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Somatic embryogenesis and indirect regeneration in Mirabilis jalapa Linn.
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 3 (2016) 3882–3891
www.materialstoday.com/proceedings
ICMRA 2016
Somatic embryogenesis and indirect regeneration in Mirabilis jalapa Linn. Gulab Khan Rohelaa, Santhosh Dameraa, Prasad Byllaa, Rajender Korrab, Sreenu Pendlib, Christopher Thammidalab a
Department of Biotechnology Kakatiya University, Warangal, Telangana, India b
Department of Boatny Kakatiya University, Warangal, Telangana, India
Abstract: Indirect somatic embryogenesis and indirect regeneration protocol was developed for the first time from leaf and nodal explants of Mirabilis jalapa Linn. Initially callus was induced in leaf and nodal explants cultured on MS medium supplemented with various concentrations of 2, 4-D, NAA and Kn, after 3 weeks of culture. Maximum amount of mean fresh and dry weight (mg) of callus(189.6 ± 0.25, 7.6 ± 0.23); (198.4± 0.34, 9.2 ± 0.23)was obtained from leaf and nodal explants, respectively when cultured on MS media supplemented with combinations of 2,4-D(3 mg/L) and NAA(5mg/L).At high concentrations of NAA (5 mg/L), somatic embryos are induced from the nodal explants after 4 weeks of culture. Shoot buds were induced from the leaf and nodal calluses when cultured on combinations of TDZ & BAP after 3 weeks of culture. Maximum number of shoot buds (08 ± 0.17 & 06 ± 0.09) were produced from leaf and nodal calluses cultured on MS + TDZ (0.5mg/L) +BAP (2 mg/L).When micro shoots were transferred to rooting media, the best rooting percentage (100%) was obtained on MS + IAA (1.0 mg/L) + IBA (2.0 mg/L). The raised plantlets when transferred to soil for acclimatization, 75 % plantlets survived in field conditions. © 2016 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of International conference on materials research and applications-2016.
Keywords: Somatic embryogenesis, callus, regeneration, plantlets.
1.
Introduction:
Mirabilis jalapa Linn is one of the important ornamental plant species which produces flowers in different types of colors. Additionally, an individual flower can also have different colors. A single flower can be in any of the color or may have a combination of colors. It is a bush type plant with potential medicinal compounds present in their
2214-7853© 2016 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of International conference on materials research and applications-2016.
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leaves and root system. Mirabilis jalapa Linn has been exported from the Peruvian Andes in 1540. Mirabilis jalapa exists in different forms; some of them have tuberous roots which makes them tosurvive the dry conditions. The flowers usually open from late afternoon onwards; hence the name Four–O-Clock plant is used as a common name.Carl Correns used Mirabilis as a model plant for his studies on cytoplasmic or maternal inheritance in the starting of 20th century. In his study, he found that the plants variegated leaves character is being coded by certain factors outside the nucleus. Correns proposed that leaf color in Mirabilis was passed on by uniparental mode of inheritance. (Miko, 2008).Similarly flower colour in Mirabilis jalapa Linnis studied as an example for incomplete dominance. In 2001, Mirabilis jalapa Linn reported as a model system for studying floral senescence by Gookin.
1.1 Medicinial Importance: Mirabilis jalapa’s flower possess useful compounds such as betaxanthins (Piattelli et.al., 1965), which is used for physical fitness of human body, swimming, or any other fitness that strengthens your body. Rotenoids which are present in the roots (Fen et.al.,2002) can be used as insecticidal and anti-inflammatory compounds (Bairwa et.al., 2013). Kaladhar et.al., 2010 and Shaik et.al., 2012, has reported the phytoconstituents found in Mirabilis Jalapa.A number of active compounds were reported from different organs of Mirabilis jalapa Linn (Kaladhar et. al., 2010) including antiviral compounds likeribosome-in activating protein (RIP), anti-fungal compounds, antimicrobialproteins and rotenoids showing inhibition of HIV-1 reverse transcriptase (Kaladhar et. al., 2010). Siddique et.al., 1990; has reported the
presence of stigmasterol, ursolic acid, oleanolic acid and brassicasterol in the
methanolic extract of the aerial parts of Mirabilis jalapa Linn and are responsible for antimicrobial activity against bacterial and fungal organisms.Isoflavone, a roteinoid and a dehydrorotenoid, compounds are responsible for the antimicrobial activity against Candida albicans was reported by Yang et.al., 2001.Antimicrobial activity is reported against multidrug-resistant Staphylococcus aureus by Michalet et .al., 2007. This plant species is reported as a hyper accumulator of cadmium metal by Yu and Zhou, 2009. From the above data it is evident that -
Mirabilis jalapaLinn has horticulture importance because of its wide cultivation as an ornamental plant
-
Used as a model plant for genetical studies and studying floral senescence
-
Possesses medicinally important compounds such as Betaxanthins, Rotenoids, important fatty acids, important plant sterols, antiviral activity compounds like ribosomal inactivating protein (RIP), Antimicrobial peptides, Anti helmenthic compounds, free radical scavenging properties and also hyper accumulator of cadmium metal.
1.2 Limitations: 1) Poor seed germination 2) Scanty information on in vitroseed germination of Mirabilis jalapa Linn 3) Scanty information on in vitroculture of Mirabilis jalapaLinn through indirect regeneration methods.
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1.3 Objectives: In view of the above importance and limitations of Mirabilis jalapa Linn, the following objectives were chosen in the present study 1) In vitro seed germination 2) Callus induction from various explants 3) Regeneration of shoots from callus 2.
