IN VITRO PROPAGATION OF ELAEOCARPUS SPHAERICUS

Abstract

The present study involves in-vitro clonal propagation of Elaeocarpus sphaericus through axillary bud sprouting and
subsequent shoot regeneration. Optimal proliferations of axillary shoot were induced on MS medium supplemented
with BAP (1.5 mg/l) and KN (1.5 mg/l). Optimal shoot multiplication was induced on MS medium fortified with BAP
(1.5mg/l), KN (1.5 mg/l) and CH (100 mg/l). MS medium supplemented with 1.0 mg/l BAP and 50 mg/l ADS was used for routine sub-culturing of multiple shoots at periodic interval of every 3 weeks. Of the 6 treatments (3 each of IBA and NAA) imposed for rooting of shoots, ½ MS+ 1.0 mg/l IBA induced 80% rooting. Plants were hardened in vitro and acclimatized in hardening chamber and subsequently transferred to field.

Full text

172 The Indian Forester [February
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Hkkj) izkIr dh xbZA blds vykok blus n'kkZ;k fd 30 ls-eh- x 30 ls-eh- ikni ls ikni vkSj iafDr ls iafDr vUrjky esa [kkn mipkj ds lHkh ekeyksa
esa vf/dre mRiknu vfHkfyf[kr fd;k x;kA References
Azizi P., Khomame A.M. and Mirsoheil M. (2008). Influence of cow manure vermicompost on growth of Dieffenbachia. Eco. Env. and Cons.,
14 (1):1-4.
Atiyeh R.M., Arancon N.C., Edwards A. and Metzger J.D. (2000a). Influence of Earthworm - processed pig manure on the growth and yield of
greenhouse tomatoes. Bioresource Technology, 75: 175-180.
Joy P.P. (2003). Agrotechnological practices for quality crud drug production in Nilappana (Curculigo orchioides Gaertn.). Ph.D. Thesis. Kerala
Agricultural University.
Kumar R. (2006). Effect of planting time of cuttings on growth of cutting of Populus deltoides under nursery conditions. Indian Journal of
Forestry, 29 (1): 381-383.
Minhas R.S. and Sood A. (1994). Effect of inorganic and organics on yield and nutrient uptake by three crops in rotation on in acid alfisol.
Journal of Indian Society of Soil Science, 42: 257-260.
MDI (2001-2011) Met. Dept. of India (2001-2011). Mohanbari Station, Dibrugarh, Assam.
Nazimudeen S.K., Ramaswamy S. and Khameswaran L. (1978). Effect of Andrographis paniculata on snake venom induced death and its
mechanism. Indian J. Pharm. Sci., 40:133-134.
Pratibha G., Korwar G.R., Palanikumar D. and Jois V. (2007). Effect of planting materials, fertilizers and microsite improvement on yield and
quality of henna (Lawsonia inermis) in Alfisols of semi-arid regions. Indian Journal of Agricultural Sciences, 77(11): 721-725.
Pandey A.K. and Patra A.K. (2001). Cultivation of Kalmegh (Andrographis paniculata) in the Satpura Region of Madhya Pradesh. Souvenir,
National Seminar on Com. Cult. Pross. and Mark. of Medicinal and Aromatic Plants, JNKVV, Jabalpur (M.P.), India: 86-87.
Rezainejad Y. and Afyuni M. (2000). The evaluation of organic matters effect on soil chemical characteristics, elements uptake by corn and its
yield. Journal of Sciences and Techniques of Agricultural and Natural Resources, 4:19-27.
Reddy M.B., Reddy K.R. and Reddy M.N. (1988). A survey of medicinal plants of Chenchu tribes of Andhra Pradesh. India. Int. J. Crude Drug
Res., 26(4): 189-196.
Saikia L.R. and Uapdhyaya S. (2007). Influence of different soil supplements on the growth and dry matter production of indigenous
medicinal plant Andrographis paniculata Nees. Eco. Env. and Cons., 13(2): 395-401.
Singh V. and Singh R.K. (2006). Effect of season, time of planting and plant density on the growth, yield and andrographolide content of
Kalmegh (A. paniculata Nees.) under North Indian condition. Int. J. of Plant Science, 1(1):6-9.
Sharma R. ( 2004). Agrotechniques of medicinal plants. Daya Publishing House, Delhi- 110035, p: 264.
Tiwari J.P. (2001). Status of medicinal and aromatic plants in Madhya Pradesh and future perspective. Souvenir, National Seminar on Com.
Cult. Pross. and Mark. of Medicinal and Aromatic Plants, JNKVV, Jabalpur (M.P.), India: 5-15.
