Salinity Management in Glycine Max L.
Using Cytokinin from Rhizobacteria Isolated
from Mines and Dump Sites
Sarita Sharma, Rathod Zalak R and Meenu S Saraf*
Department of Microbiology and Biotechnology, University School of Sciences, Gujarat University, India
Submission:April 30, 2022;Published:May 17, 2022
*Corresponding author:Meenu S Saraf, Department of Microbiology and Biotechnology, University School of Sciences, Gujarat University, Ahmedabad- 380009, Gujarat, India
How to cite this article: Sarita S, Rathod Zalak R, Meenu S S. Salinity Management in Glycine Max L. Using Cytokinin from Rhizobacteria Isolated from
Mines and Dump Sites. Curr Trends Biomedical Eng & Biosci. 2022; 20(5): 556047. DOI:10.19080/CTBEB.2022.20.556047
Salinity stress is one of the most damaging abiotic stresses, and it is quickly spreading over the world. It has a major negative impact on plant health and yield, resulting in massive losses. The current work looks at the salt tolerance of rhizobacteria SHHMZ4, SMHMZ46, and SMHMP23 isolated from mines and landfills. These isolates grew in Nutrient broth with NaCl concentrations ranging from 0% to 20% and were able to synthesis cytokinin under salty conditions (2 percent w/v NaCl) and generated 19, 23, and 20 μg/ml, respectively. To confirm cytokinin biosynthesis, TLC and FTIR analyses of isolated cytokinin and reference standard kinetin were performed. Plant investigations in vitro were also performed to establish the efficiency of the cytokinin-producing rhizobacteria. According to the findings of this study, PGPR has a significant function in enhancing salt tolerance in plants and can be utilized as a biofertilizer to promote crop growth in saline areas.
Keywords: Phytohormone; Cytokinin; Salt tolerant PGPR; Rhizobacteria; Salt stress
Drought and salt, which create osmotic stress and impede crop growth and development, have a significant impact on crop productivity. Salinity is a negative state that occurs in arid and semi-arid areas all over the world. It is one of the most serious environmental concerns endangering the food security of the world’s growing human population, resulting in a yearly loss of 1-2 percent of arable land . Plants’ cellular processes are altered by salinity, resulting in a variety of physiological, morphological, biochemical, and molecular changes . The accumulation of toxic ions such as Na+ and Cl– in cells, as well as improper nutrient absorption and oxidative stress damage, all have a significant impact on plant growth and development [3,4]. When plants are exposed to high salt stress, the rhizosphere acts as a reservoir for plant growth-promoting rhizobacteria (PGPRs), which can aid plant acclimation and growth. Through both direct and indirect impacts on plant growth, PGPRs play a crucial role in promoting plant development even in stressful settings. Among the direct mechanisms include phytohormone production (for example, cytokinin), enhanced nitrogen fixation, phosphate solubilization, HCN release, and so on [5,6]. Bacterial species such as Alcaligenes, Azospirillum, Arthrobacter, Klebsiella, Acinetobacter, Bradyrhizobium, Bacillus, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Pseudomonas, Serratia, and Rhizobium have already been found as plant growth promoters [7-9].
Cytokinins are phytohormones that play a significant role in the cell cycle and impact a number of developmental programmed . Endogenous cytokinin levels are exceedingly low in plant tissues . The ability of rhizobacteria to produce phytohormones such as cytokinin is a key role in plant growth. Cytokinin is widely distributed in higher plants, algae, and bacteria, and it is also produced by plant-associated microbes [12,13]. This hormone is vital in the development of plant cancers . Cytokinin regulates cell division, cell enlargement, and tissue expansion, which improves plant growth and yield and plays an important role from seed germination to leaf and plant senescence [4,15], as well as harmonizing important physiological processes throughout the plant’s lifecycle [16,17]. The current research focuses on the discovery and characterization of cytokinin-producing rhizobacteria isolated from the Zawar mines in Udaipur, Rajasthan, and the Pirana waste site in Ahmedabad, Gujarat. We highlight the efficacy of salt-tolerant rhizobacteria for cytokinin biosynthesis, as well as their aptitude as an alternative ecofriendly bio-enhancer for enhanced crop production in salt-stressed situations, in this research.
We obtained 51 isolates from the Zawar mines in Udaipur,
Rajasthan, and 40 isolates from the Pirana waste site in
Ahmedabad, Gujarat, in previous research. Three isolates
(SMHMZ4, SMHMZ46, and SMHMP23) were shown to be highly
resistant to heavy metals (Cd, Ni, and Pb) [18-20]. Under salt
stress conditions, this rhizobacteria was tested for salt resistance
and the ability to produce Phytohormones (Cytokinin).
