B I O D I V E R S I T A S ISSN: 1412-033X
Volume 7, Nomor 3 Juli 2006
Halaman: 297-301
♥ Correspondence address:
Jl. Tentara Pelajar 3A Bogor 16111.
Tel. +62-251- 337975, Fax. +62-251-338820
e-mail: borif@indo.net.id
R E V I E W:
In Vitro Selection and Somaclonal Variation for Biotic and
Abiotic Stress Tolerance
ENDANG GATI LESTARI
♥
Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD), Bogor 16111.
Received: 22
nd
May 2006. Accepted: 29
th
June 2006.
ABSTRACT
As an alternative technology, plant improvement through somaclonal variation is expected to support conventional breeding. New superior
variants with a better performance and more attractive texture could be obtained through this method. To enhance genetic variation, both
physical and chemical treatments such as gamma ray (Co
60
) and Ethyl Methane Sulphonate (EMS) compound could be applied. In
particular for vegetative propagated plants, in vitro induced mutation is the most effective method to improve variation. For obtaining the
desired characteristic of plant, in vitro selection is the best method due to its capability to manipulate the variation to the expected result.
Therefore, by applying the selection agent to the media, plant tolerance to both abiotic and biotic could be acquired. Generally, the
tolerance at the callus level at the specific selection agent is positively correlated with the tolerance at the plant level. At this point, PEG
(polyethylene glycol) and manitol is chemical compound useful for drought tolerance, fusaric or filtrate is for fusarium wilt, A1Cl
3.
6H
2
O is for
Al tolerance.
© 2006 Jurusan Biologi FMIPA UNS Surakarta
Key words: somaclonal variation, in vitro selection, induced mutation.
INTRODUCTION
Plant improvement through somaclonal variation and in
vitro selection is some techniques of in vitro culture for
obtaining plant genotype tolerance to the biotic or abiotic
stress, such as drought, high salinity, Al stress, acid soil,
and disease tolerance (Ahmed et al., 1996; Yusnita et al.,
2005). In addition, the plants are expected to have some
desired characters such as having bigger fruit size, more
interesting flower texture, more delicious taste and higher
production (Pedrieri, 2001; Ahloowalia and Maluszynski,
2001; Witjaksono, 2003). Ahloowalia (1986) states, that
somaclonal variation would give advantages if it increases
genetic variation, particularly the character which is not
obtained at the mother plant. Because of its low genetic
variation, particular plants which are only vegetative
propagation or self-pollination, desired characters could be
obtained through somaclonal variation.
In vitro selection is one of somaclonal variation method.
Its effectiveness and efficiency are due to its ability of
changing the plant to the desired character, either by
applying a selection agent on the culture media or by giving
particular condition to change the somaclone with the
required character (Van den Bulk, 1991; Karp, 1995).
Somaclonal variation occurs at the plant resulted from cell
regeneration during in vitro culture period, generally are not
originated from the axilar shoot or tip-shoot. Somaclonal
variation are caused by several factors, such as: by
genotype and polyploidy level, environment during the
growing period, the applied growth regulator, culture period
and the applied procedure (Maluszynski et al., 1995) and
the presence of selection agent.
Somaclonal variation occurs among the population of
plant resulted from in vitro culture. It is apparently caused
by gene amplification, the alteration of a basic couple,
transposing migration, methylation transform, chromosome
instability, chromosome inversion, one spot mutation,
translocation, ploidy change, restructuring or deletion
(George and Sherington, 1984; Phillips et al., 1990; Dennis,
2004; Kumar and Marthur, 2004). The high range of
mutation is called chimera, pleytrophy, genetic instability
and epigenetic variation. Epigenetic variation is the one
when the resulted change was so unstable that the resulted
expression returns to its origin. The new mutant could be
created through induced mutations by using chemical
mutagen such as ethylene scimine (ES), diethyl sulphonate
(DES), ethyl methane-sulphonate (EMS), and the azida
group. Physical mutagen frequently used is x-rays, gamma-
rays (Co
60
), fast neutron (nf) and thermal neutron (Nth).