Materials 2.1 Plant Material: Mirabilis jalapaLinn species with red, yellow, pink and white coloured flowers were collected separately from Bhimaram area of Hanamkonda City, Warangal District, Telangan. They were used in the present investigation. 2.2 Seed Material: Seed material was collected from Mirabilis jalapa Linn from Bhimaram area of Hanamkonda, Warangal. They were used in the present study. For in vitro seed germination, fresh seeds were collected and inoculated on to MS basal media 2.3 Explants 2.3.1
Hypocotyl (ca 1 cm long) and cotyledon ((ca 1cm) explants were obtained from 2 weeks old seedlings of Mirabilis jalapaLinn were used in the present study.
2.3.2
Leaf (ca 1 cm), stem (internode) (1 cm long) , nodal ( 1 cm long ) and shoot tip (1 cm long ) explants were collected from Mirabilis jalapa Linn (2 months old) plants with pink coloured flowers, which were present atBhimaram area of Hanamkonda, Warangal..
2.3.3
Callus: Callus (100 mg of fresh weight) was induced from leaf and stem (node) explants were used in the present study for indirect regeneration.
3.
Methods 3.1 Preparation of MS medium: One litre (1000 ml) MS medium was prepared and the pH of the medium was adjusted using either 0.1 N HCl or 0.1 N NaOH to 5.8 before gelling the medium. The medium was solidified with 0.8% agar, by melting on water bath. The hot molten medium was distributed in to culture tubes uniformly and mouths covered tightly with cotton plugs. The culture tubes along with media were autoclaved at 121ºC /15 lbs pressure for 20 minutes then allowed to cool and the culture tubes kept for slanting. 3.2 Surface sterilization of explants: Leaf (ca 1 cm) and nodal explants were washed with 1.0% detergent (Tween-20%) for 6 min. The explants were
surface sterilized with mercuric chloride (0.1%) for 2
minutes and washed with sterile distilled water for 3-4 times and then placed on sterile filter paper to absorb the adhering water droplets. 3.3 Aseptic techniques for sterile transfer: All operations were made under sterile laminar airflow cabinet. The laminar airflow cabinet was switched on for 30 minutes with UV light to make sterile conditions inside the laminar air flow
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3.4 Inoculation:The culture tubes containing MS medium were kept horizontally, toreduce the contaminations. By using forceps explants were inoculated into the culture tubes and were immediately closed with cotton plugs. 3.5 Incubation: The inoculated culture tubes were incubated at 25±2ºC under 16 hours light followed by 8 hours dark period. Cultures were maintained at light intensity of 3000 Lux provided by florescent lamps. 3.6 Statistical Analysis: Research data obtained in various experiments in the study was statistically analyzed by using SPSS Version 17 and means were compared using Tukey’s tests at the 5% level of significance. All means are presented with SE. 4.
Results 4.1 In vitro Seed Germination Fresh seeds of Mirabilis jalapa Linn cultivar pink coloured flower, was collected from 6 months old plants. These seeds were used initially for In vivo seed germination studies, which has given 0% in vivo germination. This poor in vivo seed germination can be overcome by the plant by protecting it as a underground stem during the dry and hot conditions during summer. For in vitro seed germination studies, we inoculated the fresh seeds on to basal Murashige and Skoogs media. After 2 weeks of duration, only 2% of seeds have germinated (Plate 1). 4.2Callus induction& Somatic embryogenesis Callus was induced in leaf and nodal explants of Mirabilis jalapaLinn (Plate 2A & 2B) cultured on MS medium supplemented with various concentrations of 2, 4-D, NAA and Kn, after 3 weeks of culture (Tables 1-3). On 2,4-D supplemented media, maximum amount of mean fresh and dry weight (mg) of callus (186.7 ± 0.44, 9.7 ± 0.61) & (138.6 ± 0.13, 4.9 ± 0.31) was observed in leaf and nodal explants cultured on 5 mg/L 2,4-D (Table 1 ). On NAA supplemented media, maximum amount of mean fresh and dry weight (mg) of callus(136.7 ± 0.24, 7.5 ± 0.14) & (155.8 ± 0.34, 7.8 ± 0.19) was observed in leaf and nodal explants cultured on 5 mg/L NAA, respectively (Table 2). At high concentrations of NAA (5 mg/L), somatic embryos are induced from the nodal explants after 4 weeks of culture (Plate 2C & 2D)On 2,4-D + NAA supplemented media, maximum amount of mean fresh and dry weight (mg) of callus (189.6 ± 0.25, 7.6 ± 0.23); (198.4± 0.34, 9.2 ± 0.23) was observed in leaf and nodal explants cultured on 3mg/L 2,4-D + 5mg/L NAA (Table 3). Overall maximum amount of mean fresh and dry weight (mg) of callus (189.6 ± 0.25, 7.6 ± 0.23); (198.4± 0.34, 9.2 ± 0.23) was obtained from leaf and nodal explants, respectively when cultured on MS media supplemented with combinations of 2,4-D(3 mg/L) and NAA(5mg/L).
