expected to play an increasingly
significant role in commercial development on new products for regulating plant growth.
Fruit juice, as one of plant-derived chemicals, is studied on its effects on plant growth in vitro
in containing salt's MS media. Plant variation of cultivars studied before wad showed in their
response to the growth regulators. The highest number of roots per shoot was obtained on MS
media supplemented with1.0 mg/L dole 3-butyric acid (IBA). Regenerated plants were
successfully acclimatized and eventually transferred to the green house with higher survival
rate. All the plants appeared morphologically uniform with normal leaf form, shape and
growth pattern. The described protocol may be useful to establish micro propagation industry
of potato that to help in overcoming difficulties of conventional vegetative propagation for
the crop species (Khadiga, Elaleem et al. 2009). Multiplication velocity of planets differed in
the amount of growth regulators. The lemon juice and conducted milk are used in the
vegetative production for potato planets in vitro (hassen 2011). Top sprouts of desert variety
tubers was cut and cultured in M.S media that contains different lemon juice and coconut
milk concentration (1,2 ml/L),with 2mg/L BA as control (hassen 2011). It is also indicated
that the use of pollen extracts, licorice and lemon juice in media of had effects on plant
growth (Al-Kaaby and Hussein 2000). The addition of 6 cm3/L lemon juice had an
encouraging effect on callus induction and growth. PGRs compounds and cultivars may
produce the better results in comparison with others. There are also different reports that
interaction of growth regulator and cultivar are significant. The main aim of this study is to
get optimal level lemon juice. For reduce a cost without impact on the efficiency of the
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process of micro propagation. Was lemon juice (Table 2),is one of the solutions to this
problem as an alternative to commercial industrial growth regulators who increased their use
of private studies in tissue culture, the remnant of this paper is organized as follows;
Materials and method are explained in next section then the results and discussion are
presented, finally the conclusions are given.
2. MATERIALS AND METHOD
This study was conducted in Institute and Flowers (IVF), Chinese Academy of Agricultural
Sciences (CAAS). Three potato varieties, Zhongshu 3 (early maturity), Zhongshu 20 (late
maturity) and Atlantic (mid-maturity) are used in this study .The virus-free seedling was
obtained from potato Research Centre. The nutrient media, was consisted of MS media
(Murashige and Skoog 1962) for following ingredients: 4.43 g /L of MS powder, the medium
contained 30 g /L sucrose and 5.89g /L, agar the pH of the media was adjusted to 5.8 ± 0.01
with 0.1 N either NaOH or 0.1 N HCl before adding plant agar prior to autoclaving (Bhatia. 2005).
2.1. Culture Media and Conditions
Required doses of growth regulators were added alternative to commercial industrial growth
regulators lemon juice to the normal MS media (Table 2), (0, 1, 2 and 3 ml/L) respectively as
included in below .Containing according to a potato nodal segment culture protocol. In
addition, cut seedling a length of 1-2 cm (Fig 1). And Planting culture explants and prepared
and sterilized under a laminar air flower with filtration method. Using a tongs length media
sterilized vertical 3 pots/each variety, 15 explants /each pot, planted in pots containing
control (standard media without the growth regulator) and of concentrations from lemon juice
for each three varieties and closed the pots by cover. And they were incubated under
controlled environmental conduit of 18°C ± 2°C. 16/8 light /dark cycles and the illumination
intensity at 1500 lx(Table 1), (Fig 1). The data are registered for the experiment after the 30
days from the date of planting and take the averages of the studied characteristics: (length of
plant, length of root, branch number, leaf number, leaf size, number of nodes and wet
weight).
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Table 1. Preparation four treatments used are in this experiment
Treatment
Media Quantity
0
Standard MS media without
the growth regulators. 3 pots/each variety, 15 plantlets/each pot.
1
Standard MS media with
1ml/L lemon juice.
3 pots/each variety, 15 plantlets/each pot.