Through in vitro selection to numerous mutant or genetic
material is possible (Maluszynski et al., 1995). Likewise, in
vitro selection could also be conducted at the population
such as cell and callus and shoot resulted from
regeneration in the small area and in the controlled
condition. Application of the selecting agent in the culture
media, in vitro method is very advantageous. Only such a
tolerant plant is capable to grow that to obtain the plant with
the desired character does not take a long time (Biswas et
al., 2002). The achievement of in vitro selection technique
to obtain the tolerant plant requires the availability of: (i)
'2,ELRGLYG
BIODIVERSITAS Vol. 7, No. 3, Juli 2006, hal. 297-301
298
high variation of cell, (ii) easy application of in vitro selection
method, (iii) regeneration method of tolerant cell
(Widoretno, 2003), and (iv) the desired character to be
inherited (Yusnita, 2005).
IN VITRO SELECTION FOR FUSARIUM DISEASES
TOLERANCE
The availability of the appropriate selection agent is the
determining factor for the optimum result. Generally, the
selection agent used is fungi culture filtrate or the familiar
toxin such as oxalate acid and fusaric acid (Matsumoto et
al., 1995). Fusaric acid (C
10
H
13
O
2
N) is a metabolite product
from many strain of Fusarium oxysporum and applied as
“selecting agent” for the cell culture and callus culture to
inhibit the fungi germination. Based on the reality, toxin or
filtrate can be used for selecting agent because there is a
relationship between toxin tolerance and disease tolerance.
Application of fusaric acid or filtrate for in vitro selection
has frequently been applied for tomato, banana (Mariska et
al., 2005), and abaca (Damayanti, 2002; Sukmadjaja et al.,
2003). The result of the experiment showed that the new
regenerate resulted from toxin tolerant cell or tissue is also
tolerant to disease. Accordingly, Jin et al. (1993) obtained
filtrate culture from Fusarium solani, which has pathogenic
characteristic, inhibit the callus culture growth of soybean
that was susceptible to death syndrome disease.
Fusaric acid produced by Fusarium oxysporum is a
toxically organic component functioning among others: to
inhibit cytocinine oxidation, to restrict respiration process at
mitochondria, as well as to ATP at the membrane plasma
and polyvinyl oxidation activity. In short, it inhibits growth
and regeneration (Sukmadjaja et al., 2003). According to
Mariska et al. (2005) the fusaric acid application at a rate of
45 mg/l on the green and yellow “Ambon” banana (Musa
paradisiaca Sp.) was capable of inhibiting the controlled
propagation for 80%. Meanwhile, shoot radiated at the
dosage of 5,7.5 and 10 gray inhibit 60, 60 and 26%. Using
Fusarium oxysporum conidia in rice culture of 5 g/5 kg soil
and 2.5g/5 kg soil, the resistance test conducted at the
green house, showed that several number of plants
remained green of being tolerance to the disease because
of radiation with the dosage of 7,5 gray, without selection
and selection using 30 ppm fusaric acid, and 10 gray
without selection and selection using 40% fusaric acid. The
highest percentage of the tolerant plant was produced by
the irradiation treatment with fusaric acid selection.
Greenhouse test of the abaca somaclone by
Sukmadjaja et al. (2003) showed a correlation between
pathogenic fusarium and fusaric acid tolerance. After
inoculation with conidia, the highest percentage of survived
plant was obtained from plant selected with fusaric acid 15-
30 ppm followed by filtrate 50% cross selection, as much as
75% either from the application of 10
4
or 10
5
. Those plants
are creating from the irradiated explants with the dosage of
1 Krad. The use of 15-30 fusaric acid at the plant without
radiation produced 25% tolerant plants (Table 1). For this
reason, induced mutation treatment tends to produce
mutant with increasing disease tolerance. The physical
mutagen treatment with gamma rays combined with in vitro
selection will increase the disease tolerance plant.
