CHAPTER-6
INFLUENCE OF BIOINOCULANT TREATED PLANTS, GROWTH
REGULATORS AND NUTRIENTS ON VASE LIFE REGULATION OF
SOME SELECTED ORNAMENTAL FLOWERING PLANTS
6.1 INTRODUCTION AND REVIEW OF LITERATUR E
Vase life of cut flower is most attractive and economic component of c ut flower (Kazami and
Ameri, 2012). Consumers of cut flowers demand high quality flowers at the time of purchase
and a guaranteed long vase life so that aesthetic value of the flowers will last. The global
trade of cut flowers and potted plants is worth US $40 billion per annum (Ezhilmathi et al.,
2007). Cut flowers make up about one-third of the value of the global ornamental horticulture
market (Gupta et al., 2006). Commercial floriculture is one of the most profitable agroindustry in the world and in the floriculture industry, the quality of flowering crops is limited
by its longevity, which is influenced by senescence (Wani et al., 2012). Wilting and
senescence of the petals determine the longevity of the flower. So, in the commercial use of
cut flowers it is usually the life span of petals, which determine effective life of a flower. The
market loss of cut flowers due to inefficient postharvest management in India is estimated to
be around 20-40% (Waheeduzzama et al., 2006). The techniques of prolonging the vase life
of cut flowers are a great asset to the growers and users (Nair et al., 2003). Beauty and fresh
look of the flower can be retained only for a few days even when some chemical
preservatives are used to prolong their vase life (Nirmala et al., 2008).
The longevity or potential vase life of flower is determined by the environmental
conditions under which the flowers are produced and post harvest factors under which
flowers are kept. The factors affecting quality of cut flowers generally are: depletion of
carbohydrate, ethylene production, water relation and xylem blockage (Marandi et al., 2011),
temperature and relative humidity. Two major events which take place in senescing petals are
increase in respiration and hydrolysis of cell components. The enzymatic changes found
during petal senescence are associated with these two processes. Increase in activity of
peroxidase enzyme was found in petals of Tulip (Carfantan and Danssant, 1975). The
increase activity of peroxidases is related to increase in free radicals and peroxides which
ultimately react with cellular constituents (Fridovich, 1975) and is involved in increase in the
petal senescence (Frenkel, 1975; Mishra et al., 1976; Brennan and Frenkel, 1977) and also in
ethylene production (Beauchamp and Fridovitch, 1970). During the course of petal
senescence there is a sharp decline in the levels of macromolecules components suc h as
starch (Ho and Nichols, 1997) and proteins (Halevy and Mayak, 1979; Borochov and
Woodson, 1989; Stephenson and Rubinstein, 1998; Viera et al., 2010).
Flowers are one of the most important adornments of the world with their valuable
aesthetic, environmental, economic and medicinal properties (Varu and Barad, 2008).
Gerbera is one of the ten most popular cut flowers in the world and according to global
trends in floriculture, it occupies the fourth place in cut flowers (Choudhary and Prasad,
2000; Safa et al., 2012). Besides floral arrangements, Gerbera is widely used in bouquets
(Nair et al., 2003). The problem with Gerbera cut flowers is the short postharvest life
(Mohammadiju et al., 2014). Mencarelli et al. (1995) found that occurrence of stem break in
Gerbera could be due to ethylene production and associated with early senescence.
Chrysanthemum is again one of the most important flowers and ranked as the second most
economically important cut flower in the world, after Rose (Kafi and Ghahsareh, 2009). The
most important postharvest problem in Chrysanthemum is its inability to absorb water which
leads to flower wilting. Water interaction is the main factor determining the quality and
survival of cut flowers (De Silva, 2003). The water balance of flowers is disrupted both by
the blockage of transporting vessels of the flowers as well as by reduction of the cell
capacity to retain water (Constanta et al., 2013). Air bubbles in the Chrysanthemum stem
vessels reduce the quality of flowers. Marigold is an important loose flower in India, used for
making garland, religious offering and social functions (Syamal et al., 2006). However, these
flowers have relatively short vase life as well as shelf life and finding methods to increase
flower longevity is of great importance (Mashhadian et al., 2012). Arbuscular Mycorrhizal
fungi play an important role in increasing vase-life of cut flowers and shelf life of loose
flower by reducing ethylene production (Besmer and Koide, 1999) and increasing water
absorption. Plant growth regulators like kinetin, salicylic acid and sucrose also play an
impotant role in checking the petal senescence.
There are several reports on post harvest treatment of cut flowers to increase the vase
life by using growth regulators [kinetin (KN) and salicylic acid (SA)], sugar, macronutrients,
micronutrients etc. Salicylic acid is well known phenol that can decrease ROS (Reactive
Oxygen Species) with increasing antioxidant enzyme activity (Ansari and Mishra, 2007).