2 Standard MS media with
2ml/L lemon juice.
3 pots/each variety, 15 plantlets/each pot.
.
3 Standard MS media with
3ml/ L lemon juice.
3 pots/each variety, 15 plantlets/each pot.
Figure 1: Stages multiplication and plant growth in the laboratory under normal MS media
and concentrations from lemon juice.
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Table 2: The nutritional value per 100g in raw lemon without peel.(U.S. Department of
Agriculture 2013).
Nutritional value per 100 g
Protein 1.1 g
Thiamine (vit. B1) 0.04 mg(3%)
Riboflavin (vit. B2) 0.02 mg (2%)
Niacin (vit. B3) 0.1 mg (1%)
Pantothenic acid (B5) 0.19 mg (4%)
Vitamin B6 0.08 mg (3%)
Folate (vit Vitamin C. B9) 11 mg (3%)
Choline 5.1 mg (1%)
Vitamin C 53 mg (64%)
Calcium 26 mg(3%)
Iron 0.6 mg (5%)
Magnesium 8 mg (2%)
Manganese 0.03 mg (1%)
Phosphorus 16 mg (2%)
Potassium 138 mg (3%)
Zinc 0.06 g (1%)
2.2. Statistical analysis
Data are analysed using the Statistical Analysis System ( SAS. 2010.) program. Plantlets
samples tested are repeated three times under each treatment three samples are duplicated
in each tested.
3. RESULTS
3.1. Length of plants
Plant length was affected significantly by different lemon juice level on the whole (Table 3).
Nevertheless, with the increase of lemon juice concentration, plant length of each variety
showed not same reaction to lemon juice. Plant length of Atlantic was affected most
significantly that it increased 2.18 cm in MS media with 3 ml/L lemon juice in comparison to
normal media. While, lemon juice induced slight effects on plant length of Zhongshu 3 that
just increased 0.67 cm in comparison to the normal media .The results indicated that effects
of lemon juice on plant length differ from potato varieties. The plant morphology of each
variety is shown as in (Fig 2, A, B and C).
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Table 3. Effects of different lemon juice concentrations on plant length (cm).
Variety Plant length (cm) Mean Difference
between 0 and 3
Lemon juice concentration(ml/L)
0 1 2 3
Zhongshu 3 7.99 8.04 8.4 8.66 8.27 0.67
Zhongshu 20 8.22 8.05 9.65 10.06 8.99 1.84
Atlantic 6.13 6.87 7.56 8.31 7.22 2.18
Mean 7.45 7.65 8.54 9.01 --- 1.56
Note: LSD, variety 0.359* , level 0.415*, variety× level 0.719* . *significant at P<0.05.
( A) Zhongshu 20 (B) Zhongshu 3 ( C) Atlantic
3.2. Length of Root
Results showed in (Table 4) in the Zhongshu 20 surpassed significantly on Atlantic and
didn’t surpass on variety Zhongshu 3, where it didn’t record any significant differences,
where it recorded the highest rate for root's length 10.02cm, and the lowest rate of described
9.08 cm. As best responses to regeneration were at 3ml/L concentration recorded a significant
difference on the rest of the concentration recording 10.40 cm, and lowest length for roots
was at (0) concentration, has recorded less value 8.70 cm, and as is the case in plant height,
for lemon juice slight effects on root's length of Zhongshu 3 that just increased 0.69cm in
comparison to the normal MS media. Maximum percentage of difference between 0 and 3
more length of root formations was recorded of Zhongshu 20 and Atlantic from lemon juice, (
Fig2, A,B and C).
0 1ml/L 2ml/L 3ml/L 0 1ml/L 2ml/L 3ml/L 0 1ml/L 2ml/L 3ml/L
Fig 2. Effects of three concentration of lemon juice (0, 1, 2 and 3 ml/L), on potato varieties (A) Zhongshu
20variet
y,
(
B
)
Zhon
g
shu 3 variet
y
and
(
C
)
Atlantic variet
y
.
