Plant Tissue Cult.

12(2) : 167-172, 2002 (December)

PTC
Regeneration of Plantlets Through Somatic
Embryogenesis from Nucellus Tissue of Citrus
macroptera Mont. var. anammensis ('Sat Kara')

M. N. Miah, Sahina Islam
1
and Syed Hadiuzzaman
2


Institute of Life Sciences, National University, Gazipur, Bangladesh

Key words : Nucellus, Somatic embryogenesis, Plantlet regeneration,
Citrus macroptera.

Abstract
Somatic embryogenesis and plantlet regeneration were achieved in callus
cultures of nucellus derived from undeveloped ovule of immature fruits of
Citrus macroptera. Four types of media were used but only modified MS medium
supplemented with malt responded well. Calli produced in malt supplemented
MS medium were embryogenic in nature. After transfer of this embryogenic
callus in hormone free MS medium, somatic embryos were developed.
Independent plantlets were developed from these somatic embryos by further
subculture in the same medium. After five weeks of culture the plantlets were
transferred to pot soil mixed with cow-dung derived biogas slurry and survived
well.

Introduction
Citrus macroptera Mont. var. anammensis is commonly known as Sat Kara, the
most popular and expensive citrus fruits grown in greater Sylhet areas of
Bangladesh. It is a semi wild species and used as medicine by local tribes of
Assam, India (Ghosh, 1990). In Bangladesh, both green and mature fruits are
used for cooking and pickle preparation. The sweet flavor of fruits due to
essential oil of flavedo parts may be used in flavoring purposes in perfume
industries. Every year, Bangladesh earns handsome amount of foreign currency
by exporting these fruits especially to UK, USA and Middle East countries. Like
other citrus species Sat Kara is also propagated conventionally by means of
seeds, grafting and budding methods. Due to present demand of this fruit in
both local and foreign markets it is necessary to develop a suitable protocol for
mass propagation from existing elite cultivars.
-----------------------------------------
1
IFRD, BCSIR, Dr. Kudrat-E-Khuda Road, Dhanmondi, Dhaka-1205, Bangladesh.
2
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.
168 Miah et al.
The process of somatic embryogenesis is a suitable method of micro-
propagation and has the potential of mass propagation commercially at low cost
per unit. Conventional breeding methods with woody perennial crops have
hampered because of a large generation gap of 6 - 8 years before they can be
assessed together with the difficulty associated with working on tissue of mature
origin. Although there is no available information from Citrus macroptera yet,
successful somatic embryogenesis from nucellus tissues of different citrus
species was achieved by several researchers (Rangan and Murashige 1969;
Bitters et al. 1972; Kochba et al. 1972; Starrantino and Russo 1980; Pasqual et al.
1984; Chen et al. 1990 and Gill et al. 1994. Moreover, the nucellus is a non-
vascularized tissue of maternal origin and can be regarded as a pocket of
juvenile tissue in an otherwise adult plant (Button and Kochba 1977).
The present investigation was undertaken to develop the in vitro suitable
protocols for micropropagation of Sat Kara plants from nucellus through somatic
embryogenesis.

Materials and Methods
Four to five weeks old immature fruits were collected from Citrus Research
Station, BARI, Jaintapur, Sylhet. Fruits were washed thoroughly under running
tap water to reduce dust and surface contaminants. Then they were dipped in
90 % alcohol for one minute followed by 4 % sodium hypochlorite solution for 10
min and finally rinsed four times with sterile distilled water under laminar
airflow cabinet. The fruits were then placed on an autoclaved ceramic tile and
cut open by sharp sterilized knife and then undeveloped ovules/immature
seeds were separated. For somatic embryogenic callus induction the seeds were
cut by scalpel and nucellus halves were separated and cultured on a semi-solid
modified MS medium supplemented with 500 mg/l malt extract called it somatic
embryogenic medium1 (SEM1) after Tisseret and Murashige (1977). At the same
time nucellus halves were cultured into MS + 0.1 mg/l 2,4-D + 0.05 mg/l Kn
(SEM2), MS + 0.5 mg/l 2,4-D + 0.1 mg/l Kn (SEM3) and MS + 1 mg/l 2,4-D + 0.5
mg/l Kn (SEM4) media. The nucellus halves were taken in conical flasks and
containing five samples with three replicates for each medium.The responded
calli were further subcultured on same media and simultaneously into hormone
free MS medium.
The pH of all media was adjusted to 5.7 before addition of agar and
sterilized by autoclaving for 20 minutes at 1.05 kg/cm
2
(15 psi) pressure at
121_C. An amount of 10 gm/l agar (BDH) was used for SEM1 and 0.8 gm/l for
other three media. The flasks containing explants were incubated on culture
Regeneration of Plantlets Through Somatic Embryogenesis 169
racks. The cultures were maintained at 25_C 2 under the cool white
fluorescent lights for 16 hr photoperiod.