MgSO4, NaCl, Na2HPO4, CaCl2, and KH2PO4 were bought
from Fine Chemicals (P) Ltd. in New Delhi, India. While the
microbiological media viz. tryptone yeast broth, nutrient broth,
agar powder, pre-coated TLC plates, casamino acid, thiamine,
and biotin were obtained from SRL Pvt. Ltd, Mumbai, India, the
methanol, ethyl acetate, kinetin, sodium hypochlorite, carboxymethyl-
cellulose, and glycerol were obtained from Hi Media
Laboratories, Mumbai, India.
The tolerance of the rhizobacterial isolate to NaCl was
examined in nutrient broth (NB) medium with different NaCl
concentrations (0, 2.5, 5, 7.5, 10, 12.5, 15, 20, 22.5, and 25%
(w/v)). 50 μl of overnight grown rhizobacterial culture was
inoculated in 5 ml of NB medium and incubated at 30 oC in a
rotary shaker (Redmi, India) at 200 rpm for 24 hours. A UV-Vis
spectrophotometer was used to evaluate the optical density of the
rhizobacterial cultures at 600 nm (Systronics 166). To determine
the level of salt tolerance, the Minimum Inhibitory Concentration
(MIC) techniques were utilized. The MIC values are the lowest
salt concentrations at which rhizobacterial growth is inhibited.
To determine the lowest inhibitory concentration of all selected
isolates, cultures were allowed to grow on N-agar plates treated
with salt (NaCl). The initial concentration of salt was 1%, and
it was gradually increased by 1% until no viable colonies were
observed. One loopful of activated culture was streaked onto a
Nutrient agar plate containing 1% NaCl and incubated at 37 oC for
24 to 48 hours. The following day, a loop of these incubated plates
was streaked into a Nutrient agar plate containing 2% NaCl and
incubated for 24 hours at 37 oC.
The Minimum Inhibitory Concentration (MIC) approaches
were used to assess the level of salt tolerance. The MIC values are
the lowest salt concentrations that inhibit rhizobacterial growth.
Cultures were allowed to grow on N-agar plates treated with salt
to determine the lowest inhibitory concentration of all selected
isolates (NaCl). The starting concentration of salt was 1%, and
it was subsequently increased by 1% until no viable colonies
were seen. One loopful of activated culture was streaked onto a Nutrient agar plate containing 1% NaCl and incubated at 37 oC for
24 to 48 hours. The next day, a loopful of these incubated plates
was streaked into a Nutrient agar plate containing 2% NaCl and
incubated at 37 oC for 24 hours. This process was continued until
a salt concentration was reached at which no viable bacterial
growth could be observed [21,22].
Preparation of the inoculum: An inoculum of activated
culture was transferred to fresh M9 media supplemented with 0.2
percent casamino acids, 0.01 percent thiamine, and 2 pg biotin
per liter and cultivated for 5 days at 282 oC at 200 rpm (Remi,
India). An aliquot of 0.1 percent (v/v) activated culture turbidity
with an optical density of 0.90.1 at 600 nm was used as inoculum
. A Systronics 166 spectrophotometer was used to measure
the growth rate at 600 nm [17,24].
Cytokinin production: For cytokinin production, fresh M9
medium supplemented with 0.2 percent casamino acids, 0.01
percent thiamine, 2 pg of biotin, and 2% (w/v) was used . 250
ml Erlenmeyer flask with 100 ml M9 medium inoculated with 1
ml activated inoculum of salt tolerant rhizobacteria and incubated
for 5 days at 282 oC at 200 rpm (Remi, India). The experiment was
conducted out three times. After 72, 96, and 120 hours, cytokinin
production was quantified spectrophotometrically at 665 nm. M9
medium injected with a single virus served as the control [17,24].
Extraction of crude cytokinin: After 72, 96, and 120 hours,
the rhizobacterial cells were isolated from the supernatant by
centrifugation for 15 minutes at 4 oC (Remi, India). The cell free
supernatant was filtered using a 0.45 μm filter (Hi-media, India).
Cytokinin was extracted three times with ethyl acetate. The
extracted insoluble fraction was diluted in 1 ml of HPLC grade
methanol and stored at -20 oC until further analysis [17,24].
Thin layer chromatography of extracted cytokinin: Thin
layer chromatographic separation was achieved with a small
volume (10 l) of ethyl acetate extract (sample) and standard
cytokinin (kinetin) using mobile phase n-butanol: acetic acid:
water (12:3:5 v/v/v) and was observed under UV light (254 nm)
using a UV transilluminator (Biorad, India) [17,24].
FTIR characterization of extracted cytokinin: FTIR spectra
obtained as described by utilizing BRUCKER Alpha ECO-ATR
(Attenuated Total Reflectance) with 16 scan and 4000-400 cm-1
using extracted cytokinin dissolved in HPLC grade methanol
confirmed the molecular identification of cytokinin produced by
rhizobacteria [17, 25].