Kazemi et al. (2011) found that the treatment of salicylic acid increased water uptake in
Carnation cut flower and increased vase life. Kazemi and Ame ri (2012) also reported
increased vase life of cut Gerbera by SA. The improved vase life by SA treatment as
preservative solutions might be due to their role in inhibiting the microbial growth and
preventing bacterial plugging (Kazemi and Ameri, 2012). SA when applied as a preservative
solution decrease lipid peroxidation and delay senescence (Jamshidi et al., 2012). The vase
life of many flowers has been prolonged when kinetin or cytokinins have been added to
holding solution (Mayak and Halevy, 1980; Van Staden et al., 1990). The uptake of sucrose
via vase solution maintained level of total soluble sugars in the petals and also stimulate the
water uptake by enhancing osmotic driving force of the cells for water intake (Ho and
Nichols, 1997). The adequate supply of sucrose to the petals facilitated the continuation of
the metabolic activities to the optimum during vase life and stimulated the synthesis of lipids
and restricted the oxidation of unsaturated fatty acids (Alka et al., 2006), and effectively
lowered the lipid peroxidation and delayed the cell death of petal cells. Awad et al. (1986)
reported that placing flower in sucrose solution enhanced the longevity of Calendula and
Zinnia.
Beneficial microbes like Arbuscular mycorrhizal fungi (AMF), Phosphate solubilizing
bacteria (PSB) and Trichoderma viride play an important role in increasing flower quality as
well as vase life. AM fungus is able to suppress ethylene production by affecting phenolic
metabolism of plant (Vierhelig et al., 1994). Trichoderma spp. has been found to have
stimulatory effects on AM fungi (Calvet et al., 1992, 1993). In addition, Trichoderma also
enhances plant growth (Windham et al., 1986; Naseby et al., 2000) and increases resistance
against plant pathogens (Chet, 1990; Tanwar, 2013). Phosphate solubilizing bacteria increase
the availability of phosphorus in soil through the secretion of phosphatase enzyme which
leads to transfer of organic phosphorus to their available forms (Abou El-yazeid et al, 2007).
Consequently, it enhances phosphorus absorption and accumulation in plant tissues which
ultimately improve the yield quality of the crops. Increased vase life in Gerbera has been
reported with the use of AM fungi by Narayana Gowda (2003). Srivastava and Govil (2005)
revealed that biofertilizers like AM fungi, PSB and Azotobacter improved floral characters as
well as vase life of Gladiolus as compared to control. Bhatia et al. (2004) reported that
inoculating the media with AM fungi increased bud size and ultimately size of flower in
Carnation. Bhalla et al. (2006) observed that the flowering parameters like size of flowers
and vase life improved with the addition of AM fungi, PSB and Azospirrilim to the growing
media. Mycorrhizal fungi significantly increased flower diameter, flower fresh and dry
weights of Marigold plants as compared to non mycorrhizal plants (Abdul-Wasea et al.,
2011). Scagel (2004a) found that flowers of AM fungi inoculated plants generally lasted
longer than non-mycorrhizal plants. Padmadevi et al. (2004) reported that phosphate
solubilizing bacteria, AM fungi along with inorganic nutrients and growth regulators brought
about significantly higher effects on flowering attributes in Anthurium. Wen (1991) reported
that Gerbera plants colonized by Glomus mosseae produced flowers, which lasted three days
longer than flowers of non-mycorrhizal plants in the vase solution. Chrysanthemum plants
inoculated with AM fungi with 50 per cent recommended NPK increased the vase life of
flower compared to plants receiving only recommended NPK. Similarly, in Gerbera,
maximum vase life (12 days) was observed in flowers harvested from plants treated with AM
fungi (Seetha, 1999). Scagel and Schreiner (2006) found increase in longevity of
Zantedeschia flowers by inoculating with AM fungi. Chauhan and Kumar (2007) observed
that AM fungi with higher level of nitrogen significantly increased the flower diameter of
Calendula. Bhalla et al. (2007) reported that flower size and vase life of Carnation flower
increased when treated with phosphate solubilizing microorganism, Azospirillum and
inorganic fertilizers. Meir et al. (2010) observed an increase in the vase life of Eustoma
grandiflorum due to AM fungi. The corms of Gladiolus when inoculated with PSB and
Azotobacter, significantly improved the floral characters and vase life (Chaudhry et al.,
2013). Inoculation of Freesia and Harlequin flower with AM fungi increased protein and
amino acid content (Scagel, 2003; 2004a). Krishna and Bagyaraj, (1983) and Vazquez et al.
(2001) found that AM fungi increased protein content in Brodiaea laxa. Liu et al. (2011)
found that physiobiochemical parameters of Marigold plants such as soluble sugar content,
soluble protein content and antioxidant enzyme activity, could be improved by AM fungi.
Keeping in view the above information, the present investigation was undertaken to
compare the efficiency of two AM fungi (Glomus mossease and Acaulospora laevis) alone
and in combination with Pseudomonas fluorescence, Trichoderma viride, growth regulators
and nutrients on the vase life of Chrysanthemum, Gerbera and shelf life of Tagetes flowers.