Upadhyaya S. and Saikia L.R. (2012). Influence of Eco-friendly Manures on Leaf Biomass Production of a Traditional Medicinal Plant
Adhatoda vasica Nees. under upper Brahmaputra Valley Agroclimatic Zone. Journal of Agricultural Science and Technology, A 2:115-
119.
be encouraging as the farmers will be benefitted from Detail work by using more types of organic manures will
this practice than some other staple crops of this zone. be the aim of further investigation. IN VITRO PROPAGATION OF ELAEOCARPUS SPHAERICUS
J.M.S. CHAUHAN, PRABHA BISHT, MONIKA PANWAR AND AJAY THAKUR
Tissue Culture Discipline, Genetics and Tree Propagation Division
Forest Research Institute, Dehradun (Uttarakhand), India
E-mail : chauhanjms@icfre.org
ABSTRACT
The present study involves in-vitro clonal propagation of Elaeocarpus sphaericus through axillary bud sprouting and
subsequent shoot regeneration. Optimal proliferations of axillary shoot were induced on MS medium supplemented
with BAP (1.5 mg/l) and KN (1.5 mg/l). Optimal shoot multiplication was induced on MS medium fortified with BAP
(1.5mg/l), KN (1.5 mg/l) and CH (100 mg/l). MS medium supplemented with 1.0 mg/l BAP and 50 mg/l ADS was used for
routine sub-culturing of multiple shoots at periodic interval of every 3 weeks. Of the 6 treatments (3 each of IBA and
NAA) imposed for rooting of shoots, ½ MS+ 1.0 mg/l IBA induced 80% rooting. Plants were hardened in vitro and
acclimatized in hardening chamber and subsequently transferred to field.
Key words: Elaeocarpus sphaericus, In-vitro propagation, Nodal explant, Axillary shoot sprouting.
Introduction The natural as well as planted areas of Eleocarpus
have been decreasing gradually due to household and
Elaeocarpus sphaericus (Gaertn.) K. Schum. (Syn: industrial use. Nut collection for beads has caused the
Elaeocarpus ganitrus Roxb.), commonly known as shrinkage of the seed bank in the soil. Besides, poor seed
'Rudraksha' belongs to family Elaeocarpaceae. It is a large viability has adverse affects on the regeneration of
evergreen broad-leaved tree found in tropical ever green species, pushing it to threatened category. The use of
forests and preferably grows in suitable climatic regions
0biotechnology on trees has now opened up new
with temperature ranges of 25-30 C. The beneficial possibilities for rapid mass multiplication of existing
powers of Elaeocarpus beads have been well known for stocks of germplasm as well as conservation of
mental diseases, epilepsy, asthma, hypertension, medicinally important plants/plant parts (Bajaj, 1986;
arthritis and liver diseases. An HPTLC densitometry Haissig et al., 1987; Agarwal and Gupta, 1991; Gupta and
method has been developed to estimate quercetin in E. Agrawal, 1992).
sphaericus beads so that plant can be standardized on The reports available are pertaining to studies on
the basis of its bioactive marker (Singh et al., 2013). pharmacological activities of E. sphaericus (Dasgupta et
According to ayurvedic medical system, wearing of al., 1984; Singh and Nath, 1999; Bhattacharya et al.,
Elaeocarpus can have a positive effect on the heart and 1975; Singh et al., 2000; Kumar et al., 2008; Sakat et al.,
nerves attributing to its electromagnetic properties. The 2009) and regeneration status and seedling survival of
Elaeocarpus extracts are also known to exhibit rudraksh (Khan et al., 2004).
antimicrobial, antifungal and pharmacological activities. The present investigation was undertaken to find
The leaves of the species are considered medicinally out optimal conditions for in-vitro propagation through
important due to the presence of secondary metabolites axillary bud culture that can help in conservation and
like alkaloids, flavanoids, tannins, steroids, saponins, production of true-to-type plants on large scale to ensure
terpenoids and cardiac glycosides. Flavanoids are used in genetic stability.
the management of anxiety (Shah et al., 2010; Kumar et Material and Methods
al., 2011; Garg et al., 2013). Wearing Garbh Gauri E. Plant material and sterilization
sphaericus helps women who have problems in
conceiving a child and are prone to abortion. E. Young healthy shoots of E. sphaericus were
sphaericus is also useful for women suffering from collected from 3 year old tree growing at Non-Wood
hysteria and coma (Swami et al., 2010). The tree, Forest Product (NWFP) nursery of Forest Research
Institute, Dehradun. Nodal segments measuring 2.0 - 2.5
therefore, is of immense traditional and medicinal value cm were cut and washed in running tap water to remove
creating interest for extensive scientific research in this the dust particles followed by several washings in liquid
direction (Nain et al., 2012).
Indian Forester, 141 (2) : 173-177, 2015
http://www.indianforester.co.in ISSN No. 0019-4816 (Print)
ISSN No. 2321-094X (Online)

In vitro propagation of Elaeocarpus sphaericus 175
2015]
174 The Indian Forester [February
detergent (Teepol, 5-10 drops/100 ml.) in a vial under washings with sterilized distilled water in laminar flow
gentle agitating conditions. Subsequently, they were cabinet to remove the traces of sterilant. Prior to
washed with surfactant Tween-20 (2 drops/100 ml inoculation proximal and distal ends of nodal explants
solution) for 10 minutes and after washing under running were trimmed slightly to remove tissues that have
tap water, fungicide treatment (2% Bavistin, 50% imbibed sterilants.
carbendazim WP) for 40 minutes was given. Nodal Culture media and incubation conditions
explants were surface sterilized by soaking in Mercuric For induction and shoot multiplication MS
Chloride (0.1%) for 10 minutes followed by repeated
medium (Murashige and Skoog, 1962) having 3% sucrose experiments. All the cultures were incubated in a culture
0 0
was used. The medium was gelled with 0.7% room at 25 C ±2 C for 16 hours in light (illuminated by 40
bacteriological agar. The pH of medium was adjusted to watt cool white fluorescent tubes, 1200 lux) and 8 hours
5.8 by using 1N NaOH or 1N HCl prior to addition of agar. in dark.