The formation of saline soil: The sandy loamy soil came
from the agricultural areas of Jagatpur, near Gota in Ahmedabad,Gujarat, India. At a rate of 100 mM/kg, this soil was treated with
salt (NaCl). A soil sample was transported to IFFCO in Gandhinagar,
Gujarat, India, for measurement of soil properties. This number
was determined using the average NaCl content in saline soil,
which ranged from 48 to 111 mM [26,27].
Seed bacterization and Pot studies: Soya bean seeds (Glycine
max L.) were used in this study. The seeds were surface sterilized
for 2 minutes in 70% ethanol and 5 minutes in 2% sodium
hypochlorite before being washed ten times in sterile distilled
water. The selected powerful isolates SMHMZ4, SMHMZ46, and
SMHMP23 were grown in M9 medium at 282 oC for 5 days. Surface
sterilized seeds were soaked overnight at room temperature
in culture inoculated M9 media including sterilized carboxymethyl-
cellulose (1 percent CMC) as an adhesive, then air dried
for further research. As a control, seeds were treated with sterile
distilled water amended with CMC alone. For the pot study, 35 (D)
27.5 CM pots were used, and 1000 gm of sterile soil amendment
containing 100 mM NaCl/Kg soil was sown in each pot with 10
seeds. The plant’s vegetative profile was monitored after 30 days
in the pot. The experiment was carried out in triplicate .
Plant analysis: Plant growth parameters such as fresh
weight, dry weight, root and shoot length, pigments, and proline
concentration were measured after 30 days of treatment. The DW
was calculated after 72 hours of drying in an 80 oC hot air oven
According to Bates et al. the proline content of the leaves was
determined . The concentration of samples was calculated
with reference to standard graph of proline prepared in the range
of 10-100 μg ml-1.
The experiment used a randomized block design. All
experiments were conducted in triplicate. Each treatment’s results
were assumed as an arithmetic mean with standard error. Data
were subjected to one-way analysis of variance (ANOVA) followed
the DMRT (Duncan’s Multiple Range Analyze) by using IBM SPSS
Statistics version 22. (SPSS Inc. Chicago, USA). The Levene test
was applied to evaluate variance homogeneity
A standard method was used to examine the physicochemical
parameters of soil samples. Table 1 summarizes the findings.
Higher pH and chloride levels suggested alkaline soil. The organic
and inorganic content of soil has a significant impact on the
structure of the microbial community .
Salt tolerance rhizobacteria screening: All of the
rhizobacterial isolates tested positive for NaCl tolerance in this
study. Many scientists’ observations are consistent with the
findings of our inquiry [17,26,27]. The rhizobacterial isolates
SMHMZ4, SMHMZ46, and SMHMP23 grew in NB medium at NaCl concentrations ranging from 0 to 22.5 percent (Figure 1).
The relative growth of these isolates in medium supplemented
with varying amounts of NaCl is depicted in Figure 1. Higher
salt concentrations of up to 22% were tolerated by SMHMZ4,
SMHMZ46, and SMHMP23. For all isolates, the minimum
inhibitory concentration was 23 percent NaCl.
The quantification of cytokinin secretion using a
spectrophotometer, as described by [17,24]. All three isolates
(SMHMZ4, SMHMZ46, and SMHMP23) produced cytokinin at
17.67, 23.23, and 18.26 μg/ml, respectively, under conventional
test conditions (Figure 2). Shah et al. 2020 revealed that their microorganisms RM3 produced 18.1 μg/ml cytokinin in M9
medium under salt stress. In a peptone-rich growth medium,
Karadeniz et al. found that the plant growth regulators auxin,
gibberellin, cytokinin, and abscisic acid were synthesized as
primary and secondary metabolites by their bacteria Proteus
mirabilis, P. vulgaris, Klebsiella pneumoniae, Bacillus megaterium,
B. cereus, and Escherichia coli .
TLC plates were spotted with an ethyl acetate fraction of
cytokinin and developed with an n-butanol:acetic acid:water
(12:3:5 v/v/v) mobile phase. A blue speck was seen under UV light
that recognized the cytokinin molecule (Figure 3). The extracted
cytokinin sample in the lane had the same Rf value as the cytokinin
reference standard (Kinetin). The findings are consistent with
prior findings from our lab and other authors [17,24,30].
The FTIR graph in Figure 4 indicates the presence of strong
peaks in the stretching f region. The presence of cytokinin, as well
as additional compounds with different functional groups created
by SMHMZ46 under salt stress and capable of supporting plants
in alleviating salt stress, was revealed by a full overlap of standard
and sample peak locations. In a similar investigation, Shah et al.
found that their strain RM3 produced cytokinin in the presence
of 670 mM NaCl . DS Hart and colleagues published an FTIR
analysis of cytokinin in aqueous solution to determine its stability
at different pH levels .
