Journal

Journal of Applied Horticulture, 20(2): 103-111, 2018 Appl

Some natural extracts from plants as low-cost alternatives for

synthetic PGRs in rose micropropagation

Urmi Chauhan, Anil Kumar Singh, Divyesh Godani, Satish Handa, Praveen S. Gupta,
Shivani Patel and Preetam Joshi*
Department of Biotechnology, Shree M & N Virani Science College, Rajkot (India) 360005. *E-mail: [email protected]

Abstract
Effect of various plant extracts during in vitro culture of rose (Rosa hybrida L. cv. bush rose), with the objective of replacing synthetic
Plant Growth regulators (PGRs) to reduce the production cost, was studied. Test extracts included sweet lime juice, orange juice, sweet
corn extract, tomato fruit extract and coconut water. Significant increase in shoot multiplication (15.41±1.12 shoots/explant), shoot
length (3.66±0.08 cm), fresh weight (7.48±0.71 g) and dry weight (1.68±0.075 g) was observed when coconut water (@10 % v/v) was
used in the standard MS medium. Addition of tomato fruit extract in the MS medium did not show any noteworthy effect on growth in
rose micropropagules. Total chlorophyll and other biomolecules varied with the change in the type and concentration of plant extract.
Highest accumulation of biomolecules was recorded on coconut water (@ 10 % v/v) supplemented MS medium followed by sweet
corn extract and orange juice. Although tomato fruit extract (@10 % v/v) enhanced the total chlorophyll biosynthesis but at the same
time depressed the accumulation of other biomolecules. Treatment of plant extract was given in two different ways; a) incorporation in
the medium prior to autoclaving (PrA) and b) post-autoclaving addition of filter sterilized extract (PoA). No significant changes were
noted in growth when mode of application was changed. To know the physiological pandemonium in the cells, peroxidase and IAA-
oxidase activity was noted. No abnormal changes in the activity of these enzymes were recorded in the propagules grown on different
plant extracts. The total cost of synthetic 6-benzylaminopurine (BA) can be reduced upto 98 % by replacing it with natural plant extract.
Key words: Rose micropropagation, Synthetic PGRs, natural plant extract, 6-benzylaminopurine, growth, low-cost alternatives

Introduction during past two decade (Singh and Pal, 2015).

Rose is a woody perennial shrub which is native to China but due There are several factors which add to the production cost of
to its high demand and economic importance, is now grown all tissue culture grown plantlets. Foremost of these is the cost of
over the world. The genus Rosa belongs to family Rosaceae which chemicals and glassware (which are often very expensive) used in
include more than hundred species and enormous commercial medium preparation. Several studies were carried out for lowering
down the production cost of plantlets. These reports were mainly
cultivars. Multiplication of rose in conventional methods, is
focused on use of some cheaper gelling agents like Xanthagar
generally done by grafting and cutting. But, this method is very
(Jain-Raina and Babbar, 2011), corn and potato starch (Mohamed
slow and risk of disease and environmental hazard is always
et al., 2010), isabgoal (Agrawal et al., 2010), Guar-gum (Babbar
there (Otiende et al., 2016). To overcome this, a number of tissue
et al., 2005), Cotton fiber (Moraes-Cerdeira et al., 1995), use of
culture protocols, which guarantee disease free plantlets, have
low grade inorganic salt (Ogero et al., 2012; Dhanalakshmi and
been developed as a potential tool for rapid and mass propagation
Stephan, 2014), use of low grade table sugar as carbon source
in rose (Nikbakht et al., 2005). Micropropagation is not only (Tyagi et al., 2007; Agrawal et al., 2010). The main focus of the
a gambit for rapid propagation of plants but also overtures researchers was on replacing gelling agent and sucrose as carbon
elimination of pathogen and endows the researcher with scope source with some cheaper options, just because both of these
for development of new cultivars (Joshi and Purohit, 2011). components share major cost in medium preparation. Total cost
The last two decades were revolutionary years for commercial contributed alone by agar-agar (gelling agent) and sucrose (tissue
tissue culture and the technology in its real sense transformed in culture grade), in standard MS medium preparation, is 49.61 %
profitable business, particularly for ornamental and horticultural and 38.49 % respectively. Beside this, synthetic PGR like 6-BA
plants. Contrary to this, the technology has several limitations. (7.78 %) and other inorganic and organic component (4.12 %)
Micropropagation is a cost intensive technology as compared to also significantly contribute to the production cost of standard
the conventional methods of plant propagation. It needs several MS medium which was ignored in this direction. Some natural
types of skills, accompanied by sophisticated instruments and plant extracts contain important substances same as PGR or which
controlled environmental conditions which make it capital- exhibit PGR like activities. The growth promoting activity of
intensive industry. Hence, in some cases the unit cost per plant coconut water under in vitro condition has long back reported as
becomes exorbitant. This has restricted the growth of these path breaking discovery by Van Overback et al. (1941). Beside
industries in developing countries like India and only a few these important substances, the natural extracts also contains
funded institutions and big companies sustained in this race while some important amino acids, proteins, carbohydrates, lipids
small units were dropped out, which has tremendously increased and primary and secondary products which positively influence
Journal of Applied Horticulture (www.horticultureresearch.net)
104 Low-cost alternatives in rose micropropagation