Somaclonal variation and in vitro selection applied to
peanut has produced somaclone R1 and R2 which are
resistant to stem-rot disease caused by Sclerotium. Induced
with S. rolfsii in the plastic house, several plant has more
full pods and disease resistant than their mother plant
(Yusnita, 2005). Another example of selecting agent is DON
(dioxynivalenol), toxin produced by pathogenic fungi to
produce wheat which is tolerant to scab disease (Yang et
al., 1998) and fungi culture filtrate containing toxin obtained
from mango which is tolerant to anthracnose disease
(Collectrotrichum glocorporioides Pens) (Jayasandar et al.,
2000). Several plants resulted from somaclonal variation
and in vitro selection is presented at Table 2.
IN VITRO SELECTION FOR AL TOLERANCE
In vitro selection for acid soil and Al toxic tolerance could
be applied with AlCl
3.
6H
2
O as the selection component on
the low acid media as much as pH 4 (Short et al., 1987).
Those method of application has resulted several Al
tolerance plants such as in rice (Van Sint Jan et al.,1997),
soybean (Mariska, 2003), and tobacco (Yamamoto et al.,
1994). Van Sint Jan (1997) shows that one of three Al
tolerance was obtained from unselected callus using Al,
while the others were selected with 250 and 1000 µmol
from the total Al. The Al tolerant plants could not be
determined from selection result because of the somaclonal
variation.
Hutami et al., 2001 attained the somatic embryo
structure of soybean which is capable of proliferation after
selection at the media containing Al and low pH, mainly
which was originated from radiation at 4 gray in Wilis and
Sindoro. Stress condition caused by Al results in the
decreasing capability of growth and the development of
somatic cell. It happened in the tobacco (Yamamoto et al.,
1994), and rice (Purnamaningsih et al., 2001; Edi, 2004).
From in vitro soybean selection using Al and gamma
ray, several acid tolerant somaclones are obtained. Until the
5
th
generation (G5), soybean lines with 60-65 pods is
acquired, of which the stems are higher than their control
plants (Sindoro). In this research, it is shown that there is a
positive correlation between the mass of somatic cell
tolerant to Al and low pH and their tolerance to acid field
(Mariska, 2003). Al toxicity to selection media could be
emerged by modifying macro nutrient MS, i.e. by increasing
NH
4
NO
3
, CaCl
2
(2H
2
O) and decreasing KH
2
PO
4
and the
application of Fe which was not chelated by EDTA
(Purnamaningsih et al., 2001).
In vitro selection in rice varieties (Rojolele and T309)
using 100, 200, 300, 400 and 500 ppm Al, showed that the
higher Al concentration given to the media, the less callus
could regenerate. Equally, no callus regenerated at Al 500
ppm. Based on the nutrient test and using acid soil in the
green house (Edi, 2004), shoot resulted from callus tolerant
to selection media showed the tolerance characteristic to Al
(Purnamaningsih et al., 2001). As indicated by Taylor
(1995) somatic embryo cells remained developed in the Al
and low pH media because of the tolerance characteristic at
the cell level. It is similar to Rath’s research (1996) that only
the tolerant cells survive in the media containing selection
component. From several researches above, it could be
concluded that cell selection is potential for producing new
genotype adapted to the environmental stress (Adkin et al.,
1995). In vitro selection for drought tolerance
In vitro selection for drought tolerance commonly uses
PEG as selection component. This method has been
applied to several plants (Bouslama and Scapaugh, 1994).
Petcova et al. (1995) and Lestari et al. (2005) found a
positive correlation between the capability of germinating
seed at the media containing PEG and the growth of the
LESTARI – In vitro selection for stress tolerance
299
Table 1. Percentage of survived plant resulted from in vitro selection with F oxysporum conidia suspension in the green house after 30
days (Sukmadjaya et al., 2002).
