FNCA Guidelines― On Chitosan PGP Application

for Rice, Chilli and Other Crops ―Edited by:
Masao Tamada1), Mitsumasa Taguchi2) ,Kamaruddin Bin Hashim3), Quoc Hien Nguyen4)
1) Takasaki Advanced Radiation Research Institute, Japan Atomic Energy Agency2) Quantum Beam Science Center, Japan Atomic Energy Agency
3) Malaysian Nuclear Agency
4) Vietnam Atomic Energy Institute CONTENTS CONTRIBUTERS .......................................................................................i
PREFACE ................................................................................................ iii
Part 1. Plant Growth Promoter (PGP) ..................................................... 1
1.1 What is PGP? ........................................................................................ 1
1.2 Effect of PGP on Plants .......................................................................... 1
1.3 PGP Preparation by Radiation Processing ............................................ 8
1.3.1 Chitosan.......................................................................................... 8
1.3.2 Degradation by Radiation Processing ......................................... 10
1.4 Advantages of PGP Prepared by Radiation Processing ...................... 12
1.5 References ............................................................................................. 12Part 2. PGP (Degraded chitosan) ............................................................. 14
2.1 Preparation (Citing Preparation Guideline, Additive, etc.) ................ 14
2.2 Preservation .......................................................................................... 18
2.3 References ............................................................................................. 19
Part 3. Rice .............................................................................................. 22
3.1 Cultivations of Rice .............................................................................. 22
3.2 Recommended Application ................................................................... 23
3.2.1 Application Protocol for MR 219 and Mutant MR 219-9 and219-4 ............................................................................................... 24
3.3 Effect of Seed Treatment ...................................................................... 25
3.4 Effect of Concentration ....................................................................... 26
3.5 Effect of Molecular Weight ................................................................... 28
3.6 Effect of Foliar Spray Frequency ....................................................... 28
3.7 References ............................................................................................. 28
Part 4. Chilli ............................................................................................ 29
4.1 Cultivations of Chilli ............................................................................ 29
4.2 Chilli TM 999 variety ........................................................................... 32
4.2.1 Effect of Concentration ................................................................ 35
4.2.2 Effect of Molecular Weight ............................................................ 38
4.2.3 Foliar Spray ................................................................................. 42
4.3 Application Protocol for Kulai Hybrid F1 S469 ................................... 42
4.3.1 Effect of Concentration ................................................................ 43
4.3.2 Effect of Molecular Weight ............................................................ 47
4.4 References ............................................................................................. 48
Part 5. Q&A ............................................................................................. 49
5.1 General .................................................................................................. 49
Final Remarks ......................................................................................... 50
APPENDIX. Data sheet obtained in each country................................... 51

CONTRIBUTORSBANGLADESH
Dr Salma SULTANA
Bangladesh Atomic Energy Commission (BAEC)CHINADr Guozhong WU
Shanghai Institute of Applied Physics (SINAP)
INDONESIADr Darmawan DARWIS
Ms Tita PUSPITASARI
National Nuclear Energy Agency (BATAN)JAPANDr Masao TAMADADr Mitsumasa TAGUCHIDr Naotsugu NAGASAWA
Japan Atomic Energy Agency (JAEA)
MALAYSIADr Kamaruddin Bin HASHIM
Dr Marina Binti TALlB
Ms Maznah Binti MAHMUD
Malaysian Nuclear Agency (Nuclear Malaysia)
PHILIPPINES
Ms Charito Tranquilan ARANILLA
Philippine Nuclear Research Institute (PNRI)THAILANDDr Phiriyatorn SUWANMALA
Thailand Institute of Nuclear Technology (TINT)
VIETNAMDr Quoc Hien NGUYENi

Vietnam Atomic Energy Institute (VINATOM)
Secretariat:
Nuclear Safety Research Association (NSRA)
5-18-7 Shin-bashi, Minato-ku, Tokyo 105-0004 JAPANii

PREFACE
Plant growth promoter (PGP) is a substance which enhances thegrowth of plants. If PGP is applied to crops, the increase of crop yield causes
economical impact in agricultural production. Such PGP can be produced byradiation processing of polysaccharides such as chitosan and carrageenan
which are originated from crustacean shelf and seaweed, respectively. Hence,electron accelerator application project in Forum for Nuclear Cooperation in
Asia (FNCA) has investigated the PGP synthesis as an application ofradiation degradation of natural polymers and revealed the effect of PGP
originated from chitosan and carrageenan on various crops since 2006. These
positive data of yield increase in size and weight of edible crop part have beenshared among the 10 participating countries of Bangladesh, China, Indonesia,
Japan, Kazakhstan, Malaysia, Mongolia, Philippines, Thailand, and Vietnam
in FNCA. Especially, it was found that PGP originated from chitosan induced
elicitor activity against fungal disease. Chemical pesticide may be replacedby environmentally-friendly PGP. To promote the technology transfer of PGP,
the accumulated data of crop yield should be opened to end-users like farmers
in each country. These years our group obtained the PGP data for rice andchilli which are beneficial crops in Asia countries.
This guideline is composed of 5 chapters and appendix; Chapter 1 isintroduction of PGP to understand what PGP is, effect of PGP on plants,
preparation by radiation processing, and advantages of PGP prepared byradiation processing. Chapter 2 dealt with preparation, physical and chemical
properties, dilution before usage, preservation. End-users can get brief
information of production process and handlings of PGP. Chapter 3 and 4 areprotocols of timing in plant growth stage, PGP concentration, foliar sprayfrequency, etc. for rice and chilli, respectively. Chapter 5 is Q&A for generalmatters and cases of rice, and chilli. PGP effects on various crops exclusive
rice and chilli are listed as appendix.
This guideline is expected to be benefit for technology transfer of
PGP prepared by radiation processing to end-users as well as sustainableagriculture with economic growth in the Asia region.
Masao TAMADA
Project Leader of Japan

iii

iv

1. Plant growth promoter (PGP)
1.1 What is PGP?
PGP is classified as natural growth stimulant which is non-toxic andsoluble in water that enhances the growth of plants and also has elicitor
effect against plant disease. It is improved the overall health growth anddevelopment of plants that determine the crop quality and productivity. As
a result, increase in yield which benefits farmers and agriculture production
including floriculture and horticulture crops.
PGP can be either synthetically produced or obtained from the biologicalderivatives like seaweed for example carrageenan and alginate, andcrustacean shelf like chitin/chitosan. PGP from biological derivative is more
effective and safe to be used by end user and no hazardous to consumer. It
could be easily applied through foliar spray on field crops, fruits, vegetables
and flowers to maximize the productivity. PGP either stimulates productionof plant hormones in plant or itself content of plant hormones that accelerate
the plant growth.
Plant hormones is an organic substance, naturally produces in plant
which affects plant growth. There are several plant hormones such as auxins,
cytokinins and gibberellins that act as regulator for plant growth. Chitosan
contains plant hormones such as indol acetic acid (auxin), cytokinin (kinetin
and zeatin) and gibberelic acid which promote plant growth.
1.2 Effect of PGP on Plants
PGP from crustacean shelf or seaweed has an effect on overall
growth of plants. It has capability to enhance or accelerate the rate of growthand maturation of crops or plants without interfering natural physiologicalactions. For example, the existence of auxin hormone in PGP will stimulate
cell elongation in stem through lengthwise growth of plants, and root
initiation on stem cuttings and lateral root development in tissue culture. It
also helps in fruit development by delays fruit ripening, inhibit leaf fall andlateral branching. In the case of cytokinin plant hormones exist in PGP, it
will stabilize protein and chlorophyll content in the leaf which produce green
leaves, prevent yellowing of leaves. It promotes the conversion of etioplasts
into chloroplasts via stimulation of chlorophyll synthesis. Cytokinin issynthesized in roots and move upward in the xylem to leaves and fruits. It
stimulates morphogenesis development of shoot initiation/bud formation in１

tissue culture. In conjunction with auxin and cytokinin will promote cell
division in plant. Gibberellin is another plant hormone which has been
identified in PGP to promote plant growth. It mostly found in seeds, young
leaves and in roots. It move upward from the root and promote growth ofmain stems of plant. The PGP will accelerate the growth of plant throughcell propagation on stems and roots, increase chlorophyll in green leaves andenhances maturating with early flowering, which increase fruit yield ofplant or crops.

ChilliControl

Oligochitosan

Figure 1.1. Effect of PGP on carrot and chilli.
One of the most important defenses of plant tissues against disease
infection is the ability to produce phytoalexin which is antibiotics
synthesized in response to pathogens or elicitors. Phytoalexins can inhibit
the growth and development of phytopathogens at an effective concentration.
Momilactones which are phytoalexins in rice leaves were firstly reported by
Cartwright et al. [1]. Agrawal et al. also indicated that momilactone A
induced in rice leaves increased greater than 10,000 ng/50 mg after 72 htreatment of chitosan (Fig. 1.2) [2].

2Figure 1.2. Phytoalexin momilactone A content in rice leaves treated withchitosanRodiguez et al. studied to treat seeds of rice (Oryza sativa L.) withchitosan and hydrolyzed chitosan for induction of defense response against
blast disease caused by Pyricularia grisea [3]. Results revealed thatseedlings obtained from seeds treated with chitosan and hydrolyzed
chitosan (oligochitosan) showed stronger resistance to blast disease
compared with non-treated plants (positive control). More details on
elicitation effect of oligochitosan for plants can be referred to the paper
reviewed by Ying, Zhao and Du (see Table 1.1) [4].
Table 1.1. The effect of oligochitosan on plant diseases control (survey inChina)
Plants

Diseases

Controlrates (%)

TobaccoTobacco mosaic virus, Potato virus

76.49
87.5

Panax
notoginseng

Virus diseaseh

Tulip

Peronospora

84.24
88.6

–

Piper nigrum L.

Mosaic virus

73.54
81.3

–

–

>90

3

Tomato

Solani, infestans, virus disease, bacterial 84.47
wilt
88.24

–

Cucumber

Peronospora

78.96
82.65

–

PepperVirus diseases, blight, anthracnose,
78.58 – 90
Pepper phytophthora blight

Egg plant

Virus diseases

93.18
100

Chinese cabbage

Erwinia carotovora subsp

78.62 – 85–

Asparagusplettuce Peronospora

45.80
62.3

Wax gourd

Blight84.81 – 95

Cauliﬂower

Black rot

63.60
64.2

–

Geen cucumber

Blight

45.50
57.6

–

CowpeaVirus diseases

31.13
58.8

–

PapayaMosaic virus

70
96

–

Watermelon

Black rot, Didym ella bryoniae, light, 81.71
virus diseases
85.40

–

Muskmelon

Powdery mildew

71.34
86.26

–

BananaBunchy top

83.70
94.6

–

Apple

Mosaic virus, Venturia inaequalis

76.68
93.85

–

SoybeanVirus diseases

75.10
100

–

Cotton plant

Cotton yellow wilt

85.50
87.2

–

Maize

Sphacelotheca Reiliana, corn northern 23.90
leaf blight, corn southern leaf blight
45.35

–

Rice

Rice blast

71.41
92.0

–

PeanutVirus diseases

23.90
26.5

–

4

–

By foliar spraying of oligochitosan with concentration of 20-50 ppm on
peanut leaves, the productivity increased to 20-40% compared with the nontreated control (Table 1.2 and Fig. 1.3) [5]. The result indicated thatoligochitosan exhibits growth-promotion effect on peanut. Similarly,
soybean seeds coated with oligochitosan (0.4%), the productivity also
increased remarkably at about 30% compared with the control [6]. In
addition, the rate of rust disease on soybean was lower than that of the nontreated control.
Table 1.2. Effect of oligochitosan on productivity of peanut
Oligochitosan,ppm

0 (ctrl)

20

30

40

50

Productivity, kg
For control, %

3.05
100

3.64
119.3

3.74
122.6

4.29
140.7

3.81
124.8

Figure 1.3. Peanut treated with oligochitosan: 0, 20, 30, 40 and 50 ppm
Hien et al. studied the elicitation and growth promotion effect ofoligochiotsan for sugarcane and rice. Results showed that oligochitosan withmoleculer weight (Mw) 6000-10,000 exhibited the most effective elicitationand growth promotion for plant [7]. The optimum oligochitosan
concentrations by spraying were 30 and 15 ppm for sugarcane and rice,
respectively. The disease index of Ustilgo scitaminea and Collectotrichum
falcatum on sugarcane was reduced respectively to 44.5 and 72.3% compared
to control (100%). The productivity of sugarcane increased about 13% (71.4
tons/ha compared to 63.2 tons/ha for control). The disease index of5