2
Medium was sterilized by autoclaving at 1.0 kg/cm Hardening and acclimatization of Plantlets: Tissue
0
(121 C) pressure for 15 minutes culture raised plantlets were hardened in- vitro (inside
0 0
For induction of shoots MS media supplemented culture room, temp. 25 C + 2 C) in gem bottles
with BAP (0.5 mg/l- 1.5 mg/l) either alone or in containing, sterilized vermiculite. Plantlets were watered
1
combination with KN (0.5 mg/l – 1.0 mg/l) and in with / MS macro salt solution. Plantlets were
5
combinations with auxins either IAA or NAA (0.2 mg/l - transferred to 9 inch size poly bags containing soil: sand:
0.5 mg/l) were used. MS medium supplemented with farm yard manure in 1:1:1 for acclimatization to ex-vitro
BAP (1.5 mg/l) in combination with KN (1.5 mg/l) and environment and watered with tap water daily.
additive casein hydrolysate (CH) at a concentration of 50 Results and Discussion
mg/l -100 mg/l was used to study the effect of casein The sterilization procedure adopted resulted in
hydrolysate on the rate of shoot multiplication. 80% aseptic and responsive cultures. The effectiveness of
For in-vitro rooting of shoots ½ strength MS 0.1% HgCl in surface disinfection of explants of tree
2
solidified medium with 3% sucrose and rooting species has earlier been reported in Quercus robur
hormones (IBA and NAA) was used. Data pertaining to (Puddephat et al., 1997), Citrus lemon (Rathore et al.,
average number of shoots and average shoot length was 2007), Eucalyptus F1 hybrid (E. citriodora x E. torelliana)
recorded after a period of 6 weeks. Sub-culturing was (Bisht et al., 2000) and Eucalyptus tereticornis (Bisht et
carried out at periodic interval of 3 weeks using MS al., 2002). Bud break occurred in nodal explants only
medium supplemented with BAP (1.0 mg/l) and CH (50 after 4 weeks of inoculation. 90% explants have shown
mg/l). Data on rooting of shoots were recorded after a bud break in MS + BAP (1.5 mg/l) + KN (1.5 mg/l) with
period of 30 days. average shoot number 1.50±0.40 and average shoot
Ten replicates were maintained for each treatment length 1.30±0.23cm (Table 1, Fig. 1. a). Whereas MS +
during culture establishment, multiplication and rooting BAP (1.5 mg/l) + KN (1.0 mg/l) gave 60% bud break with
Table: 1 : Effect of PGRs on shoot induction from nodal explants of E. sphaericus.
S. No. MS medium + Plant growth regulators
concentrations (mg/l) Percent
bud-
break
Observations after 6 weeks (mean ± SE)
BAP KN IAA NAA Number of shoots Shoot length (cm)
Control 0.0 0.0 0.0 0.0 00 - -
1 0.5 0.0 0.0 0.0 10 0.10±0.10 0.02±0.02
2 1.0 0.0 0.0 0.0 10 0.10±0.10 0.04±0.04
3 1.5 0.0 0.0 0.0 40 0.50±0.22 0.12±0.06
4 0.5 0.5 0.0 0.0 30 0.30±0.15 0.10±0.06
5 0.5 1.0 0.0 0.0 60 0.80±0.25 0.18±0.06
6 0.5 1.5 0.0 0.0 30 0.40±0.22 0.17±0.11
7 1.0 0.5 0.0 0.0 30 0.40±0.22 0.20±0.11
8 1.0 1.0 0.0 0.0 40 0.90±0.38 0.44±0.19
9 1.0 1.5 0.0 0.0 30 0.50±0.27 0.42±0.22
10 1.5 0.5 0.0 0.0 50 0.90±0.35 0.68±0.24
11 1.5 1.0 0.0 0.0 60 1.10±0.35 0.54±0.17
12 1.5 1.5 0.0 0.0 90 1.50±0.40 1.30±0.23
13 1.5 1.5 0.2 0.0 60 0.90±0.28 0.63±0.19
14 1.5 1.5 0.5 0.0 50 0.80±0.33 0.51±0.19
15 1.5 1.5 0.0 0.2 60 1.00±0.33 0.94±0.27
16 1.5 1.5 0.0 0.5 60 0.80±0.25 0.66±0.19
Table: 2 : Effect of additive casein hydrolysate on Shoot multiplication from nodal explants of E. sphaericus.