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Table 4.Effects of different concentrations of lemon juice on length of root (cm).
Variety length of root (cm) Mean Difference
between 0 and 3
Lemon juice concentration(ml/L)
0 1 2 3
Zhongshu 3 9.33 9.99 10.37 10.02 9.93 0.69
Zhongshu 20 9.25 9.75 9.68 11.36 10.01 2.11
Atlantic 7.51 9.22 9.77 9.81 9.08 2.30
Mean 8.70 9.65 9.94 10.40 --- 1.70
Note: LSD: Variety 0.410* , Level 0.473* , Variety x Level 0.820* * (P<0.05)..
3.3. Number of root
(Table 5), showed that the Zhongshu 20 surpassed on the other significantly superiority,
where it recorded highest rate for the root's number, which has reached to 5.68in the control,
while the lowest rate has recorded by Atlantic, which is 4.98.for transaction showed that the
level 2ml/L of Zhongshu 20 surpassed significant superiority on the rest of the transactions,
which has recorded the highest number 6.13, also level 1ml/L Zhongshu 3 have recorded the
lowest number of roots. Whereas it was, application of lemon juice was not effective for
increasing the number of roots for Zhongshu 20 and improving the quality as compared to
other the concentrations that recorded -0.56.
Table 5. Effects of different concentrations of lemon juice on the number of roots
Variety number of roots Mean Difference
between 0 and 3
Lemon juice concentration(ml/L)
0 1 2 3
Zhongshu 3 5.13 4.40 5.57 5.35 5.11 0.22
Zhongshu 20 5.84 5.46 6.13 5.28 5.68 -0.56
Atlantic 5.02 4.66 5.20 5.04 4.98 0.02
Mean 5.33 4.84 5.63 5.22 --- -0.11
Note: LSD: Variety 0.270* , Level 0.312* , Variety x Level 0.541* * (P<0.05).
3.4. Number of leafs
(Table 6), we saw the significant increase in the number of leafs compared to the control.
On the other hand, at the level of 3ml/L for the three varieties, Zhongshu 20 surpassed on
the rest of the varieties and recorded the highest rate for a leave's number, which recorded to
11.66, While, Atlantic variety has recorded the lowest rate for a leave's number, which has
reached to 9.56(Fig 2, Aand C).Zhongshu 20 have recorded at level 3 ml/L and significant
difference on the rest levels which have reached to 12.62, didn’t show the difference for
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lemon juice concentration, and in comparison to the normal media induced slight effects on
the number of leafs of Zhongshu 20 that just increased 0.62 cm .
Table 6.Effect of different concentrations of lemon juice on a number of leafs.
Variety number of leafs Mean Difference
between 0 and 3
Lemon juice concentration(ml/L)
0 1 2 3
Zhongshu 3 9.60 10.75 10.95 11.13 10.61 1.53
Zhongshu 20 12.00 9.68 12.33 12.62 11.66 0.62
Atlantic 8.44 10.11 9.53 10.17 9.56 1.73
Mean 10.01 10.18 10.94 11.31 --- 1.30
Note: LSD: Variety 0.432*, Level 0.499*, Variety x Level 0.865* * (P<0.05).
3.5. Number of node
Zhongshu 20 surpassed significantly on the rest of transaction as shown in (Table 7), whereas
the highest rate recorded 8.52 nodes. There is no significant difference between Zhongshu20,
Zhongshu3. The Atlantic recorded the lowest rate 7.27. The results didn’t indicate that there
are presents a difference between the third level and comparative transaction, whereas level
2ml/L recorded lowest rate for the number, which is 7.08 node(Fig 2, A, B and C). As the
results showed that there was no effect for lemon juice concentration of nodes the Atlantic
that recorded -2.27at difference between control and lemonjuice concentration.
Table 7.Effects of different concentrations of lemon juice on the number of nodes.