Pyricularia grisea on rice was reduced to 53.0% for leaf and 34.1% for neckof bloom (panicle) compared to control (100%). The productivity of riceincreased for 11-26% (6.0-6.8 tons/ha compared to 5.4 tons/ha for control).
The obtained results indicated that oligochitosan is promising to use as a
biotic elicitor for plant particularly for sugarcane and rice(Table 1.3).
Table 1.3. The effect of oligochitosan on disease index and productivity for
rice
Conc.ofLeaf DI
Panicle DI
Productivity
oligochitosan,
ppm
Index, % for
Ratio, % forTons/ha Increase,Ctrl, %
Ctrl, %%
15
30
60
Ctrl (H2O)

14.4 b
15.3 b
16.0 b
22.9 a

62.88
66.81
69.87
100

10.0 c12.0 c15.3 c29.3 a

34.1
40.9
52.2
100

6.8 bc
6.0 c
6.3 bc
5.4 a

25.93
11.11
16.67
-

More recently, Ma et al. studied to treat wheat seeds with oligochitosan
by soaking seeds in 0.0625% oligochitosan solution for 5 h [8]. The results inTable 1.4 showed that chlorophyll content increased by treating seeds witholigochitosan. It suggested that seeds treatment with oligochitosan had abeneficial effect on photosynthesis. They also confirmed the positive effect ofoligochitosan in improving the plant growth and plant’s capacity of tolerance
to salt stress [8].
Table 1.4. Effect of oligochitosan on growth parameters and chlorophyll
content of wheat seedlings
Control
NaCl
Oligochitosan Oligomer(0.0625%)(150(0.0625%)
NaCl (150 mM)
mM)
Shoot
length, cm

10.62 0.54 a,b

7.81 
0.47 c11.45  0.51 a

9.46  0.61 b

Rootlength, cm

14.98 0.62 b

10.3 
0.53 c17.94  0.49 a

14.71  0.37 b

Dry weight(g/plant)

0.35 
0.02 b

0.28 
0.01 c0.42  0.02 a

0.34  0.02 b

Chl-a, mg/g
FW0.87 
0.07 b0.69 
0.02 c0.97  0.02 a

0.91  0.01 b

6

Figure 1.4. Field test of elicitation and growth promotion effect ofoligochitosan produced by -irradiation method on rice in Vietnam
In addition, El-Sawy et al. studied on effect of radiation degradedchitosan on growth of faba bean plant [9]. They concluded that the degradedchitosan has positive effects not only on plant growth but also on the
productivity of faba bean. Furthermore, a large field test (32 ha) ofoligochitosan on rice has been carried out in Malaysia [10]. Oligochitosan
used for this field test was produced by continuous flow 60Co -ray irradiation
system which can be suitably applied on large scale production.
Table 1.5. Results of field test of oligochitosan on rice in Malaysia
Treatment

Average yield,kg

For contl. 1

For contl. 1

Contl. 1 (water)

143

-

-

Contl.
2(fungicide)
Oligochitosan 40
ppm

163

13.9%

-

163

13.9%

0.0%

7

Seed soaking in
100
ppm
oligochitosan +spraying 40 ppm

181

26.6%

11.0%

The results in Table 1.5 indicated that oligochitosan could be used toreplace fungicide, and the combination of seed soaking and spraying
oligochitosan on rice plant showed the better effect on increase of the yield.
(Fig. 1.5)
Contl.

OligoCTS

Contl.

OligoCTS

Figure 1.5. Field test of oligochitosan on rice
Furthermore, it was also reported that using 40 ppm oligochitosan intissue culture media promotes significant growth of pineapple (Fig. 1.6). The
effect of oligochitosan on reduction of disease and insect infection, andgrowth promotion on agarwood has been carrying out.

Figure 1.6. Effect of oligochitosan on tissue culture of pineapple

1.3 PGP preparation by Radiation Processing
8

1.3.1 Chitosan
Chitosan is linear aminopolysaccharide derived from chemical alkali
deacetylation process of chitin. Chitin is natural polymer polysaccharide
abundantly found in the outer skeleton of insects, crabs andshrimps.(Fig.1.7) Limitation solubility of chitin in the common solvent
hindered it commercial application. Most of chitin/chitosan is produced fromthe food industrial waste of shrimp and crabs. Chitosan has shown to havewide potential applications in treatment of industrial waste water, healthcare, food industry and agriculture.

Figure 1.7. Molecular structures of chitin and chitosan.

91.3.2 Degradation by Radiation Processing
Radiation processing technology by -rays has been identified asversatile tool in controlling the process of degradation of chitosan andeconomical way in term mass production, in the preparations of PGP (Fig.
1.8). The degradation of chitosan can be carried out in the form of powder orin solution form by dissolving 3% chitosan in 2% acetic or lactic acid.
Irradiation of chitosan in solid state needs very high dose and followed byfractionation process to produce oligochitosan. However, irradiation of
chitosan in solution state will only need low dose less than 20 kGy to form
oligochitosan compare to powder form. Irradiation of chitosan solution by rays can be carried out either in batch type using drum or continuous flow
system using column.

Figure 1.8. Mixing tank for preparation of chitosan (left), and irradiation
vessels (center and right).

Addition of hydrogen peroxide about 0.1% to 0.3% into the chitosan
solution prior to irradiation reduced further radiation dose needed forformation of oligochitosan with average molecular weight 10 kDa.

10

Figure 1.9. Oligochitosan solution
Chitosan like other natural polymers such as cellulose, carrageenan
and alginates predominantly undergo scission or degradation when exposed
to ionizing radiation to form low molecular weight chitosan.(Fig.1.9)
Ionization radiation such as -rays and electron beam, can be used toperform the degradation process.
High molecular weight (HMw) chitosan has limitation to be used in
the agriculture application as PGP due to it less solubility in weak acid.
However, oligochitosan with molecular weight about 10 kDa is soluble inwater and has been identified as PGP and give elicitor effect to resist disease
infection on plant. Further degradation of HMw chitosan into oligochitosan
needed much higher irradiation dose which is not economical for productionof oligochitosan.
Irradiation of HMw chitosan in solution form of acetic or lactic acidhas been identified to improve the degradation process to oligochitosan at
low irradiation dose. Study has shown that by adding 0.1 to 0.3% hydrogen
peroxide in the HMw chitosan solution will further reduce radiation doseneeded to produce oligochitosan.(Fig.1.10)

11

Figure 1.10. Synergistic effect of hydrogen peroxide on degradation of 3%
chitosan solution by -ray irradiation
1.4 Advantages of PGP prepared by Radiation Process
Degradation process of chitosan to produce PGP can also be carried out
by chemical or enzymatic process. However, radiation processing technology
has more advantages over chemical and enzymatic processes and preferable
process for degradation of chitosan due to several reasons, such asi) process of degradation can be performed at room temperature,ii) easy to control the degree of degradation by varying irradiation dose,iii) practical for large production and cost effective.

1.5 References[1] Cartwright, D., Langcake, P., Pryce, R.J., Leworthy, D.P., Ride, J.P., 1977.
Chemical activation of host mechanisms as a basis for crop protection.
Nature, 27, 511-513.
[2] Agrawal, G.K., Rakwal, R., Tamogami, S., Yonekura, M., Kubo, A., Saji,H., 2002. Chitosan activates defense/stress response(s) in the leaves of
Oryza sativa seedlings, Plant Physiol. Biochem., 40, 1061-1069.
12

[3] Rodriguez, A.T., Ramirez, M.A., Cardenas, R.M., Hernandez, A.N.,
Velazquez, M.G., Bautista, S., 2007. Induction of defense response of
Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating
seeds with chitosan and hydrolyzed chitosan. Pest. Biochem. Physiol.,89, 206-215.
[4] Yin, H., Zhao, X., Du, Y., 2010. Oligochitosan: A plant diseases vaccine-A
review. Carbohydr. Polym. 82, 1-8.
[5] Dzung, N.A., Thang, N.T., 2005. Study of oligoglucosamine effect on thegrowth and development of peanut plants, J. Biology (in Vietnamesewith English abstract), 27, 50-54.
[6] Dzung, N.A., Thang, N.T., 2002. Effect of oligoglucosamine prepared byenzyme degradation on the growth of soybean, In K. Suchiva, S.
Chandrkrachang, P. Methacanon, & M.G. Peter V (Eds.), Advance inChitin Science, 463-467.
[7] Hien, N.Q., The, D.T., Phu, D.V., Duy, N.N., 2010. Preparation of biotic
elicitor for rice and sugarcane by gamma irradiation, progress report of
IAEA RC No. 14773/R0.
[8] Ma, L., Li, Y., Yu, C., Yang, Y., Li, X., Li, N., Chen, Q., Bu, N., 2010.
Alleviation of exogenous oligochitosan on wheat seedlings growth undersalt stress, Protoplasma. DOI: 10.1007/s00709-011-0290-05.
[9] El-Sawy, N.M., Abd El-Rehim, H.A., Elbarbary, A.M., Hegazy, E.A., 2010.
Radiation-induced degradation of chitosan for possible use as a growthpromoter in agricultural purposes. Carbohydr. Polym., 79, 555-562.
[10] Hashim, K., Khairul, Z., 2012. Field test of oligochitosan on rice in
Malaysia, FNCA workshop on radiation processing of natural polymers,
30. Jan – 2 Feb., Manila, Philippines.

13Part 2. PGP (Degraded Chitosan)
2.1 Preparation (Citing Preparation Guideline, Additive, etc.)
Chitosan is a linear aminopolysaccharide (polyglucosamine) derived
from chitin, a naturally abundant mucopolysaccharide, poly β-(14)-linked
N-acetyl-D-glucosamine by alkali deacetylation.(Fig. 2.1)

O

H

H
CH2OH
H H
H H
O
OH
O
NH
CH2OH
H
H OC
H
CH3
NH2

H

OH

O

~30%

~70%

Figure 2.1. The molecular structure of chitosan with degree ofdeacetylation of ~70%

Rawmaterials

ChitinChitosanFigure 2.2. The major sources from shrimp, crab shells and squid pens forchitin and chitosan production (IAEA/RCA RTC, 19-23 Oct., 2009,
Bangkok, Thailand)
Chitosan has a wide range of applications, such as in waste water
treatment, in medicine and cosmetics, in food and functional food and in
agriculture [1-4]. The production of chitin/chitosan is currently based on
crab and shrimp shells discarded as a food industry waste. The global
annual estimate of shellfish processing discards is more than one million
metric tons [1]. Thus, disposal of shellfish wastes has been a challenge formost of the shellfish-processing countries. Therefore, production of value14

added products such as chitin/chitosan, oligomers and their derivatives forutilization in different fields is of utmost interest.(Fig.2.2)
Study on preparation of low molecular weight (Mw) chitosan andoligochitosan by irradiation (-rays, electron beam) has been carrying out inmany radiation processing research centers due to their beneficial
properties and potential applications in different fields. The weight-average
Mw from 10,000 to about 100,000 is considered for low Mw chitosan, while
Mw of oligochitosan is generally less than 10,000. Chitosan prepared fromchitin by the deacetylation process has generally high Mw which in manycases limits its applications.
The low Mw chitosan and its oligomer have some special biologicalproperties which are different from that of the ordinary high Mw chitosansuch as antioxidant property [5,6], antimicrobial property [7-10], antitumor
activity [11], immunity stimulation for animal [12,13] and for plants [14-16].
Comprehensive information of biological activities of chitosan andoligochitosan can be referred to the articles reviewed recently by Kim &Rajapakse [17] and Xia et al. [18]. A variety of techniques including chemicaland enzymatic hydrolysis, radiation degradation processes can be used toprepare low Mw chitosan and its oligomer as described by Makuuchi [19].
However, radiation (-rays, electron beam) is a useful tool for degradation ofpolymer from the viewpoint of environmentally friendly processing
technology [20].
8

MW × 105

6

4

2

0
0

50

100

150

200

Dose, kGy

Figure 2.3. Changes in molecular weight of chitosan irradiated by -rays inpowder and flake form in air with doses
15

Accordingly, the low Mw chitosan can be conveniently prepared byirradiation of high Mw chitosan in powder or flake form [14, 21-23](Fig.2.3).
Ulanski & Rosiak reported Gd-values (scission/dose of 100 eV)at 0.9, 1.1 and1.3 scissions/100 eV for chitosan powder irradiated in vacuum, in air and inoxygen, respectively [21]. However, Czechowska-Biskup reported the higher
Gd value for chitosan irradiated in air (Gd = 0.6 mol/J eq. to 5.79
scission/100 eV) [24]. The reason for the difference in Gd values may be due
to many factors that needs further study to identify.
1.2

