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Pak. J. Bot., 47(1): 305-310, 2015.

EMBRYOGENIC CALLUS INDUCTION, SOMATIC EMBRYOGENESIS, REGENERATION


AND HISTOLOGICAL STUDIES OF KINNOW MANDARIN
(CITRUS RETICULATA BLANCO L.) FROM NUCELLAR
EMBRYO AND EPICOTYL REGION
SYEDA KAHKASHAN KAZMI1, SAIFULLAH KHAN1,3*, NURUL KABIR2, AMEER AHMED MIRBAHAR1,
MARIAM RAZIQ4 AND NAHEED KAUSER1
1
Biotechnology Wing, H.E.J. Research Institute of Chemistry,
2
Dr. Panjwani Center for Molecular Medicine and Drug Research,
International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
3
Department of Agriculture and Agribusiness Management, University of Karachi, Pakistan
4
Department of Biomedical Engineering, Sir Syed University of Engineering and Technology Karachi, Pakistan.
*
Corresponding author e-mail: drsaif65@gmail.com; Phone:(92-21) 99261693

Abstract

Citrus is one of the most dominant horticultural fruit crop in the world. The objective of this study was to develop an
efficient protocol for invitro embryogenic callus induction and regeneration of Kinnow mandarin. In this study, different
concentrations of 2,4-D (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/L) along with constant 0.5mg/L BAP were used to check the
effective response of epicotyl segments and nucellar tissues on callus induction and regeneration. It was observed that MS
medium containing 2.5 mg/L 2,4-D and 0.5mg/L BAP supplemented with 0.5gm/L malt extract was suitable for
embryogenic callus induction from both epicotyl segments and nucellar embryonic tissues. Callus embryogenesis was
achieved on simple MS medium where as regeneration was obtained on MT medium containing 0.5mg/L BAP and 0.5mg/L
Kinetin along with 0.5gm/L malt extract. It was observed that the nucellar embryos are the best explants for efficient callus
induction and regeneration. In order to ensure the efficient regeneration ability of nucellar callus histological studies were
performed, which showed that nucellar embryos and its calli have more chloroplasts as compared to epicotyl segments,
which enhanced regeneration ability of nucellar embryos.

Key words: Indirect regeneration, Nucellar embryos, Toluidine Blue O staining

Introduction Frost, at the Citrus Research Center, University of


California, Riverside, USA in 1915. It was introduced,
Among the economically important fruit crops citrus acclimatized and released by researchers working at the
is one of the most widely used. Citrus belongs to the campus of the University of Agriculture Faisalabad
family Rutaceae and are native to Southeast Asia (Khan, 1992). Due to Kinnow mandarin’s exceptional
(Indonesia and China), but now extensively grown almost taste, easy peeling property, beautiful bright orange color
throughout the world under tropical and sub-tropical and pleasant fragrance, it was readily accepted by the
conditions where the soil and climatic conditions are growers (Khan, 1992). Kinnow mandarin export can be
favorable for its growth and yield (Shah, 2004). Its annual increased by introducing a seedless variety because in
production in the world is about 105 million tons international market such varieties are in great demand
(Randhawa et al, 2012). The top six citrus producing and Pakistani Kinnow variety have 20-25 seeds per fruit.
countries are China, Brazil, United States of America, There is a report about retaining seedless varieties in
India, Mexico and Spain (Anon., 2013). In addition to it Pakistan (Khalil et al., 2011), but its propagation is not
citrus fruits are nutritious and are good source of very fast through traditional methods of propagation. To
carbohydrates, dietary fiber, vitamins as well as minerals. overcome this problem, it is necessary to propagate
Its flavor is among the most popular fruit flavors for Kinnow mandarin through tissue culture techniques, such
beverages. They are equally precious for people with as direct and indirect micropropagation from callus
micronutrient deficiencies as well as those who are culture and efficient regeneration so that large number of
concerned about the problems of over nutrition, obesity plants can be obtained in less time. This is a substantial
and diet-related chronic diseases. Thus citrus is an ideal current need of time to conduct the research and develop
component of low-fat, sodium-restricted diets (Economos high temperature and disease resistant as well as salt and
& Clay, 1998). drought tolerant varieties, especially root stocks are
Citrus fruits account for about 40% of the total fruit needed to develop and propagate on a large scale to give
production in the Pakistan where it is cultivated over an support and sustainability to this important crop. For these
area of 194,500 ha with an annual production of about aspects of research, micropropagation by using tissue
1982.2 million tons (Pakistan Bureau of Statistics, 2010- culture technique is the best choice.
11). More than 95% of the citrus fruits are produced in the There are various previous reports available for direct
Punjab province and 70% of the entire crop plantation is and indirect micropropagation of citrus varieties including
Kinnow mandarin (Niaz et al, 2004). Kinnow mandarin is micropropagation of rough lemon through callus
a hybrid between the two varieties, namely Citrus nobilis formation and regeneration in the presence of 2,4-D and
and Citrus deliciosa. This cross was made by Dr. H.B BAP was performed by (Ali & Mirza, 2006). Two years
306 SAIFULLAH KHAN ET AL.,

