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Archives of Applied Science Research, 2014, 6 (4):124-128
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ISSN 0975-508X
CODEN (USA) AASRC9

The reduction of mimosine content in Leucaena leucocephala (petai belalang)

leaves using ethyl methanesulphonate (EMS)
Mohamed Zaky Zayed1, Fasihuddin Badruddin Ahmad2, Mohamed Abdallah Zaki3,
Wei-Seng Ho1* and Shek-Ling Pang4
1

Forest Genomics and Informatics Laboratory (fGiL), Department of Molecular Biology, Faculty of Resource
Science and Technology, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak
2
Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Kota
Samarahan, Sarawak
3
Department of Animal Production, Faculty of Agriculture, Alexandria University, Alexandria, Egypt
4
Applied Forest Science and Industry Development (AFSID), Sarawak Forestry Corporation, Kuching, Sarawak
*email: wsho@frst.unimas.my
_____________________________________________________________________________________________
ABSTRACT
A study was conducted to reduce the mimosine content of Leucaena leucocephala leaves by using ethyl
methanesulphonate (EMS). The seeds were treated with three different doses of EMS (i.e., 0.1%, 0.3% and 0.6%)
aiming at reducing the mimosine content and improving the nutritive value of L. leucocephala. The mimosine
content, crude protein and crude fiber were decreased with the increasing of EMS doses. The effect of 0.6% EMS
treatment was much more beneficial as compared to 0.1% EMS or 0.3% EMS in the reduction of the mimosine
content in L. leucocephala leaves. Mimosine content was successfully reduced from 1.6% to 0.2% or a reduction of
87.5% obtained in 0.6% EMS treated samples. This result has led to the identification of a new L. leucocephala
clone with reduced mimosine content from the 0.6% EMS treated samples. Livestock feed should not contain more
than 30% of L. leucocephala as the mimosine can cause hair loss and stomach problems in animals. This limitation
can be overcome by using this newly identified L. leucocephala clone. The livestock feed now can contain more than
30% of L. leucocephala leaves and still have all the Leucaena benefits. However, treatment with 0.6% EMS
decreased the nutritive values of L. leucocephala leaves, but the crude protein of 0.6% EMS treated samples
(18.69%) was still higher than Medicago sativa (alfalfa) with 14.83% crude protein. With this clone, it assists the
foresters or farmers to reduce the livestock feed price significantly in future.
Keywords: Leucaena leucocephala, ethyl methanesulphonate (EMS), mimosine, mutagenesis, nutritive value, clone
_____________________________________________________________________________________________
INTRODUCTION
Leucaena leucocephala (Lam) de Wit. or locally known as petai belalang belongs to family leguminosae. It is a
perennial leguminous tree native to Central America with a wide distribution in the tropics and subtropics, and
suitable for growing in marginal and sub-marginal lands with a wide assortment of uses. It is cultivated for
multipurpose uses, e.g. forage or fodder, lumber, fence posts, fuelwood, charcoal, pulp and to soil improvement [1].
L. leucocephala has been planted widely in agroforestry applications. It is useful for forming erosion barriers,
windbreaks, for shade and support, green manure and cut fodder. The seed is used to derive various medicines like
stomach diseases, use of contraception and abortion. All parts of the plant are edible to animals, including leaves,
young stem, flowers, young and mature pods, and seeds [2]. Leucaena foliage (leaflets plus stems) contains both
nutrients and roughage and makes a ruminant feed roughly comparable to alfalfa forage. It is a rich source of protein
(15-38%), produce up to 20 metric tons of dry matter per ha and the foliage is highly digestible (60-70%) [3]. The
leaves are also a rich source of carotenoids and vitamins. L. leucocephala is often being described as the alfalfa of