Results and Discussion
Four types of somatic embryogenic media were used to observe the embryogenic
callus induction and plant regeneration from nucellus explants. Time for callus
initiation was between 8 and 15 days in all media. The results obtained from
this experiment are presented in Table 1. Among all the media tested, calli
developed in SEM1 were only embryogenic in nature. These calli were loose
light green and having good growth (Fig. 1). Calli obtained in other media were
compact and initially green but with the lapse of time they turned brown.
Embryogenic calli when transferred to hormone free MS medium somatic
embryos were developed (Fig. 2). Complete plantlets were developed from these
somatic embryos by further subculture in the same hormone free MS medium
(Fig. 3).

Table 1. Effects of different somatic embryogenic media on somatic embryogenesis of C.
macroptera.

Name of
media
% of
ncellus
responde
d
Days to
callus
initiation
Nature of
response
Response for
somatic embryos
SEM1
MS + 500 mg/l malt
extract
73.33 7 - 12 Loose, light green
callus having good
growth
Somatic
embryos
developed
SEM2
MS + 0.1 mg/I 2,4-D
+ 0.05 mg/l Kn
50.00 9 - 15 Compact callus,
initially green but
brown later. No
further growth
Nil
SEM3
MS + 0.5 mg/I 2,4-D
+ 0.1 mg/l Kn
41.66 8 - 13 Compact callus,
initially green but
brown later. No
further growth
Nil
SEM4
MS + 1 mg/I 2,4-D
+ 0.5 mg/l Kn
41.66 8 - 13 Compact, whitish
swelling only. No
further growth
Nil

Regeneration of plants via somatic embryogenesis has been preferred as a
method for multiplication of viable germplasm in many woody plants (Bonga
1987). The somatic embryogenic callus and plantlets were obtained from
nucellus of many citrus species on malt supplemented SEM1 medium. Tisserat
and Murashige (1977) obtained that this medium was suitable to develop a
protocol for somatic embryogenesis from nucellus of citrus species. However,
some of the researchers used MT (Murashige and Tucker 1969) medium
170 Miah et al.
supplemented with malt extract for somatic embryogenesis from nucellus of
sweet orange (Pasqual et al. 1984), and some selected species of citrus (Pimental
and Villegas 1993).



Figs. 1 - 5 : Regeneration of plantlets through somatic embryogenesis in Citrus macroptera.
1. Compact embryogenic callus from nucellus after four weeks of culture in malt
supplemented modified MS. 2. Development of somatic embryos in callus after four
weeks of subculture in basal MS. 3. Development of plantlets from somatic embryos
after second subculture in basal MS. 4. Complete plantlet with shoot and root
developed from somatic embryos. 5. Single established plant in pot soil.