MW × 105

1.0
0.8

5%

0.6

1%

3%

0.4
0.2
0
0

8

16

24

32

40

48

Dose, kGy

Figure 2.4. Changes in molecular weight of chitosan in acid solution
irradiated with -rays
Oligochitosan has been produced in most cases by irradiation of
chitosan in solution [14, 25, 26]. Gd values were found out to be of 0.058,
0.08 and 0.10 mol/J for 1%, 3% and 5% chitosan solution, respectively,
based on the results of molecular weight presented in Fig. 2.4. Wasikiewicz
reported higher Gd value (0.353 mol/J) for 1% chitosan solution [26]. The
reason may be the difference of deacetylation degree of chitosan used forstudy, because chitin is more stable to degradation than chitosan.
The cost of enzymatic hydrolysis process for production ofoligochitosan is generally higher than that of oxidative degradation process[19]. However, oxidative degradation has disadvantages mainly due tochemical structure changes caused by the formation of carboxyl groups,
deamination and even breakage of glucoside ring [27]. In addition, chemical
16

process has some drawbacks due to low production yields and higher
pollution risk to environment [17]. In general, radiation degradation toprepare oligochitosan needs high dose so that it is not convenient for large
scale production.
Time (hour)

Mw  103

100

0

6

12

18

24
1% H2O2
-ray
-ray & 1% H2O2

80
60
40
20
0

0

8

16

32

40

Dose (kGy)

Figure 2.5. Changes in molecular weight of  chitosan treated with H2O2, rays and H2O2/- rays versus treatment time and dose (dose rate: 1.33
kGy/h)
Thus, in order to degrade chitosan effectively, synergistic degradationby combination of hydrogen peroxide (H2O2) with -rays [28-30], ultraviolet
light [31] and microwave [32] has been studied. According to the results
reported by Kang et al. [28] and El-Sawy et al. [29], chitosan in thesuspension or paste form mixed with high concentration of H2O2 (10-30%)
was degraded by -ray irradiation to prepare oligochitosan in high dose
range from 20 to more than 100 kGy. Furthermore, according to the resultsof the synergic effect of -rays and H2O2 for degradation of β-chitosan insolution reported by Hien et al. [30], oligochitosan with different Mw lessthan 10,000 can be produced in low dose range up to 16 kGy with theaddition of a small amount of 1% H2O2 into 5%  chitosan solution so that it
contributes to reduce the cost of production (Fig. 2.5).
However, according to the experiences in setting up large scale ofoligochitosan manufacture by -ray irradiating chitosan solution (200litters/batch in Vietnam, 500 litters/batch in Indonesia and 1,000 litters/one
run in Malaysia), it was recognized that the highly viscous chitosan solution(>3%) is not suitable for processing. Therefore, study to prepare17oligochitosan with different Mw less than 10,000 by -ray irradiation of 3%
chitosan solution containing H2O2 with various concentration (0.25 - 1%) has
been carried out [33]. The influence of varying dose on the changes ofmolecular weight and radiation degradation yield of chitosan wasinvestigated. The synergistic degradation effect induced by -rays and H2O2
was also calculated based on the percentage of the reduction (100 
Mw/Mw0) of chitosan molecular weight [30].

Proposed Procedure for Production of Oligochitosan









Chitosan (powder and/or flake form) with DD 70-90% and Mw 1 – 2 
105 g/mol.
Dissolving chitosan with concentration of 30 g/L in 1.5% lactic acid.
Keep standing overnight and then filtering through stainless steel net(100 mesh).
Adding H2O2 into chitosan solution with final concentration of about
0.5%.
Irradiation of chitosan/H2O2 solution at average dose of about 10-15
kGy.
Neutralization of irradiated chitosan solution by NaOH 2 M to pH 5.05.5.
Adding preservative (0.2% sodium benzoate).
Packaging final product: biotic elicitor for plants.

Characteristics of the biotic oligochitosan plant growth promoter andelicitor:
Oligochitosan concentration: 30 g/L (30,000 ppm)
Molecular weight (Mw): 5 – 10  103 g/mol.
pH ~ 5.0-5.5
Appearance: yellowish liquid
Expire date: 2 years
2.2 Preservation
In order to maintain the qualitative stability and for long timestorage in ambient condition of oligochitosan solution, appropriate
preservatives should be used mainly from the infection of the certain fungus
18species. Typical preservative agents are suggested to add to oligochitosan
solution: sodium benzoate (0.2% w/v), potassium sorbate (0.2% w/v) oralternatively ethyl alcohol (40% v/v).
2.3 References[1] Knorr, D., 1991. Recovery and utilization of chitin and chitosanin food processing waste management. Food Technology, 114-122.
[2] Hirano, S., 1992. Chitin biotechnology applications. Biotechnology
Annual Review 2, 237-258.
[3] Shahidi, F. et al., 1999. Food applications of chitin and chitosan. Food
Science & Technology 10, 37-51.
[4] Kumar M.N.V.R., 2000. A review on chitin and chitosan applications.
Reactive & Functional Polymers 46, 1-27.
[5] Tomida, H., Fujii, T., Furutani, N., Michihara, A., Yasufuku, T., Akasaki,K., et al., 2009. Antioxidant properties of some different molecularweight chitosans. Carbohydr. Res. 344, 1690-1696.
[6] Feng, T., Du, Y., Li, J., Hu, Y., Kennedy, F.J., 2008. Enhancement ofantioxidant activity of chitosan by irradiation. Carbohydr. Polym. 73,
126-132.
[7] Liu, N., Chen, X.G., Park, H.J., Liu, C.G., Liu, C.S., Meng, X.H., Yu, L.J.,
2006. Effect of MW and concentration of chitosan on antibacterial activityof Escherichia coli. Carbohydr. Polym. 64, 60-65.
[8] Zheng, L.Y., Zhu, J.F., 2003. Study on antimicrobial activity of chitosan
with different molecular weights. Carbohydr. Polym. 54, 527-530.
[9] Jeon, Y., Park, P., Kim, S., 2001. Antimicrobial effect ofchitooligosaccharides produced by bioreactor. Carbohydr. Polym. 44, 7176.
[10] Matsuhashi, S., and Kume, T., “Enhancement of antimicrobial activityof chitosan by irradiation” J. Sci. Food Agric. 73 (1997) 237-241.
[11] Qin, C.Q., Du, Y.M., Xiao, L., Li, Z., and Gao, X.H., 2002. Enzymic
preparation of water-soluble chitosan and their antitumor activity. Int.
J. Biol. Macromol. 31, 111-117.
[12] Luo, L., Cai, X., He, C., Xue, M., Wu, X., Cao, H., 2009. Immune response,stress resistance and bacterial challenge in juvenile rainbow trouts
Oncorhynchus mykiss fed diets containing chitosan-oligosaccharides.
Curr. Zool. 56, 416-422.
19

[13] Huang, R.L., Deng, Z.Y., Yang, C.B., Yin, Y.L., Xie, M.Y., Wu, G.Y., et al.,2007. Dietary oligochitosan supplementation enhances immune statusof broiler. J. Sci. Food Agric. 87, 153-159.
[14] Hien, N.Q., 2004. Radiation degradation of chitosan and some biologicaleffects. Radiation Processing of Polysaccharides, IAEA-TECDOC-1422,
IAEA, Vienna, Austria, 125-136.
[15] Rodriguez, A.T., Ramirez, M.A., Cardenas, R.M., Hernandez, A.N.,
Velazquez, M.G., Bautista, S., 2007. Induction of defense response of
Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating
seeds with chitosan and hydrolyzed chitosan. Pest. Biochem. Physiol. 89,
206-215.
[16] Yin, H., Zhao, X., Du, Y., 2010. Oligochitosan: A plant diseases vaccineA review. Carbohydr. Polym. 82, 1-8.
[17] Kim, S.K., Rajapakse, N., 2005. Enzymatic production and biologicalactivities of chitosan oligosaccharides (COS): A review. Carbohydr.
Polym. 62, 357-368.
[18] Xia, W., Liu, P., Zhang, J., Chen, J., 2011. Biological activities of chitosan
and chitooligosaccharides. Food Hydrocolloids 25, 170-179.
[19] Makuuchi, K., 2010. Critical review of radiation processing of hydrogeland polysaccharide. Radiat. Phys. Chem. 79, 267-271.
[20] Haji-Saeid, M., Safrany, A., Sampa, M.H.O., Ramamoorthy, N., 2010.
Radiation processing of natural polymers: The IAEA contribution.
Radiat. Phys. Chem. 79, 255-260.
[21] Ulanski, P., Rosiak, J.M., 1992. Preliminary study on radiation-inducedchanges in chitosan. Radiat. Phys. Chem. 39, 53-57.
[22] Chmielewski, A.G., Migdal, W., Swietoslawski, J., Swietoslawski, J.,
Jakubaszek, U., Tarnowski, T., 2007. Chemical-radiation degradation ofnatural oligoamino-polysaccharides for agricultural application. Radiat.
Phys. Chem. 76, 1840-1842.
[23] Zainol, I., Akil, H.M., Mastor, A., 2009. Effect of -irradiation on thephysical and mechanical properties of chitosan powder. Mater. Sci. Eng.
C 29, 292-297.
[24] Czechowska-Biskup, R., Rokita, B., Ulanski, P., Rosiak, J.M., 2005.
Radiation-induce and sonochemical degradation of chitosan as a way toincrease its fat-binding capacity. Nucl. Instr. Meth. Phys. Res. B 236,
383-390.

20

[25] Choi, S.W., Ahn, J.K., Lee, W.D., Byun, W.M., Park, J.H., 2002.
Preparation of chitosan oligomers by irradiation. Polym. Degrad. Stab.
78, 533-538.
[26] Wasikiewicz, M.J., Yoshii, F., Nagasawa, N., Wach, A.R., Mitomo, H.,
2005. Degradation of chitosan and sodium alginate by gamma radiation,
sonochemical and ultraviolet methods. Radiat. Phys. Chem. 73, 287-295.
[27] Qin, C.Q., Du, Y.M., Xiao, L., 2002. Effect of hydrogen peroxide
treatment on the molecular weight and structure of chitosan. Polym.
Degrad. Stab. 76, 211-218.
[28] Kang, B., Dai, D.Y., Zhang, Q.H., Chen, D., 2007. Synergic degradationof chitosan with gamma radiation and hydrogen peroxide. Polym.
Degrad. Stab. 92, 359-362.
[29] El-Sawy, N.M., Abd El-Rehim, H.A., Elbarbary, A.M., Hegazy, E.A.,
2010. Radiation-induced degradation of chitosan for possible use as a
growth promoter in agricultural purposes. Carbohydr. Polym. 79, 555562.
[30] Hien, N.Q., Phu, D.V., Duy, N.N., Lan, N.T.K., 2012. Degradation of
chitosan in solution by gamma irradiation in the presence hydrogen
peroxide. Carbohydr. Polym. 87, 935-938.
[31] Wang, S.M., Huang, Q.Z., Wang, Q.S., 2005. Study on the synergetic
degradation of chitosan with ultraviolet light and hydrogen peroxide.
Carbohydr. Res. 340, 1143-1147.
[32] Shao, J., Yang, Y., Zhong, Q., 2003. Study on preparation ofoligoglucosamine by oxidative degradation under microwave irradiation.
Polym. Degrad. Stab. 82, 395-398.
[33] Duy, N.N., Phu, D.V., Anh, N.T., Hien, N.Q., 2011. Synergistic
degradation to prepare oligochitosan by -irradiation of chitosan solution
in the presence of hydrogen peroxide, Radiat. Phys. Chem., 80, 848-853.

21

Part 3. Rice
3.1 Cultivations of Rice
Preparation of planting is divided into 4 main steps, based on the farmer
practices recommended by FELCRA Berhad, the fully goverment owned
company administered by Malaysian Ministry of Rural and Regional
Development which own largest area of rice plantation in Peninsular
Malaysia. They are :i.
Management of rice straw
After harvesting, rice straw is left sun-dried. Additionally, herbicide isapplied to enhance the decomposition of straw. Then the straw was cut
using hoe or flail mower. The straw was then burnt in a direction opposite
the normal wind flow in order to provide perfect combustion. The burning
procedure also helps to kill the pest (e.g snails) in the soil.
ii.
Land smoothing
In Malaysia, land smooting procedure is classed into two categories
which are minor and major land smoothing. The minor land smoothingis done in every season before planting, while major land smoothing isdone once in 3-4 seasons of planting. The equipments are usually usedduring land smooting are rotovator and tractor backbucket. The
rotovator is used to cut the high spots while tractor backbucket is used
to filling the low spots and levelling/smoothing the land.
iii.
Ploughing
Plouging is for land grading and levelling before planting. Besides,
ploughing also promotes elimination of wild weed and reduces thegermination of weedy rice (locally known as padi angin), which induces
crop losses by reducing the yield and quality of the rice. The rotovator isused to break the soil into smaller soil granules. Meantime, theherbicides and pesticides are applied during ploughing to get rid of thewild weed and pests (e.g snails). The Niplow harrow is used at the end of
the ploughing procedure for final land grading and leveling before startplanting.
iv.
Planting
In Malaysia, there are three types planting methods i.e direct seeding in
wet condition, direct seeding in saturated condition and transplanting.
All these methods require germination enhancer during seed treatment(seed soaking) to increase the percentage of seed germinate. Most of the22commercial products contain very strong acidic solution such as nitricacid as seed germination enhancer.