S. No. MS medium + Plant growth regulators
concentrations (mg/l) Observations after 6 weeks (Mean ± SE)
BAP KN CH Number of shoots Shoot length (cm)
Control 1.5 1.5 0.0 8.1±1.18 1.14±0.18
1 1.5 1.5 50 10.7±1.42 1.33±0.19
2 1.5 1.5 100 12.5±1.18 1.30±0.10
Fig. 1 (a-f) : In vitro propagation of Elaeocarpus sphaericus
Bud break in nodal ex-plants Multiple shoots formation
Multiplication of shoot In vitro rooting of shoot
In vitro Hardening of plantlet Ex-vitro transferred plants

In vitro propagation of Elaeocarpus sphaericus 175
2015]
174 The Indian Forester [February
detergent (Teepol, 5-10 drops/100 ml.) in a vial under washings with sterilized distilled water in laminar flow
gentle agitating conditions. Subsequently, they were cabinet to remove the traces of sterilant. Prior to
washed with surfactant Tween-20 (2 drops/100 ml inoculation proximal and distal ends of nodal explants
solution) for 10 minutes and after washing under running were trimmed slightly to remove tissues that have
tap water, fungicide treatment (2% Bavistin, 50% imbibed sterilants.
carbendazim WP) for 40 minutes was given. Nodal Culture media and incubation conditions
explants were surface sterilized by soaking in Mercuric For induction and shoot multiplication MS
Chloride (0.1%) for 10 minutes followed by repeated
medium (Murashige and Skoog, 1962) having 3% sucrose experiments. All the cultures were incubated in a culture
0 0
was used. The medium was gelled with 0.7% room at 25 C ±2 C for 16 hours in light (illuminated by 40
bacteriological agar. The pH of medium was adjusted to watt cool white fluorescent tubes, 1200 lux) and 8 hours
5.8 by using 1N NaOH or 1N HCl prior to addition of agar. in dark.
2
Medium was sterilized by autoclaving at 1.0 kg/cm Hardening and acclimatization of Plantlets: Tissue
0
(121 C) pressure for 15 minutes culture raised plantlets were hardened in- vitro (inside
0 0
For induction of shoots MS media supplemented culture room, temp. 25 C + 2 C) in gem bottles
with BAP (0.5 mg/l- 1.5 mg/l) either alone or in containing, sterilized vermiculite. Plantlets were watered
1
combination with KN (0.5 mg/l – 1.0 mg/l) and in with / MS macro salt solution. Plantlets were
5
combinations with auxins either IAA or NAA (0.2 mg/l - transferred to 9 inch size poly bags containing soil: sand:
0.5 mg/l) were used. MS medium supplemented with farm yard manure in 1:1:1 for acclimatization to ex-vitro
BAP (1.5 mg/l) in combination with KN (1.5 mg/l) and environment and watered with tap water daily.
additive casein hydrolysate (CH) at a concentration of 50 Results and Discussion
mg/l -100 mg/l was used to study the effect of casein The sterilization procedure adopted resulted in
hydrolysate on the rate of shoot multiplication. 80% aseptic and responsive cultures. The effectiveness of
For in-vitro rooting of shoots ½ strength MS 0.1% HgCl in surface disinfection of explants of tree
2
solidified medium with 3% sucrose and rooting species has earlier been reported in Quercus robur
hormones (IBA and NAA) was used. Data pertaining to (Puddephat et al., 1997), Citrus lemon (Rathore et al.,
average number of shoots and average shoot length was 2007), Eucalyptus F1 hybrid (E. citriodora x E. torelliana)
recorded after a period of 6 weeks. Sub-culturing was (Bisht et al., 2000) and Eucalyptus tereticornis (Bisht et
carried out at periodic interval of 3 weeks using MS al., 2002). Bud break occurred in nodal explants only
medium supplemented with BAP (1.0 mg/l) and CH (50 after 4 weeks of inoculation. 90% explants have shown
mg/l). Data on rooting of shoots were recorded after a bud break in MS + BAP (1.5 mg/l) + KN (1.5 mg/l) with
period of 30 days. average shoot number 1.50±0.40 and average shoot
Ten replicates were maintained for each treatment length 1.30±0.23cm (Table 1, Fig. 1. a). Whereas MS +
during culture establishment, multiplication and rooting BAP (1.5 mg/l) + KN (1.0 mg/l) gave 60% bud break with
Table: 1 : Effect of PGRs on shoot induction from nodal explants of E. sphaericus.
S. No. MS medium + Plant growth regulators
concentrations (mg/l) Percent
bud-
break
Observations after 6 weeks (mean ± SE)
BAP KN IAA NAA Number of shoots Shoot length (cm)
Control 0.0 0.0 0.0 0.0 00 - -
1 0.5 0.0 0.0 0.0 10 0.10±0.10 0.02±0.02
2 1.0 0.0 0.0 0.0 10 0.10±0.10 0.04±0.04
3 1.5 0.0 0.0 0.0 40 0.50±0.22 0.12±0.06
4 0.5 0.5 0.0 0.0 30 0.30±0.15 0.10±0.06
5 0.5 1.0 0.0 0.0 60 0.80±0.25 0.18±0.06
6 0.5 1.5 0.0 0.0 30 0.40±0.22 0.17±0.11
7 1.0 0.5 0.0 0.0 30 0.40±0.22 0.20±0.11
8 1.0 1.0 0.0 0.0 40 0.90±0.38 0.44±0.19
9 1.0 1.5 0.0 0.0 30 0.50±0.27 0.42±0.22
10 1.5 0.5 0.0 0.0 50 0.90±0.35 0.68±0.24
11 1.5 1.0 0.0 0.0 60 1.10±0.35 0.54±0.17
12 1.5 1.5 0.0 0.0 90 1.50±0.40 1.30±0.23
13 1.5 1.5 0.2 0.0 60 0.90±0.28 0.63±0.19
14 1.5 1.5 0.5 0.0 50 0.80±0.33 0.51±0.19
15 1.5 1.5 0.0 0.2 60 1.00±0.33 0.94±0.27
16 1.5 1.5 0.0 0.5 60 0.80±0.25 0.66±0.19
Table: 2 : Effect of additive casein hydrolysate on Shoot multiplication from nodal explants of E. sphaericus.