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Table 1: Callus induction in Leaf and Nodal explants cultured on MS medium supplemented with various concentrations of 2, 4-D, in Mirabilis jalapa Linn
Explant
Morphogenetic Response
Callus
MS + 2,4-D mg/L
Mean fresh weight (mg) Leaf 1.0 2.0 White callus 141.3 ± 0.12d 3.0 White callus 157.5 ± 0.25c 4.0 Friable white callus 158.0 ± 0.61b 5.0 Brown callus 186.7 ± 0.44a Nodal 2.0 White callus 87.1 ± 0.13g 3.0 Friable White callus 103.1 ± 0.29f 4.0 Brown callus 111.4 ± 0.51e 5.0 Brown callus 138.6 ± 0.13d Data represents average of three replicates. Each replicate consists of 25 cultures. Data scored at the end of 3 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at=0.05
Mean dry weight (mg) 5.4± 0.31d 6.3 ± 0.04c 8.5 ± 0.16b 9.7 ± 0.61a 2.1 ± 0.11g 3.2 ± 0.14f 4.2 ± 0.13e 4.9 ± 0.31d
Table 2: Callus induction in Leaf and Nodal explants cultured on MS medium supplemented with various concentrations of NAA, in Mirabilis jalapa Linn
Explant Leaf
Nodal
Morphogenetic Response MS + NAA mg/L 1.0 2.0 3.0 4.0 5.0 2.0 3.0 4.0 5.0
Callus
-
Mean dry weight (mg) -
121.0 ± 0.12b 127.4 ± 0.15c 136.7 ± 0.24d 102.5 ± 0.14a 121.5 ± 0.14b 135.6 ± 0.32d 155.8 ± 0.34e
4.5 ± 0.31a 5.2 ± 0.22b 7.5 ± 0.14d 4.2 ± 0.21a 4.8 ± 0.16b 6.2 ± 0.10c 7.8 ± 0.19d
Mean fresh weight (mg) White callus Friable white callus Friable brown callus White callus Friable White callus Brown callus Brown callus with Somatic embryos after 3 weeks of cultures
Data represents average of three replicates. Each replicate consists of 25 cultures. Data scored at the end of 3 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at = 0.05 Table 3: Callus induction in Leaf and Nodal explants cultured on MS medium supplemented with various concentrations and Combinations of 2, 4-D & NAA, in Mirabilis jalapa Linn
Explant Leaf
MS + NAA mg/L MS + 2,4-D mg/L
Morphogenetic Response
3.0 1.0 3.0 2.0 White callus 3.0 3.0 Friable white callus 3.0 4.0 Friable white callus 3.0 5.0 Brown callus Node 3.0 2.0 White callus 3.0 3.0 Friable White callus 3.0 4.0 Friable Brown callus 3.0 5.0 Brown callus Data represents average of three replicates. Each replicate consists of 25 cultures. Data scored at the end of 3 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at=0.05
Callus Mean fresh weight (mg) 113.2± 0.13e 141.2 ± 0.31c 153.0 ± 0.23b 189.6 ± 0.25a 121.3 ± 0.11d 123.3 ± 0.31d 141.3 ± 0.24c 198.4± 0.34a
Mean dry weight (mg) 3.8± 0.41e 4.6 ± 0.24d 6.7 ± 0.71c 7.6 ± 0.23b 3.2 ± 0.12e 4.3 ± 0.21d 6.3 ± 0.12c 9.2 ± 0.23a
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4.3 Regeneration of shoots from Callus (Indirect Organogenesis) Primary callus (friable and creamy white in colour) of leaf and nodal explants of Mirabilis jalapa were induced maximally on 3mg/L 2, 4-D. When these primary calluses were subcultured on the same medium, callus turned fragile and there is an increase in fresh and dry weights of callus (Table 4). Shoot buds were induced from the leaf and nodal calluses when cultured on combinations of TDZ & BAP after 3 weeks of culture (Table 5), Fig 1& Fig 2, Whereas TDZ and BAP in alone has not resulted in shoot induction form both leaf and nodal calluses.Maximum number of shoot buds (08 ± 0.17 & 06 ± 0.09) (Fig 1) were produced from leaf and nodal calluses (Plate 3B & 3C), cultured on MS + TDZ (0.5mg/L) +BAP (2 mg/L). When the concentration of TDZ is increased beyond 0.75 mg/L in combination with 2mg/L of BAP, it resulted in decrease in no. of shoot buds production (Table 5)(Fig 1& Fig 2). Table 4: Sub culturing of leaf and stem (nodal) calluses on MS supplemented with various concentrations of 2, 4-D in Mirabilis jalapa Linn Callus initiated on
Leaf MS+ 2,4-D (3 mg/L) Stem (Node) MS+ 2,4-D (3 mg/L)
Primary culture Mean Fresh weight in Mean Dry weight in (mg) (mg) 157.5 ± 0.25c 6.3 ± 0.04c
103.1 ± 0.29f
Sub-culture Mean Fresh weight in Mean Dry weight in (mg) (mg) 196.5±0.25a 11.4±0.12a
3.2 ± 0.14f
147± 0.26e
5.8 ± 0.29d
Data represents average of three replicates. Each replicate consists of 10 cultures. Data scored at the end of 2 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at=0.05
Table 5: Shoot bud differentiation from Sub-cultured leaf and nodal calluses cultured on various concentrations of TDZ and BAP in Mirabilis jalapa Linn Sub-cultured Callus
TDZ (mg/L)
Leaf
0.10 0.25 0.50 0.75 1.00
BAP (mg/L) 2.00 2.00 2.00 2.00 2.00
Stem (Nodal)
0.10
2.00
-
0.25 0.50 0.75
2.00 2.00 2.00
01 ± 0.23c 06 ± 0.09a 02 ± 0.16b
1.00 2.00 Data represents average of three replicates. Each replicate consists of 10 cultures. Data scored at the end of 2 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at=0.05
No. of shoot buds 03 ± 0.06c 08 ± 0.17a 04 ± 0.10b -
-
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Fig 1: Shoot bud differentiation from Sub-cultured leaf callus cultured on various concentrations of TDZ and BAP in Mirabilis jalapa Linn
Fig 2: Shoot bud differentiation from Sub-cultured Nodal callus cultured on various concentrations of TDZ and BAP in Mirabilis jalapa Linn
4.4 Rooting In vitro shoots obtained from indirect regeneration through callus were inoculated onto rooting media which consists of various concentrations and combinations of IAA and IBA. The best rooting percentage (100%) was obtained on MS + IAA (1.0 mg/L) + IBA (2.0 mg/L) (Plate 3D). 5.