6.2 MATERIALS AND METHODS
In the present investigation, three plants viz Chrysanthemum indicum, Gerbera jemesonii,
and Tagetes erecta were used. Flowers of C. indicum and G. jemesoni were harvested with a
sharp scalpel in the morning hours. The scapes were cut under water to prevent cavitation and
immersed in the bucket full of water. These were brought immediately to the laboratory and
basal few centimeters of scapes were recut under water to obtain uniform length. Flowers of
different bioinoculant treatments (As described in chapter- 5) were immersed in conical
flasks containing 100 ml distilled water while flowers without any bioinoculants (control)
were further immersed in 100 ml of different concentrations of K inetin i.e. KN (37.50µM,
3.75µM), Salicylic acid i.e. SA (37.50µM, 3.75µM), Sucrose (0.1mM) and Sodium chloride
(0.1mM). The flasks containing cut flowers were kept at room temperature in the laboratory
to study the various parameters. Flowers of T. erecta were cut with scalpel and brought into
the laboratory without dipping in water and kept as loose flower under laboratory conditions.
The experiment was run in triplicate and samples were taken at 0 th, 8th and 16th day of stages
for C. indicum and T. erecta and 0th, 6th and 12th day for Gerbera, according to their longevity
for biochemical analysis.
Preparation of Plant Growth Regulators and Nutrients
Kinetin (KN), Salicylic acid (SA), Sucrose and Sodium chloride (NaCl) were tested for their
efficiency in enhancing the vase life and improving the flowers quality along with various
changes in some biochemical features.
Selected concentrations are as followsKinetin (KN)
KN-1=37.50 µM and KN-2=3.75 µM
Salicylic acid (SA)
SA-1=37.50 µM and SA-2=3.75 µM
Sucrose (S)
S=0.1 mM
Sodium chloride (NaCl)
NaCl=0.1mM
Firstly, stock solutions were prepared from which various dilutions were made at the
time of experiment. The stock solution of KN [C 10H9N5O (Mol. wt. 215.2)] having a
concentration 37.50 µM was prepared by dissolving 8.07 mg KN in 15 ml warm double
distilled water (DDW) and raised the volume to 1000 ml. 3.75 µM KN was prepared by
taking 25 ml volume from the stock and raised it to 250 ml with DDW. Stock solution of SA
[C7 H6 O3 (Mol wt. 138.121)] was also prepared in the same manner. 37.50 µM concentration
was prepared by dissolving 5.1795 mg in 15 ml DDW and raised to 1000 ml with DDW,
followed by the preparation of 3.75 µM SA by taking 25 ml volume of stock solution and
raising the volume 250 ml with DDW. Sucrose [C 12 H22O 11 (Mol. wt. 342.30)] solution of 0.1
M was prepared by dissolving 34.23 gm in DDW and raised it to 1000 ml with DDW. 0.1 M
solution of sodium chloride [NaCl (Mol. wt. 58.44)] was prepared by dissolving 5.84 mg into
100 ml of DDW.
OBSERVATIONS
Visible effect
External appearance and freshness of flowers during the course of vase life was recorded
daily.
Assessment of flowe r diameter
Flower diameter was determined as the mean of two perpendicular measureme nts across a
flower at 0th , 8th and 16th day for C. indicum, T. erecta and 0th, 6th and 12th for G. jamesonii.
Fresh weight of flower
Fresh weight of three randomly selected flowers of T. erecta was recorded.
Longevity
The longevity or average vase life of the flowers was counted from the day of transfer of
spikes to holding solutions to the day of termination when the visible signs of senescence
appeared.
Volume of holding solution absorbed
The volume of holding solution absorbed by the flowers of C. indicum and G. jamesonii was
calculated by measuring the volume of solution at the end of experimental set up and
subtracting it from the initial quantity of the vase solution kept in the flasks.
Bioche mical Analyses
Samples were taken for various biochemical estimations such as protein, total sugar and
reducing sugars. Specific activity of peroxidase enzyme was also measured.
Estimation of total soluble sugars and re ducing sugar
Sugar was calculated by Anthrone method of Hart and Fisher (1971). 100 mg sample was
extracted in 10 ml of DDW and centrifuged at 5000 rpm for about 10 min in a Remi
centrifuge (R-8C). Residue was discarded, supernatant was collected, raised the final volume
to 50 ml with DDW and pH was noted. Three ml of the extract was taken in triplicate in test
tubes and kept on boiling water bath at 100 o C temperature for five minutes. Six ml of
anthrone reagent, prepared by mixing 0.5 gm anthrone in 250 ml of 98% concentrated H 2SO 4
was added slowly along the wall of test tube while test tubes were placed on bo iling water
bath. Gentle shaking was done to mix the content and blue green colour is formed devoid of
any turbidity. Test tubes were again placed on boiling water bath and after five minutes all
test tubes were cooled under tap water or ice water. Blank was prepared by mixing three ml
of DDW and six ml of anthrone reagent. It was kept on same water bath with reaction set till
blue green colour appeared. Absorbance of all the samples was noted at 600 nm in uv- vis
spectrophotometer (Specord-205, Analytic Jena, Germany).
The remaining extract (35 ml) was hydrolyzed with 10 ml of HCl (50%). Hydrolysis
was carried out at room temperature for a day and next day same pH was set with 6N NaOH
as it was on previous day. This solution was raised up to 100 ml with DDW. Now three ml of
each extract was taken in triplicate and put on boiling water bath for five minutes followed by
transfer of six ml of anthrone reagent in each test tube. Absorbance was taken at 625 nm in
the uv-vis spectrophotometer (Specord-205, Analytic Jena, Germany) after cooling the test
tubes under tap water. Amount of reducing sugars were calculated against a standard curve of
glucose, as described later in this chapter.
