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Development of An Efficient in Vitro Regeneration Protocol For Fig (Ficus Carica L.)
Development of An Efficient in Vitro Regeneration Protocol For Fig (Ficus Carica L.)
S.S. Dhage, V.P. Chimote*, B.D. Pawar, A.A. Kale, S.V. Pawar and A.S. Jadhav
State Level Biotechnology Centre, Mahatma Phule Krishi Vidyapeeth, Rahuri-413722, Maharashtra, India.
*E-mail: vivekchimote@rediffmail.com
Abstract
7KHSUHVHQWLQYHVWLJDWLRQZDVXQGHUWDNHQWRGHYHORSDQHI¿FLHQWin vitroUHJHQHUDWLRQSURWRFROLQIRXU¿JFXOWLYDUVviz., Poona Fig,
Brown Turkey, Conadria and Deanna. Highest shoot tip establishment was observed in Deanna (100 %), followed by Conadria (79.2
%) and Brown Turkey (76.7 %) on MS medium supplemented with 2.5 mg/L 6-benzylaminopurine (BAP), 0.5 mg/L gibberellic acid
(GA3). Establishment of shoot tips was very poor in cultivar Poona Fig (11.7-13.3 %). Further inoculation of shoots on MS medium
VXSSOHPHQWHGZLWKPJ/LQGROHEXW\ULFDFLG,%$UHVXOWHGLQERWKPXOWLSOHVKRRWLQJDVZHOODVURRWLQJ6LJQL¿FDQWQXPEHURI
newly formed shoots were observed in Conadria (4.7) and Deanna (3.8) as against in Brown Turkey (1) and Poona Fig (0.6). Highest
root induction was observed in Conadria (73.3 %), followed by Deanna (52.2 %), Brown Turkey (26.7 %) and Poona Fig (24.4 %).
7KHVHUHVXOWVFRQ¿UPHGWKDWWKHVKRRWEXGHVWDEOLVKPHQWDQGPXOWLSOHVKRRWLQGXFWLRQLQ¿JLVKLJKO\JHQRW\SHVSHFL¿F$VWKHUHVSRQVH
of popular cultivar Poona Fig to shoot tip culture was very poor, tender leaf explants were further used for regeneration study. Optimum
regeneration was observed using MS medium supplemented with 4.0 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) for callusing; 7
PJ/WKLGLD]XURQ7'=DQGPJ/ĮQDSKWKDOHQHDFHWLFDFLG1$$IRUVKRRWLQJDQGPJ/,%$IRUURRWLQJ
Key words: )LJUHJHQHUDWLRQJHQRW\SHVSHFL¿FVKRRWWLSFXOWXUHPXOWLSOHVKRRWLQJURRWLQJ
,QWURGXFWLRQ been optimized (Fraguas et al., 2004). Thus, the objective of our
study was to optimize in vitroUHJHQHUDWLRQSURWRFROLQ¿JXVLQJ
7KHFRPPRQ¿JFicus carica L.) is well known for its nutritive shoot tips and leaves.