Transplanting of rice seedling

Direct seeding in water

Figure 3. 1. Photos of palnting.

3.2 Recommended Application
Nuclear Malaysia has done research on application of oligochitosan on
local rice plant, MR 219, for more than 5 years and the research still goingon. Oligochitosan is applied at 3 different levels, which are :i.
Seed – seeds are soeked in the oligochitosan solution atconcentration of 100 ppm for 24 hours.
ii.
Seedling – seedlings are sprayed with oligochitosan 40 ppm atday 3, 7 and 14 after sowing. After first 3 weeks at nursery stage(either on the field or nursery shed), the seedlings are transferedto the real field.
iii.
At the field – the oligochitosan is sprayed at the field 4 times (Day20, 30, 40 & 70) at concentration of 10, 20, 40 and 100 ppm. Spraytiming is 2 days prior normal fertilizing. The oligochitosan helps
to strengthen the stem of seedlings, promotes growth of new
seedlings from the mother plant, enhances the growth of fruit
panicles and boosting fruit quality by maintaning the strength andhealth of panicle and plant overall(protect from blast or
bacterial attack).

23

Figure 3. 2. Photos of Seed soaking (left) and treated rice seed for seedling(right).

3.2.1 Application Protocol for MR 219 and Mutant MR 219-9 and MR 219-4
Rice MR 219 is one of the most planted rice variety by Malaysian farmers.
It was developed by MARDI obtained from the cross between of MR 137 x
MR 151 and been released on January, 9th 2001. The characteristics of MR
219 are :a. has short maturation period (105 – 111 days)b. highly resistance to bacterial, leaf blight, blast and brown plant
hopper.
c. long grades grain
d. low amylose content (21.4%) – gives soft texture of cooked rice.
e. able to produce high yield (10 Mt/ha) – requires high fertilizer andgood water management
Meanwhile MR219-4 and MR 219-9 are the mutant varieties obtained
from the -ray irradiation of MR 219 at dose 300 Gy. It is developed byresearchers from Nucler Malaysia and UPM (Putra University of Malysia).
They copy the characteristics of MR 219 and exhibit other special
characteristics such as :a. Longer panicle
b. High percentage of filled grainsc. Can adapt in flooded and water stress regimes (aerobic soil)
The application method of oligochitosan on MR 219-4 and MR 219-6 aresame as for MR 219. The method is described below :24

a. Seed treatmenti.
The oligochitosan solution 100 ppm is prepared by dissolving
5ml oligochitosan of 20,000 ppm for preparation of 1 L
solution
ii.
Seeds are soked in the oligochitosan solution 200 ppm for 24hours. Make sure all the seeds are well-immersed in theoligochitosan solution.
iii.
Then the seeds are dripped for 24 hours.
b. Seedling stage (foliar)i.
The oligochitosan solution 40 ppm is prepared by dissolving
2 ml oligochitosan of 20,000 ppm for preparation of 1 L
solution.
ii.
The oligochitosan is sprayed onto the seddling.
iii.
The application is done once a week for 3 weeks (Day 3, 7 and
14).
c. At the field (foliar)i.
The oligochitosan solution 40, 60 and 100 ppm is prepared bydissolving 2 ml, 3 ml and 5 ml of oligochitosan of 20,000 ppm
respectively for preparation of 1L solution.
ii.
The oligochitosan solution is sprayed onto the plants.
iii.
The application is done on each 2 days prior normal
fertilizing. Total application is 4 times (Day 20, 30, 40 & 70)
starting on day 3 after plants are transferred procedure.

3.3 Effect of Seed Treatment
Table 3.1 shows the percentage of seeds germinate after treated witholigochitosan 100 ppm at different soaking duration. On day 1, soaking
duration of 15 min., 30 min. and 60 min. give less than 50% of germinatedseeds. For 90 min. and 120 min. the total of germinate seeds are more than
50% but the germination rate is still very low. The seed soaking for 24 hgave the highest percentage of seeds germinate i.e more than 95%. Based on
the farmers’ experience, the soaking duration of rice seeds is recommendedfor 24 h and not less than that. It is because the rice husk is very hard andseed treatment requires longer time to make sure the solution treatment
can penetrate into the inner layer of the husk. Meanwhile for seed with has
25soft outer layer such as Chilli seed, the soaking period should be around 1
h to 2 h and if too long the quality of seeds become deteriorated.
Table 3. 1 Effect of soaking duration on rice seed germination
Oligochitosan
100 ppm
15 min30 min60 min90 min

120 min24 hrs

Day 1

Day 2

Day 3

Day 4

Day 7

a
b
c

47
53
38

85
89
84

91
89
88

91
91
88

93
93
92

Average

46

86

89.3

90

92.7

a
b
c

53
42
40

85
81
75

85
86
84

90
89
85

92
91
92

Average

45

80.3

85

88

91.7

a
b
c

34
28
32

77
60
60

82
78
77

89
80
81

96
92
89

Average

31.3

65.7

79

83.3

92.3

a
b
c

56
52
52

86
82
75

93
89
84

95
90
88

95
92
88

Average

53.3

81

88.7

91

91.7

a
b
c

60
53
60

84
83
87

90
93
92

92
92
94

95
96
95

Average

57.7

84.7

91.7

92.7

95.3

a
b
c

95
93
98

97
98
99

99
99
99

99
99
99

99
99
99

Average

95.3

98

99

99

99

3.4 Effect of Concentration
Figure 3.3. illustrates the germination rate of the rice seeds after
treated with oligochitosan at different concentration. On day 1, the highest
26germination rate is treatment oligochitosan 100 ppm which is more than82%. The lowest germination rate is oligochitosan 200 ppm treatment which
is only 61.7% . Based on this result, oligochitosan 100 ppm treatement is
considered to be the best treatment for seed germiantion stage.
As for seedling or nursery stage, oligochitosan 40 ppm treatment isselected to be used. It is because the effect of oligochitosan 40 ppm on
germination as well as the growth of seedling shows no significant differencewith oligochitosan 100 ppm treatment.

Total Germinated Seeds (%)

100

90
82.3
80

78.3
74.7

70

89

91
89.7 89.7

85.7

88.3

84
81.3

9392.7
90.7

90.7

94.3
93.3
92.7
92.7
90.7

87.7

20ppm
40ppm

84.3

80ppm
100ppm77

200ppm

71.3

61.7
60
Day 1

Day 2

Day 3
Days

Day 4

Day 5

Figure 3. 3. The germination rate of rice seeds after treated witholigochitosan at different concentration

Figure 3. 4 Foliar application of oligochitosan on the field27

3.5 Effect of Molecular Weight
In rice project, we never study the effect of oligochitosan molecularweight on the growth of rice. During study, we used oligochitosan with single
Mw i.e Mw = 10 000 g/mol. The study by El-Sawy and friends (2010) has
shown that degraded chitosan has better effect on the growth of faba bean
compare to chitosan.
3.6 Effect of Foliar Spray Frequency
Bittelli et al. (2001) reported foliar application of chitosan on pepper
plants can reduce 26-43% of plant transpiration or water loss while
maintaing biomass production and yield. Chitosan has the self- film forming
ability, this property induces formation of thin film on the outer layer of theplant skin. This thin film controls the plant transpiration.
In our case, we recommend to use foliar application of chitosan oncein a week for the seedling under the shed (nursery stage). At the early stage
chitosan applied for the purpose to enhance the production of early leaf. For
the mature plants, e.g Chilli – foliar application of chitosan on mature plants
has to be done at least (optimum) twice a week for the growth enhancement
and protection purposes. As mentioned before, chitosan thin film whichobtained after foliar application can protect the plants against disease or
microbes. The foliar application twice a week should be continued until
harvest time. This is because besides protecting the plants chitosan thin film
also provide protection to the fruits as well. The chitosan thin film which
formed on the fruit skin preserves the fruit from deterioration easily.
3.7 References[1] El-Sawy, N.A., El-Rahim, H. A., El-Barbary, A. M. and Al-Sayed, A. H.
2010. Carbohydrate Polymers,79, 555-562.
[2] Bittelli, M., Flury, M., Campbell, G. S and Nichols, E. J. 2001.
Agricultural and Forest Meteorology, 107(3), 167-175.

28

Part 4. Chilli
4.1 Cultivations of Chilli
Cultivation of Chilli was adopted from the results of researchcollaboration between National Nuclear Energy Agency (BATAN) and
Development Planning Agency of Kerinci Regency, Jambi province. Chilli
cultivation divided into three main steps. They are:
1. Preparation of land
In the cultivation of Chilli, land preparation should come first,
followed by preparation of the seed or seedling. In preparation of land,
the following steps should be done:a. Management of soil
Soil management is done by digging the ground to clear the land
from debris roots of old crop marks and all kinds of weeds that grow.
Land plowed or dug as deep as 40 cm to improve soil aeration andto eliminate the intruder organism plant which hiding in the
ground. After land cultivated, prepared the raised beds,120 cm
width and 30 cm height, and the distance between beds is around
80 cm. The length of each bed is 12 m or adjusted with the trench
width. Create good drainage because red Chilli plants are notresistant to water logging. After preparing the beds, the soil wasleft around 10-14 days up to dry.
b. Soil acidity (pH)
Soil pH should be checked. The ideal pH of soil is around 6.5-7.5(neutral). If soil pH below 6, add agricultural lime (dolomite) withconcentration depending on initial pH. If the soil is very acid, asmuch as 2 ton/ha of dolomite should be applied. Dolomite is addedin the same time with soil digging and beds preparation.
c. Fertilization
Compost or manure fertilizers with concentration 20 ton/ha wasapplied on the beds and mixed homogeneously. Instead of organic
fertilizer, the basal dressing, a well-mixed fertilizer containing
29

urea (300 kg/ha), SP36 (250 - 300 kg/ha), and KCl (potassium
chloride) (250 kg/ha) was also applied. The beds was then irrigatedaccordingly in order to dissolve fertilizer
d. Installation of Silver Black Plastic Mulc
Around 12 rolls of mulch are needed per hectare. The mulch issilver on top, and black on the underside. The silver side reflects
the light and repels insects, the dark underside controls weeds).
The mulch should be laid on the soil in the middle of the day andin full sunlight. The heat will soften the plastic, so it is easier tostretch. The plastic mulch should be spread tightly over the soil,
with the silver side facing upwards. The plastic should be pulled
tightly down over the sides of the bed, and fastened in place using
thin pieces of bamboo. These are about 40 cm long. They are bent
in half, and pushed into the soil to hold the edges of the mulch atintervals of 50 cm. A cross is cut in the plastic mulch for a planting
hole where each plant will grow. Installation of silver black plastic
mulch is shown in Figure 4.1.

Figure 4.1. Preparation and installation of silver black plastic mulch.
2. Seedlings
Choose the quality of seed and growing medium. The medium used to
grow the seedlings is a mixture of fertile soil and manure, at a ratio of 1:1.
Seedlings are grown in a polyethylene (plastic) bag, or in a well-prepared30

seed bed. To prepare the seed bed, the soil is cleaned of weeds and rubbish,and then ploughed. Clods of earth should be broken up by hand. The
seedlings should have plenty of light, but should be shaded from direct sun
by a simple structure. It has a roof of rice straw or similar materials. To
prevent seedling from insect attack, insect screen can be used. The seedlings
should be watered every day, or as needed. Before seedling, the seed was
immersed in a mixture of warm water (50oC) or Previcur N (1 ml) dan
bactomycin (1 ml) solution and oligochitosan with concentration of 25-50
ppm for 3 h. Remove the floating seed. Take out the seeds from the solution
and dried under sun light for one day. Seed was then transplanted into
prepared polybag, one seed for one polyethylene bag. At 10 days seedling,oligochitosan with concentration 50 ppm was sprayed. When the seedlingsare 20 - 24 days old (i.e. 20 - 24 days after sowing) they are ready to betransplanted to the beds as can be seen in Figure 4.2.