Protein estimation
Total protein was estimated by method of Bradford (1976). In this study, 100 mg plant
material was warmed in 10 ml of 80% ethanol on water bath for 2-5 minutes, and then it was
allowed to cool at room temperature. After cooling, it was homogenized with the same
ethanol using pestle and mortar. Homogenate was centrifuged at 5000 rpm for 10 minutes.
The residue was re-extracted with five ml of five per cent perchloric acid and centrifuged
again for 10 minutes at 5000 rpm. After discarding the supernatant, the residue was reextracted with five ml of 1N NaOH and dissolved it by keeping in water at a temperature of
40-50oC for 20 to 30 minutes. After 30 minutes, it was centrifuged again at 5000 rpm for 2030 minutes and supernatant was stored in refrigerator as a protein source.
Preparation of protein binding dye (Coomassie brilliant blue G-250)
10 mg of Coomassie brilliant blue G-250 dye was dissolved in five ml of ethyl alcohol. 10 ml
of Orthophosphoric acid was added to it with vigorously shaking. The final volume was
raised to 100 ml with DDW. Now, in a test tube 0.2 ml of enzyme extract and 0.8 ml of DDW
was taken to make it one ml. Then five ml of dye was added in each test tube. Absorbance
was taken at 595 nm in a uv-vis spectrophotometer (Specord-205, Analytic Jena, Germany).
Blank was prepared without enzyme extract. One ml of DDW was taken instead of enzyme
along with five ml of dye. The protein content of sample was determined against a standard
calibration curve, drawn earlier with bovine serum albumin (BSA, Sigma product, USA),
described later in this chapter in detail.
Measure ment of Pe roxidase enzyme activity
The peroxidase activity was measured by the method of Maehly (1954). For this 100 mg of
plant material was homogenized with ice cold phosphate buffer (pH-7.0). Phosphate buffer of
pH-7.0 was prepared by adding 3.89 ml of 100 mM KH2 PO 4 and 6.11 ml of 100 mM
Na2 HPO4 for total volume of 10 ml buffer. Homogenized material was centrifuged at 5000
rpm for 10 minutes. Supernatant was taken out as enzyme source. The react ion set was
prepared by mixing two ml of enzyme source, phosphate buffer, guiacol (20 mM) and
hydrogen peroxide (10 mM) in sequence. In the blank set two ml of enzyme source, two ml
phosphate buffer and four ml of double distilled water (DDW) were added. Reaction and
control sets were incubated undisturbed for 10 minutes at room temperature. The absorbance
was taken at 420 nm in uv-vis spectrophotometer (Specord-205, Analytic Jena, Germany).
Specific activity of peroxidase was expressed in terms of mg protein per 10 min. Protein was
estimated from the same extract following the procedure of Bradford (1976) as mentioned
above.
CALIBRATION CURVES
Protein estimation using bovine serum albumin (BSA)
Five mg of BSA was dissolved in 50 ml of DDW and different volumes of BSA solution
were taken such as 100µl, 200µl, 300µl, 400 µl, 500µl, 600µl, 700µl and 800µl in test tubes
using micropipettes. Total volume of the solutions was raised to one ml with DDW, added
five ml of Coomassie brilliant blue G-250 dye in each test tube and kept it undisturbed for 10
minutes at room temperature. After 10 minutes absorbance was recorded at 595 nm in the uvvis spectrophotometer (Specord-205, Analytic Jena, Germany). A graph was plotted taking
different concentrations of BSA on X-axis and absorbance on Y-axis.
Fig. 6.1 Calibration curve for the estimation of protein in terms of Bovine Serum
Albumin (BSA).
The quantification of total soluble sugars and reducing sugar by using Glucose:
10 mg glucose was measured and dissolved in 10 ml of DDW. Now different concentrations
of glucose solution were prepared by taking 10µl, 20µl, 30µl, 40µl, 50µl, 60 µl, 70 µl, 80µl,
90µl and 100µl of stock solution and raising the volume to three ml with DDW in each test
tube. All test tubes were kept on boiling water bath for five minutes fo llowed by carefully
pouring of six ml anthrone reagent. Anthrone reagent was prepared by dissolving 0.5 gm of
anthrone in 250 ml 98% H2 SO 4. After pouring of anthrone reagent, test tubes were again
heated for 5 min. on boiling water bath and then cooling was done. The absoebance was
taken at 600 in uv-vis spectrophotometer. Graphs were plotted taking concentration of
glucose along X-axis and absorbance at 600 along Y-axis.
Fig. 6.2 Calibration curve for the quatitative estimation of total sugars in terms of
glucose
Fig. 6.3 Calibration curve for the quantitative estimation of reducing sugars in terms of
glucose
Experime ntal analysis
All results were analyzed using Analysis of Variance (ANOVA), followed by post hoc test
through computer software SPSS 11.5 version. Means were ranked at
0.005 level of
6.3 RESULTS AND DISCUSSION
The senescence of flower petals are associated with a series of morphological, physiological
and biochemical changes. Morphological changes include changes in the visible effects of a
cut flower like shape, size, diameter, colo ur etc. Physiochemical and biochemical changes
include an increase in hydrolytic enzymes, degradation of macromolecules and loss of
cellular compartmentalization.