S. No. MS medium + Plant growth regulators
concentrations (mg/l) Observations after 6 weeks (Mean ± SE)
BAP KN CH Number of shoots Shoot length (cm)
Control 1.5 1.5 0.0 8.1±1.18 1.14±0.18
1 1.5 1.5 50 10.7±1.42 1.33±0.19
2 1.5 1.5 100 12.5±1.18 1.30±0.10
Fig. 1 (a-f) : In vitro propagation of Elaeocarpus sphaericus
Bud break in nodal ex-plants Multiple shoots formation
Multiplication of shoot In vitro rooting of shoot
In vitro Hardening of plantlet Ex-vitro transferred plants

In vitro propagation of Elaeocarpus sphaericus 177
2015]
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orZeku vè;;u esa d{kh; dyh vadqj.k vkSj ijorhZ izjksg iqutZuu ds }kjk byhvksdkiZl LiQsfjdl dk ik=k Dyksuh; izc/Zu 'kkfey gSA ch-
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rhu lIrkg ds fu;rdkfyd vUrjky ij cgq izjksgksa ds fu;fer mi&lao/Zu ds fy, 1-0 mg/l ch-,-ih- vkSj 50 mg/l Ads ds lkFk ewyksRifÙk ds
fy, ykxw fd, x, 6 mipkjksa (vkbZ-ch-,- vkSj ,u-,-,- ds izR;sd ds rhu&rhu) esa ls 1@2 MS$1.0 mg/l vkbZ-ch-,- us 80 izfr'kr ewyksRifÙk izsfjr
dh gSA ikniksa dks ik=k esa dBksjhd`r fd;k vkSj dBksjhdj.k pSEcj esa n'kkuqdwfyr djds {ks=k esa gLrkUrfjr fd;k x;kA
References
Agrawal V., Gupta S.C. (1991). In vitro plantlet development from 25-year-old trees of Populus euramericana - a hybrid tree. Plant Science,
78:99-105.
Agrawal V., Prakash S. and Gupta S.C. (2002). Effective protocol for in vitro shoot production through nodal explants of Simmondsia
chinensis. Biol. Plant, 45: 449-453.
Ahmad N. and Anis M. (2005). In vitro mass propagation of Cucumis sativus L. from nodal segments. Turk. J. Bot., 29: 237-240.
Bajaj Y.P.S. (1986). Biotechnology of tree improvement for rapid propagation and biomass energy production. In: Biotechnology in
agriculture and forestry, vol. 1, Trees, 1. (Bajaj, Y.P.S. ed.) Berlin: Springer-Verlag: 1-23.
Bhattacharya S.K., Debnath P.K., Pandey V.B. and Sanyal A.K. (1975). Pharmacological investigation on Elaeocarpus ganitrus. Planta Med.,
23: 174–177.
Bisht P., Joshi I., Chauhan J.M.S., Sharma S.K. and Bagchi S.K. (2000). Micropropagation of A 23-year-old candidate plus tree of Eucalyptus
tereticornis sm. Ind. Jour. forestry, 23(2):149-154.
Bisht, Prabha, Sharma, V.K. and Uniyal, D.P. (2002). Clonal propagation of Maure Eucalyptus F1 hybrid (E. citriodora Hook. x E. torelliana F.V.
Muell.). Ind. Jour. forestry, 25(4):481-485.
Claudebo M., Gendraud M. and Franclet A. (1988). Roles of phenolic compounds on micropropagation of juvenile and mature clones of
Sequoiadendron giganteum : influence of activated charcoal. Sci. Hort.,34: 283-291.
Dasgupta A., Agarwal S.S. and Basu D.K. (1984). Anticonvulsant activity of the mixed fatty acids of Elaeocarpus ganitrus Roxb. (Rudraksh).
Indian J. Physiol. Pharmacol, 28: 245-286.
Garg K., Goswami1 K. and Khurana G. (2013). A Pharmacognostical Review on Elaeocarpus Sphaericus. Int. J. Pharm. Sci., 5(1): 3-8.
Gupta S.C., Agrawal V. (1992). Micropropagation of woody taxa and plant productivity. In: Role of biotechnology in agriculture. (Prasad, B.N,
Chimire, C.P.S. and Agrawal, V.P. eds.) New Delhi: Oxford Publishing Co. Pvt. Ltd., 37-52.
Haissig B.E., Nelson N.D. and Kidd G.H. (1987). Trends in the use of tissue culture in forest improvement. Biotechnology, 52-57.
Khan M.L., Bhuyan P., and Tripathi R.S. (2004). The conservation status of Rudraksh (Elaeocarpus ganitrus Roxb.) in tropical wet evergreen
forests of Arunachal Pradesh. Current Science, 86: 646-650.