the growth of micropropagules under in vitro conditions. The earlier. The treated shoots were further sub-cultured on the same
artificial MS medium, which are mostly used in plant tissue plant extract containing fresh medium at a gap of three weeks up
culture, contain all the essential inorganic and organic salt, to six cycle i.e., a period of 126 days. All the above treatments
chelating agents, carbon source, growth regulators and water. But, were repeated thrice and six replicates were set for each
the exact nutritional requirements differ from plant to plant and experiment. At the end of experiment the micropropagules were
within the same plant differs from cell to cell. The natural plant taken out of culture bottle and were subjected to measurement of
extract, which are produced through many complex metabolic various growth parameters and biochemical analysis.
pathways, contains several metabolites that may act as potent
Measurement of growth parameters: Average shoot length,
growth enhancers, which a synthetic medium is completely
total shoot number and total biomass in terms of fresh and dry
lacking. We hypothesized that the addition of such ready prepared
weight were measured. For measurement of biomass (fresh weight
metabolites, in the form of natural extract in the growth medium,
and dry weight), propagules obtained from each treatment were
can substitute the need of PGRs and thus can lower the production
taken out and the fresh weight was measured using an electronic
cost as well at the same time promote the growth under culture
top pan balance. For dry weight calculation, after measuring the
conditions. In order to increase application of tissue culture
fresh weight those fresh shoots were kept in an oven at 62˚C for
technology in rose cultivation, innovative approaches are needed
drying till constant weight.
to lower the cost of micropropagules production. The aim of the
current study was to develop an efficient and affordable method Chlorophyll contents: The chlorophyll contents were calculated
for the micropropagation of rose. as per the method described by Arnon (1949). 500 mg of shoots
were weighed and ground in pestle and mortar with 80 % acetone
Materials and methods under dark conditions. Extracts were centrifuged at 10, 000
Explant preparation and culture establishment: Shoot cultures rpm and the supernatant was used to measure absorbance on
of Rosa hybrida L. cv. bush rose were established according to the spectrophotometer (UV-1800 Shimadzu, Japan).
protocol described by Carelli and Echeverrigaray (2002). Healthy Total phenols: The total phenol content was measured as per the
plants were procured from local nursery and maintained in green method described by Mahadevan (1975) using Folin Ciocalteu’s
house of college. Mature nodal segments, containing axillary reagent. 500 mg of shoots were weighed and crushed in pestle
buds, were selected, cut and rinsed with tap water to remove the and mortar in 70 % methanol. The extracts were centrifuged at
dust particles followed by two washes with detergent and then 10, 000 rpm for 15 minutes. The clear supernatants were used
sterilization by dipping in 70 % ethanol for one min. Further for quantitative determination of total phenol content. For each
sterilization of explant was done by treatment with 1 % Sodium reaction 500 μL methanolic extract was taken in a test tube and to
hypochlorite for 10 min followed by three to four time washing this 1.0 mL suitably diluted (1:1 ratio of reagent and DDW) Folin
with sterile distilled water. Aseptic inoculation of explant was Ciocaltaeu’s reagent was added followed by 2.0 mL of Na2CO3
done on the standard Murashige and Skoog’s (1962) medium (20 % w/v) solution. The test tubes were heated in boiling water
containing 3.0 mg L-1 BAP, 0.01 mg L-1 NAA, 0.8 % agar and bath with intermittent shaking for about 1.0 min. Tubes were
3.0 % sucrose. After initial establishment of cultures, regular subsequently cooled under running tap water. The blue colored
sub-culturing was done every three weeks interval. Cultures were product was diluted to 25 mL by adding DDW and the percent
kept under standard growth room conditions maintained at 28±2 transmittance was measured at 650 nm in a spectrophotometer
°C temperature and a 16 h light/8h dark cycle providing 45μ mol (UV-1800 Shimadzu, Japan). The total phenol concentration in
m-2s-1 photon flux density. each sample was estimated with the help of standard curve prepared
Experimental design: We tried a range of natural plant extracts using different concentrations (10-100 μg) of caffeic acid.
viz. orange juice, coconut water, tomato fruit extract, sweet corn Total carbohydrates: Quantitative estimation of total
extract and sweet lime juice, in varied concentration (3-15 % ) carbohydrate content was carried out as per method described
to study its regulatory effects on growth of micropropagules. by Tandon (1976). In vitro derived propagules were homogenized
Plant extracts were added to standard multiplication medium in 0.1 M phosphate buffer (pH 7.0) and the homogenates were
of rose devoid of PGR. For comparison, both positive and centrifuged at 10, 000 rpm for 15 min. For each reaction 15 μL of
negative controls were also kept, where the cultures were grown supernatant was mixed with 4.0 mL of 0.2 % Anthrone reagent (in
on medium containing PGRs and medium devoid of PGRs, conc. H2SO4) and placed in boiling water bath for five minutes.
respectively. Since the natural plant extract contain metabolites, The absorbance was recorded at 610 nm wave length. The total
which may be thermo-labial and their effect may vary with the carbohydrate contents were determined using standard curve
autoclaving, two different ways were adopted to incorporate the prepared from various concentrations of glucose.
plant extract. In first approach, plant extracts were incorporated
in the nutrient medium prior to autoclaving (pre-autoclaving) Total protein: Quantitative estimation of total protein was
while in the second case, filter sterilized plant extracts were added performed as per Bradford’s method (1976). One mL of the
aseptically in the medium after autoclaving (post-autoclaving ), suitably diluted crude tissue extract (the supernatant) was mixed
when the temperature of medium was brought down to about 50 with 5.0 mL of Coomassie Brilliant Blue G–250 dye (Bradford
°C. Each culture bottle contained ca. 50 mL of semi-solid medium reagent) and transmittance of the resultant solution (coloured
and were caped tightly to avoid contamination. The pH of medium complex) was read with spectrophotometer (UV-1800 Shimadzu,
was always adjusted to 5.8 before autoclaving. Each culture bottle Japan) at 595 nm. The amount of protein was determined using
was aseptically inoculated with a cluster of five shoot (ca. 1.5 cm) standard curve prepared using various concentrations of albumin
and kept under standard growth room conditions as mentioned protein.
Journal of Applied Horticulture (www.horticultureresearch.net)
 Low-cost alternatives in rose micropropagation 105