Percentage of living plant
Conidia concentration per ml
Dosage of Radiation (Krad) Early selection Cross selection
10
3
10
4
10
5
0 Without fusaric acid selection (ppm)
Filtrate (%)
0-0
15-10
30-45
Filtrate (%)
50
50
50
0
0
25
-
0
0
25
-
0
0
25
-
1 Without fusaric acid selection (ppm)
Filtrate (%)
0-0
15-10
30-45
Filtrate (%)
50
50
50
0
45
70
75
60
0
50
60
75
40
0
40
60
75
30
Table 2. Several disease tolerant plants resulted from somaclonal and in vitro selection (Yusnita, 2005).
Plant In vitro culture system for Selecting agent Resistant to
Potato Callus culture Fungi Filtrate culture
Phytophtora
Tomato Callus from leaf explants - Fusarium oxysporum f.sp. infestan lycopersici ras 2
Papaya Shoot culture -
Phytoptora palmivora
Soybean Embryonic culture - Fusarium oxysporum f.sp cubense ras 4
Banana Multiple bud clumps Fusaric acid Fusarium sp. ras I
Mango Somatic embryo culture Fungi Filtrate culture
Coleetotricum gloesporoides
Strawberry Morphogenetic callus Rhizoctonia fragariae and Botrytis cemerea
Apple Shoot culture Fungi Filtrate culture
Phytophtora cactorum
Wheat Morphogenic callus Fungi Filtrate culture Fusarium graminearum and Fusarium culmorum
Table 3. Shows PEG, root penetration, proline content tests and grain production after drought stress (Lestari, 2005)
Treatment (radiation
and selection)
Genotype
20% PEG
examination
Root
penetration
test
Proline
nmol/g
Weight of
grain/panicle
Grain/pani
cle%
Decreasing
(%) filled
grain
Notes
Control IR 64 NS NE 17.07 TP TP 100 NT
0 gray (20% PEG) IR64-1 G P 169.6 8.90 62 44 NT
“ IR64-2 G P 140.1 5.51 51 52 NT
5 gray (0%PEG) IR64-17 G P 202.40 0 0 100 NT
“ IR64-3 G P 176.78 23.53 49 17 Tolerant
“ IR64-3.1 NS NE NE NE NE NE NT
“ IR64-3.2 G P 204.45 26.0 58 26 Tolerant
“ iR64-3.3 NS NE NE NE NE NE NT
“ IR64-4.1 G P 150.0 18.97 55 31 Tolerant
“ IR64-4.2 G P 267.2 20.0 53 29 Tolerant
“ IR64-5.1 G NP NE NE NE NE NT
“ IR64-5.2 NS NE NE NE NE NE NT
“ IR64-5.3 G NP NE NE NE NE NT
“ IR64-5.4 NS NP NE NE NE NE NT
“ IR64-7.1 G P 180.18 18.08 52 25 Tolerant
“ IR64-7.2 G P 147.69 19.43 56 30 Tolerant
“ IR64-8.2 NS NP NE NE NT NT NT
“ IR64-8.3 G NP NE NE NT NT NT
“ IR64-11 G P 160.35 12.9 49 36 Tolerant
“ IR64-11.1 G P 156 20.65 61 37 Tolerant
7 gray ()% PEG) IR64-18 NS NP NE NE NT NT NT
“ IR64-19 G NP NE NE NT NT NT
“ IR64-22 G NP NE NE NT NT NT
“ IR64-23 G P 113.6 NE NE 100 NT
Notes: * = drought-stress tolerance indication, NS: no sprouting, NE = not examined, NP = not penetrating, NT = no tolerant, ND = no
producing. G= germination, P = penetrating.
BIODIVERSITAS Vol. 7, No. 3, Juli 2006, hal. 297-301
300
plants under stress condition. It is similar to Dragisga et al.