Kumar G., Karthik L. and Rao B., Kokati V. (2011). Antimicrobial activity of Elaeocarpus sphaericus Roxb (Elaeocarpaceae): An in vitro study.
Elixir Bio. Tech., 40 (2011): 5384-5387.
Kumar T., Sathish S.S., Palvannanb T. and Bharathi Kumar V. M. (2008). Evaluation of Antioxidant Properties of Elaeocarpus ganitrus Roxb.
Leaves. Iranian Journal of Pharmaceutical Research, 7 (3): 211-215.
Murashige T. and Skoog F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15:473-97.
Nain J., Garg K. and Dhahiya S. (2012). Analgesic and Anti-Inflammatory Activity Of Elaeocarpus Sphaericus Leaf Extract. Int. J. Pharm.
Pharm. Sci., 4(1): 379-381.
Puddephat I.J., Alderson P.G. and Wright N.A. (1997). Influence of explants source, plant growth regulators and culture environment on
culture initiation and establishment of Quercus robur L. in vitro. Journal of Exp. Botany., 48(309): 951-962.
Purohit S.D., Kukda G., Sharma P. and Tak K. (1994). In vitro propagation of Wrightia tomentosa through enhanced axillary branching. Plant
Science, 103: 67-72.
Quraishi A. and Mishra S.K. (1998). Micropropagation of nodal explants from adult tree of Cleistanthus collinus. Plant Cell Rep., 17:430-433.
Ran Y. and Simpson S. (2005). In vitro propagation of the genus Clivia. PCTOC, 81:239-242.
Rathore J.S., Rathore M.S., Singh M., Singh R.P. and Shekhawat N.S. (2007). Micropropagation of mature trees of Citrus limon. Indian J.
Biotechnology, 6:239-244.
Sakat S.S., Wankhede S.S., Juvekar A.R., Mali V.R. and Bodhankar S.L. (2009). Antihypertensive effect of aqueous extract of Elaeocarpus
ganitrus Roxb. seeds in renal artery occluded hypertensive rats. International Journal of PharmTech Research, 1(3): 779-782.
Shah G., Shri R., Mann A., Rahar S. and Panchal V. (2010). Anxiolytic effects of Elaeocarpus sphaericus fruits on the elevated plus-maze model
of anxiety in mice. International Journal of PharmTech Research, 2(3):1781-1786.
Singh, B., Ishar M., Pal S. and Sharma A. (2013). Estimation of Quercetin, an Anxiolytic Constituent, in Elaeocarpus sphaericus. J.
Pharmacognosy and Phytochemistry, 1 (6):117-121.
Singh R.K. and Nath G. (1999). Antimicrobial activity of Elaeocarpus sphaericus. Phytother Res., 13(5): 448–450.
Singh R.K., Bhattacharya S.K. and Acharya S.B. (2000). Studies on extracts of Elaeocarpus sphaericus fruits on in vitro rat mast cells.
Phytomedicine, 7(3): 205-7.
Srangsam A. and Kanchanapoom K. (2007). Establishment of in vitro Culture of Musa AA Group 'Kluai Sa' and Musa AA Group 'Kluai Leb Mue
Nang'and the Analysis of Ploidy Stability. Science Asia, 33: 437-442.
Swami G., Nagpal N., Rahar S., Singh P., Singla S., Porwal A. and Kapoor R. (2010). Elaeocarpus sphaericus: Medical and Scientific facts. Der
Pharmacia Lettre, 2(1): 297-306.
average of 1.10±0.35 shoots with average shoot length propagation from mature trees has always been difficult
0.54±0.17 cm. After 3 weeks, the shoots regenerated due to several inherent problems such as establishment
from nodal explants were transferred to fresh medium of of aseptic cultures, severe microbial contamination and
the same composition to establish initial stock of shoots browning of explants due to accumulation of secondary
(Fig. 1. b.) The shoots were subsequently used for further metabolites (Claudebon et al., 1988; Agrawal and Gupta,
in vitro shoot multiplication (Fig. 1. b). 1991; Purohit et al., 1994; Quraishi and Mishra, 1998;
Agrawal et al., 2002). Data recorded on rooting
For shoot multiplication, additive CH proved experiments reveal that of the six treatments (three
beneficial resulting in increased number and length of concentrations each of IBA and NAA) imposed for
shoots (Table 2). MS medium containing BAP (1.5 mg/l) + inducing rooting in shoots produced in-vitro, 80 per cent
KN (1.5 mg/l) and CH (100mg/l) proved to be the best rooting was induced in IBA (1.0 mg/l) with average 3.4
combination giving an average of 12.5±1.18 shoots with roots per shoots with average root length 2.7 cm after 30
mean shoot length 1.30±0.10 cm, as recorded after 6 days followed by 60 per cent in IBA (0.5mg/l) and NAA
weeks (Figure 1. c). At 50 mg/l CH concentration, average (1.0mg/l) with average roots per shoot 2.8 and 2.2 with
10.7±1.42 shoots with shoot length 1.33±0.19 cm was average root length 1.8 and 1.9 respectively. IBA was
obtained. Positive effect of casein hydrolysate in shoot found more effective than NAA in inducing roots.