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the tropics. It provides high protein forage for ruminants such as cattle, water buffalo, sheep and goats which
increases milk production [4].
Leucaena foliage has been used as a rich source of protein for both ruminants and non-ruminants. However, its
leaves contain mimosine that is toxic when ingested by animals. Reported toxicity signs in fish, shrimp and cattle
were reduced growth and feed efficiency, and increased mortality [2, 5, 6]. The nutritive value of the L.
leucocephala leaf meal can be improved if most of the mimosine is removed or degraded to a relatively less toxic
compound. Mimosine is a free amino-acid very often present in certain legumes which include L. leucocephala.
Mimosine and its degradation product 3-hydroxy-4(1H)-pyridone (DHP) are both toxic when ingested by both
ruminants and non-ruminants [2]. The animal varies in their ability to tolerate dietary levels of L. leucocephala
according to their ability to break down mimosine to DHP and degrade DHP. Some of the L. leucocephala cells that
harbour mimosine contain the enzyme which is necessary to convert mimosine to DHP [7]. Generally, less than 30%
L. leucocephala meal of diets is considered safe for feeding cattle without signs of illness but it should not be for
more than six months otherwise the result may be general illness, production of goiters, loss of hair, and reduced
fertility. Meanwhile non ruminants do not tolerate rations that contain more than 5% to 10% L. leucocephala [8].
The mutation assisted breeding can play an important role in crop improvement either directly or by supplementing
the conventional breeding. The direct use of mutation by ethyl methanesulphonate (EMS) is a valuable approach to
plant breeding, particularly when it is desired to improve one or a few characters in a well-adapted variety. EMS has
been reported to be the most effective mutagen and typically it produces only point mutations [9]. EMS is the most
commonly used mutagen in plants as it causes a high frequency of nucleotide substitutions without substantial
killing, as detected in different genomes [10]. Thus, the present study was aimed to produce L. leucocephala plant
with reduced mimosine content through EMS induced mutations.
MATERIALS AND METHODS
The mutagenic treatments
The completely randomized design containing four replicates was used in this study. Each replicate contained four
treatments and 120 seeds. The seeds were pre-soak in distilled water for one hour and then air dried before soaked in
different EMS concentrations in petri dishes for three hours. Laboratory temperature during the treatment was 21 oC,
whereas the relative humidity was about 59%. Four different concentrations of EMS were used, i.e. 0.0% (distilled
water, control), 0.1%, 0.3% and 0.6% EMS. After the treatment, seeds were washed in distilled water for 15 minutes;
air dried and then soaked in 100oC water for 20 seconds followed by soaking for 24 hours to 48 hours in 25 oC water.
The treated seeds were then sown in seed trays.
Mimosine content
The fresh leaves of L. leucocephala were harvested at the age of seven months after planting. 1 g of the fresh leaves
was added with 10 ml of 0.1 N HCl in a flask. The mixture was vortexed for 1-2 minutes and then extracted for 24
hours. HPLC (Shimadzu CTO-2A) UV-Vis detector analysis was performed on analytical column C18 (4.6 X 150
mm, 5 mM) at 60oC. A total of 20l of sample solution was injected into the column and eluted with a mobile phase
of 0.2% (w/v) orthophosphoric acid, and detected using UV at 280 nm based on the procedure of Puchala et al. [11].
Nutritive value
Leaves samples were ground into fine powder by using a grinder. The samples were analyzed for dry weight (DW),
crude protein (CP), crude fat (EE), crude fiber (CF), organic matter (OM) and total ash (TA) as described by the
Association of Official Analytical Chemists (AOAC) [12]. Moisture was determined by drying the samples at 105oC
overnight and the loss in weight was reported as a percentage of moisture. Nitrogen free extract (NFE) in the
samples were calculated using the following equation: NFE = 100- (Moisture + CP + EE + CF). Gross energy (GE)
was calculated as 5.65, 9.45 and 4.12 Kcal per 100 gram of protein, lipid and carbohydrate, respectively.
RESULTS AND DISCUSSION
A total of 64 L. leucocephala seedlings were analysed in the present study. Mimosine content in L. leucocephala
was analysed by using the HPLC method and the results are given in Table 1. Mimosine content was 1.6%, 0.8%,
0.7% and 0.2% in the control (0.0%), 0.1%, 0.3% and 0.6% EMS treated samples, respectively for L. leucocephala.
Mimosine content decreased significantly with the concentration of EMS used in this study. The mimosine content
recorded was the highest (1.6%) in 0% EMS (control) and the lowest (0.2%) in 0.6% EMS treated samples. The
present results were nearly similar to those reported by EL-Ashry et al. [13], with mimosine content of 1.92% for L.
leucocephala leaves (untreated sample) at the age of seven months. Similar results were also reported by
Mutayobaet al. [14], with mimosine content of 1.89% for L. leucocephala leaves.

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Table 1: The Mimosine content in L. leucocephala leaves
EMS treatment (%) Mimosine content (%) Mimosine content reduction (%)
Control, 0.0%
1.6a
0.0
0.1%
0.8b
50.0c
0.3%
0.7b
56.3b
c
0.6%
0.2
87.5a
LSD0.05 means in the same column within each item having different superscripts are
significantly different (p< 0.05)