Total proline: Proline in the plant samples was determined June 1, 2016). Major tissue culture raised ornamental plants,
following the method described by Bates et al. (1973). Proline produced by these industries for international market are Ficus,
was extracted from 1.0 g fresh tissue using 10 mL of aqueous Rose, Spathiphyllums, Syngoniums, Philodendrons, Nerium,
sulphosalicyclic acid (3.0 %). The homogenate was centrifuged Alpenia, Yucca, Cordylines, Pulcherrima, Sansevieria, Gerbera,
at 10, 000 rpm for 15 min. To 2.0 mL of suitably diluted fresh Anthuriums, Statis, Lilies, Alstromeria etc. The pace of growth of
extract, was added 2.0 mL of glacial acetic acid and 2.0 mL these industries has fallen in recent past, as evident by report from
of freshly prepared ninhydrin (1.25 g ninhydrin dissolved in a Department of Agriculture and Processed Food Products Export
mixture of 30.0 mL warm glacial acetic acid and 20.0 mL of 6 Development Authority, Government of India, which says that
M O–phosphoric acid and used within 24 h when stored at 4°C. export in floriculture was 27121.86 metric ton in the year 2012-13,
The mixture was heated in boiling water bath for 1 h. The reaction which was dropped to 22947.27 metric ton in the year 2014-15
was terminated by transferring the reaction tubes to ice and to it (http://agriexchange.apeda.gov.in/indexp/exportstatement.aspx.
was added 4.0 mL of toluene followed by stirring on a cyclomixer Accessed June 1, 2016). The expansion of these industries is
(Remi) for 20-30 sec. After thawing it to room temperature, mainly hampered by high production cost of plantlets.
the toluene layer (upper one, pink in colour) was allowed to There are several reports where trials for lowering the production
separate. The percent transmittance was measured at 520 nm and cost of plantlets has been done but these were mainly focused on
the proline concentration was determined from standard curve replacing the gelling agent and carbon source, while the PGRs
prepared using different concentrations of L-proline. also contribute significant cost in production. In the present
Peroxidase: Peroxidase was determined by the method study, we tried some natural plant extract viz. orange juice,
given in Worthington Enzyme Manual (Anonymous, 1972). coconut water, tomato extract, sweet corn extract and sweet lime
The rate of degradation of hydrogen peroxide (H2O2) by the juice as alternative source of costlier synthetic PGRs. In order
enzyme with O–dianisidine as hydrogen donor was determined to study the effect of above mentioned plant extract, several
spectrophotometrically by measuring the rate of colour growth parameters viz. number of shoot, shoot length, fresh
development at 460 nm. To a 3.0 mL cuvette, 2.7 mL of 0.1 M weight and dry weight were recorded and compared with control
phosphate buffer (pH 7.0), 0.1 mL of O–dianisidine (1.0 mg/ml micropropagules (both grown on with or without standard PGRs).
dissolved in 30 % methanol) and 0.1 mL enzyme extract were The growth of micropropagules varied when type, concentration
added. At the end, 0.1 mL of H2O2 (0.1 M) was added and mixed and mode of application of plant extracts were changed. Number
quickly. The change in per cent transmittance was recorded at 15 of shoot were found to be highest (15.41±1.12), when coconut
sec intervals for 3.0 min. water was added at 10 % (v/v) concentration while sweet lime
juice showed marginal increase in shoot number among the
IAA–oxidase: The IAA–oxidase activity was determined using other extracts tested (Table 1). Addition of plant extracts after
0.5 ml, 1.0 mM dichloroindophenol (DCP), 0.5 mL (1 mM) the autoclaving of medium (post-autoclaving) always showed
manganese chloride (MnCl ), 0.2 mL indole acetic acid (150 mg increased number of shoot at the corresponding concentration
2
in 1.0 mL concentration) and made up to 2.0 mL with phosphate in all the cases. This may be due to the obvious reason because
buffer and 0.1 mL enzyme extract, using method of Srivastava since high temperature degrades certain thermo labial compounds
and Van Huystee (1973). The reaction mixture was incubated at present in the extract. Among the concentrations of various
37 °C in dark. After an hour, 2.0 mL of Salkowaski's Reagent (1.7 extracts tested, 10 % (v/v) concentration was found to be best
g FeCl3.6H2O dissolved in 12 mL distilled water, in an ice bath, in terms of increase in shoot number except for sweet lime juice
240 mL of H2SO4 added very slowly) was added to terminate the where 15 % (v/v) concentration showed better results. Higher
reaction in the tubes. The per cent transmittance of the mixture concentration (15 % v/v) always hampered the growth in all
was measured at 530 nm after 1 h incubation. The amount of the cases and this may be due to the fact that with the increased
IAA oxidized was calculated with the help of standard curve of concentrations, number of inhibitory molecules in the extracts
IAA. The activity of IAA oxidase is represented as amount of also increases which subsequently interfere in growth (Arteca,
IAA oxidized by each gram fresh weight of tissues in one hour. 2013). Besides the growth regulators, the addition of various
plant extract also increase mineral concentration in the medium
For all the above analysis, three replicates were used and each
which indirectly support in increase of shoot number (Niedz et
reaction was repeated thrice. Suitable blanks were maintained
al., 2015). The shoot numbers in treated plants were comparable
wherever required. A statistical analysis was done to check the
with the numbers obtained in control plants, where standard
validity of data.
PGRs were used (Table 1). Similarly, significant increase in
shoot length was also recorded with the addition of different
Result and Discussion plant extracts. Highest increase in shoot length was observed
Plant tissue culture is key sector of agri-biotechnology industry with sweet corn extract (4.39 cm @ 15 %) which was higher than
in India and floriculture lead the market, supplying mostly its corresponding control plant (4.1 cm; Table 1). Both orange
the plantlets in the international market of 150 countries all juice and sweet lime juice exerted similar effect with respect
over the world (Malhotra, 2012). Till the end of April 2016, to shoot length which was comparatively lower than coconut
Department of Biotechnology (DBT), Government of India water. Although supplementation of coconut water significantly
under the “National Certification System for Tissue Culture increased the shoot length but it did not equate with control
Raised Plants (NCS-TCP), has recognized 96 plant tissue culture plants, supplemented with standard PGR. In case of fresh and
industries, which was initially only one in the year 1985 (http:// dry weight, coconut water gave better results followed by sweet
dbtncstcp.nic.in/html/content/Recognized_TCPU’s.pdf. Accessed corn and orange juice. Coconut water at 10 % (v/v) concentration
Journal of Applied Horticulture (www.horticultureresearch.net)
106 Low-cost alternatives in rose micropropagation