multiplication has also been highlighted in Curcumas
sativa (Ahmad and Anis, 2005), Cilia saps. (Ran and Plantlets were hardened in- vitro (inside culture
0 0
Simpson, 2005), Musa saps. (Srangsam and room, temp. 25 C + 2 C) in gem bottles containing,
1
Kanchanapoom, 2007). MS medium supplemented with sterilized vermiculite. Plantlets were watered with / MS
5
1.0 mg/l BAP and 50mg/l Ads (CH) was used for routine macro salt solution. Hardened plantlets were transferred to
sub culturing of multiple shoots at an interval of every 3 9 inch size poly bags containing soil: sand: farm yard manure
wks. By this procedure 5 sub-cultures have already been in 1:1:1 for acclimatization to ex-vitro environment and
carried out. The rate of shoot formation per culture watered with tap water daily. No mortality of plants was
th
increased 8-10 folds after 5 subculture. In vitro observed though the growth rate was slow.
Table: 3: Effect of different auxins and their concentrations on rooting of in vitro regenerated shoots of Eleocarpus sphaericus after 30
days. S. No. ½ strength MS medium
+PGR (mg/l) Rooting % Average number of
roots per shoot
(Mean ± SE)
Average length of
roots(cm)
(Mean ± SE) IBA NAA
1. - - - - -
2. 0.2 - 20 2.0 ± 0.21 1.2 ± 0.13
3. 0.5 - 60 2.8 ± 0.13 1.8 + 0.13
4. 1.0 - 80 3.4 ± 0.16 2.7 ± 0.15
5. 0.2 - - -
6. 0.5 40 1.6 ± 0.16 1.3 ± 0.15
7. 1.0 60 2.2 ± 0.13 1.9 ± 0.10
176 The Indian Forester [February

In vitro propagation of Elaeocarpus sphaericus 177
2015]
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References
Agrawal V., Gupta S.C. (1991). In vitro plantlet development from 25-year-old trees of Populus euramericana - a hybrid tree. Plant Science,
78:99-105.
Agrawal V., Prakash S. and Gupta S.C. (2002). Effective protocol for in vitro shoot production through nodal explants of Simmondsia
chinensis. Biol. Plant, 45: 449-453.
Ahmad N. and Anis M. (2005). In vitro mass propagation of Cucumis sativus L. from nodal segments. Turk. J. Bot., 29: 237-240.
Bajaj Y.P.S. (1986). Biotechnology of tree improvement for rapid propagation and biomass energy production. In: Biotechnology in
agriculture and forestry, vol. 1, Trees, 1. (Bajaj, Y.P.S. ed.) Berlin: Springer-Verlag: 1-23.
Bhattacharya S.K., Debnath P.K., Pandey V.B. and Sanyal A.K. (1975). Pharmacological investigation on Elaeocarpus ganitrus. Planta Med.,
23: 174–177.
Bisht P., Joshi I., Chauhan J.M.S., Sharma S.K. and Bagchi S.K. (2000). Micropropagation of A 23-year-old candidate plus tree of Eucalyptus
tereticornis sm. Ind. Jour. forestry, 23(2):149-154.
Bisht, Prabha, Sharma, V.K. and Uniyal, D.P. (2002). Clonal propagation of Maure Eucalyptus F1 hybrid (E. citriodora Hook. x E. torelliana F.V.
Muell.). Ind. Jour. forestry, 25(4):481-485.
Claudebo M., Gendraud M. and Franclet A. (1988). Roles of phenolic compounds on micropropagation of juvenile and mature clones of
Sequoiadendron giganteum : influence of activated charcoal. Sci. Hort.,34: 283-291.
Dasgupta A., Agarwal S.S. and Basu D.K. (1984). Anticonvulsant activity of the mixed fatty acids of Elaeocarpus ganitrus Roxb. (Rudraksh).
Indian J. Physiol. Pharmacol, 28: 245-286.
Garg K., Goswami1 K. and Khurana G. (2013). A Pharmacognostical Review on Elaeocarpus Sphaericus. Int. J. Pharm. Sci., 5(1): 3-8.
Gupta S.C., Agrawal V. (1992). Micropropagation of woody taxa and plant productivity. In: Role of biotechnology in agriculture. (Prasad, B.N,
Chimire, C.P.S. and Agrawal, V.P. eds.) New Delhi: Oxford Publishing Co. Pvt. Ltd., 37-52.
Haissig B.E., Nelson N.D. and Kidd G.H. (1987). Trends in the use of tissue culture in forest improvement. Biotechnology, 52-57.
Khan M.L., Bhuyan P., and Tripathi R.S. (2004). The conservation status of Rudraksh (Elaeocarpus ganitrus Roxb.) in tropical wet evergreen
forests of Arunachal Pradesh. Current Science, 86: 646-650.
Kumar G., Karthik L. and Rao B., Kokati V. (2011). Antimicrobial activity of Elaeocarpus sphaericus Roxb (Elaeocarpaceae): An in vitro study.
Elixir Bio. Tech., 40 (2011): 5384-5387.
Kumar T., Sathish S.S., Palvannanb T. and Bharathi Kumar V. M. (2008). Evaluation of Antioxidant Properties of Elaeocarpus ganitrus Roxb.