6.3.1 Influence of bioinoculant treated plants, growth regulators and nutrients on vase
life regulation of C. indicum (Tables-6.3.1a, 6.3.1b, 6.3.1c, 6.3.1d, Plate-6.1)
Visible effect
Flower senescence was characterized by change in petal colour from whitish yellow to dark
brown. In the present study it was found that the cut flower petals were fresh at 0 th day stage
in all the treatments and it exhibited shrinkage after 4 th day stage in the flowers placed in
solution of NaCl and after 7th day in control (without any bioinoculation). O n 12 th day stage,
flowers of all the treatments showed shrinkage except in treatments G+A+T, G. mosseae, A.
laevis, G+T and kinetin (KN-1). Flowers pretreated with G+A+T before placing in DDW
were fresh even at 15th day stage and only a little signs of shrinkage was observed.
Floral diameter
As depicted in Table-6.3.1a that initially at 0 th day maximum floral diameter was observed in
flowers pretreated with G+A+T (14.33±0.30) followed by consortium treatme nt of
G+A+T+P (14.30±0.15), G+P (14.26±0.25), A+P (13.90±0.30) and least was recorded in
control (10.23±0.25). Plants pretreated with AM fungi registered an increase in floral
diameter as compared to untreated flowers (control). Floral diameter decreased as senescence
proceeded. After 8th day, there was comparatively less decrease in flower diameter in
treatment G+A+T (12.43±0.56), followed by G+A+T+P (12.40±0.56) and on 16th day stage
also, results followed the same pattern.
Longevity
The average life of Chrysanthemum flower is about 7-10 days. In the present investigation,
treatment G+A+T showed maximum vase life (15.66±0.57 days) followed by G+T
(15.33±057), G. mossae (14.66±0.57) and KN-1 (14.33±0.57) (Table- 6.3.1b).
Volume of holding solution absorbed
The flowers pretreated with AM fungi showed high absorption of distilled water as compared
to flowers kept in different holding solution containing Kinetin, Salicylic acid and Sucrose. It
is evident from the Table-6.3.1b, that the volume of absorbed so lution was maximum in
flower pretreated with G+A+T (12.00±0.10) followed by G+T (11.86±0.15), G. mosseae
(11.80±0.17) and KN-1 (11.80±0.17).
Peroxidase activity
As envisaged from Table-6.3.1a, there was increment in peroxidase activity of
Chrysanthemum with increase in the holding days from 0 th day to 16th day. This is due to
entering of flowers in senescence stage as soon as they are cut from the plant system. At 0 th
day, peroxidase activity was actually found to be less in flowers harvested from AM
inoculated plants as compared to flower taken from control. Triple inoculation of G+A+T
flowers showed less peroxidase activity from 0.024±0.004 (0th day stage ) to 0.123±0.004
(16th day stage) as compared to flower placed in other solutions. Maximum peroxidase
activity was observed in flowers placed in NaCl (0.307±0.003 to 0.672±0.02) at 0th and 16th
day stage. Flower of control plant also showed fast increment in peroxidase activity from
0.240±0.026 (0 th day) to 0.630±0.006 (16th day). Among all the plant growth regulators,
Kinetin (KN-1) was found to be the most effective hormone in decreasing increment in
peroxidase activity (0.300±0.010, 0 th day, 0.328±0.005, 16th day).
Protein content
Table-6.3.1c clearly showed that at 0 th day, all the preinoculated plants with various
bioinoculants contain high amount of protein content as compared to control (non
inoculated) and maximum protein content was recorded in triple combination of G+A+T
(2.330±0.010) followed by G+T (2.196±0.025) and least amount was present in control
(1.243±0.051). Further, a decrease in protein content was recorded from 0 th day to 16th day
stage as petal senescence increases but the treatment G+A+T showed less decrease in protein
content from 0 th day stage (2.330±0.010) to 16th day stage (1.736±0.090) as compared to
other preinoculated plants and growth regulators.
Total s ugar and reducing sugar
Alteration in total soluble sugar and reducing sugar in C. indicum revealed a increment with
progress of senescence in cut flower (Table-6.3.1d). Chrysanthemum flower pretreated with
bioinoculants and flowers which were treated with growth regulators showed reduce
increment in sugar content as compared to control one. Least increment in total sugar and
reducing sugar was noticed in triple combination of G+A+T. The increment in total sugar
from 0th day to 16th day in G+A+T was 0.170±0.040 and 0.370±0.020 respectively. Similarly,
increase in reducing sugar was 0.074±0.004 (0th day) to 0.195±0.003 (16th day).
6.3.2 Influence of bioinoculant treated plants, growth regulators and nutrients on vase
life regulation of G. jamesonii (Tables-6.3.2a, 6.3.2b, 6.3.2c, 6.3.2d, Plate-6.2)
Visible effect
Flower were fresh at 0 th day in all the treatments and it showed shrinkage after 3 rd day in the
treatment of NaC l and after 5th day in control (without any bioinoculation). On 9 th day,
flowers of all the treatments showed shrinkage except in the flowers pretreated with
combination of G+T, G+A+T and flower placed in vase solution of sucrose. Flowers
pretreated with G+T before placing in DDW were fresh even at 11 th day and only a little
signs of shrinkage was observed. So, it was found that flower from those plants which were
pretreated with dual combination of G+T and flower treated with sucrose showed delayed
senescence as compared to the other flowers grown in simple traditional sand: soil mixture
and treated with other growth nutrients and regulators.