Table 1. Effect of different plant extract on in vitro growth of rose micropropagules

Plant Extract Plant extract No. of shoots (Mean) ±SD Length of shoots (cm) ±SD Fresh weight (g) ±SD Dry weight (g) ±SD
concentration PrA PoA PrA PoA PrA PoA PrA PoA
( %)
Orange juice 3 8.12 ± 0.73 8.98 ± 1.11 1.81 ± 0.04 1..89 ± 0.04 4.18 ± 0.10 4.62 ± 0.06 0.95 ± 0.031 1.04 ± 0.036
5 9.25 ± 0.86 10.22 ± 1.17 1.92 ± 0.06 2.11 ± 0.05 4.78 ± 0.20 5.28 ± 0.04 1.07 ± 0.038 1.18 ± 0.039
10 12.36 ± 1.23 13.15 ± 1.16 2.98 ± 0.05 3.12 ± 0.06 6.37 ± 0..42 6.78 ± 0.48 1.44 ± 0.056 1.51 ± 0.059
15 10.89 ± 1.03 11.95 ± 1.14 3.12 ± 0.11 3.55 ± 0.09 5.63 ± 0.32 6.20 ± 0.42 1.27 ± 0.046 1.40 ± 0.051
Coconut water 3 9.14 ± 1.23 9.05 ± 0.76 1.85 ± 0.03 2.10 ± 0.04 4.72 ± 0.19 4.65 ± 0.07 1.06 ± 0.036 1.04 ± 0.036
5 12.14 ± 1.16 13.21 ± 1.02 3.11 ± 0.09 3.55 ± 0.07 6.29 ± 0.56 6.82 ± 0.51 1.41 ± 0.048 1.41 ± 0.051
10 14.45 ± 1.22 15.41 ± 1.12 3.22 ± 0.10 3.66 ± 0.08 7.48 ± 0.71 7.44 ± 0.74 1.68 ± 0.075 1.67 ± 0.066
15 14.26 ± 1.16 14.82 ± 1.19 3.16 ± 0.09 3.56 ± 0.04 7.35 ± 0.69 7.28 ± 0.72 1.65 ± 0.052 1.63 ± 0.055
Tomato 3 7.89 ± 0.66 7.23 ± 0.28 1.84 ± 0.04 1.92 ± 0.04 4.08 ± 0.12 3.73 ± 0.08 0.92 ± 0.031 0.84 ± 0.026
extract 5 8.55 ± 1.16 9.82 ± 0.79 2.21 ± 0.06 2.32 ± 0.05 4.43 ± 0.16 5.08 ± 0.20 1.02 ± 0.032 1.15 ± 0.031
10 7.36 ± 0.49 8.73 ± 0.26 2.36 ± 0.07 2.51 ± 0.05 3.79 ± 0.09 4.05 ± 0.12 0.83 ± 0.025 0.91 ± 0.033
15 7.78 ± 0.87 8.21 ± 0.21 2.19 ± 0.04 2.41 ± 0.04 3.98 ± 0.14 3.72 ± 0.11 0.89 ± 0.056 0.83 ± 0.027
Sweet corn 3 8.24 ± 0.19 9.66 ± 0.77 3.08 ± 0.09 3.66 ± 0.10 4.25 ± 0.19 4.98 ± 0.29 0.95 ± 0.029 1.12 ± 0.044
extract 5 10.42 ± 0.96 11.74 ± 1.09 3.42 ± 0.09 3.82 ± 0.12 5.38 ± 0.31 6.22 ± 0.42 1.21 ± 0.038 1.39 ± 0.057
10 12.27 ± 1.03 13.98 ± 1.06 4.21 ± 0.13 4.36 ± 0.14 6.33 ± 0.41 6.72 ± 0.54 1.42 ± 0.051 1.51 ± 0.059
15 11.19 ± 1.18 13.04 ± 1.20 4.35 ± 0.14 4.39 ± 0.15 5.78 ± 0.45 6.76 ± 0.66 1.30 ± 0.044 1.52 ± 0.062
Sweet lime 3 6.28 ± 0.06 7.10 ± 0.11 1.83 ± 0.04 1.95 ± 0.04 3.24 ± 0.08 3.66 ± 0.12 0.73 ± 0.019 0.82 ± 0.030
juice 5 6.36 ± 0.13 7.23 ± 0.19 1.91 ± 0.06 1.98 ± 0.04 3.29 ± 0.10 3.78 ± 0.21 0.75 ± 0.020 0.85 ± 0.034
10 8.49 ± 0.93 7.92 ± 0.16 2.06 ± 0.06 2.25 ± 0.06 4.41 ± 0.17 4.11 ± 0.23 0.98 ± 0.056 0.91 ± 0.042
15 9.47 ± 0.10 9.60 ± 0.10 2.02 ± 0.06 2.27 ± 0.05 3.33 ± 0.11 3.40 ± 0.14 0.75 ± 0.021 0.74 ± 0.034
(+) ve control (with PGRs) 16.06 ± 2.11 16.17 ± 3.32 4.10 ± 0.12 4.15 ± 0.05 9.32 ± 0.95 9.35 ± 0.86 2.10 ± 0.096 2.08 ± 0.086
(-) ve control (without PGRs) 6.12 ± 0.38 6.32 ± 0.48 1.79 ± 0.04 1.81 ± 0.07 3.14 ± 0.08 3.18 ± 0.10 0.71 ± 0.018 0.72 ± 0.021
LSD (P=0.05) 12.12 12.22 2.32 2.30 3.85 3.78 0.0786 0.7617
LSD (P=0.01) 14.24 14.32 3.66 3.62 5.51 5.75 0.0953 0.0951
CV 30.21 30.19 32.18 30.19 42.26 40.22 6.51 6.32
SE Standard Error; CD Critical Difference; CV Coefficient of variation SD Standard Deviation; PrA and PoA represent the MS medium where plant
extract were added prior to autoclaving and post-autoclaving through sterile filters, respectively.