value and are consumed both as fresh and process dried. Figs are
ULFKVRXUFHRIFUXGH¿EUHPLQHUDOVYLWDPLQ.DQGDQWLR[LGDQWV Materials and methods
compounds (Vallejo et al., 2012). It is native to Southwest Asia
and the Mediterranean region (from Afghanistan to Portugal). Preparation of explants: $[LOODU\VKRRWWLSVRIIRXU¿JFXOWLYDUV
:RUOGZLGH¿JLVZLGHO\FXOWLYDWHGZLWKDQDUHDRIODNKKD viz., Poona Fig, Brown Turkey, Conadria and Deanna as well as
and production of 10.93 lakh tonnes harvested in 2012. However tender leaves of Poona Fig were collected in morning hours from
despite of huge demand both cultivation area (5500 ha) and healthy mature plants from experimental orchard of the All India
production (19,000 tonnes) is very low in India (FAOSTAT, Coordinated Research Project Arid Zone Fruits, Mahatma Phule
([RWLFFXOWLYDUVGRQRW¿QGIDYRUZLWK,QGLDQIDUPHUVDV Krishi Vidyapeeth, Rahuri. These explants were sterilized with
WKH\DUHPRUHSURQHWRQHPDWRGH7KHVHHGVRI¿JDUHQRQYLDEOH 0.1, 0.2, 0.3 % (w/v) mercuric chloride (HgCl2) or 4 % (w/v)
KHQFHWKH¿JVDUHSURSDJDWHGWKURXJKYHJHWDWLYHDSSURDFKHV sodium hypochlorite (NaClO) solution for 3, 5, 7, 9 min. They
However, these propagation approaches are slow and limited. ZHUHIXUWKHUULQVHG¿YHWLPHVZLWKVWHULOHGLVWLOOHGZDWHU
Development of clonal propagation methods have numerous Shoot tip culture establishment: Initially shoot tip culture was
potential applications e.g., plant transformation, germplasm attempted in cv. Poona Fig. However, very poor bud breaking
conservation, synthetic seeds and mutation breeding (Ji et al., was observed. In order to check whether initial poor results
2011). Micropropagation has been successfully employed for LQFY3RRQD)LJZDVGXHWRJHQRW\SHVSHFL¿FUHVSRQVHWKUHH
rapid multiplication of genetically identical and superior quality other fig cultivars i.e. Brown Turkey, Conadria and Deanna
planting material in many fruit crops. In vitro propagation in were further included in the present study. Sterilized shoot tips
¿JVHUYHVWKHSXUSRVHRIPDVVVFDOHSURGXFWLRQRIKLJKTXDOLW\ were inoculated on Murashige and Skoog (MS) basal medium
planting material (Rout et al., 2006). In vitroUHJHQHUDWLRQLQ¿J (Murashige and Skoog, 1962) supplemented with 2.5 or 3.5
using various explants such as shoot tips (Murithi et al., 1982; mg/L 6-benzylaminopurine (BAP), 0.5 mg/L gibberellic acid
Haelterman and Docampo, 1994; Gella et al., 1998; Hepaksoy and (GA3), 100 mg/L ascorbic acid and 150 mg/L citric acid. They
Aksoy, 2006), nodal explants (Fraguas et al., 2004), leaves (Kim were initially kept in dark for a week and then incubated at 25 °C
et al., 2007; Dhage et al., 2012; Soliman et al., 2010) and apical temperature with 16 h light period and 8 h dark period.
buds (Kumar et al., 1998; Gella et al., 1998) has been reported.
Multiple shoot induction and rooting: Individual shoots from
'HYHORSPHQWRIDQHI¿FLHQWUHJHQHUDWLRQSURWRFROLVHVVHQWLDO shoot tip induction medium were further transferred to MS
for successful in vitro SURSDJDWLRQDQGWUDQVIRUPDWLRQLQ¿J medium with either of 1/2/3/4 mg/L of indole-3-butyric acid
Fig is recalcitrant in its production of adventitious shoots (Kim (IBA), 100 mg/L ascorbic acid, 150 mg/L citric acid and 0.2 %
et al., 2007) and factors affecting shoot proliferation have not activated charcoal for further induction of shoots and roots.
'HYHORSPHQWRIDQHI¿FLHQWin vitroUHJHQHUDWLRQSURWRFROLQ¿JFicus carica L.) 161
concentrations (0.2-0.4 %) and 4 % NaClO proved to be more (0-1.0 additional shoots/explant). Multiple shoots may be likely
toxic leading to browning and death of leaf explants. due to carry over effect of previous medium.