Acclimatization Plantlets with 2–4 well developed leaves and roots were transferred to soil, 75 % plantlets survived in field conditions.
6.
Discussions: In this study leaf and nodal explants of Mirabilis jalapa Linncultured on MS basal medium did not produce any callus, but when cultured on various concentrations of 2,4-D, NAA either individually or in
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combinations produced morequantity of callus, after 3 weeks of culture. Though the rate of callus growth was negligible in first week, the entire explant was covered with healthy callus after 3 weeks.Major portion of callus that developed from leaf and nodal explants was friable to compact & green coloured but became brown when cultured for more than six weeks on the same medium at high concentrations of auxins. The callus that developed on leaf and nodal explants was creamy white in colour. There is scanty information on callus induction from Mirabilis jalapa Linn. Xinjia Xu et. al., 2005 has reported callus induction from cotyledon and epicotyl explants of Mirabilis jalapa Linn on 2,4-D and IAA supplemented media, similarly Tamer et.al., 1997 has also reported callus induction from Mirabilis jalapa Linn from leaf explants in Kinetin supplemented media, where as we have induced the callus from explants like leaf and nodal explants on 2,4-D and NAA either in alone or in combinations. In vitro plantlet production via indirect organogenesis from leaf calluses of Mirabilis jalapa Linn were induced on MS + TDZ + BAP combination. Zaccai et. al., 2007, has reported the direct and indirect shoot induction from nodal explants by using 2mg/L Benzyladenine + 2mg/L Zeatin + 1mg/L Indole Acetic acid, whereas in our study we have employed TDZ for production of shoots from leaf and nodal callus.Xinjia Xu et. al., 2005 has also reported the production of shoots by using TDZ plant growth regulator, but they reported direct shoot induction form petioles of cotyledon explants, whereas in our studies we have induced shoots indirectly from leaf callus by employing combination of TDZ & BAP. Xinjia Xu et. al., 2005, in their report has not reported regarding no. of shoots produced directly from the petioles of cotyledon explants, whereas in our study we have regenerated 08 ± 0.17 shoots indirectly from leaf callus when cultures on MS + TDZ (0.5mg/L) +BAP (2 mg/L).
7.
Conclusion: This is the first report on induction of callus and somatic embryos from nodal explants in Mirabilis jalapa. Compared to earlier reports, we have regenerated more no. of shoots (08 ± 0.17) indirectly from leaf callus when cultured on MS + TDZ (0.5mg/L) +BAP (2 mg/L). PLATE-2
PLATE-1 A
A
B
C
B
C
In vitro seed germination of Mirabilis jalapa Linn. on MS basal media
D
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Callus induction in leaf and nodal explants of Mirabilis jalap Linn
(A)
Mirabilis jalapa Linn Seeds
(B)
Seedling (10 days old)
A)
Callus induction in Leaf explants cultured on MS + 2.4-D (3 mg/L)
(C)
Seedling (20 days old)
B)
Callus induction in Nodal explants cultured on MS + NAA (2 mg/L)
Induction of Somatic Embryos from Nodal callus in Mirabilis jalapa Linn C)
Induction of single oval shaped somatic embryo from nodal callus after 2 weeks of culture, on MS media supplemented with NAA 5mg/L
D)
Induction of two somatic embryos from nodal callus after 3 weeks of culture, on MS media supplemented with NAA 5mg/L
PLATE-3 B
A
D
E
C
In vitro plant regeneration from leaf callus of Mirabilis jalapa Linn. A)
Single Shoot bud induction in leaf callus cultured on MS + TDZ (0.10 mg/L) + BAP (2.0 mg/L)
B)
Multiple Shoot bud induction (03 ± 0.06) in leaf callus cultured on MS + TDZ (0.25 mg/L) + BAP (2.0 mg/L)
C)
Multiple Shoot buds (08 ± 0.17) induction in leaf callus cultured on MS + TDZ (0.50 mg/L) + BAP (2.0 mg/L)
D)
Micro shoot transferred on to rooting media (MS + IAA (1.0 mg/L) + IBA (2.0 mg/L) Rooted individual plantlet Complete plantlets transferred to pots containing sterile soil for hardening
F
E) F)
References
1.
Gookin, T. 2001. Evaluation of Mirabilis jalapa as a model system for studying floral.
2.
Hutchinson J, 1969. Evolution and phylogeny of flowering plants. London.
3.
Kaladhar D.S.V.G.K, Siva Kishore Nandikolla. Antimicrobial studies, Biochemical and image analysis in Mirabilis lalapa Linn. International journal of pharmacy & Technology 2010 Vol.2 (3), pp683-693.
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Khemraj Bairwa, Ishwari N. Singh, Somendu K. Roy, Jagdeep Grover, Amit Srivastava, and Sanjay M. Jachak (2013).J. Nat. Prod. 2013, 76, 1393−1398.
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Michele Zaccai, Guixia Jia, Xinlu Chen,Oksana Genis, Danit Feibin,Revital Gesua (2007). Regeneration and transformation system in Mirabilis jalapa. Scientia Horticulturae,Volume 111, Issue 3, 5 February 2007, Pages 304–309.
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Mirabilis jalapa Marvel Of Peru PFAF Plant Database". Pfaf.org. Retrieved 2012-07-31.
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Mohammad Shamim Ahmad, Abdul Rauf, Jamal Mustafa and Mohammad Osman Sheikh (1984), An 8-hydroxyoctadeca-cis-11,14dienoic acid from Mirabilis jalapa seed oil. Phytochemistry, 1984, Volume 23, Issue 10, Pages 2247–2249.