Figure 4.2. Preparation of seed and seedling of chilli.
3. Planting
Two rows of seedlings are planted in each bed. Seedlings should beplanted 60 cm apart, with 60 cm between the rows. Transplanting is best
done in the early morning or early evening, when it is cool. Before
transplanting, the root is immersed in oligochitosan 50 ppm. After planting,watered the plant as needed with low-pressure spray. Seven days after
planting, stake from bamboo with height 100-150 cm was plugged in the soil
closed to Chilli plant. (Fig. 4.3) One month after planting, plant stems underthe main branch tied to the stake.

31

Figure 4.3. Installation of bamboo stake.
Supplementary fertilization and maintenance play an importantrole in Chilli planting. A well-mixed fertilizer containing of NPK, Super
bionik D, Super Bionik B, and compost was applied at 3, 6, 9 and 12 weeks
after planting. Oligochitosan is sprayed to leaf and all part of plant withconcentration of 50 ppm every 10 days starting from 15 days after planting.

4.2 Application Protocol for Chilli TM 999 variety
Based on our result on chilli plants variety TM 999 at Kerinci
Regency, Jambi Province, the application of oligochitosan is applied at 3
different levels, which are :i. Seed: seeds are soaked in the oligochitosan solution at concentrationof 50 ppm for 3 h.
ii. Seedling: during seedlings stage, oligochitosan is sprayed only oncewith 50 ppm at 10th day after sowing. After first 3 weeks at nursery
stage, the seedlings are transferred to the real field.
iii. At the field: the oligochitosan is sprayed to the leaf and all part ofplants every 10 days starting from 15 days after planting.

32

Oligochitosan is used along of chilli planting cycle. Other pesticides
may also be used to prevent diseases.

Chilli or red pepper or chilli (Javanese) is a fruit and plant memberof the genus Capsicum. The fruit can be classified as vegetables and spices,
depending on how it is used. As a seasoning, spicy chillies are very popular
in Southeast Asia including Indonesia as a food flavor enhancer. Chilli TM
999 is one of hybrid type chilli which is the most planted chilli in Kerinci,
Jambi Province. This type of chilli Hybrid comes from Hungnong, Korea.
The application method of oligochitosan on Chilli TM 999 is as follows:d. Seed treatment
iv. The oligochitosan solution 50 ppm is prepared by dissolving 1 mL
oligochitosan of 50,000 ppm for preparation of 1 L solution
v. Seeds are soaked in the oligochitosan solution 50 ppm for 3-5 hours.
Make sure all the seeds are well-immersed in the oligochitosan solution.
vi. Floating seeds are taken out and discarded. only the seeds that sink(quality seeds) are used for seedlingvii. Dry the quality seeds under sunrise for one day.
e. Seedling stage (foliar)
iv. The oligochitosan solution 50 ppm is prepared by dissolving 1 mL
oligochitosan of 50,000 ppm for preparation of 1 L solution.
v. At 10th day after seedling, oligochitosan with concentration 50 ppm
was sprayed onto the seedling.
vi. When the seedlings are 20 - 24 days old (i.e. 20 - 24 days after sowing)
they are ready to be transplanted to beds
f. At the field
iv. The oligochitosan solution 50 ppm is prepared as described above.
v. The oligochitosan solution is sprayed onto the plants (leaf, trunk andbranch).
vi. The application is done every 10 days starting from 15th day afterchilli transplanted to the field. If an insecticide is needed, it is applied
at 5 days before applying of oligochitosan.
vii. Oligochitosan can be used until chilli is harvested.
4.2.1.Effect of Concentration
33

Research on the effect of oligochitosan concentration to chilli plants
has been done at Kerinci Regency, Jambi Province under collaboration
between National Nuclear Energy Agency and Development Planning
Agency of Kerinci District, Jambi province. Hybrid TM 999 chilli variety wasused. Cultivation system as mention above was adopted in this experiment.
Oligochitosan is produced by irradiating of chitosan using -rays with doseof 75 kGy. Oligochitosan prepared by BATAN with three different
concentrations at 50, and 100 ppm and 0 ppm as control were used. For
each concentration of oligochitosan, 80 chilli plants were planted in raised
beds, which have 12 m length and 60 cm distance between plants. Each
concentration has three replications, so every oligochitosan concentrationhas 240 plants. Oligochitosan was applied by spraying to leaf and trunk at
every 10 days. Insecticides were used when it is needed. Several parameterssuch as plant height, number of branch, trunk circumference were measured
at 30 and 45 days after panting, and yield in every harvesting times as wellas total yield were also calculated. For measurement of plant height, numberof branch, trunk circumference, the results are the average value from 15
plants for each concentration of oligochitosan while for calculation yield ofeach harvesting and total yields of each treatment, the results are from total
yield of 240 plants for every harvesting. Chilli fruits which show
homogeneous red color were harvested. Normally harvesting conducted
every 4 to 5 days.
The results show that at 30 days after planting (DAP), number ofbranch, trunk circumference and plant height were highest value for plants
treated with oligochitosan 50 ppm whereas the lowest for control plant. After
45 DAP number of branch, trunk circumference and plant height show thesame results as shown in Table 4.1. It was found that at 25 DAP, the plants
have disease which the symptom are the curling of leaves, their small size,
shortened internodes and general dwarfing of the plant which assumes abushy appearance, leaves are of pale (light yellow) color and roll downwards.
This disease probably attacked by Cucumber Mosaic Virus transmitted byaphids and whitefly. To prevent spread out of diseases, insecticides wereapplied for all plants. However, control plants still showed severe diseases
while plants treated by 50 and 100 ppm oligochitosan significantly reduced
diseases. These results showed that plants treated by chitosan moreresistant to diseases. By continue applying oligochitosan as scheduled, chilli
plants treated with 50 and 100 ppm recovered from diseases while control
34

plant still does not recover.
In Table 4.2, yield of chilli at different harvesting times versus ochitosan concentration. Chilli without oligochitosan treatment only can be
harvested up to 14 times in one planting cycle, while chilli treated witholigochitosan harvested up to 24 times. Chilli treated with 50 ppm
oligochitosan showed highest total yield in every harvesting times as can beseen in Tables 4.1 and 4.2. Total yield of chilli without oligochitosan
treatment (control), treated with 50 and 100 ppm harvested for eachtreatment from 240 plants are 82, 292.3, and 251.4 kg, respectively. It is canbe concluded that the best concentration of oligochitosan for chilli is 50 ppm.
Table 4.1. Effect of oligochitosan concentration on the average plants height,
number of branch and trunk circumference at 30 and 45 days after planting
O-chitosan
concentration,
ppm

Plant heightnumber of branch

trunk
circumference

At 30 days after planting
0

45

8.4

4.7

50

70

10

6

100

60

9.5

5.4

At 45 days after planting
0

72

10.4

6

50

92

13

7

100

85

12

7

35

Table 4.2. Yield of chilli at different harvesting times versus oligochitosan
concentration. Each chitosan concentration has 240 plants
Harvesting times

Yield (kg) at different oligochitosan concentration
0 ppm

50 ppm100 ppm

1

0.17

0.23

0.2

2

0.35

0.7

0.6

3

1.2

2.8

1.8

4

3.5

7

5.2

5

9.3

12.3

10.6

6

11

19

16

7

6.6

11,8

9.1

8

10

26

26.2

9

4.5

8.8

9.2

10

10

24

20.5

11

8

23

20

12

11

15.4

15.1

13

5

9.8

10

14

2

16

15

15

0

15.2

14

16

0

16

13.4

17

0

16.5

12

18

0

17

15

19

0

15

9

20

0

17

15

21

0

18

15

22

0

23

20

23

0

3.7

0

24

0

1.2

0

TOTAL

82.86

292.3

251.4

36

4.2.2. Effect of Molecular Weight
Chitosan obtained from deacetylation of chitin was irradiated with-rays at doses of 25, 50, and 100 kGy with dose rate of 6 kGy/h. Then
irradiated chitosan was diluted by using acetic acid to get concentration of
5 % (b/v) named concentrated oligochitosan. The viscosity average molecularweight (Mv) was measured by using viscosimetry Oswald method. The
viscosity average molecular weight of oligochitosan decreased with anincrease in irradiation dose as shown in Table 4.3.
Table 4.3. Effect of irradiation dose on viscosity average molecular weight ofchitosanDose (kGy)

Mv (kDa)

0

51.7

25

12.7

50

9.67

100

2.09

The concentrated oligochitosan with various molecular weights thendiluted with tap water with ratio of 1:1000 (v/v) to get 50 ppm oligochitosan
for foliar spray. The Capsicum annum plant was watered using 50 ppm ofoligochitosan once in two days. The height and dried weight of plants wereobserved at 30 days after planted. The result in Table 4.4 shows that theheight of Capsicum annum plants was increasing with decreasing molecularweight (Mv) of irradiated chitosan which used for watering plant. The
highest degree of increasing of plant is 79.7% obtained by watering plantwith oligochitosan having Mv of 2.09 kDa which irradiated at dose of 100
kGy. The same result has observed at dried weight of plant as presented inTable 4.5.

37

Table 4.4. Effect of irradiated chitosan on height of capsicum annum
plant
Height of Plant (cm)
No

1
2
3

Mv,
kDa
51.74212.713
9.665

No of plant

Average

Degree ofincreasin
g
(%)

1

2

3

4

5

6

7

8

9

10

34

36

35

37

33

34

35

39

34

34

35.1±1.3
4

30.4

37

38

39

37

38

39

38

37

36

38

37.7±0.7
6

40.0

45

45

44

46

47

44

45

46

47

48

45.7±1.1
0

69.8

48

48

47

49

50

48

47

48

49

4

2.09

48

48.2±0.6
8

79.7

5

Control
26 28 28 29 25 26 27 25 28 27(water
)26.9±1.1
20

Table 4.5. Effect of irradiated chitosan on dried weight of capsicum
annum plantNo

Mv, kDaThe average of dried weight(g)Degree of increasing(%)

1

51.742

10.8

31.7

2

12.713

12.2

48.7

3

9.665

15.8

92.6

4

2.09

16.2

97.5

5

Control(water)

8.2

0

The disease index of Capsicum annum and Capsicum frutescens
plants shown in Table 4.6. The disease index decrease from 20 to 0.83% forCapsicum annum plant after treated by oligochitosan irradiated with 100
kGy. The decreasing of disease index from 12.5 to 2.08% also observed on
38Capsicum frutescens plant.
Table 4.6. Effect of oligochitosan on disease index of capsicum annum andcapsicum frutescens plants
1. Capsicum annum

TreatmentNosampleHeight of
plant
(cm)

With Oligochitosan:
virus.

Amountof shoots(pieces)

Note

Total plants are 240. Two plants were attack by

1

46

59

2

44

29

3

35

72

4

57

146

5

54

68

6

48

24

7

54

119

8

50

60

9

59

83

10

54

55

50.1

71.5

Average

Intensityof virus
attack
(%)

0.832/240 x100%= 0.83 %Without Oligochitosan: Total plants are 200. Forty plants were attack by
virus
1

39

30

2

36

28

3

56

29

4

51

32

5

50

62

6

35

28

7

42

32

8

40

30

9

34

30

10

30

25
39

20

40/200 x100% = 20%

Average

41.3

32.6

2. Capsicum frutescensWith oligochitosan: Total plants are 240. Five plants were attacked by
virus
1

48

10

2

32

4

3

38

4

4

29

5

5

47

10

6

62

12

7

26

6

8

25

1

9

29

12

10

63

33

39.9

9.7

Average

2.08

5/240 x100%= 2.08%

Without Oligochitosan: Total plants are 200. Twenty five plants wereattack by virus

Average

1

20

4

2

12

4

3

38

9

4

40.5

4

5

40

6

6

25

4

7

25

5

8

30

5

9

25

4

10

38

7

29.35

5.2

40

12.5

25/200x100%=12.5%

4.2.3. Foliar SprayThe oligochitosan solution 50 ppm is prepared as described previously. The
oligochitosan solution is sprayed onto the plants (leaf, trunk and branch).
The application is done every 10 days starting from 15th day after chillitransplanted to the field. If an insecticide is needed, it is applied at 5 days
before applying of oligochitosan. Oligochitosan can be used until chilli isharvested.
4.3 Application Protocol for Kulai Hybrid F1 S469
Chilli Kulai (Capsicum annum) is originated from Taiwan. The
characteristic of Chilli Kulai are :i.
High percentage of seed germinationii.
Plant height : 80 cm – 110 cm
iii.
Fruith length : 10-13 cm
iv.
Fruit weight : 12-14 g/fruit
Meanwhile Chilli Kulai Hybrid F1 469 exhibit better charactersitics than
Chilli Kulai, such as :i.
Very high percentage of seed germination : >80%
ii.
Plant height : average 35 cm (fast growing, uniform growth
rate)
iii.
High yield : 25 g/fruit (able to produce up to 180 fruits per tree)
iv.
Fruit length : 17 cm with thick layer of the outer skinv.
Resistant to disease
The method of oligochitosan application on Chilli plant is described below:
a. Seed treatmenti.
The oligochitosan solution 100 ppm is prepared by dissolving
5ml oligochitosan for preparation of 1 L solution.
ii.
Seeds are soaked and immersed in the ooligochitosan solution
for 2 hours.
iii.
Then the seeds are dripped and plant on the next day on wet
peat moss (tray).
b. Nurseryi.
As normal practice, the seedling are kept under nursery shed
for about 30 days.
41

ii.