The nutritive values of L. leucocephala leaves are summarized in Table 2. The crude protein ranged from 18.69% in
0.6% EMS to 23.48% in the control samples, the crude fiber ranged from 9.61% (0.6% EMS) to 18.77% (0.0%
EMS), the crude fat ranged from 2.66% (0.6% EMS) to 4.86% (0.1% EMS), the total ash ranged from 8.91% (0.6%
EMS) to 9.65% (0.3% EMS), the nitrogen-free extract ranged from 36.24% (0.0% EMS) to 51.21% (0.6% EMS)
and the gross energy ranged from 313.25 kcal/100g (0.0% EMS) to 349.12 kcal/100g (0.1% EMS). The nutritive
value in the control samples showed the highest in crude protein and crude fiber, meanwhile the lowest in crude
protein, crude fiber, crude fat and total ash for 0.6% EMS samples of L. leucocephala. Crude protein was decreased
with the increasing doses of EMS. The crude protein was the highest (23.48%) in the control and the lowest (18.69%)
in 0.6% EMS treated samples. This result is comparable to Medicago sativa (alfalfa) with the nutritive value of
14.83% crude protein, 27.23% crude fiber, 2.15% crude fat, 6.27% total ash, 90.24% organic matter, 39.76%
nitrogen-free extract, 267.92 kcal/100g gross energy and 55.35 mg cp/kcal P/E ratio as reported by Babth et al. [15].
This result is also in good agreement with other published results [13, 16, 17, 18, 19, 20, 21, 22]. They found that
the nutritive values for L. leucocephala were in the range of crude protein (20% - 31%), crude fiber (5% - 14%),
crude fat (6% - 13%), nitrogen free extract (35% - 55%), crude ash (4% - 9%) and dry matter range (87% - 95%).
Table 2: The nutritive values of L. leucocephala leaves
CP
CF
EE
TA
OM
NFE
GE
P/E
(%)
(%)
(%)
(%)
(%)
(%)
(kcal/100g) (mg cp/kcal)
0.0
23.48a 18.77a 3.31b 9.09b 90.89b 36.24c
313.25b
74.96a
0.1
20.52b 10.94c 4.86a 9.11b 90.88b 45.45b
349.12a
58.78c
b
b
a
a
b
c
b
0.3
20.50
17.16
4.11
9.65
90.34
38.92
315.01
65.08b
0.6
18.69c
9.61c
2.66c 8.91c 91.08a 51.21a
341.72a
54.69d
LSD0.05 means in the same column within each item having different superscripts are
significantly different (p< 0.05)

EMS treatment (%)

L. leucocephala has been known as a high potential fodder from several centuries. Its nutritional value is comparable
or superior with that of M. sativa (alfalfa) with high -carotene content [23]. The leaves of L. leucocephala are most
commonly used to feed chicken and pigs, and processed in a pelleted form for freshwater fish. The dry matter
digestibility (DMD) of L. leucocephala was 57.7% and crude protein based on dry matter was 29.5% [24]. Several
reports showed that L. leucocephala could be a substitute for the imported protein supplements fed to animals. In
Queensland, Australia, a very high live weight gain of animals was recorded by using L. leucocephala leaves [4, 8,
25, 26]. Dairy cows produce higher milk yield and fat content when they are fed with L. leucocephala compared to
similar cows fed on pasture and ammoniated rice straw in grass-based diet [27]. Feeding cows and buffaloes on L.
leucocephala foliage at 10% of their diets produce higher milk yield by 20% than that of the control group [28].
However, livestock feed should not contain more than 30% of L. leucocephala. For instance, inclusion of 20% to 25%
of fresh L. leucocephala leaves in diet resulted up to 55% mortality of female and young rabbits [29, 30] or
decreased in growing and performance for rabbits [31]. These low performances may be due to the toxicity of
mimosine or poor amino acid digestibility [32].
The present study has revealed that 0.6% EMS treatment was much more beneficial as compared to 0.1% EMS or
0.3% EMS in reducing the mimosine content in L. leucocephala leaves. Mimosine content was successfully reduced
from 1.6% to 0.2% or a reduction of 87.5% in this 0.6% EMS treated samples, and therefore a new clone of L.
leucocephala (0.6% EMS) with reduced mimosine content and high crude protein (18.69%) was identified from this
study. As reported earlier, livestock feed should not contain more than 30% of L. leucocephala as the mimosine can
cause hair loss and stomach problems. However, this limitation can be overcome by using this newly identified L.
leucocephala clone. The livestock feed now can contain more than 30% of L. leucocephala leaves and yet it still
have all the Leucaena benefits. However, treatment with 0.6% EMS decreased the nutritive value in the L.
leucocephala samples, but the crude protein percentage (18.69%) was still considered high as forage quality
according to Khamseekhiew et al. [33]. They found that L. leucocephala is the most widely used species as a
valuable fodder for enhancing animal production in the tropics and the crude protein of edible materials of L.
leucocephala ranged from 14% to 30%. In fact, the crude protein percentage of 0.6% EMS treated samples was
higher than M. sativa (alfalfa) with 14.83% crude protein.

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CONCLUSION
To the best of our knowledge, this is the first report on the reduction of mimosine content in L. leucocephala leaves
using EMS-induced mutations. Mimosine content has decreased significantly with the increasing doses of EMS as
observed in the present study. The effect of 0.6% EMS treatment was much more beneficial as compared to 0.1%
EMS or 0.3% EMS in reducing the mimosine content, and this result has led to the identification of a new L.
leucocephala clone from the 0.6% EMS treated samples. The forage quality of L. leucocephala as livestock fodder
can be improved now by using this newly identified clone and it can be used as a single fodder without any problem
for ruminant and non-ruminant animals. The livestock feed now can contain more than 30% of L. leucocephala
leaves. In addition to that, it also assists the foresters or the farmers to reduce the livestock feed price in future.
Acknowledgments
This work is part of the joint Industry-University Partnership Programme, a research programme funded by the
Sarawak Forestry Corporation (SFC), Sarawak Timber Association (STA), Ministry of Higher Education (MoHE)
and Universiti Malaysia Sarawak under grant no. 02(DPI09)832/2012(1), RACE/a(2)/884/2012(02) and
GL(F07)/06/2013/STA-UNIMAS(06).
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