9.
Piattelli, L. M. L. Minale and R.A. Nicolaus (1965). Betaxanthins from Mirabilis jalapa, Phytochemistry, 1965, Volume 4, Issue 6, Pages 817–823
10. Serge Michalet, Gilbert Cartier, Bruno David, Anne-Marie Mariotte, Marie-Geneviève Dijoux-franca, Glenn W. Kaatz, Michael Stavri and Simon Gibbons, Bioorganic & Medicinal Chemistry Letters (2007). N-Caffeoylphenalkylamide derivatives as bacterial efflux pump inhibitors. Volume 17, Issue 6, Pages 1755–1758.
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mirabilis jalapa. linn.
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13. Siddiqui S., Siddiqui B.S., Adil Q. and Begum S. (1990). Constituents of Mirabilis jalapa.
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Halima (2012). Free radical scavenging and antibacterial activity of
mirabilis jalapa linn using in vitro models. Asian J Pharm Clin Res, Vol 5, Issue 3, 2012, 115-120.
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ScienceDirect Materials Today: Proceedings 3 (2016) 3882–3891
www.materialstoday.com/proceedings
ICMRA 2016
Somatic embryogenesis and indirect regeneration in Mirabilis jalapa Linn. Gulab Khan Rohelaa, Santhosh Dameraa, Prasad Byllaa, Rajender Korrab, Sreenu Pendlib, Christopher Thammidalab a
Department of Biotechnology Kakatiya University, Warangal, Telangana, India b
Department of Boatny Kakatiya University, Warangal, Telangana, India
Abstract: Indirect somatic embryogenesis and indirect regeneration protocol was developed for the first time from leaf and nodal explants of Mirabilis jalapa Linn. Initially callus was induced in leaf and nodal explants cultured on MS medium supplemented with various concentrations of 2, 4-D, NAA and Kn, after 3 weeks of culture. Maximum amount of mean fresh and dry weight (mg) of callus(189.6 ± 0.25, 7.6 ± 0.23); (198.4± 0.34, 9.2 ± 0.23)was obtained from leaf and nodal explants, respectively when cultured on MS media supplemented with combinations of 2,4-D(3 mg/L) and NAA(5mg/L).At high concentrations of NAA (5 mg/L), somatic embryos are induced from the nodal explants after 4 weeks of culture. Shoot buds were induced from the leaf and nodal calluses when cultured on combinations of TDZ & BAP after 3 weeks of culture. Maximum number of shoot buds (08 ± 0.17 & 06 ± 0.09) were produced from leaf and nodal calluses cultured on MS + TDZ (0.5mg/L) +BAP (2 mg/L).When micro shoots were transferred to rooting media, the best rooting percentage (100%) was obtained on MS + IAA (1.0 mg/L) + IBA (2.0 mg/L). The raised plantlets when transferred to soil for acclimatization, 75 % plantlets survived in field conditions. © 2016 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of International conference on materials research and applications-2016.
Keywords: Somatic embryogenesis, callus, regeneration, plantlets.
1.
Introduction:
Mirabilis jalapa Linn is one of the important ornamental plant species which produces flowers in different types of colors. Additionally, an individual flower can also have different colors. A single flower can be in any of the color or may have a combination of colors. It is a bush type plant with potential medicinal compounds present in their
2214-7853© 2016 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of International conference on materials research and applications-2016.
Rohela et.al./ Materials Today: Proceedings 3 (2016) 3882–3891
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leaves and root system. Mirabilis jalapa Linn has been exported from the Peruvian Andes in 1540. Mirabilis jalapa exists in different forms; some of them have tuberous roots which makes them tosurvive the dry conditions. The flowers usually open from late afternoon onwards; hence the name Four–O-Clock plant is used as a common name.Carl Correns used Mirabilis as a model plant for his studies on cytoplasmic or maternal inheritance in the starting of 20th century. In his study, he found that the plants variegated leaves character is being coded by certain factors outside the nucleus. Correns proposed that leaf color in Mirabilis was passed on by uniparental mode of inheritance. (Miko, 2008).Similarly flower colour in Mirabilis jalapa Linnis studied as an example for incomplete dominance. In 2001, Mirabilis jalapa Linn reported as a model system for studying floral senescence by Gookin.
1.1 Medicinial Importance: Mirabilis jalapa’s flower possess useful compounds such as betaxanthins (Piattelli et.al., 1965), which is used for physical fitness of human body, swimming, or any other fitness that strengthens your body. Rotenoids which are present in the roots (Fen et.al.,2002) can be used as insecticidal and anti-inflammatory compounds (Bairwa et.al., 2013). Kaladhar et.al., 2010 and Shaik et.al., 2012, has reported the phytoconstituents found in Mirabilis Jalapa.A number of active compounds were reported from different organs of Mirabilis jalapa Linn (Kaladhar et. al., 2010) including antiviral compounds likeribosome-in activating protein (RIP), anti-fungal compounds, antimicrobialproteins and rotenoids showing inhibition of HIV-1 reverse transcriptase (Kaladhar et. al., 2010). Siddique et.al., 1990; has reported the
presence of stigmasterol, ursolic acid, oleanolic acid and brassicasterol in the
methanolic extract of the aerial parts of Mirabilis jalapa Linn and are responsible for antimicrobial activity against bacterial and fungal organisms.Isoflavone, a roteinoid and a dehydrorotenoid, compounds are responsible for the antimicrobial activity against Candida albicans was reported by Yang et.al., 2001.Antimicrobial activity is reported against multidrug-resistant Staphylococcus aureus by Michalet et .al., 2007. This plant species is reported as a hyper accumulator of cadmium metal by Yu and Zhou, 2009. From the above data it is evident that -
Mirabilis jalapaLinn has horticulture importance because of its wide cultivation as an ornamental plant
-
Used as a model plant for genetical studies and studying floral senescence
-
Possesses medicinally important compounds such as Betaxanthins, Rotenoids, important fatty acids, important plant sterols, antiviral activity compounds like ribosomal inactivating protein (RIP), Antimicrobial peptides, Anti helmenthic compounds, free radical scavenging properties and also hyper accumulator of cadmium metal.