resulted in increase of fresh weight of propagules upto 7.48 g, plants. In contrary to total carbohydrates, incorporation of
which was comparable with that of control plant (Table 1). No even low concentration of all the test extracts (i.e., 3 % v/v),
morphological abnormalities, callusing or rooting were observed resulted in higher accumulation of phenol content as compared
in treated plant at this multiplication stage. to the controls. This could be explained on the basis of presence
of externally supplied glucose (present in plant extracts)
Incorporation of different plant extract at different concentrations
medium, which induce the accumulation of phenols in tissue
evoked varied responses in rose propagules, in terms of
culture propagules (Ozyigit et al., 2007). Similar observation
biochemical parameters. It was interesting to note that no
was recorded in scarlet rose by Amorim et al. (1977) where
remarkable changes were observed in terms of biomolecules or
exogenous supply of glucose in culture medium resulted in
enzyme activities, when the mode of application of plant extract
increased accumulation of phenols. Another important reason
was changed. Although addition of these extracts, in sterile
of increased phenol accumulation is due to increased proline
condition, after the autoclaving of the medium (PoA), resulted
biosynthesis. It is clearly observed in Table 3 that accumulation of
into slight increase of these biomolecules in most of the cases
phenol and proline are correlated and directly proportionate. Our
(except the proline), but it did not make any significance. In all
results are in accordance with observation of Shetty (2004) that
the test extracts, except sweet lime juice, the chlorophyll contents
proline-linked pentose phosphate pathway stimulate shikimate
increased with the increase in extract concentration and became
and phenylpropanoid pathways and this leads to the stimulation
equivalent to the control plants. The highest chlorophyll content
of phenol biosynthesis in cell. There are two views on role of
was recorded in the propagules grown on medium containing
phenols under in vitro growth and development of plants. One
10 % (v/v) tomato fruit extract. In contrast, post autoclaving
opinion points that the phenols depress in vitro proliferation and
addition of plant extracts did not show any significant increase
growth of plants while others talk about the opposite (López et al.,
in total chlorophyll contents (Table 2). Steady increase in total
2001). Role of phenols in controlling interaction between PGRs
carbohydrate and phenols contents was observed in the rose
and averting the abscisic acid promoted cell senescence under
propagules when grown on medium supplemented with lower
in vitro conditions has also been reported (Feucht and Treutter
concentrations (5 % v/v and 10 % v/v) of all the tested plant
1996) which are in agreement of our observation and hypothesis
extracts. Further increase in concentrations of plant extracts
that increase in phenol is not hampering growth of propagules.
caused decline in amount of both total phenols and carbohydrates
but were still higher than their corresponding controls (Table Proline is another important metabolite that accumulates under
3). Although significant increase in carbohydrate contents was various stress conditions (Qin et al., 2011). Addition of natural
recorded at 15 % (v/v) concentration of sweet corn extract, but extract resulted into increased accumulation of proline. With
at 5 % (v/v) concentration, addition of coconut water proved the increasing concentration of all the test extracts, proline
better compared to all of the test samples and even the control accumulation also increased (Table 3). Highest increase in proline
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 Low-cost alternatives in rose micropropagation 107