Variety number of nodes Mean Difference
between 0 and 3
Lemon juice concentration(ml/L)
0 1 2 3
Zhongshu 3 7.40 7.46 7.35 7.55 7.44 0.15
Zhongshu 20 7.64 7.51 8.26 10.68 8.52 3.04
Atlantic 8.91 7.93 5.62 6.64 7.27 -2.27
Mean 7.98 7.63 7.08 8.29 --- 0.31
Note: LSD: Variety 0.354*, Level 0.409*, Variety x Level 0.708* * (P<0.05).
3.6. Number of branch
The results in (Table 8), observed that there are no differences between Zhongshu 20 and
Zhongshu 3. While the Atlantic is recorded the lowest rate for the branch's number, which
has reached to 1.18. For level's lemon juice, control (0) recorded significant difference on
the level 2ml/L and 3ml/L, while there is no significant difference between control
treatments (0) and where this transaction recorded highest rate for the branch's number,
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which has reached to 1.21 branch. Transaction results indicated that the Zhongshu 20
significantly on the rest of control treatments at the level (0), which recorded the highest
rate of the branch's number, which has reached to 1.93also Atlantic recorded lowest rate at
the level (0) which is reached to 1.11. There was no effect for lemon juice concentration on
Zhongshu 20 and Zhongshu 3 in a number of branches, however has shown that slight
effects on Atlantic that recorded just increased 0.17lemon juice concentration.
Table 8. Effects of different concentrations of lemon juice on the number of branches
Variety number of branches Mean Difference
between 0 and 3
Lemon juice concentration(ml/L)
0 1 2 3
Zhongshu 3 1.26 1.17 1.24 1.11 1.20 -0.15
Zhongshu 20 1.93 1.48 1.24 1.26 1.48 -0.67
Atlantic 1.11 1.20 1.15 1.28 1.18 0.17
Mean 1.43 1.29 1.21 1.22 --- -0.21
Note: LSD: Variety 0.133* , Level 0.153*, Variety x Level 0.266* * (P<0.05).
3.7. Weight of plant
The results indicated in (Table 9), for wet weight, Atlantic variety surpassed on the Zhongshu
3, which recorded the highest rate recorded 110.10 mg, while there is no revealed any
difference between Zhongshu 20 and Atlantic. Also Zhongshu 3 recorded the lowest rate for
wet weight 98.03 mg. For concentration, Zhongshu 3 is recorded the lowest rate of lemon
juice, and has reached significant difference on control treatment (0) reached to 101.14 mg.
For treatments, Zhengzhou 20 at third-level significant difference which has reached 125.02
mgon Zhongshu 3 varieties, while there is a significant difference with Atlantic and
concentration 3ml/L .The results of comparing a difference between 0 and 3 level, 3 ml/L
between concentration on wet weight of plant of Zhongshu 3 showed that there is not a
significant effect were reached -2.42.
Table 9 . Effects of different concentrations of lemon juice on wet weight of plant (mg).
Variety weight of plant (mg) Mean Difference
between 0 and 3
Lemon juice concentration(ml/L)
0 1 2 3
Zhongshu 3 101.06 95.06 97.35 98.64 98.03 -2.42
Zhongshu 20 95.60 100.53 116.93 125.02 109.52 29.42
Atlantic 106.77 108.04 107.73 117.84 110.10 11.07
Mean 101.14 101.21 107.34 113.84 --- 12.70
Note: LSD: Variety 7.744*, Level 8.943* , Variety x Level 15.49* * (P<0.05).
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3.8. Size of leaf
The Atlantic is surpassed significantly on the rest of varieties, which has recorded the highest
rate 3.17, whereas no significant differences between Zhongshu 3 and Zhongshu 20 as shown
in (Fig 2, A, B and C) and (Table 10), For lemon juice, there was no longer any significant
difference inter MS of transaction, level 2ml/L recorded the highest rate for Zhongshu 20,
which have reached 4.33, also Zhongshu 3 recorded control at (0) level lowest rate has
reached to 2.00. However, the percentage response was found varying, depending on the
concentration of lemon juice concentration in the MS media. From the difference between 0
and 3, it is clear that with an increase in the concentration of lemon juice, there was a gradual
increase the response simple for size of leaf.