1.2 Limitations: 1) Poor seed germination 2) Scanty information on in vitroseed germination of Mirabilis jalapa Linn 3) Scanty information on in vitroculture of Mirabilis jalapaLinn through indirect regeneration methods.
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1.3 Objectives: In view of the above importance and limitations of Mirabilis jalapa Linn, the following objectives were chosen in the present study 1) In vitro seed germination 2) Callus induction from various explants 3) Regeneration of shoots from callus 2.
Materials 2.1 Plant Material: Mirabilis jalapaLinn species with red, yellow, pink and white coloured flowers were collected separately from Bhimaram area of Hanamkonda City, Warangal District, Telangan. They were used in the present investigation. 2.2 Seed Material: Seed material was collected from Mirabilis jalapa Linn from Bhimaram area of Hanamkonda, Warangal. They were used in the present study. For in vitro seed germination, fresh seeds were collected and inoculated on to MS basal media 2.3 Explants 2.3.1
Hypocotyl (ca 1 cm long) and cotyledon ((ca 1cm) explants were obtained from 2 weeks old seedlings of Mirabilis jalapaLinn were used in the present study.
2.3.2
Leaf (ca 1 cm), stem (internode) (1 cm long) , nodal ( 1 cm long ) and shoot tip (1 cm long ) explants were collected from Mirabilis jalapa Linn (2 months old) plants with pink coloured flowers, which were present atBhimaram area of Hanamkonda, Warangal..
2.3.3
Callus: Callus (100 mg of fresh weight) was induced from leaf and stem (node) explants were used in the present study for indirect regeneration.
3.
Methods 3.1 Preparation of MS medium: One litre (1000 ml) MS medium was prepared and the pH of the medium was adjusted using either 0.1 N HCl or 0.1 N NaOH to 5.8 before gelling the medium. The medium was solidified with 0.8% agar, by melting on water bath. The hot molten medium was distributed in to culture tubes uniformly and mouths covered tightly with cotton plugs. The culture tubes along with media were autoclaved at 121ºC /15 lbs pressure for 20 minutes then allowed to cool and the culture tubes kept for slanting. 3.2 Surface sterilization of explants: Leaf (ca 1 cm) and nodal explants were washed with 1.0% detergent (Tween-20%) for 6 min. The explants were
surface sterilized with mercuric chloride (0.1%) for 2
minutes and washed with sterile distilled water for 3-4 times and then placed on sterile filter paper to absorb the adhering water droplets. 3.3 Aseptic techniques for sterile transfer: All operations were made under sterile laminar airflow cabinet. The laminar airflow cabinet was switched on for 30 minutes with UV light to make sterile conditions inside the laminar air flow
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3.4 Inoculation:The culture tubes containing MS medium were kept horizontally, toreduce the contaminations. By using forceps explants were inoculated into the culture tubes and were immediately closed with cotton plugs. 3.5 Incubation: The inoculated culture tubes were incubated at 25±2ºC under 16 hours light followed by 8 hours dark period. Cultures were maintained at light intensity of 3000 Lux provided by florescent lamps. 3.6 Statistical Analysis: Research data obtained in various experiments in the study was statistically analyzed by using SPSS Version 17 and means were compared using Tukey’s tests at the 5% level of significance. All means are presented with SE. 4.
Results 4.1 In vitro Seed Germination Fresh seeds of Mirabilis jalapa Linn cultivar pink coloured flower, was collected from 6 months old plants. These seeds were used initially for In vivo seed germination studies, which has given 0% in vivo germination. This poor in vivo seed germination can be overcome by the plant by protecting it as a underground stem during the dry and hot conditions during summer. For in vitro seed germination studies, we inoculated the fresh seeds on to basal Murashige and Skoogs media. After 2 weeks of duration, only 2% of seeds have germinated (Plate 1). 4.2Callus induction& Somatic embryogenesis Callus was induced in leaf and nodal explants of Mirabilis jalapaLinn (Plate 2A & 2B) cultured on MS medium supplemented with various concentrations of 2, 4-D, NAA and Kn, after 3 weeks of culture (Tables 1-3). On 2,4-D supplemented media, maximum amount of mean fresh and dry weight (mg) of callus (186.7 ± 0.44, 9.7 ± 0.61) & (138.6 ± 0.13, 4.9 ± 0.31) was observed in leaf and nodal explants cultured on 5 mg/L 2,4-D (Table 1 ). On NAA supplemented media, maximum amount of mean fresh and dry weight (mg) of callus(136.7 ± 0.24, 7.5 ± 0.14) & (155.8 ± 0.34, 7.8 ± 0.19) was observed in leaf and nodal explants cultured on 5 mg/L NAA, respectively (Table 2). At high concentrations of NAA (5 mg/L), somatic embryos are induced from the nodal explants after 4 weeks of culture (Plate 2C & 2D)On 2,4-D + NAA supplemented media, maximum amount of mean fresh and dry weight (mg) of callus (189.6 ± 0.25, 7.6 ± 0.23); (198.4± 0.34, 9.2 ± 0.23) was observed in leaf and nodal explants cultured on 3mg/L 2,4-D + 5mg/L NAA (Table 3). Overall maximum amount of mean fresh and dry weight (mg) of callus (189.6 ± 0.25, 7.6 ± 0.23); (198.4± 0.34, 9.2 ± 0.23) was obtained from leaf and nodal explants, respectively when cultured on MS media supplemented with combinations of 2,4-D(3 mg/L) and NAA(5mg/L).