Shoot tip culture establishment: Browning due to oxidation of *HQRW\SHVSHFL¿FUHVSRQVHWR,%$WUHDWPHQWZDVREVHUYHGLQ
phenolic compounds, released from the cut ends of the explants is terms of shoot length after 30 days of culture. Higher shoot length
a major problem during in vitro cultures of woody plant species. was observed in Conadria (37.6-50.2 mm) and Deanna (25.0-42.7
In preliminary experiments it was observed that browning of mm), as compared to Poona Fig (14.0-23.0 mm) and Brown
explants hindered explant establishment in cultivar Poona Fig. Turkey (10.1-22.0 mm). Average number of leaves / shoots after
The shoot tips that survived showed poor growth in terms of 30 days of culture, was highest in Deanna (9.0) at 2 mg/L IBA
shoot elongation and leaf emergence. There was high-browning followed by Conadria (8.1) at 1 mg/L IBA concentration. Even in
in both shoot buds and leaf tissues. Therefore in the present other IBA treatments, high leaf bearing was observed in Conadria
study antioxidants such as ascorbic acid and citric acid were used (5.7-6.5) and Deanna (4.7-7.5). However leaf bearing was very
in all medium to reduce browning. Soliman et al. (2010) used poor in Poona Fig (1.0-2.2) and Brown Turkey (1.0-2.3), with
different antioxidants such as polyvinyl-pyrrolidone, citric acid best treatment being 3 mg/L IBA in both cases.
and ascorbic acids to control browning.
Highest rooting percentage was observed in MS medium
During shoot tip culture, it was observed that establishment of supplemented with 1 mg/L IBA in all genotypes i.e. Conadria
H[SODQWVZDVVLJQL¿FDQWO\LQÀXHQFHGE\JHQRW\SHDQGJURZWK (73.3 %), followed by Deanna (52.2 %), Brown Turkey (26.7
regulator used (Table 1). Highest shoot tip establishment was %) and Poona Fig (24.4 %) (Table 3). However, in Brown
observed on MS medium supplemented with 2.5 mg/L BAP Turkey shoots exhibited only aerial rooting. Cultivar Conadria
and 0.5 mg/L GA3. However in Conadria optimum shoot tip also showed significant rooting at 2 mg/L and 3 mg/L IBA
establishment was observed on MS medium supplemented with concentrations. However, there was almost no rooting in rest of
3.5 mg/L BAP and 0.5 mg/L GA3. Establishment was higher in genotype-IBA treatment combinations. Earlier, there are reports
three genotypes i.e. Deanna (62.5-100 %), Conadria (79.2-87.5 RIURRWLQJLQ¿JDWYDU\LQJOHYHOVRI,%$i.e. 0.5 mg/L IBA and
%) and Brown Turkey (53.3-76.7 %). However, response to shoot NAA (Danial et al., 2014); 1 mg/L IBA (Yakushiji et al., 2003;
tip establishment was very poor in Poona Fig (11.7-13.3 %). BAP Soliman et al., 2010); 2 mg/L IBA (Kumar et al., 1998) and
KDYHEHHQUHSRUWHGWREHXVHIXOLQ¿JVKRRWHVWDEOLVKPHQWDQG hormone free medium (Yakushiji et al., 2003; Kim et al., 2007).
proliferation (Kumar et al., 1998; Kim et al., 2007). Mustafa and
In vitro regeneration using leaf explants in cv. Poona Fig:
Taha (2012) reported enhanced shoot multiplication and callus
Poona Fig is very popular local cultivar in western India due to its
IRUPDWLRQIURPVKRRWWLSH[SODQWVRIGLIIHUHQW¿JFXOWLYDUVXVLQJ
JRRGÀDYRUHDVLO\UHPRYDEOHVNLQDQGVRIWVHHGV+RZHYHUWKLV
2.5 mg/L BAP. Hepaksoy and Aksoy (2006) used combination
variety showed very poor response to shoot tip culture therefore
of BAP, GA3 and IBA for in vitro shoot tip culture as well as in
OHDIH[SODQWVIURP¿HOGZHUHXVHGIRUUHJHQHUDWLRQVWXG\0RVW
PXOWLSOLFDWLRQPHGLXPLQ¿J*HQRW\SHVSHFL¿FUHVSRQVHGXULQJ of the reports published are based on use of leaf explants derived
in vitroFXOWXUHLQ¿JKDVDOVREHHQUHSRUWHGHDUOLHU+HSDNVR\DQG IURPVKRRWWLSFXOWXUHLQ¿J<DNXVKLMLet al., 2003; Soliman et
Aksoy, 2006; Kim et al., 2007; Dhage et al., 2012). al., 2010; Dhage et al., 2012).