old callus cultures have been used to continuously five seeds were placed in each jar, where the jars were
generate genetically uniform Citrus acida plantlets placed under dark condition at 25ºC. After three days
(Chakraverty & Goswami, 1999). Callus cultures have seeds were germinated with 100% germination potential,
also been grown from leaf, epicotyl, cotyledon and root as epicotyl region of each seedling became elongated.
segments of in vitro grown nucellar seedlings of Citrus During germination under dark condition it was observed
reticulata Blanco ‘Local Sangtra’ by using very high that nucellar embryos were sprinkled out from some seeds
concentration of NAA (10mg/l) and kinetin 0.5mg/l (Gill also reported by (Koltunow et al., 1996).
et al., 1995). Effect, of different carbohydrates on somatic
embryogenesis of citrus plants is reported by using calli Callus formation: After four weeks, germinated
from ‘Ponkan’ mandarin (Citrus reticulata, Blanco), seedlings had elongated epicotyl region cut into segments
‘Cravo’ mandarin (C. reticulata), ‘Itaboraí’ sweet orange to be initiated on MS media supplemented with growth
(C. sinensis L. Osbeck.), ‘Valencia’ sweet orange (C. regulators having different concentrations of 2,4- D (0,
sinensis) by (Ricci et al., 2002). 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/L) along with constant 0.5
In some citrus species including Kinnow mandarin, mg/L BAP with 0.5 gm/L malt extract at PH 5.7. Media
multiple embryos have been found in addition to zygotic codes assigned were MS0, MS1, MS2, MS3, MS4, MS5,
embryos representing the phenomenon called nucellar MS6. Nucellar embryos, which were sprinkled out from
polyembryony. Polyembryonic seed formation in citrus is seeds, were removed and cut into segments and placed on
one of the apomictic processes that was found to occur in same media combinations as for epicotyl segments
the ovules of angiospermic species (Koltunow, 1993). described above. All cultures were incubated at 25±2oC
During polyembryonic seed formation, non-zygotic with a 16/8 hrs photoperiod. After three days callus
nucellar embryos start growing directly from maternal formation from epicotyl region and nucellar embryonic
nucellar wall surrounding the embryo sac while the zygotic tissues was observed. During next three weeks callus
embryo development. These nucellar embryos carry the induction data was recorded and scored for statistical
same genetic makeup as it is present in their female parent analysis.
(Koltunow et al., 1996). A significant role in the evolution
of cultivated forms can be played by tetraploid genotypes Callus regeneration: After callus induction, nucellar
arising from chromosome doubling of nucellar cells of embryonic tissues were placed on simple MS medium for
apomictic citrus as parents in interploid crosses or directly somatic embryogenesis for four weeks and epicotyl calli
as new rootstocks (Aleza et al., 2011). were placed on MS media supplemented with 2mg/L 2,4-
Genetic transformation through callus is quite easy D and 0.5mg/L kinetin for somatic embryogenesis as
and high frequency transgenic plants can be obtained described by (Jain et al, 1995).
from callus since in the case of direct regeneration, Plant regeneration was achieved by transferring
frequency of untransformed plants is quite high. callus on MT media supplemented with various
Furthermore genetic transformation by protoplast combinations of BAP (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0mg/L)
isolation and fusion, agro bacterium mediated genetic and constant kinetin with 0.5mg/L. MT1 had 0.5mg/L
transformation, induced mutation through gamma rays or kinetin only, while each combination was fortified with
chemical mutagenesis can also become efficient through 0.5g/L malt extract. Media codes assigned were MT0,
callus formation and regeneration. Nucellar embryos or MT1, MT2, MT3, MT4, MT5, MT6, MT7, MT8. Jars
nucellar tissues and epicotyl segments can give an were placed in a growth room with 16 hrs photoperiod at
efficient callus induction and regeneration systems. The 25±2ºC, during next four weeks data was recorded for
aim of this research is to induce embryogenic callus from number of shoots and their length.
epicotyl segments and nucellar embryos from seeds of
Kinnow mandarin and compare the regeneration ability of Rooting of plantlets: Regenerated plantlets with healthy
callus obtained from both sources on histological basis. shoots were transferred to rooting media containing half
strength MT basal medium supplemented with growth
Materials and Methods regulators IBA 0.5mg/L and NAA 0.1mg/L along with 0.5
gm/L activated charcoal and 30 gm/L sucrose (Liu, 2005).
Seeds sterilization and germination: Seeds were The cultures were incubated at 25 ± 2°C while under white
collected from commercially available export quality fluorescent tube lights having photoperiod for 16/8 hrs.
Kinnow mandarin. Prior to sterilization, the seeds were
After four weeks in rooting media the plantlets
washed thoroughly under running tap water for 5 min and
developed full roots, and they were transferred in the
dipped in commercially available washing liquid for 10
small plastic pots to acclimatize in the green house under
min. Then they were sterilized in 70% commercial bleach
containing 2 drops of tween-20 as a wetting agent in humid environment. This procedure has been successfully
200ml distilled water for 20 minutes. The seeds were later repeated many times for micropropagation of Kinnow
washed by four rinses with autoclaved distilled water to mandarin plants.
remove the traces of bleach. After sterilization seed coats
were removed with scalpel. Statistical analysis: Data was collected for callus
Simple MT media formulated by (Murashige & induction as well as regeneration of both kinds of
Tucker, 1969) supplemented with 50gm/L sucrose and explants and was analyzed with one way analysis of
0.5gm/L malt extract at pH 5.7 adjusted using 1N HCl variance using Minitab software version 11 and bar
and 1N KOH was used for germination of seeds. Four to graphs were constructed.
EMBRYOGENIC CALLUS INDUCTION, SOMATIC EMBRYOGENESIS, REGENERATION AND HISTOLOGICAL 307