A limited number of treatments were used in this experiment to induce
somatic embryogenesis in C. macroptera from nucellus. Successful embryogenic
calli were developed only from SEM1 medium. In case of auxin-cytokinin
supplemented media initially calli were induced but these were not
Regeneration of Plantlets Through Somatic Embryogenesis 171
embryogenic. But, Pasqual and Ando (1988) used 2,4-D and Kn for somatic
embryogenesis from nucellus of sweet orange cv. valecnia and obtained some
somatic embryogenic callus.
Single primary root growth (Fig. 4) was observed in each plantlet and the
mean height of plantlets was 13 mm. Rajdan (1993) mentioned that embryos
might germinate on agar medium without any growth regulators. However,
Chen et al. (1990) observed GA
3
was effective for embryoid to plantlet
regeneration. The plantlets thus obtained through somatic embryogenesis
were transferred into soil mixed with biogas slurry 1 : 1 ratio (Fig. 5) and the rate
of survival was 100%.

Acknowledgement
A financial grant in the form of a scholarship to the first author (MNM) by the
National University, Gazipur, Bangladesh to accomplish this work is gratefully
acknowledged.

References
Bitters WP, Murashige T, Rangan TS and Nauer E (1972) Proc. 5th Conf. Intern. Org.
Citrus. Viral. Univ. Press. Gainesville. pp. 261 - 271.
Bonga JM (1987) Clonal propagation of mature trees. Problem and possible solution. In:
Bonga, J. M and Durzan, D.J (eds) Cell and Tissue Culture in Forestry. 249-271,
Martinus Nijhoff Publishers, Boston.
Button J and Kochba J (1977) Tissue culture in the Citrus Industry. In: Reinert, J. and
Bajaj, Y.P.S. (Eds.), Applied and Fundamental Aspects of Plant Cell, Tissue and
Organ Culture, Springer-Verlag, Barlin-Heidelberg. pp. 70 - 72.
Chen RZ, Li GG, Zhang LY and Li KL (1990) A preliminary study of the factors effecting
embryogenesis and plant regeneration from nucellus callus of C. reticulata cv.
ponkan. Acta-Botanica. Austrosinica. No. 6 : 75 - 80.
Ghosh SP (1990) Citrus. In: Fruits - Tropical and Subtropical, T.K. Bose and S.K. Mitra
(Eds.), Naya Prokash, Calcutta, India. pp. 63 - 131.
Gill MIS, Sing Z, Dhillon SS and Gosal SS (1994) In: Propagation of tropical and
subtripical crops. Bose, T.K., Mitra, S.K., Sadhu, M.K. and Das, P. (Eds.), Naya
Prakash, Calcutta, India. 219 p.
Kochba J, Spigel-Roy P and Safran H (1972) In: Propagation of tropical and subtripical
crops. Bose, T.K., Mitra, S.K., Sadhu, M.K. and Das, P. (Eds.), Naya Prakash,
Calcutta, India. 213 p.
Murashige T and Tucker DPH (1969) Growth factor requirements of citrus tissue culture.
In. Proc. Ist. Intl. Citrus Symp. Riverside (1968-1969) 3 : 1155 - 1161.
172 Miah et al.
Pasqual M, Cromoko OJ and Ando A (1984) In: Anais do VII Congresso Brasileiro de
Fruticultura Florianpolis, Brazil. Empressa Catariense de Pesquisa Agropecnaria
S.A. 1: 402-7.
Pasqual M and Ando A (1988) In: Propagation of tropical and subtripical crops. Bose,
T.K., Mitra, S.K., Sadhu, M.K. and Das, P. (Eds.), Naya Prakash, Calcutta, India.
212 p.
Pimental RB and Villegas VN (1993) Induction of somatic embryogenesis in selected
Citrus species. Philipine Journal of Crop Science. 18 : 12.
Rajdan MK (1993) An Introduction to Plant Tissue Culture. Oxford and IBH Publisher,
New Delhi, India. 94 p.
Rangan TS and Murashige T (1969) Proc. Ist. Int. Sy. Citrus Symp. Riverside. 1: 225-29.
Starrantino A and Russo F (1980) In: Propagation of tropical and subtripical crops. Bose,
T.K., Mitra, S.K., Sadhu, M.K. and Das, P. (Eds.), Naya Prakash, Calcutta, India.
213 p.
Tisseret B and Murashige T (1977) Probable identity of substances in Citrus that repress
asexual embryogenesis. In vitro 13 : 785 - 789.