The oligochitosan solution at concentration of 40 ppm issprayed on the seedling once in a weekc. At fieldi.
The seedlings are transfered to the field (in plastic bag).
ii.
The oligochitosan solution at concentration of 100 ppm isspray to the plants for twice a week. Recommended on day 1
and day 4 of the week, in the morning.
4.3.1 Effect of Concentration
Data are means of ten replicates. Different superscripts in thesame column are significantly different between the treatments at 5% levelaccording to Duncan’s new Multiple Range Test (DMRT).
The results in Table 4.7 and Figure 4.5 show that oligochitosan
enhanced the height of the Chilli plants. The optimum concentration ofoligochitosan for height increase of Chilli plants was 80 ppm. The Chilli
plants treated with 80 ppm of oligochitosan showed significant differences,
statistically, in Chilli height over the control plants.
Table 4.7: Effect of Oligochitosan on the Height of Chilli Plants.
Concentration of
Oligochitosan (ppm)

Chilli Height
1 month (cm)

2 months (cm)05.51 ± 0.78b

9.25 ± 1.40c

20

6.00 ± 1.18ab

15.81 ± 1.76b

30

6.04 ± 1.05ab

16.56 ± 2.22ab

40

6.19 ± 1.15ab

17.95 ± 3.39ab

80

6.73 ± 0.77a

18.53 ± 3.05a42

Figure 4.5. Effect of oligochitosan on the height of chilli plants.
Table 4.8: Effect of Oligochitosan on Chilli’s Productivity.
Conc.

Total
weight ofchilli (g)Totalnumber ofchilliTotal
number ofgreen chilli

Total
number of
red chilli

Weight per
chilli (g)061.10 ±
54.21c101.10 ±78.86b93.20 ±
77.43b

7.90± 9.52b

0.56±0.13b

20

83.10 ± 63.31bc 114.10 ±65.13b104.20 ±71.40ab

9.90±
16.03b

0.79±060ab

30

129.80 ±34.80ab167.70 ±51.78ab147.60 ±32.81ab20.10±26.5
4ab0.12ab40131.00 ±26.13ab168.40 ±77.12ab152.80 ±63.13ab9.80±6.77b

0.84±0.23ab80

180.10 ±82.18a192.40 ±95.36a158.60 ±75.62a39.60±61.4
1a0.99± 0.23a

0.79±

Table 4.8 and Figure 4.6 show the effect of oligochitosan on chilli’s
production. The results showed that the treatment of chilli plants witholigochitosan enhanced the productivity. Total weight of chilli, total numberof chilli, total number of green chilli, total number of red chilli and weightper chilli increased significantly for the chilli plants treated with 80 ppm
oligochitosan compared to the control group, as can be seen in Figures 4.7 –4.10. It was found that there was 34% increase in total weight of chilli
compared to the control. Chilli plants treated with oligochitosan displayed43

an ability to protect aphid infection. It was found that the untreated chilli
plants were badly damaged by aphid infection as shown in Figure 4.7. The
untreated chilli plants exhibited various aphid damages such as decreased
growth rates, mottled leaves, yellowing, curled leaves and finally death. The
removal of sap creates a lack of vigour in the chilli plant and aphid saliva is
toxic to chilli plants. Aphids frequently transmit disease causing organisms
like plant viruses to chilli plants. Additionally, oligochitosan advanced
flowering time. At 70 days after seedling, chilli plants treated witholigochitosan already gave chilli, while the untreated chilli plants just began
flowering. The harvest time of chilli plants treated with oligochitosan was
three weeks shorter than the untreated chilli plants.

Figure 4.6. Effect of oligochitosan on total weight of chilli.

Figure 4.7. Effect of oligochitosan on total number of chilli.

44

Figure 4.8. Effect of oligochitosan on total number of green chilli.

Figure 4.9. Effect of oligochitosan on total number of red chilli.

Figure 4.10. Effect of oligochitosan on weight per chilli.

45

Figure 4.11. Effect of oligochitosan on aphid inflection.
4.3.2 Effect of Molecular Weight
Table 4.9 demonstrates the effect oligochitosan with different Mw on
growth of Kulai Chilli Hybrid seedlings. Oligochitosn with Mw at 6.5 kDashows highest germination rate i.e 85.2% among oligochitosans.
Oligochitosan at 6.5 kDa show insignificant difference compare tooligochitosan at 18 kDa on height of plant and number of leaf. The
oligochitosan with Mw at 38.8 kDa suppressed the growth of seedlings dueto the lowest number in germination (%), height of plants and number ofleaf. Based on this result, it be concluded that oligochitosan with Mw value
below 20 kDa indicates positive effect on the growth of seedlings and plants.

Table 4.9. Effect of oligochitosans with different Mw on growth of Kulai
Chilli Hybrid seedlings.

46

Mw, kDa

Germination(%)

Height, cm

No. of leaf

Control (Water)

69.1

9.1 + 1.733

4.145 +1.12

6.5

85.2

9.4 + 1.744

4.44 + 1.11

1880

9.2 + 1.629

4.53 + 0.99

38.8

56.3

7.974 +
1.51

3.57 + 0.94

4.4 References[1] Ershov, BG, Isakova, OV, Rogoshin, SV, Gamazazade, AI, Leonova, EU.
Doul, Akad. Nauk SSR 1987; 295: 1152.
[2] Kim, C., Choi, J., Chun, H., Choi, K., 1997. Polymer Bulletin, 38, 387393.
[3] Yalpani, M., Johnson, F., Robinson, L., 1989. Chitin and Chitosan.
Newyork: Elsevier.
[4] Gatot, R., Kadariah, Sunarni, A., Iramani, D., Susilawati, S., 2005.
Study on Irradiation of Condition Chitosan for Growth Promoter of Red
Chilli (Capcinum Annum) Plant. Proceeding of The 6th ITB-UKM
Joint Seminar on Chemistry, 6, 328-332.
[5] Gatot, R., Kadariah, Sunarni, A, Iramani, D., Susilawati, S., 2005.
Irradiated Chitosan as Growth Promotes for Potato Plants (Corleus
Tuberosus Renth). Proceeding of PATIR-BATAN, 165-168.
[6] Evan, E.E., Kent, S.P.J., 1962. Histochem., Cytochem. 10, 24.
[7] Olsen, R., Schwartzmiller, D., Weppner, W., Winandy, R., 1989. Chitin
and Chitosan. New York, Elsevier.
[8] Matsuhashi, S., Kume, T., 1997. J. Sci. Food Agric. 73, 237-241.
[9] Dzung, N.A, Khanh, V.T.P, Dzung, T.T., 2010. Carbohydrate Polymers.
in press.
[10] El-Sawy, N.M, Abd El-Rehim, H.A, Elbarbary, A.M, Hegazy, E.A.,
2010. Carbohydrate Polymers. 79, 555-562.

47

Part 5. Q&A
5.1 General
Question: Is oligochitosan (PGP) applicable to all kinds of plants?
Answer: In general, PGP can be used for many kinds of plants but theproper concentration, foliar spray, etc. should still be
determined.

Question: Is oligochitosan (PGP) safe to use for plants and theenvironment?
Answer: It is absolutely safe.

Question: Where can I get oligochitosan (PGP) prepared by radiation
processing?
Answer: It can be bought from Thailand, Vietnam, Indonesia andMalaysia.

Question: How much does oligochitosan (PGP) costs?
Answer: The cost of PGP is around USD 10-15 per litre for concentrationof 20,000 to 50,000 ppm.

Question: How many litres of oligochitosan (PGP) are needed for 1 hectare?
Answer: It depends on the kind of plant. In the case of chilli, it needsaround 6 litres of PGP per hectare.

Question: What is the shelf-life of oligochitosan (PGP)?
Answer: The shelf life of PGP is two years.

48

Final Remarks
This guideline was complied by formulating the outcomes of electron
accelerator application project from 2006 under the framework of FNCA.
The previous guideline entitled “FNCA Guideline on Development ofHydrogel and Oligosaccharides by Radiation Processing” has been opened
since 2006. This guideline dealt with preparation protocols of hydrogel forsoil conditioner and oligosaccharides for plant growth promoter (PGP) using
radiation technology. FNCA participant countries as well as non-FNCA
countries have followed the described protocols to produce the standard
performance materials of Hydrogel and Oligosaccharides. The present
guideline was described for agricultural users of PGP. The users can get the
information of characteristic, proper use against rice, chilli, etc. when they
adopt PGP to enhance the yield of farm products. It is a great pleasure forall contributors and editors that PGP prepared by radiation processing willcontribute to the socioeconomic benefit in the agriculture in Asian countries.

49

APPENDIX. Data sheet obtained in each country
BANGLLADESHPGP Guide Sheet
Fill-in Dateyy/mm/dd

2014 /04/21

CountryBANGLADESH

Name of Plant

TomatoSpecies
Planting time,
seasonDecember, 2013

Harvest time,
seasonApril, 2014

Location(Temperature,
Humidity etc.)

BINA sub-station Ishurdi District, Bangladesh

Groundconditions,
Climate, etc.

Winter (Temperature 19-22°C)

Product
information
Material

ChitosanMolecular
weight (kDa)

Below 10,000

Measurement
methodIntrinsic Viscosity

InitialConcentration(ppm)
other additive
Methodology

50Cultivations

The experiment was laid out in randomized complete
block.
Design with 3 replicates. The unit plot size was 4 m
×3 m and spacing was 50 cm ×50 cm.

Recommend
applications
Germination
Timing
Foliar spray(Germination
stage)
Concentration(ppm)

0, 50, 75, 100 ppm, sprayed at vegetative andreproductive stage.

Foliar spray(Field stage)
Concentration(ppm)

PGP Guide Sheet
Fill-in Dateyy/mm/dd

2014 /04/21

CountryBANGLADESH

Name of Plant

Maize

Species

BARI maize-9

Planting time,
seasonDecember, 2013

Harvest time,
seasonApril, 2014

Location(Temperature,
Humidity etc.)

Farmer’s field of Rangpur district, Bangladesh

51

Groundconditions,
Climate, etc.

Winter (Temperature 19-22°C)

Product
information
Material

ChitosanMolecular weight(kDa)

Below 10,000

Measurement
methodIntrinsic Viscosity

InitialConcentration(ppm)
other additive
Methodology
Cultivations

The experiment was laid out in randomized
complete block design with 3 replicates. The unit
plot size was 3.9 m × 4.9 m and plant spacing was
70 cm × 30 cm. The grain yield will record on plotbasis and will convert in tones/hectare.

Recommend
applications
Germination
Timing

Foliar sprayed four times starting from 20 days
after sowing with 15 days interval.

Foliar spray(Germination
stage)
Concentration(ppm)

0, 50, 75, 100 & 125 ppm

Foliar spray(Field stage)
Concentration
(ppm)

52

53

CHINAPGP Guide Sheet
Fill-in Dateyy/mm/dd

2014/04/20

Country

China

Name of Plant

cucumberSpecies

North Cucumis sativus

Planting time,
seasonMay, summer

Harvest time,
seasonJuly, summer

Location(Temperature,
Humidity etc.)

Zhejiang Province, Humidity 50-80%, temp. 20-37
degree C

Groundconditions,
Climate, etc.

alkali red-yellow soil/ subtropical monsoon climate

Product
information
Material

low molecular weight chitosan

Molecular weight
(kDa)

20-30

Measurement
methodviscosity and GPC

InitialConcentration(ppm)

50,000 ppm (5 wt%)

other additive

surfactant, small amount of ethanol

MethodologyxCultivations

seedling in pot and transplanting.

54

Recommend
applications

promoter & disease resistantGermination
Timingany stage

Foliar spray(Germination
stage)

possible but not tested

Concentration
(ppm)

20-30

Foliar spray(Field stage)

yes

Concentration
(ppm)

40

Effect of foliar
spray frequency
on Plant Growth& Elicitor（crop yields.
length, weight,
etc. of Sprayed
and Control )Spraying for the first time after blossom. Sprayedevery 2 weeks and total 4 times in field, gainincrease 22.8% and the direct fruit rate 90%(control: 40%); sugar degree of cucumber center
was increased by about 2.7%. The leaves of plant
were much greener compared with the control.
Different concentrations of chitosan (20, 40, 100ppm) were tested and the optimal concentration
was 40 ppm.