Number of leaves per shoot also varied with genotype and Browning was observed in all media, of which least browning
culture medium used (Table 1). Cultivar Poona Fig showed poor (18.7-28.0 %) was observed on medium containing only
leaf bearing with only 17.7 % shoots having multiple leaves as 2,4-dichlorophenoxy acetic acid (2,4-D), while highest incidence
compared to other three genotypes (52.5-95 %). There was not of browning (58.7-68.0 %) was observed on both combinations
much effect of genotype and growth regulator on days to bud RI%$3DQGĮQDSKWKDOHQHDFHWLFDFLG1$$'D\VUHTXLUHGIRU
sprouting (6.1-7.0 days) and leaf emergence (7.1-7.9 days). callus treatments also varied with treatments, ranging from 35.3-
Earliest bud sprouting (6.1 days) and leaf emergence (7.1 days) 45.7 days. Late callus formation (48.3-49.0 days) was observed on
was observed in Conadria genotype. Callus formation at the base medium containing combinations of kinetin and 2,4-D.
was observed in Brown Turkey and Poona Fig after 12-16 days.
Among all treatments, calli induction frequency varied from
Multiple shoot formation was observed only in Deanna (41.7 and 21.3 % to 89.3 % (Table 3). Highest (89.3 %) and earliest callus
60.0 %) and Conadria (52.5 and 39.17%) at BAP concentrations
of 2.5 mg/L and 3.5 mg/L, respectively with GA3 (0.5 mg/L). Table 3. Callus formation in cv. Poona Fig after 60 days of inoculation
However, no multiple shooting was observed in genotype Poona Hormonal concentration Callusing Browning Days to callus
Fig and Brown Turkey in either treatment. Shoot tip culture (mg/L) (%) (%) initiation
studies clearly indicated that Poona Fig was very poor in response BAP NAA
to initial in vitro morphogenesis as compared to other genotypes. 8.0 8.0 21.3±0.92 68.0±1.4 45.7±0.88
10 .0 10.0 60.0±1.64 58.7±0.77 40.3±0.88 a
Multiple shoot induction and rooting: Individual shoots
2,4-D Kinetin
obtained from shoot tip culture were transferred to rooting 2.0 0 66.7 ±1.32 28.0±1.5 41.7 ±0.33 a
medium containing four different IBA combinations (1-4 mg/L). 2.0 0.2 48.0±1.64 40.0±1.4 49.0±0.58 b
However, multiples shoots were observed in addition to rooting. 4.0 0 89.3±2.38 18.7±0.99 35.3±0.33
5HVSRQVHWRVKRRWPXOWLSOLFDWLRQZDVKLJKO\JHQRW\SHVSHFL¿F 4.0 0.4 41.3±0.77 41.3±0.77 48.3 ±0.33 b
(Table 2). Higher new shoot induction was observed in Conadria LSD (P=0.05) 4.75 3.60 1.87
(2.7-4.7 additional shoots/explant) and Deanna (2.5-3.8 additional
*All values are means ±SE. Mean values in each column/row followed
shoots/explant). Negligible new shoot induction was observed in by the same lower-case letter(s) are not significantly different (P < 0.05)
Poona Fig (0-0.6 additional shoots/explant) and Brown Turkey by the CRD test.
'HYHORSPHQWRIDQHI¿FLHQWin vitroUHJHQHUDWLRQSURWRFROLQ¿JFicus carica L.) 163
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