Floral diameter
As depicted in Table-6.3.2a, on the 0 th day maximum floral diameter was observed in those
flasks, which contain the flowers of pretreated plants with G+A+T+P (11.23±0.25) followed
by G+T (10.76±0.20), G+A+P (10.36±0.15), G+A+T (10.33±0.25 ) and least was recorded
in control (6.33±0.15). It was found that plants pretreated with bioinoculants showed an
increase in floral diameter as compared to untreated flowers (control). Floral diameter
showed a gradual decrease as petal senescence proceeded. After 6 th day, there was
comparatively less decrease in flower diameter in treatment G+A+T+P (10.56±0.15),
followed by G+T (9.96±0.25), and on 12th day stage also, results followed the same pattern.
Longevity
The vase life of Gerbera flower is about 6-8 days. In the present investigation, flowers
pretreated with dual combination of G+T showed maximum vase life (11.66±0.57) followed
by flowers treated with sucrose (11.33±0.57) and least vase life was recorded in the flowers
placed in solution of NaCl (6.66±0.57) as envisaged from Table-6.3.2b.
Volume of holding solution absorbed
It is evident from the Table-6.3.2b that the volume of absorbed solution was maximum in
flowers pretreated with G+T (21.53±0.50) followed by consortium of G+A+T+P
(21.06±0.11) and flowers treated with sucrose (20.43±0.15).
Peroxidase activity
As depicted in Table-6.3.2a there was increment in peroxidase activity of Gerbera from 0th
day to 12th day. At 0th day, peroxidase activity was found to be less in flowers harvested from
inoculated plants as compared to flower taken from control (non-inoculated). Dual
inoculation of G+T pretreated flowers showed less increment in peroxidase activity from
0.037±0.004 (0th day stage ) to 0.097±0.005 (12th day stage) as compared to flower placed in
other solutions. Maximum peroxidase activity was recorded in flowers placed in vase
solution of NaCl (0.199±0.002 to 1.816±0.015) at 0th day and 12th day stage respectively.
Flower of control (non- inoculated) placed in DDW also showed fast increment in peroxidase
activity from 0.199±0.005 (0th day) to 1.024±0.004 (12th day stage). Among all the plant
growth regulators, sucrose was found to be the most effective hormone in decreasing
peroxidase activity from 0.191±0.002 (0 th day stage) to 0.301±0.003 (12th day stage).
Protein content
As depicted in Table-6.3.2c that protein content was increased in all the treatments as
compared to control. Application of plants earlier treated with various bioinoculants and
growth regulators were found to reduce the breakdown of protein during senescence on
progressive day. Least decrease in protein content from 0 th day (3.236±0.035) to 12th day
stage (2.136±0.090) was recorded in dual combination of G+T and highest breakdown was
recorded in the flowers treated with NaCl as well as in control flowers (non-inoculated).
Total s ugar and reducing sugar
Amount of total sugar and reducing sugar was increased as senescence increased from 0th day
stage to 12th day stage (Table-6.3.2d). Flowers placed in the solution of sodium chloride
showed maximum increment in sugar content and decrease the vase life of flowers. However
plants which were pretreated with G+T before placing in DDW showed less increment in
total sugar from 0th day (0.230±0.020 ) to 12th day stage (0.483±0.035) as well as reducing
sugar (0.089±0.005, 0.296±0.025) at 0 th day and 12th day respectively.
6.3.3 Influence of bioinoculants on shelf life regulation of Tegetes erecta (Tables-6.3.3a,
6.3.3b, 6.3.3c, 6.3.3d, Plate -6.3)
Visible effect
Flower petals of T. erecta which were fresh at 0th day stage, exhibited shrinkage after 6 th day
stage and the process was further intensified at 8th day and 16th day. The shrinkage was
maximum in flowers placed in NaCl and flowers of control plant (non-inoculated).
Floral diameter
As depicted in Table-6.3.3a that maximum flower diameter was recorded in flower pretreated
with triple combination of
A+T+P (7.83±0.05) followed by consortium of G+A+T+P
(7.43±0.20), G+T+P (7.03±0.15) and least was recorded in control (4.90±0.10). There was
gradual decrease in flower diameter as petal senescence increased. However, flowers of
bioinoculated plants showed less decrease in flower diameter as compared to control.
Fresh weight of flower
Fresh weight of flower showed an increment in all the inoculated plants and maximum flower
weight was found in plants pretreated with combination of A+T+P (8.20±0.20) followed by
G+A+T+P (7.90±0.10) and least was found in plants of control treatment ( 5.30±0.10)
(Table-6.3.3b).
Longevity
As envisaged from Table-6.3.3b that triple combination of A+T+P (16.00±1.00 ) was most
effective in increasing longevity or vase life of flowers followed by G+A+T+P (15.00±0.00 ),
G+T+P (14.00±1.00) and minimum vase life (8.00±1.00) was recorded in the flowers of
control Tagetes plant (non- inoculated). Consortium and triple inoculation was found more
effective than dual and sole inoculation.