Histological staining: Histological studies were carried out compared to other media highly significant growth was
for both nucellar embryos and epicotyl segments along observed (p<0.001) in MS5 media containing 2.5mg/l 2,4-
with induced calli. Toluidine Blue O, a polychromatic dye, D and 0.5mg/l BAP. Similarly as in the case of nucellar
was used to stain these samples. After hand sectioning, the embryo highest calli growth were observed on MS5
samples were stained with two drops of 0.1% Toluidine medium containing 2.5mg/l 2,4-D and 0.5mg/l BAP and
blue O for ten minutes in 10ml distilled water. These analysis of variance revealed highly significant result
sections on slides were observed under Nikon TE 2000E (p<0.001). Since the calli induced from nucellar
fluorescent microscope in bright field mode using 10X and embryonic tissues were later found more embryogenic as
40X magnification and images were captured with the compare to epicotyl segments (Fig. 2B and Fig. 2D).
NIKON NIS-Elements camera and its software.
Plantlet regeneration: Nucellar calli were used for
Results
regeneration study and it was observed that MT2 media
Callus growth: Analysis of variance revealed highly containing 0.5mg/l BAP and 0mg/l kinetin induced
significant differences among the numerous callus induction statistically significant (P=0.189) highest mean number of
media (p<0.001). It was observed that cream colored calli shoots as compared to other media combinations (Graph.
were induced from both types of explants in all combinations 2). Whereas on MT3 media containing 0.5mg/l BAP and
of 2,4-D excluding MS0, MS1 and MS2 media. 0.5mg/l Kinetin the regenerated plantlets were attained
The epicotyl region showed highest calli growth in the highest mean length of shoots as shown in (Graph. 2)
the MS5 medium as shown in (Graph. 1). Thus, as having high significance level of (p<0.001).

Graph. 1. Callus growth pattern of Epicotyl segments and Graph. 2. Growth pattern for number and length of shoots
Nucellar embryonic tissue on different media. produced on different media compositions from nucellar callus.