Table 10.Effects of different concentrations of lemon juice on size of leaf.
Variety size of leafs Mean Difference
between 0 and 3
Lemon juice concentration(ml/L)
0 1 2 3
Zhongshu 3 2.00 2.33 4.00 3.66 3.00 1.66
Zhongshu 20 2.06 2.33 4.33 3.33 3.01 1.27
Atlantic 2.66 2.66 3.66 3.66 3.17 1.00
Mean 2.24 2.44 4.00 3.55 --- 1.31
Note: LSD: Variety 0.120*, Level 09.139*, Variety x Level 0.241* * (P<0.05).
4. DISCUSSION
The present investigation was conducted to find out the effects of alternative growth
regulators on direct regeneration of potato. Data on seedling visible :( length of plant, length
of root, branch number, leaf number, leaf size, number of nodes and wet weight). After 30
days, the discussions of the study have been presented below: The showed wide variation in
their response to the lemon juice concentration. The concentration 3 ml/L, recording the
highest rate for variety in qualities (plant length, root's length, number of leafs, node number
and wet weight ) amounted respectively (10.06 cm, 11.36 cm, 12.62, 10.68 and125.02 mg),
The results of the study reflected interaction effects between lemon juice concentration and
potato cultivars , this can be justified by that the different concentrations of lemon juice
plays a positive role in to promote the cell division growth, and stimulate the activity of
some enzymes, the nutrients be transported to the parts of plant more easily. And on the other
hand increase the growth of vegetative and weight at the same time. Lemon is the major
component than bioactive compounds and presence of flavonoids and vitamin C
polysaccharides and acids (Table 2). As in The results from previous research on this subject
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have that the effect of lemon juice on in vitro propagation of potato seedlings and also which
corroborates findings for this study. (Asmaiel, K. A.2002, Faridud in et al 2004, hassen
2011).And the surpassed significant superiority at 2 ml/l concentration number of roots and
leaf size, which has recorded the highest rate (6.13 and 4.33) growth regulators would effect
on growth plant in addition because of genotype effect and are widely used in vitro
multiplication through to change the behaviour the plants, thus optimizing the vegetative
growth reported by many researchers.(Prat 2004; Zhang et al., 2005; Chaudhary, Z., Afroz,
A., and Rashid, H 2007, Ghavidel et al., 2012) . It may be mentioned here that among the
three potato varieties used in the present investigation showed best zhongshu 20 variety
surpassed increment of most qualities significant superiority attributed difference genotype
the and also variable responses of different potato varieties due to genetic makeup and
physiological effects on plant cells and this result possibly is based on the hypothesis that
plant hormones responsible for cell growth the greatest cellular growth when had the
concentrations of plant growth regulators used .Where this result is in line with(A. Rabbani,
B. Askari 2001, Ehsanpour, A. A. and M. G. K. Jones 2000).
5. CONCLUSION AND RECOMMENDATION
From the above study, lemon juice growth regulators had significant influence on three
varieties from the potatoes in vitro. It gave the best response the Zhongshu 20 variety
significant surpassed from Zhongshu 3 and Atlantic varieties. As 3 ml/Llemon juice
concentration gave the best response for of the studied characteristics. Therefore, recommend
the addition of lemon juice for MS media to an increase in growth, and cost less productivity
for MS media.
ACKNOWLEDGMENT
This project is supported by the Key Laboratory of Biology and Genetic Improvement of
Tuber and Root Crop, Ministry of Agriculture, P.R.China and funded by the earmarked fund
for Modern Agro-industry Technology Research System (CARS-10).
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