Peroxidase activity
As shown in Table-6.3.1a that there was increment in peroxidase activity of Marigold flower
from 0th day to 12th day stage. Increase in peroxidase activity was associated with petal
senescence. Preinoculation of plants with various bioinoculants reduced the increment in
specific activity of peroxidase. Flowers of plant pretreated with triple combination of A+T+P
showed less increment in peroxidase activity from 0th day ((0.286±0.035) to 16th day
(2.033±0.023) followed by G+A+T+P from 0 th day (0.310±0.045) to 16th day (2.133±0.035)
and highest increment was recoded in flowers of control plant (non-inoculated) during post
harvest period.
Protein content
During the petal senescence protein content was decreased in all the treatments and less
decrease was recorded in the flowers which were earlier treated with triple combination of
A+T+P from 0th day stage (0.308±0.003) to 8 th day (0.267±0.007) and 16th day stage
(0.189±0.003). Breakdown of protein was maximum in flower which was treated with NaCl
and control (non- inoculated).
Total s ugar and reducing sugar
As depicted in Table-6.3.3d, sugar content was increased during petal senescence from 0 th
day stage to 16 th day stage and maximum increment was reported in the flower treated with
NaCl as well as in control. Treatment A+T+P showed less increment in total sugar from 0 th
day (0.030±0.002) to 16 th day stage (0.060±.002) as well as reducing sugar from 0 th day
(0.020±0.002) to 16th day (0.055±.003).
From the results, it was found that flower from those plants which were earlier treated
with bioinoculats showed delayed senescence as compared to the other flowers grown in
simple traditional sand: soil mixture and treated with other growth nutrients and regulators.
The most important function of arbuscular mycorrhizal fungi is thought to be the nutrient
absorption from the soil to enhance the crop growth and yield (Smith and Read, 1997) that
ultimately maintained the different visible effects. Dufault et al. (1990) also reported that
mycorrhizal inoculation improves the phosphorus and potassium uptake which results in
improved flower quality in Gerbera.
Decrease in flower diameter is also a sign of progress towards se nescence as observed
in this experiment and that decrease in flower diameter is due to loss of moisture content and
tissue degradation from the flowers with increase in number of days. Similarly, Gulzar (2003)
observed that the diameter of Iris germanica, Hemerocallis fulva and Petunia hybrida
flowers decreased gradually with increase in the number of days after transferring of the
scapes to various holding solutions. Floral diameter of AM treated and growth regulators
treated flower decreases at a slower rate than the control, which can be due to better uptake of
nutrients especially phosphorus and potassium during active growth period of flowers which
results in better flower quality. Bhalla et al., (2006) also observed an increase in the size of
flower diameter in Gladiolus plants inoculated with AM fungi.
In the present investigation, AM fungi treated flowers showed higher vase life. Wen
(1991) and Wen and Chang (1995) reported that colonization by mycorrhizal fungi increases
the vase life of cut flowers but the exact mechanism is still unknown. One possibility can be
the enhanced nutrient absorption especially nitrogen, potassium, zinc and copper by
colonization of roots with AM fungi and the enhanced metabolic exchanges between both the
partners and secondly it may be due to more vigour, strength and turgidity of AM inoculated
plants as compared to control (Kim et al., 2002). Pretreated plants with mycorrhiza before
transferring the flowers in vase solution may accumulate nutrients in a shorter time span so
they have, earlier in life sufficiently more nutrient supplied with nutrients to initiate flower
development (Perner et al., 2007). Minimum longevity was observed in NaCl treated flower
and in control flowers. The results of present study are in accorda nce with Nowak and
Rudnicki (1990) that high salinity level decreases flower longevity. Besmer and Koide (1999)
also showed that mycorrhizal fungi play an important role in increasing vase- life of cut
flowers by reducing ethylene production. Our results are in accordance with the results of
Srivastva and Govil (2005) concerning the effect of VAM, Azotobacter and phosphate
solubilizing bacteria (PSB) on flowering in Gladiolus and found that among various
characters studied, vase life was improved by AM, PSB and Azotobacter.
Petals of cut flowers are the main ornamental parts and the turgidity of these petals is
important for good looking product. Turgidity of petals mainly depends on water uptake.
Under low availability of water, O 2 can produce reactive oxygen species (ROS) such as
superoxide (O 2 -), H2 O2 and highly toxic hydroxyl radicals (OH-) (Larson, 1988) resulting in
DNA damage and lipid peroxidation (Egert and Tevini, 2002). Higher water uptake
subsequently increase the cut flower fresh weight (Mashhadian et al., 2012) as it is well
known that loose flowers like marigold are usually traded by weight. There was a significant
correlation between solution uptake and relative fresh weight. This is in accordance with
Knee (1992) who observed that maximum weight gain and solution uptake was correlated.