PGP Guide Sheet
Fill-in Dateyy/mm/dd

2014/04/20

Country

China

Name of Plant

pepperSpecies

red capsicum

55Planting time,
seasonMarch Spring

Harvest time,
seasonMay, Summer

Location(Temperature,
Humidity etc.)

Zhejiang Province, Humidity 50-80%, temp. 15-30
degree C

Groundconditions,
Climate, etc.

alkali red-yellow solid/ subtropical monsoon climate

Product
information
Material

low molecular weight chitosan

Molecular weight
(kDa)

20-30

Measurement
methodviscosity and GPC

InitialConcentration(ppm)

50,000 ppm (5 wt%)

other additive

surfactant, small amount of ethanol

Methodology
Cultivations

seedling in pot and transplanting.

Recommend
applications

promoterGermination
Timingany stage

Foliar spray(Germination
stage)

possible but not tested

56Concentration
(ppm)

30-40

Foliar spray(Field stage)

yes

Concentration
(ppm)

50

Effect of foliar
spray frequency
on Plant Growth& Elicitor（crop yields.
length, weight,
etc. of Sprayed
and Control )Spraying for the first time after blossom. Sprayedevery 2 weeks and total 5 times in green house. The
average length of chilli was increased by 15% andthe gain increase was 9.5%, compared to thecontrol. Virus resistance was significant. Different
concentrations (50 and 100 ppm) were tested andthe optimal was 50 ppm.

57

INDONESIAPGP Guide Sheet
Fill-in Dateyy/mm/dd

2014/03/09

Country

Indonesia

Name of Plant

Rice

SpeciesOryza sativa L (MR219)

Planting time,
season2013/08/20

Harvest time,
season2013/11/27

Location(Temperature,
Humidity etc.)

Temp (30 -33 C), >80%RH(25C)

Groundconditions,
Climate, etc.

type of soil sandy clay loam, tropical climate

Product
information
Material

Chitosan flake/powder from shrimp shell

Molecular weight(kDa)

Oligochitosan 50,000 ppm used with averagemolecular weight 7 kDa

Measurement
methodGPC

InitialConcentration(ppm)

50,000 ppm

other additive
Methodology

58

Cultivations

Field test of oligochitosan on rice was performed incollaboration with local farmer located at Pasuruan
of East Java. The rice seed soaked with 50 ppm
oligochitosan and sprayed one time on seedling tray
at 5 days after planting. After transferred to thefields and sprayed 6 times with 50 ppm and 100
ppm oligochitosan on rice plant in field with 7 daysfor one spraying time interval at vegetative period
and 5 times at generative period with 10 days’ timeinterval. The results of field test showed that rice
yields increase up 29% for 50 ppm and reached 32%
for 100 ppm of oligochitosan compared to that ofcontrol without oligochitosan. Results also indicatedthat oligochitosan can be used as plant elicitor andgrowth promoter for rice.

Recommend
applications

direct seeding method transplanting method

Germination

1. Rice seed treatment,
dilution 1 mL of 50,000 ppm
oligochitosan solution with
1000 times water (1/1000).
Rice seeds are dipped inoligochitosan solution about
12 hours and continue with
24 hours dripping.
2. Transfer seed on seedlingtray and kept it for another24 hours before transfer the
tray to seedling pond. The
seed is watering every day.
3. Spraying rice seed with50 ppm oligochitosan on
day 5.
4. Transfer the rice plant to
the rice field on day 12

Timing59

Foliar spray(Germination
stage)

Spray 50 ppm
oligochitosan on 5th day

Concentration
(ppm)

50

Foliar spray(Field stage)

Spray 50 and 100 ppm
oligochitosan on 10th, 17th,
24th, 31st, 38th and 45thdays after planting at
vegetative period and 55th,
65th and 75th days after
planting at generative
period.

Concentration(ppm)

50 and 100

Effect of foliar
spray frequency
on Plant Growth& Elicitor（crop yields.
length, weight,
etc. of Sprayed
and Control )With oligochitosan

Without oligochitosan

60

JAPANPGP Guide Sheet
Fill-in Dateyy/mm/dd

2014/1/13

CountryJapan

Name of Plant

Cyclamen persicum

Species
Planting time,
period, season

November~December

Harvest time,
period, season

November

Location(Temperature,
Humidity etc.)

green house, Temp (25~28 C), ~80%RH(25C)

Groundconditions,
Climate, etc.
Product
information
Material

Chitosan flake from Crab shells

Molecular weight(kDa)

Oligochitosan 40,000 ppm used with averagemolecular weight 10 kDa

Measurement
methodGPC

InitialConcentration(ppm)

40,000ppm

other additive(N)7: (P)8: (K)8

Methology

61

Cultivations

Field test of oligochitosan on cyclamen was
performed in collaboration with Azbio Co., Japan.
sprayed 100 ppm oligochitosan on cyclamen plant infield with 2 weeks for one spraying time interval.
Result of field test, survival ratio of cyclamen insummer improves up 15% compared to control
without oligochitosan.

Recommend
applications
Germination
Timing
Foliar spray(Germination
stage)
Concentration(ppm)
Foliar spray(Field stage)

Spray at every 2weeks from July to September

Concentration
(ppm)

100

Effect of foliar
spray frequency on
Plant Growth &Elicitor（crop yields.
length, weight,
etc. of Sprayed
and Control )Application protocol for cyclamen field
1. Dilution oligochitosan 40,000ppm with 400 times
by water to 100ppm.
2. Spraying oligochitosan at every 2 weeks.

62

63

MALAYSIAPGP Guide Sheet
Fill-in Dateyy/mm/dd

2014/1/13

Country

Malaysia

Name of Plant

Rice

Species(MR219)Planting time,
period, season

October-November

Harvest time,
period, season

February-March

Location(Temperature,
Humidity etc.)

Temp (30 -33oC),

Ground conditions,
Climate, etc.

type of soil sandy clay loam, tropical climate>80%RH(25oC)

Product
information
Material

Chitosan powder from shrimp shell

Molecular weight(kDa)

Oligochitosan 20,000 ppm used with averagemolecular weight 10.5 kDa

Measurement
methodGPC

InitialConcentration(ppm)

20,000 ppm

other additive
Methodology

64Cultivations

Field test of oligochitosan on rice was performed incollaboration with FELCRA Bhd (government owned
company in agriculture business), Sg. Burung
Agriculture Department and Malaysian Agriculture
Research Development Institution (MARDI). The
germination study on rice seed found that rice seedsoaked with 100 ppm oligochitosan follow withsprayed 3 times with 40 ppm oligochitosan shortened
seedling period to 10-12 days compare to normal
practice 15-17 days using commercial chemical
germination product. Result of field test shown thatseed soaked for 24 hours in 100 ppm oligochitosan
and 4 times spray with 100 ppm oligochitosan during
the planting period, increase rice yield up to 26%
compare to control without oligochitosan andfungicide treatment. Yield results also revealed thatoligochitosan can replace chemical fungicidecommonly used by the farmers and at the same time
give better yield than using fungicide for disease
treatment.

Recommend
applications

direct seeding method

transplanting method

Germination

Rice seed treatment:
Require amount of 20,000
ppm oligochitosan
solution was diluted with200 times water (1/200).
Rice seeds are dipped in
100 ppm oligochitosan
solution for 24 hours andcontinue with 24 hours
dripping. The treated
seed was scattered to wetpaddy field

1. Rice seed treatment:
Require amount of 20,000
ppm oligochitosan
solution was diluted with200 times water (1/200).
Rice seeds are dipped in
100 ppm oligochitosan
solution for 24 hours.
2. Transfer seedtreatment on seedlingtray and kept it foranother 24 hours before
transfer the tray toseedling pond. The seed is65

watering every day.
3. Spraying rice seedtreatment with 40 ppm
oligochitosan solution on
3rd, 8th and 13th days.
4. Transfer the rice plant
to the rice field on 12thday.
Timing
Foliar spray(Germination
stage)

noon day 3, 8 and 13

Concentration
(ppm)

no

40

Foliar spray(Field stage)

on day 3, 18, 53 and 71, 3
days before apply
fertilizer

on day 3, 18, 53 and 71
after transfer to paddy
field, 3 days before apply
fertilizer.

Concentration
(ppm)

100

100

Effect of foliar
spray frequency on
Plant Growth &Elicitor（crop yields.

Result of oligochitosan performance on plant rice
1. Germination of rice seed treated and spray witholigochitosan could be transfer to paddy field on 12
days compare to control on 15 to 17 days.
2. Root of rice plant treated and spray witholigochitosan at germination stage shown rapid
growth compare to control using commercial plant
growth promoter.
3. Rice plant treated and spray with oligochitosan
shown 26% increase in yield compare to control
without oligochitosan. Rice plant spray with fungicide
only 14% increase in yield.

length, weight, etc.
of Sprayed andControl )66

Treated with commercial productTreated with oligochitosan

Treated with oligochitosan

Treated with commercial product

67

PGP Guide Sheet
Fill-in Dateyy/mm/dd

2014/5/5

Country

Malaysia

Name of Plant

Chilli

Species

Chilli Kulai Hybrid F1 469 (Capsicum annum)

Planting time,
period, season

July to November 2013

Harvest time,
period, season

Once a week (Sept. - Nov.)

Location(Temperature,
Humidity etc.)

Temp (30 -33oC),>80%RH(25oC)

Groundconditions,
Climate, etc.
Product
information
Material

Chitosan powder from shrimp shell

Molecular weight(kDa)

Oligochitosan 20,000 ppm used with averagemolecular weight 10.5 kDa

Measurement
methodGPC

InitialConcentration(ppm)

20,000 ppm

other additive

White onion extract

Methodology

68Cultivations

Field test of oligochitosan on chilli was carried out
in collaboration with Nurezqi Enterprise
agriculture company. The germination study ofchilli plant was performed by soaking the chilli
seed either with 100 ppm oligochitosan or chemical
consist of previcure plus tonic and water as control,for 2 hours, follow with sieve and dry prior toseedling process in peatmoss. Nutrient film
technique was used for germination of young chilli
plant where plant was watering with nutrient 2
times per day for 5 minutes. During germination,
young chilli plant was sprayed with 40 ppm
oligochitosan, one a week. After 26 days, healthy
young plant was transfer to plastic bag content
coconut coir dust for field test using fertigation
system. Under fertigation system, the chilli plants
were dripped with certain amount of water andliquid fertilizer every 5 minute, on coconut coir
dust which replacing sand, in plastic container.
Chilli plant was foliar spray every week with mix
solution of 100 ppm oligochitosan with white onion
extract. The harvest of red chilli fruit was carriedout twice per week. Average yield of chilli plant
spray with mix solution of 100 ppm oligochitosan
and white onion extract is 1598g, increase 15%
compare to chemical treatment (1387g).

Recommend
applications

69

Germination

1. Chilli seed was soaked in 100 ppm oligochitosan,for 2 hours, follow with sieve and dry prior toseedling process in peatmoss.
2. The seed was laid down in the peat moss inplastic container.
3. Nutrient film technique was used for
germination of young chilli plant where plant waswatering with nutrient 2 times per day for 5
minutes.
4. During germination, young chilli plants were
sprayed with 40 ppm oligochitosan, once a week.
After 26 days, healthy young plant was transfer toplastic bag content coconut coir dust for field test
study using fertigation system.

Timing
Foliar spray(Germination
stage)

One a week

Concentration
(ppm)

40

Foliar spray(Field stage)

One a week

Concentration(ppm)

10 ppm oligochitosan plus white onion extract.

70Effect of foliar
spray frequency
on Plant Growth& Elicitor（crop yields.
length, weight,
etc. of Sprayed
and Control )Result of oligochitosan performance on chilli plantat germination stage
1. Germination of chilli seed treated with 100 ppm
oligochitosan and spray with 40 ppm oligochitosan
one a week, gave average highest plant length androot, 35.8 cm and 7.4 cm, respectively compare to
80 ppm (35.2 and 5.8 cm), 100 ppm (33.8 and 5.6cm), and control using chemical (33.3 and 5.6 cm)
and were water (32 and 4.7 cm).
Result of field test application of oligochitosan onchilli plant
1. Foliar spray of chilli plant with 100ppm
oligochitosan plus white onion extract produce
average highest yield 1598g per plant compare tochemical (1387g), 100 ppm oligochitosan plus halfchemical (1345g), 100 ppm oligochitosan (1191g),
white onion (1142g) and control water (573g).
2. Field test also reveal elicitor effect of PGP
toward insect and disease attack on the chilli plant.
Foliar spray with 100 ppm oligochitosan plus whiteonion extract shown the lowest infected only 22.3%
plant from total 9 plants compare to treatmentusing chemical, 100 ppm oligochitosan and 100
ppm oligochitosan plus half chemical which gave
33.3%, white onion 44.4% and control using water
88.8%.