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Table 1: Callus induction in Leaf and Nodal explants cultured on MS medium supplemented with various concentrations of 2, 4-D, in Mirabilis jalapa Linn
Explant
Morphogenetic Response
Callus
MS + 2,4-D mg/L
Mean fresh weight (mg) Leaf 1.0 2.0 White callus 141.3 ± 0.12d 3.0 White callus 157.5 ± 0.25c 4.0 Friable white callus 158.0 ± 0.61b 5.0 Brown callus 186.7 ± 0.44a Nodal 2.0 White callus 87.1 ± 0.13g 3.0 Friable White callus 103.1 ± 0.29f 4.0 Brown callus 111.4 ± 0.51e 5.0 Brown callus 138.6 ± 0.13d Data represents average of three replicates. Each replicate consists of 25 cultures. Data scored at the end of 3 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at=0.05
Mean dry weight (mg) 5.4± 0.31d 6.3 ± 0.04c 8.5 ± 0.16b 9.7 ± 0.61a 2.1 ± 0.11g 3.2 ± 0.14f 4.2 ± 0.13e 4.9 ± 0.31d
Table 2: Callus induction in Leaf and Nodal explants cultured on MS medium supplemented with various concentrations of NAA, in Mirabilis jalapa Linn
Explant Leaf
Nodal
Morphogenetic Response MS + NAA mg/L 1.0 2.0 3.0 4.0 5.0 2.0 3.0 4.0 5.0
Callus
-
Mean dry weight (mg) -
121.0 ± 0.12b 127.4 ± 0.15c 136.7 ± 0.24d 102.5 ± 0.14a 121.5 ± 0.14b 135.6 ± 0.32d 155.8 ± 0.34e
4.5 ± 0.31a 5.2 ± 0.22b 7.5 ± 0.14d 4.2 ± 0.21a 4.8 ± 0.16b 6.2 ± 0.10c 7.8 ± 0.19d
Mean fresh weight (mg) White callus Friable white callus Friable brown callus White callus Friable White callus Brown callus Brown callus with Somatic embryos after 3 weeks of cultures
Data represents average of three replicates. Each replicate consists of 25 cultures. Data scored at the end of 3 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at = 0.05 Table 3: Callus induction in Leaf and Nodal explants cultured on MS medium supplemented with various concentrations and Combinations of 2, 4-D & NAA, in Mirabilis jalapa Linn
Explant Leaf
MS + NAA mg/L MS + 2,4-D mg/L
Morphogenetic Response
3.0 1.0 3.0 2.0 White callus 3.0 3.0 Friable white callus 3.0 4.0 Friable white callus 3.0 5.0 Brown callus Node 3.0 2.0 White callus 3.0 3.0 Friable White callus 3.0 4.0 Friable Brown callus 3.0 5.0 Brown callus Data represents average of three replicates. Each replicate consists of 25 cultures. Data scored at the end of 3 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at=0.05
Callus Mean fresh weight (mg) 113.2± 0.13e 141.2 ± 0.31c 153.0 ± 0.23b 189.6 ± 0.25a 121.3 ± 0.11d 123.3 ± 0.31d 141.3 ± 0.24c 198.4± 0.34a
Mean dry weight (mg) 3.8± 0.41e 4.6 ± 0.24d 6.7 ± 0.71c 7.6 ± 0.23b 3.2 ± 0.12e 4.3 ± 0.21d 6.3 ± 0.12c 9.2 ± 0.23a
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4.3 Regeneration of shoots from Callus (Indirect Organogenesis) Primary callus (friable and creamy white in colour) of leaf and nodal explants of Mirabilis jalapa were induced maximally on 3mg/L 2, 4-D. When these primary calluses were subcultured on the same medium, callus turned fragile and there is an increase in fresh and dry weights of callus (Table 4). Shoot buds were induced from the leaf and nodal calluses when cultured on combinations of TDZ & BAP after 3 weeks of culture (Table 5), Fig 1& Fig 2, Whereas TDZ and BAP in alone has not resulted in shoot induction form both leaf and nodal calluses.Maximum number of shoot buds (08 ± 0.17 & 06 ± 0.09) (Fig 1) were produced from leaf and nodal calluses (Plate 3B & 3C), cultured on MS + TDZ (0.5mg/L) +BAP (2 mg/L). When the concentration of TDZ is increased beyond 0.75 mg/L in combination with 2mg/L of BAP, it resulted in decrease in no. of shoot buds production (Table 5)(Fig 1& Fig 2). Table 4: Sub culturing of leaf and stem (nodal) calluses on MS supplemented with various concentrations of 2, 4-D in Mirabilis jalapa Linn Callus initiated on
Leaf MS+ 2,4-D (3 mg/L) Stem (Node) MS+ 2,4-D (3 mg/L)
Primary culture Mean Fresh weight in Mean Dry weight in (mg) (mg) 157.5 ± 0.25c 6.3 ± 0.04c
103.1 ± 0.29f
Sub-culture Mean Fresh weight in Mean Dry weight in (mg) (mg) 196.5±0.25a 11.4±0.12a
3.2 ± 0.14f
147± 0.26e
5.8 ± 0.29d
Data represents average of three replicates. Each replicate consists of 10 cultures. Data scored at the end of 2 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at=0.05
Table 5: Shoot bud differentiation from Sub-cultured leaf and nodal calluses cultured on various concentrations of TDZ and BAP in Mirabilis jalapa Linn Sub-cultured Callus
TDZ (mg/L)
Leaf
0.10 0.25 0.50 0.75 1.00
BAP (mg/L) 2.00 2.00 2.00 2.00 2.00
Stem (Nodal)
0.10
2.00
-
0.25 0.50 0.75
2.00 2.00 2.00
01 ± 0.23c 06 ± 0.09a 02 ± 0.16b
1.00 2.00 Data represents average of three replicates. Each replicate consists of 10 cultures. Data scored at the end of 2 weeks of culture. Mean ± standard error. Mean followed by the same superscript in a column is not significantly different at=0.05
No. of shoot buds 03 ± 0.06c 08 ± 0.17a 04 ± 0.10b -
-
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Fig 1: Shoot bud differentiation from Sub-cultured leaf callus cultured on various concentrations of TDZ and BAP in Mirabilis jalapa Linn
Fig 2: Shoot bud differentiation from Sub-cultured Nodal callus cultured on various concentrations of TDZ and BAP in Mirabilis jalapa Linn
4.4 Rooting In vitro shoots obtained from indirect regeneration through callus were inoculated onto rooting media which consists of various concentrations and combinations of IAA and IBA. The best rooting percentage (100%) was obtained on MS + IAA (1.0 mg/L) + IBA (2.0 mg/L) (Plate 3D). 5.