The results of this experiment showed a higher water uptake and water maintenance in cut
flowers which were earlier treated with AM fungi and other bioinoculants. Meir et al. (2010)
found an increase in fresh flower weight of Eustoma grandiflorum due to AM fungi. Water
absorption was maximum in the earlier days, which ultimately decreases with increase in the
number of days during postharvest period. This decrease in water absorption with increase in
number of days might be due to blockage of conducting tissue and high water potential. AM
fungi inoculated plants showed increase in the holding solution absorption, which can be due
to better vascular development of AM inoculated plants and hence, absorbs large quantity of
water (Chang, 1994; Kim et al., 2002).
In the present investigation increment in peroxidase activity was observed with the
progress of senescence in control as well as in treated flowers but the degree varies.
Enhanced peroxidase activity may be associated with an increase in the level of peroxides
(H2 O2 ) and free radicals (O 2 -) which react with cellular constituents (Mishra et al., 1976) and
probably involved in promotion of petal senescence (Brennan and Frenkel, 1977; Blilou et
al., 2000). During petal senescence it is believed that AM fungi increase the superoxide
dismutase (SOD) activity which is an antioxidant enzyme (Fridovich, 1975) and which is
responsible in lowering the peroxidase activity. SOD reacts with active forms of oxygen,
resulting in their low level (Smirnoff, 1993). Also SOD constitute an important primary
defence mechanism of cells against free radicals generated under stress conditions (Bowler et
al., 1992; Foyer et al., 1994).
The finding related to sugar changes in the flowers has revealed an increment in the
concentration of both total sugar and reducing sugars with the advancement of senescence.
Increment in the amount of sugars was due to degradation of starch and already existing
sugars in the petals and this active break down occurred more intensively in stress tissue
(Halaba and Rudniki, 1986). The demand of hexose in cut flower might be satisfied by
hydrolysis of starch which can also be fulfilled by exogenous application of sucrose. Van
Meeteran et al. (1995) observed the increment in the endogenous sugars during development
of florets in Fressia hybrida. The rise in the quantity of sugar was checked by application of
sucrose, plant growth regulators as well as with preinoculation of AM fungi with other
bioinoculants. The best result was shown by AM fungi followed by sucrose and growth
regulators.
The present study also revealed the decrease in protein content during petal
senescence in all the treatments. Several authors, Celikel and Van Doorn (1995), Stephenson
and Rubinstein (1998), Wagstaff et al. (2002) and Vieira et al. 2010) worked on loss of
protein during petal senescence. Loss of protein content was checked by the application of
growth regulators as well as by preinoculation by AM fungi. From the results it is clear that
amount of protein content was already higher in those plants which were earlier treated with
various bioinoculants. Findings were consistent with the result of Liu et al. (2011), Krishna
and Bagyaraj (1983) and Vazquez et al. (2001) who studied about the increment in protein
content due to AM fungi.
Cut flowers deteriorate very quikly and hence, to maintain freshness of flowers, they
have to be treated with different plant growth regulators or nutrient solutions and even some
biostimulants like arbuscular mycorrhizal fungi alone and along with some other beneficiary
microorganisms like T. viride and P. fluorescens. It was found that pretreatment of plants in
soil with AM fungi alone or in different combination with other microbes gave better results
as compared to control and post harvest treatments of cut flower with different growth
regulators and nutrients.
Plate- 6.1
Effect of different treatments on vase life of Chrysanthemum indicum
Flask 1: Control, 2: Glomus mosseae, 3: Acaulospora laevis, 4: Trichoderma viride, 5:
Pseudomonas fluorescens, 6: G+A, 7: G+T, 8: G+P, 9: A+T, 10: A+P, 11: T+P, 12: G+A+T, 13:
G+A+T, 14: G+T+P, 15: A+T+P, 16: G+A+T+P, 17: KN-1 (37.50µM), 18: KN-2 (3.75 µM), 19:
SA-1 (37.50µM), 20: SA-2 (3.75 µM), 21: Sucrose (0.1mM), 22: NaCl (0.1mM)
A: Vase life at 0-Day
B: Vase life at 8-Day
C: Vase life at 16-Day
Plate- 6.2
Effect of different treatments on vase life of Gerbera jamesonii
Flask 1: Control, 2: Glomus mosseae, 3: Acaulospora laevis, 4: Trichoderma viride, 5:
Pseudomonas fluorescens, 6: G+A, 7: G+T, 8: G+P, 9: A+T, 10: A+P, 11: T+P, 12: G+A+T, 13:
G+A+T, 14: G+T+P, 15: A+T+P, 16: G+A+T+P, 17: KN-1 (37.50µM), 18: KN-2 (3.75 µM), 19:
SA-1 (37.50µM), 20: SA-2 (3.75 µM), 21: Sucrose (0.1mM), 22: NaCl (0.1mM)
A: Vase life at 0-Day
B: Vase life at 6-Day
C: Vase life at 12-Day
Plate -6.3
Effect of different treatments on Shelf life of Tagetes erecta
T1 : Control, T2 : Glomus mosseae, T3 : Acaulospora laevis, T4 : Trichoderma viride, T5 :
Pseudomonas fluorescens, T6 : G+A, T7 : G+T, T8 : G+P, T9 : A+T, T10 : A+P, T11 : T+P,
T12 : G+A+T, T13 : G+A+T, T14 : G+T+P, T15 : A+T+P, T16 : G+A+T+P
A: Shelf life at 0-Day, B: Shelf life at 8-Day, C: Shelf life at 16-Day