Table 2. Effect of different plant extract on chlorophyll contents in rose micropropagules grown under in vitro conditions
Plant Extract Plant extract Total chlorophyll content Chlorophyll a content Chlorophyll b content
concentration (mg g-1 FW ±SD) (mg g-1 FW ±SD) (mg g-1 FW ±SD)
( %) PrA PoA PrA PoA PrA PoA
Orange 3 0.21 ±0.0041 0.26 ±0.0039 0.11 ±0.0021 0.14 ±0.0035 0.12 ±0.0022 0.12 ±0.0162
juice 5 0.29 ±0.0203 0.25 ±0.0201 0.18 ±0.0125 0.13 ±0.0086 0.16 ±0.0100 0.11 ±0.0094
10 0.35 ±0.0223 0.29 ±0.0241 0.19 ±0.0020 0.16 ±0.0037 0.16 ±0.0092 0.14 ±0.0063
15 0.38 ±0.0234 0.31 ±0.0212 0.23 ±0.0095 0.16 ±0.0102 0.17 ±0.0111 0.15 ±0.0041
Coconut 3 0.18 ±0.0042 0.19 ±0.0048 0.10 ±0.0020 0.09 ±0.0122 0.09 ±0.0016 0.09 ±0.0049
water 5 0.22 ±0.0301 0.18 ±0.0221 0.11 ±0.0121 0.10 ±0.0133 0.08 ±0.0084 0.07 ±0.0079
10 0.26 ±0.0253 0.20 ±0.0251 0.12 ±0.0021 0.10 ±0.0111 0.11 ±0.0066 0.09 ±0.0098
15 0.25 ±0.0234 0.19 ±0.0222 0.12 ±0.0096 0.09 ±0.0166 0.10 ±0.0981 0.09 ±0.0116
Tomato 3 0.22 ±0.0041 0.19 ±0.0039 0.09 ±0.0020 0.10 ±0.0021 0.10 ±0.0033 0.09 ±0.0019
extract 5 0.30 ±0.0204 0.25 ±0.0201 0.17 ±0.0115 0.13 ±0.0125 0.15 ±0.0109 0.11 ±0.0097
10 0.39 ±0.0233 0.36 ±0.0240 0.22 ±0.0119 0.11 ±0.0020 0.16 ±0.0087 0.12 ±0.0088
15 0.38 ±0.0271 0.31 ±0.0289 0.23 ±0.0211 0.14 ±0.0095 0.19 ±0.0121 0.15 ±0.0041
Sweet corn 3 0.18 ±0.0038 0.19 ±0.0046 0.10 ±0.0019 0.10 ±0.0028 0.12 ±0.0022 0.08 ±0.0072
extract 5 0.26 ±0.0199 0.21 ±0.0220 0.14 ±0.0125 0.11 ±0.0086 0.16 ±0.0100 0.09 ±0.0140
10 0.32 ±0.0211 0.30 ±0.0235 0.16 ±0.0031 0.11 ±0.0038 0.16 ±0.0092 0.08 ±0.0032
15 0.32 ±0.0262 0.32 ±0.0310 0.16 ±0.0005 0.12 ±0.0114 0.17 ±0.0111 0.10 ±0.0071
Sweet lime 3 0.23 ±0.0032 0.23 ±0.0037 0.12 ±0.0036 0.10 ±0.0027 0.11 ±0.0044 0.12 ±0.0020
juice 5 0.21 ±0.0192 0.19 ±0.0222 0.11 ±0.0110 0.09 ±0.0093 0.09 ±0.0080 0.09 ±0.0013
10 0.23 ±0.0214 0.18 ±0.0248 0.12 ±0.0017 0.10 ±0.0104 0.10 ±0.0072 0.08 ±0.0012
15 0.22 ±0.0301 0.17 ±0.0210 0.12 ±0.0089 0.09 ±0.0113 0.10 ±0.0021 0.08 ±0.0028
(+) ve control (with PGRs) 0.28 ±0.0053 0.29 ±0.0053 0.15 ±0.0046 0.15 ±0.0092 0.13 ±0.0096 0.14 ±0.0014
(-) ve control (without PGRs) 0.20 ±0.0062 0.21 ±0.0062 0.11 ±0.0051 0.11±0.0069 0.10 ±0.0083 0.10 ±0.0078
LSD (P=0.05) 0.0181 0.0211 0.0118 0.0179 0.0145 0.0156
LSD (P=0.01) 0.059 0.062 0.038 0.082 0.0182 0.0179
CV 4.46 4.68 4.02 4.11 4.22 4.37
SE Standard Error; CD Critical Difference; CV Coefficient of variation SD Standard Deviation; PrA and PoA represent the MS medium where plant
extract were added prior to autoclaving and post-autoclaving through sterile filters, respectively.
Table 3. Effect of different plant extract on total carbohydrates, protein, phenol and proline contents in rose micropropagules grown under in vitro
conditions
Plant Extract Plant extract Total Carbohydrate content Total Protein content Total Phenol content Total Proline content
concentration (mg g -1 Fresh tissue)±SD (mg g -1 Fresh tissue) ±SD (mg g-1 Fresh tissue)±SD (mg g -1 Fresh tissue) ±SD
( %) PrA PoA PrA PoA PrA PoA PrA PoA
Orange 3 82.21±2.32 84.45±2.92 68.31±3.66 72.27±1.86 1.41 ±0.07 1.42 ±0.06 18.62 ±0.97 17.32 ±1.16
juice 5 92.26±3.27 96.85±4.12 79.82±4.56 82.53±2.41 1.87 ±0.21 1.45 ±0.01 25.78 ±1.24 20.45 ±1.11
10 105.85±3.88 110.22±4.68 75.61±3.64 82.42±2.36 2.19 ±0.08 1.69 ±0.04 34.29 ±1.03 31.69 ±2.04
15 100.78±3.63 112.45±4.20 69.75±5.82 73.21±3.12 2.12 ±0.10 1.52 ±0.02 42.23 ±2.10 33.52 ±2.03
Coconut 3 84.31±3.12 92.55±2.88 78.22±2.12 82.36±2.32 1.09 ±0.06 0.89 ±0.07 12.68 ±0.91 09.42 ±1.28
water 5 107.26±4.16 108.77±3.22 89.78±3.44 96.42±3.36 1.17 ±0.22 0.96 ±0.01 15.86 ±1.42 12.48 ±1.75
10 107.95±3.78 116.24±4.88 95.54±3.76 99.39±3.63 1.19 ±0.09 1.01 ±0.02 24.31 ±1.14 20.96 ±2.45
15 111.73±3.83 116.41±5.21 109.28±4.52 109.32±4.09 2.08 ±0.11 1.81 ±0.07 33.18 ±2.20 23.27 ±2.14
Tomato 3 78.41±2.82 82.49±3.02 57.41±1.86 62.72±1.74 1.19 ±0.06 1.69 ±0.11 16.26 ±1.90 15.63 ±1.07
extract 5 81.23±3.69 85.89±4.18 62.72±2.06 65.68±2.40 1.27 ±0.27 1.96 ±0.04 24.55 ±1.29 22.47 ±1.02
10 96.81±4.47 100.62±4.31 70.52±3.04 72.53±2.31 1.31 ±0.18 1.91 ±0.06 30.33 ±2.02 25.28 ±2.13
15 92.76±4.33 104.47±4.10 69.64±3.13 73.33±2.11 1.96 ±0.11 2.01 ±0.10 32.18 ±2.85 27.49 ±2.64
Sweet corn 3 86.41±3.22 94.58±2.77 74.23±3.11 78.61±2.38 1.08 ±0.07 0.92 ±0.06 13.58 ±0.98 08.90 ±0.79
extract 5 96.23±4.13 98.66±3.12 86.82±3.84 90.20±4.06 1.15 ±0.32 0.98 ±0.04 18.81 ±1.22 13.33 ±1.76
10 100.45±3.48 101.26±4.87 96.41±3.93 99.11±3.92 1.29 ±0.07 1.10 ±0.09 20.33 ±1.10 16.36 ±2.05
15 116.79±4.89 119.51±4.23 90.12±3.52 92.21±3.08 2.12 ±0.10 2.01 ±0.17 20.19 ±1.22 14.52 ±2.54
Sweet lime 3 77.42±1.82 80.26±1.92 60.40±1.81 66.64±2.16 1.51 ±0.11 1.62 ±0.13 27.63 ±2.07 25.23 ±2.26
juice 5 80.32±2.37 84.58±2.42 72.29±2.17 78.21±2.93 2.17 ±0.22 2.45 ±0.17 35.71 ±2.41 33.54 ±2.11
10 95.78±3.18 102.12±4.13 76.52±2.37 82.54±3.36 2.78 ±0.12 3.59 ±0.17 44.91 ±3.11 41.28 ±3.21
15 88.97±3.14 96.54±3.11 68.66±2.04 71.84±2.12 2.42 ±0.11 2.55 ±0.09 48.35 ±3.21 45.19 ±3.43
(+) ve control (with PGRs) 106.78±4.78 104.54±3.48 90.88±4.01 91.12±3.21 0.89 ±0.06 0.87 ±0.04 14.78 ±1.91 14.60 ±1.08
(-) ve control (without
75.96±2.16 77.25±3.77 52.93±2.61 50.71±3.42 0.90 ±0.07 0.89 ±0.08 22.42 ±1.97 21.61 ±1.97
PGRs)
LSD (P=0.05) 21.2436 21.3436 20.4541 21.6562 0.189 0.193 4.4770 4.5781
LSD (P=0.01) 33.3412 33.1241 30.2511 30.5517 0.324 0.339 6.2418 6.1281
CV 17.82 18.02 16.21 16.35 09.26 08.95 4.04 4.10
SE Standard Error; CD Critical Difference; CV Coefficient of variation SD Standard Deviation; PrA and PoA represent the MS medium where plant
extracts were added prior to autoclaving and post-autoclaving through sterile filters, respectively.