71Oligochitosan produce 3 chilli fruits
develop at one V stem of chilli plant

Chilli plant recovery from Mozac disease

Fertigation System of Chilli Plant

Insect attack chilli plant72

PHILIPPINESPGP Guide Sheet
Fill-in Date yy/mm/dd

2014/03/17

Country

Philippines

Name of Plant

MungbeanSpeciesVigna radiata (L .) Wilczek (Pagasa 21)

Planting time, period, season

August (wet season)

Harvest time, period, season

November

Location (Temperature, Humidity
etc.)

not recorded

Ground conditions, Climate, etc.

clay loam, tropical climate

Product information
Material

oligo-kappa carrageenan

Molecular weight (kDa)oligo-kappa carrageenan (ppm)
used with average molecularweight 7 kDa

Measurement method

GPC

Initial Concentration (ppm)

10000 ppm

other additive

None

Methodology

73

Cultivations

Pot experiment under screen-houseconditions was conducted using
different concentrations of oligokappa carrageenan (20, 40, 60, 80,
and 100 ppm) with tap water
serving as the negative control (0ppm) and a mungbean inoculant(commercial preparation of
Rhizobium spp.) served as positive
control. Results showed thatsoaking the seeds in 60 ppm oligokappa carrageenan and spraying
the same to resulting plants
proved best as the tallest plant 62
days after sowing and at harvest
time, earliest to flower, longest pod(fruit) length, and highest yield (interms of number and weight ofseeds) were obtained. Generally,
plants treated with oligocarrageenan were better than thecontrol plants (both untreated andthose treated with legume
inoculant) except for the 100-seedweight where there is no difference
among treatments.

Recommend applications

seed treatment prior to sowing

Germination

seeds are soaked in 60 ppm oligokappa carrageenan for 30 minutes
before sowing

Timing
Foliar spray(Germination stage)

two-weeks after sowing and every
2 weeks after each foliar spray
until plant maturity

Concentration (ppm)
Foliar spray

60(Field stage)74

Concentration (ppm)
Effects of seed treatment andfoliar spray on plant growth andyield- Ctrl+ CtrlCR20

Application protocol for rice field
1. Dilution oligochitosan 30,000
ppm with 1000 times by water to
30 ppm.
2. Spraying oligochitosan 4 times
on day 10, 20, 30 and 40 after 10days of planting. 400-500 L of
diluted oligochitosan applies for 1
hectare which means about 0.5L oforiginal oligochitosan withconcentration 30,000
ppm/ha/spraying time.

CR40

75

CR60

CR80

CR100

PGP Guide Sheet
Fill-in Dateyy/mm/dd

2014/03/17

Country

Philippines

Name of Plant

PeanutSpecies

Arachis hypogaea L. (Namnama)

Planting time,
period, season

OctoberHarvest time,
period, season

January

Location(Temperature,
Humidity etc.)

not recorded

Groundconditions,
Climate, etc.

clay loam, tropical climate

Product
information
Material

oligo-kappa carrageenan

Molecular weight(kDa)oligo-kappa carrageenan (ppm) used with averagemolecular weight 7 kDa

Measurement
methodGPC

InitialConcentration(ppm)

10000 ppm

other additive

None

Methodology

76Cultivations

Pot experiment under open field conditions was
conducted using different concentrations of oligokappa carrageenan (20, 40, 60, 80, and 100 ppm)
with tap water serving as the negative control (0ppm) and a peanut inoculant (commercial
preparation of Rhizobium spp.) served as positive
control. Results showed that seeds soaked inradiation-modified kappa carrageenan germinatedearlier than the controls. The tallest peanut
seedlings 16 days after sowing averaged 21.28 mm
for 60 ppm oligo-kappa carrageenan but it was not
significantly different from plants treated withother concentrations of oligo-kappa carrageenan.
At maturity, it was the tallest at 624.00 mm andsignificantly different from other treatments. The
negative control (0 ppm) and positive control (0ppm but with commercial inoculant) were short at
6.62 and 8.00 mm, respectively, 16 days aftersowing and at 409.00 mm and 442.67 mm,
respectively, at harvest time. The values are
significantly different from those treated withradiation-modified kappa carrageenan. The
plants flowered as early as 29 days after sowing(for 60 ppm) compared to 50.25 days from sowing(for negative control) and 41.5 days from sowing(positive control). An experiment using thepeanut variety Biyaya 6 that have been treatedwith 60 ppm oligo-kappa carrageenan produced
flowers as early as 29 days from sowing. With
regards to yield, 60 ppm oligo-kappa carrageenan
produced 36 pegs and 17 pods per plant.
Likewise, the length of pod, 100-seed weight, andyield per plant were significantly the greatest at 60
ppm oligo-kappa carrageenan. Number of seeds
per pod did not vary among treatments.

77

Recommend
applications

seed treatment prior to sowing

Germination

seeds are soaked in 60 ppm oligo-kappa
carrageenan for 30 minutes before sowing

Timing
Foliar spray(Germination
stage)

two-weeks after sowing and every 2 weeks after
each foliar spray until plant maturity

Concentration
(ppm)

60

Foliar spray(Field stage)
Concentration(ppm)
Effects of seedtreatment andfoliar spray on
plant growth andyield

Application protocol for rice germination
1. Rice seed treatment, dilution of 30,000 ppm
oligochitosan solution with 300 times water (1/300).
Rice seeds are dipped in oligochitosan solution withconcentration of 100 ppm about 24 hours andcontinue with 24 hours dripping.
2. Transfer seeds on seedling field.
3. After 12 for seedling growth, transfer seedlings
to rice field.
The germination study on rice seed found that riceseed soaked with 100 ppm oligochitosan overnight
the germination increased from 88.0% to 94.4% forcontrol and treated rice seeds, respectively. In
addition, fresh biomass of rice seedlings increased
from 1.18 g/seedling to 1.7 g/seedling for control
and treated rice seeds, respectively.

78

Application protocol for rice field
1. Dilution oligochitosan 30,000 ppm with 1000
times by water to 30 ppm.
2. Spraying oligochitosan 4 times on day 10, 20, 30
and 40 after 10 days of planting. 400-500 L of
diluted oligochitosan applies for 1 hectare whichmeans about 0.5 L of original oligochitosan withconcentration 30,000 ppm/ha/spraying time.

79

(-) control

20 ppm(+) control

100 ppm

60 ppm40 ppm80

80 ppmTHAILANDPGP Guide Sheet
Fill-in Dateyy/mm/dd

April 28,2014

Country

Thailand

Name of Plant

Chilli

Species

Capsicum annuum L.

Planting time,
seasonAugust-November

Harvest time,
seasonNovember-December

Location(Temperature,
Humidity etc.)

Temp (30 -35 C), >80%RH(25C)

Groundconditions,
Climate, etc.

loamy soil, tropical climate

Product
information
Material

Chitosan flake from shrimp shell

Molecular weight(kDa)

Oligochitosan 20,000 ppm used with averagemolecular weight 11.6 kDa

Measurement
methodGPC

InitialConcentration(ppm)

20,000other additivenoMethodology
Cultivations

The application of oligochitosan at 80 ppm showedsignificant effects, statistically, not only on chilli's
growth but also on chilli's productivity. The
oligochitosan also displayed ability to protect aphid
inflection in chilli plants. Additionally,81

oligochitosan can also shorten the harvest time ofchilli plants for three weeks. The treatment of Thaichilli plants with 80 ppm oligochitosan showed
positive effects on Thai chilli's growth andproductivity, compared with the untreated chilli
plants. It was found that there was 34% increase
in total weight for the chilli plants treated witholigochitosan as compared to the untreated plants.
Recommend
applicationstransplanting method

Germination

Chilli seeds were implanted in Petri dishes forthree days, and then the seedlings weretransplanted into clay plots. All chilli plants weretreated with the same amount of fertilizer (16-1616) via soil treatment at the beginning of theexperiment.

Timing
Foliar spray(Germination
stage)

no

Concentration
(ppm)

no

Foliar spray(Field stage)

The foliar spraying of oligochitosan was applied
once a week.

Concentration(ppm)

80 ppmEffect of foliar
spray frequency
on Plant Growth& Elicitor（crop yields.
length, weight,
etc. of Sprayed
and Control )The chilli plants treated with 80 ppm ofoligochitosan showed significant differences,
statistically, in terms of height, over the control
plants. It is quite obvious that the productivity ofchilli plants, in terms of the numbers of chillies,
increased with increasing concentration ofoligochitosan. The total weight of chilli, total
number of chillies and weight per chilli increased
significantly for the chilli plants treated with 80
ppm oligochitosan compared to the control group.
82

In addition to enhancement of growth andproductivity, the chilli plants treated witholigochitosan also displayed an ability to protect
aphid infection.
Application protocol for chilli
Dilution of 20,000 ppm oligochitosan solution
with water. Chilli pot test: Spraying 80 ppm
oligochitosan once a week.

83

VIETNAM
PGP Guide Sheet
Fill-in Dateyy/mm/dd

2014/1/13

Country

VietnamName of Plant

Rice

Species

Japonica

Planting time,
period, season

April ~ May

Harvest time,
period, season

Sep. ~ Oct.

Location(Temperature,
Humidity etc.)

Temp (25~28 C), 80%RH(25C)

Groundconditions,
Climate, etc.

Paddy red soil, subtoropical,

Product
information
Material

Chitosan flake from Crab and/or shrimp shells

Molecular weight(kDa)

Oligochitosan 30,000 ppm used with averagemolecular weight 10 kDa

Measurement
methodGPC

InitialConcentration(ppm)

30,000 ppm

other additive
Methodology

84

Cultivations

Field test of oligochitosan on rice was performed incollaboration with Mekong Delta Rice ResearchInstitute, Can tho City. The germination study onrice seed found that rice seed soaked with 100 ppm
oligochitosan and sprayed 4 times with 30 ppm
oligochitosan on rice plant in field with 10-15 daysfor one spraying time interval. Result of field test,
rice yield increases up from 10 to 20% compared tocontrol without oligochitosan. Results also
indicated that oligochitosan can be used as plant
elicitor and growth promoter for rice

Recommend
applications

direct seeding method

transplanting method

Germination

Rice seed treatment,
dilution of 30,000 ppm
oligochitosan solution
with 300 times water(1/300). Rice seeds are
dipped in oligochitosan
solution withconcentration of 100
ppm about 24 hours andcontinue with 24 hours
dripping.

1. Rice seed treatment,
dilution of 30,000 ppm
oligochitosan solution
with 300 times water(1/300). Rice seeds are
dipped in oligochitosan
solution withconcentration of 100
ppm about 24 hours andcontinue with 24 hours
dripping.
2. Transfer seeds on
seedling field.
3. After 12 for seedling
growth, transfer
seedlings to rice field.

Timing
Foliar spray(Germination
stage)
Concentration
(ppm)

85

Foliar spray(Field stage)

Spray on day 15, 30, 45
and 60

Rice field: Spray time of
30 ppm oligochitosan
with 10-15 days for one
spraying time interval

Concentration
(ppm)

30

30

Effect of foliar
spray frequency
on Plant Growth& Elicitor（crop yields.
length, weight,
etc. of Sprayed
and Control )Application protocol for rice germination
1. Rice seed treatment, dilution of 30,000 ppm
oligochitosan solution with 300 times water (1/300).
Rice seeds are dipped in oligochitosan solution withconcentration of 100 ppm about 24 hours andcontinue with 24 hours dripping.
2. Transfer seeds on seedling field.
3. After 12 for seedling growth, transfer seedlings
to rice field.
The germination study on rice seed found that riceseed soaked with 100 ppm oligochitosan overnight
the germination increased from 88.0 to 94.4% forcontrol and treated rice seeds, respectively. In
addition, fresh biomass of rice seedlings increased
from 1.18 g/seedling to 1.7 g/seedling for control
and treated rice seeds, respectively.

Fig.1. Increase of germination rate of rice seeds
treated with 100 mg/L oligochitosan
86Fig.2. Increase of fresh biomass of rice seedlings
when treated rice seed with 100 mg/L oligochitosan
Application protocol for rice field
1. Dilution oligochitosan 30,000 ppm with 1000
times by water to 30 ppm.
2. Spraying oligochitosan 4 times on day 10, 20, 30
and 40 after 10 days of planting. 400-500 L of
diluted oligochitosan applies for 1 hectare whichmeans about 0.5 L of original oligochitosan withconcentration 30,000 ppm/ha/spraying time.

87