Salinity has a direct impact on the physiochemical and
biological properties of soil, which has a negative impact on
plant development and productivity. Because of osmotic stress,
specific ion toxicity, nutritional imbalances, and/or a combination
of these variables, salinity has a deleterious influence on plant
growth. Several research on the application of PGPRs to alleviate
salt stress in a range of crops have been reported [21,32,33]. Our findings showed that inoculating plants with NaCl-tolerant PGPR
improved plant growth under salt stress conditions. This showed
that rhizobacteria contact reduced NaCl stress. After being
successfully treated with a cytokinin-producing bacterial culture,
fresh and dry weight of shoots and roots increased, as did shoot and
root length (Table 2). The results of a 30-day development cycle in
a pot experiment with inoculated and untreated plant samples are
shown in Figures 5 and 6. Under salt stress, un-inoculated Glycine max L. shoot and root lengths dropped significantly (p<0.05),
whereas inoculations with SMHMZ4, SMHMZ46, and SMHMP23,
as well as co-inoculation (all three bacterial strains), significantly
enhanced their lengths.
Our results revealed that inoculated plants with NaCl-tolerant
PGPR promoted superior plant growth under salt stress conditions.
This demonstrated that rhizobacteria interaction alleviated NaCl
stress. Successfully treated with a cytokinin-producing bacterial
culture, there was an improvement in fresh and dry weight of
shoots as well as roots, as well as an increase in shoot and root
length (Table 2). Figures 5 and 6 illustrate the results of a 30-day
growth cycle in a pot experiment with inoculated and untreated
plant samples. Under salt stress, the shoot and root lengths of un-inoculated Glycine max L. decreased significantly (p<0.05),
whereas inoculations with SMHMZ4, SMHMZ46, and SMHMP23,
as well as co-inoculation (all three rhizobacterial strains),
significantly increased their lengths. The growth-promoting and
increased NaCl tolerance effects of the inoculated isolates could be
attributed to Cytokinin synthesis in a NaCl-stressed environment.
Sapre et al.  used ½ MS medium supplemented with 100
mM NaCl to study the effect of PGPR strain IG 3 (Klebsiella sp.)
on the detrimental effect of NaCl on oat seedlings . Root
length, shoot dry weight, and root dry weight were substantially
(p<0.05) higher in PGPR injected plants than in controls under
stress conditions. In comparison to the control, halotolerant RM3
exhibited a favorable effect on Trigonella foenum-graecum under
saline conditions, according to Shah et al. In Triticum aestivum L.,
the action of halotolerant consortia in boosting plant growth has
also been proven .
Durum Wheat (Triticum turgidum subsp. durum) has also
been shown to have PGPR activity from halotolerant bacteria .
It has been discovered that PGPR with ACC deaminase activity
can protect against a wide range of abiotic stressors, including
NaCl stress [36-38]. It is well understood that phytohormones,
notably IAA generated by rhizobacteria, are required for root
initiation and the elongation of root lengths of lateral roots and
adventitious roots, hence supporting the host plant in maximum
nutrient absorption [39,40]. Plants seeded with PGPR strain
IG 3 and treated under NaCl stress produced significantly more
biomass than negative control plants.
In reaction to environmental stress, plants have a defence
mechanism that increases proline content. Proline could be
beneficial in avoiding membrane damage . In our study,
rhizobacteria treatment on the plants significantly increased
free proline content (p<0.05), which may contribute to the
development of ecological adaptation in the Glycine max L. plant
under stress conditions (Figure 7). According to Trivedi et al. their
endophytes boost proline content in Glycine max L. under stress
circumstances as compared to a non-inoculated control .
The current study is an important step in determining beneficial
rhizobacteria and investigating their potential to improve plant
growth under abiotic stress conditions. Plant development and
growth are mostly dependent on rhizobacteria, which encourage
plant growth. This study focuses on the production of cytokinin
by isolated rhizobacteria, a phytohormone that promotes plant
survival under abiotic stress such as salt by assisting in plant
cell proliferation and differentiation. Isolation of such varied
rhizobacterial isolates in the current study might be a beneficial
step toward better agricultural yield and production under salt
stress. The future use of these isolates with increased cytokinin
production will maximum values the development of bio-based
products it can be used as bio-enhancers.
I would like to express my sincere gratitude to my guide
Prof. (Dr.) Meenu Saraf for support and guidance and DIST-FIST
sponsored Department of microbiology and biotechnology,
University school of sciences, Gujarat university, Gujarat, India for
providing required facility.
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