Journal of Applied Horticulture (www.horticultureresearch.net)

108 Low-cost alternatives in rose micropropagation

was noticed in sweet lime extract which was almost double (27.63 sample was recorded at lowest concentration of extract which
±2.07 mg g-1 fresh tissue) compared to control plants, even at subsequently decreased with increasing concentration of extract.
the lowest concentration tested. Interestingly, the coconut water The difference in higher and lower value of enzyme activity was
at low concentration, did not show any significant increase in little and nugatory which could be explained on the fact that
proline. The probable reason behind higher accumulation of since all the propagules are in same developmental stage, i.e.,
proline is due to increased salt level, acidic compounds, phenols multiplication stage, and activity of the peroxidase depends on
and other metabolites present in test extracts and due to adaptive developmental stage which generally increases during rooting
response of plants in culture conditions. Similar observation was and hardening stage (Zeng et al., 2015). When compared with
recorded by Woodward and Bennett (2005) in in vitro cultures the control plant, the activity of peroxidase was found to be
of Eucalyptus camaldulensis where increased salt concentration almost similar which shows that addition of plant extract does
resulted in increased proline accumulation. not evoke production of reactive oxygen molecules. Similar
Studies on the carbohydrate contents shed light on the sugar observation was recorded by Sağlam (2013) in cucumber, where
metabolism and its uptake in plants. As a general observation in exogenous supply of Brassinosteroids (a group novel plant growth
all the test samples, increase in extract concentration resulted in substances, initially extracted from pollen grains of rapeseed) did
enhancement of carbohydrate level although it failed to equate the not affect the peroxidase activity. IAA-oxidase is considered to
control plants except in coconut water. Further addition of extract be responsible for the enzymatic oxidative decarboxylation of
resulted in decrease of carbohydrates accumulation. Coconut IAA and the activity of this group of enzymes has been largely
water at 5 % and 10 % (v/v) concentration exerted similar effect associated with adventitious rooting (Porfirio et al., 2016). Similar
on carbohydrate biosynthesis which was comparable to control to proxidase, IAA-oxidase activity was also found to be almost
plant. Similar observations were also recorded for protein similar in rose micro-propagules grown on different type and
accumulation. Coconut water exerted similar effect in terms of concentration of plant extracts (Fig. 2). Addition of plant extract,
protein accumulation when compared to PGR supplemented after autoclaving the medium (PoA) did not exert any significant
control plant. Since in the crude extracts of plants, the mixture effect in both peroxidase and IAA-oxidase activity in rose micro-
of growth substance, inhibitors, phenolics and several other propagules (data not shown). The results also confirms that the
metabolites are found in varied concentration. Hence these added plant extract do not contain any inhibitory molecules which
extracts could not equate the effect in the biochemical changes interfere in activities of these enzymes.
during plant development which a purified synthetic growth Generally culture media are frequently supplemented with a
hormone can do (Table 3).
range of organic extracts like protein hydrolysates, coconut
Peroxidase is known to play a role in growth and differentiation milk, yeast and malt extract, ground banana, orange juice, potato
and its high activity could be correlated to the process of extract, and tomato juice (Molnar et al., 2011). Several natural
differentiation that occurred during shoot induction (Thakar cytokinin like zeatin, zeatin riboside and C-3 and cell division
and Bhargava, 1999). No significant change was recorded with activity of these growth regulators has already been reported in
change in type of plant extract (Fig. 1). Highest activity in all test sweet corn extract. In case of Anthurium cubense, replacement
300
Control
Sweet Corn Extract
290
Tomato Extract
Coconut Water
ΔA/min/g/fresh tissue)