(1996) who obtained the similar result that PEG could be
applied after treating an osmotic stress on the in vitro selection.
Short et al. (1987) stated that in the in vitro culture, PEG
is capable to induce water stress and positively correlated
with that in the field or the green house (Damii and Hughes,
1997). PEG could be applied for stimulating drought
because it could inhibit water in such a way that no water is
provided for somatic cell, except for the callus/somatic cell
which has particular mechanism for absorbing water. The
research of Adkin et al. (1995) showed similar result. Only
the tolerant callus which bears PEG media selection could
increase its tolerance against drought stress. From the
experiment result, the second generation is tested against
the plant of callus origin selected with PEG media. Result
shows that the dry-weight of the plant is higher than that of
the mother plant.
Selective media applied with PEG could inhibit the
growth and development of the soybean explants and could
decrease the number of the formed somatic embryo
(Widoretno et al., 2003; Husni et al., 2005). The increasing
amount of PEG added to the selective media bring the
deterioration influence of PEG to somatic/callus embryo
(Widoretno et al., 2003). The result shows that the
treatment with 15% PEG will cause 90% tolerant soybean
genotype (explants B3731) remained survived. The
percentage is lower, 54% and 30% respectively, when it is
applied to the moderate genotype soybean (Tidar variant)
and the sensitive soybean genotype (MSC8606).
In vitro selection for obtaining the tolerance to the
drought stress has been conducted to green grams
mungbean (Vigna radiata L.) (Gulati and Jaiwal, 1993), rice
(Oryza sativa L.) (Adkins et al., 1995; Biswas et al., 2002;
Lestari, 2005, 2006), barley (Sorghum bicolor L.) (Duncan
et al., 1995), potato (Prakash et al., 1994) and soybean
(Widoretno, 2003; Husni et al., 2005). Husni et al., 2005
obtained three drought tolerant soybean somaclones
originated from somatic embryo which had been selected
with 20% PEG (BM 6000). During penetration test of the
root, those somaclone have faster capability of penetrating
paraffin layers than that of the previous variants
Tanggamus and Nanti.
Selection to the callus of several rice variety conducted
by Prakash et al., 1994 using 5, 10, 15 mg/l of PEG (BM
6000) successfully obtained drought tolerant plants. Five
plants from three varieties increased their tolerance to
drought. PEG as selection component significantly inhibit
callus growth and therefore it could inhibit seed germination
with drought intolerance. In vitro selection to the rice of
Gajahmungkur, Towuti and IR64 varieties conducted by
Lestari (2005; 2006) using PEG 20% produced some
tolerant plants after listing for their tolerance in green
house, i.e. five plants from Gajahmungkur, 9 from Towuti
and 8 from IR64. Those plants could produce more full
grains per panicle than that of the mother plants.
Germination test for the seed from in vitro selection
shows that the seed from IR64 (mother plant) did not
germinate at the 20% PEG solution (BM 6000); however,
the seed from other somaclone (17 somaclone) germinated
at the 20% PEG solution (Lestari, 2005). The seed from the
germinated somaclone eventually penetrated paraffin layers
at the base of the vase (Table 3). From the test, 12
somaclone could rapidly penetrate paraffin layers, in
addition to the thicker and longer size of root (Lestari et al.,
2005). Proline analysis to the plant showed that the leaf of
the control plants (IR 64) has very low proline content, as
much as 17,07 nmol/g, while the leaves from various
somaclone produce higher proline, 113.6-287.2 nmol/g
(Lestari, 2004).
CONCLUSION
Induced mutation could increase genetic variation to the
plant which eventually could give the advantage for
providing breeders with genetic material for plant selection.
In vitro culture and irradiation effectively produce new
somaclones with desired characteristics. Through in vitro
selection, by giving particular emphasize on the media,
certain expected traits could be produced.
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