Acclimatization Plantlets with 2–4 well developed leaves and roots were transferred to soil, 75 % plantlets survived in field conditions.
6.
Discussions: In this study leaf and nodal explants of Mirabilis jalapa Linncultured on MS basal medium did not produce any callus, but when cultured on various concentrations of 2,4-D, NAA either individually or in
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combinations produced morequantity of callus, after 3 weeks of culture. Though the rate of callus growth was negligible in first week, the entire explant was covered with healthy callus after 3 weeks.Major portion of callus that developed from leaf and nodal explants was friable to compact & green coloured but became brown when cultured for more than six weeks on the same medium at high concentrations of auxins. The callus that developed on leaf and nodal explants was creamy white in colour. There is scanty information on callus induction from Mirabilis jalapa Linn. Xinjia Xu et. al., 2005 has reported callus induction from cotyledon and epicotyl explants of Mirabilis jalapa Linn on 2,4-D and IAA supplemented media, similarly Tamer et.al., 1997 has also reported callus induction from Mirabilis jalapa Linn from leaf explants in Kinetin supplemented media, where as we have induced the callus from explants like leaf and nodal explants on 2,4-D and NAA either in alone or in combinations. In vitro plantlet production via indirect organogenesis from leaf calluses of Mirabilis jalapa Linn were induced on MS + TDZ + BAP combination. Zaccai et. al., 2007, has reported the direct and indirect shoot induction from nodal explants by using 2mg/L Benzyladenine + 2mg/L Zeatin + 1mg/L Indole Acetic acid, whereas in our study we have employed TDZ for production of shoots from leaf and nodal callus.Xinjia Xu et. al., 2005 has also reported the production of shoots by using TDZ plant growth regulator, but they reported direct shoot induction form petioles of cotyledon explants, whereas in our studies we have induced shoots indirectly from leaf callus by employing combination of TDZ & BAP. Xinjia Xu et. al., 2005, in their report has not reported regarding no. of shoots produced directly from the petioles of cotyledon explants, whereas in our study we have regenerated 08 ± 0.17 shoots indirectly from leaf callus when cultures on MS + TDZ (0.5mg/L) +BAP (2 mg/L).
7.
Conclusion: This is the first report on induction of callus and somatic embryos from nodal explants in Mirabilis jalapa. Compared to earlier reports, we have regenerated more no. of shoots (08 ± 0.17) indirectly from leaf callus when cultured on MS + TDZ (0.5mg/L) +BAP (2 mg/L). PLATE-2
PLATE-1 A
A
B
C
B
C
In vitro seed germination of Mirabilis jalapa Linn. on MS basal media
D
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Callus induction in leaf and nodal explants of Mirabilis jalap Linn
(A)
Mirabilis jalapa Linn Seeds
(B)
Seedling (10 days old)
A)
Callus induction in Leaf explants cultured on MS + 2.4-D (3 mg/L)
(C)
Seedling (20 days old)
B)
Callus induction in Nodal explants cultured on MS + NAA (2 mg/L)
Induction of Somatic Embryos from Nodal callus in Mirabilis jalapa Linn C)
Induction of single oval shaped somatic embryo from nodal callus after 2 weeks of culture, on MS media supplemented with NAA 5mg/L
D)
Induction of two somatic embryos from nodal callus after 3 weeks of culture, on MS media supplemented with NAA 5mg/L
PLATE-3 B
A
D
E
C
In vitro plant regeneration from leaf callus of Mirabilis jalapa Linn. A)
Single Shoot bud induction in leaf callus cultured on MS + TDZ (0.10 mg/L) + BAP (2.0 mg/L)
B)
Multiple Shoot bud induction (03 ± 0.06) in leaf callus cultured on MS + TDZ (0.25 mg/L) + BAP (2.0 mg/L)
C)
Multiple Shoot buds (08 ± 0.17) induction in leaf callus cultured on MS + TDZ (0.50 mg/L) + BAP (2.0 mg/L)
D)
Micro shoot transferred on to rooting media (MS + IAA (1.0 mg/L) + IBA (2.0 mg/L) Rooted individual plantlet Complete plantlets transferred to pots containing sterile soil for hardening
F
E) F)
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