Histological studies: Histological analysis of nucellar Discussion


embryos and epicotyl segments along with their
induced callus was performed after Toluidine blue O Epicotyl segments and nucellar tissues both
staining. It was observed that in nucellar embryos, the showed callus induction. In both cases the callus was
epidermis and chlorenchyma cells appeared bluish soft, friable and embryogenic in the media with
green in color where as vessels appeared dark purple 2.5mg/l 2,4-D and 0.5mg/l BAP. These findings are in
and parenchyma cells looked greenish blue (Fig. 3c). disagreement with (Nafees et al, 2009) since they used
BAP and 2,4-D combinations from 0 to 0.4mg/l and
In calli induced from nucellar embryos it was
also with (Chakraverty & Goswami, 1999) as they
observed that dark purple cells formed from
found 1mg/l 2,4-D and 0.5mg/l BAP (90.2%) suitable
parenchymatous cells having condensed chloroplast
for callus induction as well as 1mg/l BAP for (75.7%)
within them (Fig. 3d). In epicotyl segment the cross somatic embryogenesis. In this study it was observed
section appeared just like stem segment showing that calli from nucellar tissues were found more
epidermis with dark green color and blue colored embryogenic resulting to produce more plants as
lignified sclerenchyma as describe by (O’Brien et al, compared to calli induced from epicotyl segments (Fig.
1964). Phloem looked light brown and xylem 1E and Fig. 2E), although callus induced from
appeared dark blue in color (Fig. 3a). Callus induced epicotyls was more(Fig. 1) but less regenerative. These
from epicotyl segment appeared colorless and only findings are in contrast with (Gill et al., 1995) since
nucleus could be seen stained blue (Fig. 3b). they found epicotyl parts more embryogenic as
308 SAIFULLAH KHAN ET AL.,

compare to other plant parts. In our approach before Thus to increase leaves width for successful
putting on regeneration, nucellar callus was placed on acclimatization these shoots were placed on MT media
hormone free simple MS medium along with 0.5gm/L along with 0.5mg/l kinetin and 0.5mg/l BAP (Fig. 2).
malt extract for somatic embryogenesis, so that it During acclimatization, the farmyard manure was
readily accepts MT media for regeneration. Callus mixed with the soil to provide nutrients to the plants.
induction from other parts of the Kinnow mandarin like In Toluidine blue O staining the parenchymatous
leaves, stem segments was also tested, but leaves failed cells appeared reddish purple as described by (O’Brien
to induce callus which is in disagreement with et al, 1964). Histological study highlighted the reasons
(Chakraverty & Goswami, 1999). They reported for less regeneration ability of callus induced from
embryogenic calli from the leaves of Citrus acida Roxb epicotyl segments, as compared to that from nucellar
which may be due to cultivar difference. This embryonic tissues. Nucellar induced callus was found
phenomenon of different genetic behavior to same
intensely rich in green chloroplast bodies present in
media composition was also studied by (Mendes-da-
parenchymatous cells as shown in (Fig. 3B) at 40X
Glória et al., 1999).
During regeneration stages, many shoots magnification. Their presence is a proof of high
developed along with many thin leaves on MT media photosynthetic activity and apparently, due to this
supplemented with 0.5mg/l BAP but during characteristic plants regenerate more rapidly and are
acclimatization, these plants were not very successful. higher in number.

Fig. 1. Indirect regeneration of Kinnow mandarin from Epicotyl regions.


A. Epicotyl segments grown in a dark condition B. Epicotyl segments on callus media C. Callus growth D. Light regeneration E. Dark
regeneration F. Separated plantlets on shooting media G. Rooting of plantlets H. Acclimatization
EMBRYOGENIC CALLUS INDUCTION, SOMATIC EMBRYOGENESIS, REGENERATION AND HISTOLOGICAL 309

Fig. 2. Indirect regeneration of Kinnow mandarin from Nucellar Embryos.


A. Nucellar embryos B. Callus growth in light C. Callus growth in dark condition D. Somatic embryogenesis E. Shoots elongation F.
Regeneration from callus.

Fig. 3. Microscopic images of stained region of epicotyl segment and nucellar region of Kinnow mandarin.
A. Epicotyl segment stained section B. Embryogenic callus of epicotyls segment C. Nucellar embryo stained section D. Embryogenic
callus of Nucellar embryo.
310 SAIFULLAH KHAN ET AL.,

Acknowledgements through bud wood irradiation. Afr J. Biotechnol., 10(65):


14562-14565.
We are highly grateful to Higher Education Khan, I.A. 1992. Prodeedings of the first International seminar
on citriculture in Pakistan.
Commission of Pakistan for providing research grant for Khan, I.A. and J.W. Grosser. 2004. Regeneration and
this study. We are also thankful to all our group members characterization of somatic hybrid plants of Citrus sinensis
and especially Pablo Aleza from Laboratoriode Cultivo de (sweet orange) and Citrus micrantha, a progenitor species
Tejidos, Centro de Protección Vegetaly Biotecnología, of lime. Euphytica, 137: 271-278.
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Moncada, Valencia, Espain; her provided research formed without meiosis or fertilization in ovules. Plant
Cell., 5: 1425-1437.
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Koltunow, A.M., T. Hidake and S.P. Robinson. 1996.
Polyembryony in Citrus. Accumulation of seed storage
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(Received for publication 12 September 2013)

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