Induction Mutation of Plant

Growth Promoting Bacteria

by Cold Plasma Technology
Boonraksa Mangmee
Saisawhan Kamoi
Wasita Injai

Advisor: Dr. Kanta Sangwijit

Introduction

(A) Maize not inoculated with a Phosphorus Solubilizing Microorganism.

(B) Maize inoculated with a Phosphorus Solubilizing Microorganism
Ref : Elizabeth et al., 2017
OBJECTIVE
01

Screening of plant growth

promoting bacteria (PGPB)

02

Enhancing PGPB activity

by induce mutation using
cold plasma technology
 Methods

• Soil sampling
• Screening of Phosphate Solubilizing
Bacteria
• Bacterial Identification by 16s rRNA Gene
Sequence Analysis
• Induction Mutation of the Selected Bacteria
• Mutant screening
• Investigation of Plant Growth
 Soil sampling

Collected Dilution
Area around University of Phayao and
avocado farm

Methods
 Screening of Phosphate Solubilizing Bacteria

The bacteria was cultured on selected media (PVK agar plates)

Methods
 Bacterial Identification by 16s rRNA Sequencing

16s rRNA
Gene Cloning

DNA Gene
extraction Sequencing

Methods
 Induction
Mutation of the
Selected Bacteria

Methods
 Cold atmospheric pressure plasma jet (APPJ)

Ref : Nitipol et al., 2017

Schematic diagram of cold Bacterial samples were treated by

atmospheric pressure plasma jet. cold atmospheric pressure plasma jet.

Methods
 Bacterial treatment condition

• Gas : Ar

• Input voltage : 85 volt

• Gas flow rate of 2 L/min

• Exposure Time : 1 to 10
min

Methods
 Mutant
screening

Methods
 Investigation of plant growth

Water Control Bacterial treatment Mutant treatment

Inoculate seeds with Inoculate seeds with the Inoculate seeds with the
distilled water control bacteria mutant

Methods
 Height

Growth Root length

Parameters Size of leave

Flowering day

Productivity
Result
Isolation and Screening of Bacteria

AF28 AF03

AF15
UP01
Isolation and Screening of Bacteria

UP46

UP13

UP07
 Bacterial Identification by 16s rRNA Sequencing

isolates Phosphate pathogenesis

solubilization
AF03 + - Bacillus subtilis

AF15 + + Burkholderia ambifaria

AF28 + + Burkholderia ambifaria

UP01 + + Burkholderia contaminans

UP07 + - Bacillus subtilis

UP13 + - Bacillus subtilis

UP46 + + Staphylococuus hominis

Table 1. Identification of bacteria isolates for phosphate solubilization

 Bacterial Identification by 16s rRNA
Sequencing

AF03

UP07
UP13

Isolates AF03, UP07 and UP13 are non-pathogenic bacteria.

From 3 isolates, isolated UP13 was selected for further study.
 Bacterial Identification by 16s rRNA Sequencing

UP13 (Bacillus subtilis) was selected for induction mutation

 Screening of mutants

Bacteria were treated under Ar plasma for 1 minutes and screened

on PVK agar plates.
 Screening of mutants

Bacteria were treated under Ar plasma for 3 minutes and screened

on PVK agar plates.
 Screening of mutants

Bacteria were treated under Ar plasma for 5 minutes and screened

on PVK agar plates.
 Screening of mutants

Bacteria were treated under Ar plasma for 10 minutes and screened

on PVK agar plates.
 Sample 3 Days 5 Days 7 Days

PVK broth 5.86 5.96 5.91

PH measurement UP13 (control) 5.24 5.38 5.08

UP13-3M 15 5.28 5.02 4.97

From136 isolates which
were treated under Ar UP13-3M 16 5.11 4.93 4.84

plasma, 8 isolates were UP13-3M 18 5.26 5.17 5.05

exhibited lower pH
UP13-10M 1 5.07 4.85 4.66
compared to its control
UP13-10M 2 5.09 5.15 4.91
in PVK broth.
UP13-10M 3 5.22 4.90 5.00

UP13-10M 4 5.27 4.99 4.94

UP13-10M 14 5.21 5.13 5.02

Table 2. Quantitative examination of pH measurement and

phosphate solubilization by different isolates.
 Sample 3 Days 5 Days 7 Days

PVK broth 5.86 5.96 5.91

PH measurement UP13 (control) 5.24 5.38 5.08

UP13-3M 15 5.28 5.02 4.97

UP13-3M 16 5.11 4.93 4.84

UP13-3M 18 5.26 5.17 5.05

UP13-10M 1 5.07 4.85 4.66

UP13-10M 2 5.09 5.15 4.91

UP13-10M 3 5.22 4.90 5.00

From table 2. Isolated

UP13-10M 4 5.27 4.99 4.94
UP13-10M 4
was selected for further
UP13-10M 14 5.21 5.13 5.02
study of plant growth

Table 2. Quantitative examination of pH measurement and

phosphate solubilization by different isolates.
 Investigation of plant growth
Sample Width of leaves Height (cm) Root length (cm) Number of
(cm) sprout

H2 O 0.496 ±0.134 5.783 ±1.066 0.900 ±0.141 18

Control 0.439 ±0.108 5.785 ±0.971 2.550 ±0.353 27

Mutant 0.414 ±0.126 5.971 ±0.860 3.900 ±2.546 21

Table 3. Investigation of plant growth by measure after 14 days of planting

Thank you
Back up
 DNA fingerprinting by HAT-RAPD

Random Amplification of Polymorphic DNA

(RAPD)

Methods
 Phosphate
Sample 3 Days 5 Days 7 Days Solubilization
(mg/L)

PVK broth 5.86 5.96 5.91 35.28

UP13 (control) 5.24 5.38 5.08 83.36

UP13-3M 15 5.28 5.02 4.97 111.45

UP13-3M 16 5.11 4.93 4.84 125.28

UP13-3M 18 5.26 5.17 5.05 107.40

UP13-10M 1 5.07 4.85 4.66 144.21

UP13-10M 2 5.09 5.15 4.91 117.19

UP13-10M 3 5.22 4.90 5.00 172.51

UP13-10M 4 5.27 4.99 4.94 241.23

UP13-10M 14 5.21 5.13 5.02 123.79

Table 2. Quantitative examination of pH measurement and phosphate solubilization by different isolates.

 Investigation of plant growth

Number of
Width of leaves seedling
Sample Height (cm) Root length (cm)
(cm) (percentage of
germination)

Water 0.496 ±0.134 5.783 ±1.066 0.900 ±0.141 18 (60%)

Control 0.439 ±0.108 5.785 ±0.971 2.550 ±0.353 27 (90%)

Mutant 0.414 ±0.126 5.971 ±0.860 3.900 ±2.546 21 (70%)

Table 3. Investigation of plant growth after 14 days of planting