280
Orange
Orange Juice
Juic
Sweet Lime Juice
270
b c
b a ad cd d
ab a a ab
260 b
b c ef a
a b c a e
cd
c
Peroxidase Activity ( 

250 af

240

230

220

210

200
3% 5% 10% 15%
Plant extract concentration (%)

Fig. 1. Activity of peroxidase enzyme in micro-propagules of rose grown in vitro on the standard MS medium supplemented with
various plant extracts. Extracts were added prior to autoclaving the medium (PrA). Letters indicate significant difference by Tukey’s
Test (P < 0.05).
Journal of Applied Horticulture (www.horticultureresearch.net)
 Low-cost alternatives in rose micropropagation 109

12 Control Tomato Extract Sweet Corn Extract

c
Orange Juice Coconut Water
c
b
IAA oxidase (mg/g IAA destroyed/h/g/fresh tissue)

10 b Sweet Lime Juice

cd
ab ef d c
ad a a
e abc bc c
8 c
c c ef c
b bc

6
c

0
3% 5% 10% 15%
Plant extract concentration (%)

Fig. 2 Activity of IAA-oxidase enzyme in micro-propagules of rose grown in vitro on the standard MS medium supplemented with various
plant extracts. Extracts were added prior to autoclaving the medium (PrA) Letters indicate significant difference by Tukey’s Test (P < 0.05) .
of cytokinin with cheaper citrus fruit rind derived substance, Table 4. Cost analysis of natural substitutes of BA
Pectimorf proved better in vitro growth and 90 % survival of Component Concentration Cost/L Decrease in ( %)
plantlets during acclimatization (Montes et al., 2000). Similarly, ( % v/v) media in cost in reference
at 10 mg L-1 concentration, Pectimorf gave in vitro multiplication INR to standard
synthetic BA
rate of Spathiphyllum comparable to 0.5 mg L-1 BA concentration Synthetic PGR- BA* 0.003 Rs. 52.59 -
(Hernandez et al., 2009). Similar reports of application of orange
Sweet lime juice 5 Rs. 0.80 98.47
juice (@ 10 % v/v) in culturing explants of lemon, grapefruit,
Tomato fruit extract 5 Rs. 0.25 99.52
sweet orange and mandarian are available (Duran-vila, 1989).
Sweet corn 5 Rs. 0.40 99.23
Growth promotory role of coconut water in embryo culture of
many species was reported long back during early development Orange juice 5 Rs. 0.80 98.47
of plant tissue culture. Later it was discovered that coconut water Coconut water 5 Rs. 0.50 99.04
contains rebosyl-zeatin, which is very similar to zeatin isolated *Sigma-aldrich
from young maize endosperm (Letham, 1974). Similarly, several In the current study coconut water (10 % v/v), used as substitute
growth substances and volatile compounds in sweet lime juice for highly priced synthetic BA, was found effective for in vitro
(Maria et al., 2012) and tomato fruit extracts (Rath, 2013) have shoot multiplication of rose while both tomato extract and sweet
been reported. Plant growth-promoting substances such as auxins, lime juice showed comparatively lower shoot multiplication.
cytokinins, and betaines have also been reported in seaweed Supplementation of sweet corn and orange juice also showed
(Khan et al., 2009) and their application as cheaper option in successful multiplication and growth of propagules compared
growth of tomato seedling has also been studied (Hernández- to negative control. Although this growth did not equate with
Herrera et al., 2014).
the response received in propagules grown on MS medium
Cost analysis and comparison of different plant extract and supplemented with synthetic BA but this slight depression in
standard synthetic cytokinin has been depicted in Table 4. It growth can be compromised on the tiff of high reduction of
was concluded that replacement of commercial synthetic BA production cost. In conclusion, coconut water (10 % v/v) provided
(Sigma-Aldrich) with natural plant extract (@ 5 % v/v) can higher rate of multiplication and was cost-effective also (Table
reduce the cost up to 99 % in reference to cost of PGR. This 1) as compared to synthetic BA.
profit gets amplified with subsequent sub-culturing cycles. There
are few reports where natural plant extracts were used to replace Major apprehension of today’s plant tissue culture industry is
synthetic PGRs for lowering down the production cost. Vora the high cost for production and maintenance of large numbers
and Jasrai (2012) reported that fresh juice of sweet-lime (5 % of cultures and incessantly rising numbers of accessions. Our
v/v) provided higher rate of multiplication in in vitro cultures outcome showed that by replacing synthetic PGR, the cost of the
of banana. Similarly, coconut water in combination of MS salts medium could be reduced significantly. However, this needs to
was successfully used for regeneration of Celosia with the aim be tested for in vitro cultures of a range of plants including many
of lowering the production cost (Daud et al., 2011). more horticultural plants.
Journal of Applied Horticulture (www.horticultureresearch.net)
110 Low-cost alternatives in rose micropropagation

Acknowledgements Letham, D.S. 1974. Regulators of cell division in plant tissues. XX. The
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