Leaves. Iranian Journal of Pharmaceutical Research, 7 (3): 211-215.
Murashige T. and Skoog F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15:473-97.
Nain J., Garg K. and Dhahiya S. (2012). Analgesic and Anti-Inflammatory Activity Of Elaeocarpus Sphaericus Leaf Extract. Int. J. Pharm.
Pharm. Sci., 4(1): 379-381.
Puddephat I.J., Alderson P.G. and Wright N.A. (1997). Influence of explants source, plant growth regulators and culture environment on
culture initiation and establishment of Quercus robur L. in vitro. Journal of Exp. Botany., 48(309): 951-962.
Purohit S.D., Kukda G., Sharma P. and Tak K. (1994). In vitro propagation of Wrightia tomentosa through enhanced axillary branching. Plant
Science, 103: 67-72.
Quraishi A. and Mishra S.K. (1998). Micropropagation of nodal explants from adult tree of Cleistanthus collinus. Plant Cell Rep., 17:430-433.
Ran Y. and Simpson S. (2005). In vitro propagation of the genus Clivia. PCTOC, 81:239-242.
Rathore J.S., Rathore M.S., Singh M., Singh R.P. and Shekhawat N.S. (2007). Micropropagation of mature trees of Citrus limon. Indian J.
Biotechnology, 6:239-244.
Sakat S.S., Wankhede S.S., Juvekar A.R., Mali V.R. and Bodhankar S.L. (2009). Antihypertensive effect of aqueous extract of Elaeocarpus
ganitrus Roxb. seeds in renal artery occluded hypertensive rats. International Journal of PharmTech Research, 1(3): 779-782.
Shah G., Shri R., Mann A., Rahar S. and Panchal V. (2010). Anxiolytic effects of Elaeocarpus sphaericus fruits on the elevated plus-maze model
of anxiety in mice. International Journal of PharmTech Research, 2(3):1781-1786.
Singh, B., Ishar M., Pal S. and Sharma A. (2013). Estimation of Quercetin, an Anxiolytic Constituent, in Elaeocarpus sphaericus. J.
Pharmacognosy and Phytochemistry, 1 (6):117-121.
Singh R.K. and Nath G. (1999). Antimicrobial activity of Elaeocarpus sphaericus. Phytother Res., 13(5): 448–450.
Singh R.K., Bhattacharya S.K. and Acharya S.B. (2000). Studies on extracts of Elaeocarpus sphaericus fruits on in vitro rat mast cells.
Phytomedicine, 7(3): 205-7.
Srangsam A. and Kanchanapoom K. (2007). Establishment of in vitro Culture of Musa AA Group 'Kluai Sa' and Musa AA Group 'Kluai Leb Mue
Nang'and the Analysis of Ploidy Stability. Science Asia, 33: 437-442.
Swami G., Nagpal N., Rahar S., Singh P., Singla S., Porwal A. and Kapoor R. (2010). Elaeocarpus sphaericus: Medical and Scientific facts. Der
Pharmacia Lettre, 2(1): 297-306.
average of 1.10±0.35 shoots with average shoot length propagation from mature trees has always been difficult
0.54±0.17 cm. After 3 weeks, the shoots regenerated due to several inherent problems such as establishment
from nodal explants were transferred to fresh medium of of aseptic cultures, severe microbial contamination and
the same composition to establish initial stock of shoots browning of explants due to accumulation of secondary
(Fig. 1. b.) The shoots were subsequently used for further metabolites (Claudebon et al., 1988; Agrawal and Gupta,
in vitro shoot multiplication (Fig. 1. b). 1991; Purohit et al., 1994; Quraishi and Mishra, 1998;
Agrawal et al., 2002). Data recorded on rooting
For shoot multiplication, additive CH proved experiments reveal that of the six treatments (three
beneficial resulting in increased number and length of concentrations each of IBA and NAA) imposed for
shoots (Table 2). MS medium containing BAP (1.5 mg/l) + inducing rooting in shoots produced in-vitro, 80 per cent
KN (1.5 mg/l) and CH (100mg/l) proved to be the best rooting was induced in IBA (1.0 mg/l) with average 3.4
combination giving an average of 12.5±1.18 shoots with roots per shoots with average root length 2.7 cm after 30
mean shoot length 1.30±0.10 cm, as recorded after 6 days followed by 60 per cent in IBA (0.5mg/l) and NAA
weeks (Figure 1. c). At 50 mg/l CH concentration, average (1.0mg/l) with average roots per shoot 2.8 and 2.2 with
10.7±1.42 shoots with shoot length 1.33±0.19 cm was average root length 1.8 and 1.9 respectively. IBA was
obtained. Positive effect of casein hydrolysate in shoot found more effective than NAA in inducing roots.
multiplication has also been highlighted in Curcumas
sativa (Ahmad and Anis, 2005), Cilia saps. (Ran and Plantlets were hardened in- vitro (inside culture
0 0
Simpson, 2005), Musa saps. (Srangsam and room, temp. 25 C + 2 C) in gem bottles containing,
1
Kanchanapoom, 2007). MS medium supplemented with sterilized vermiculite. Plantlets were watered with / MS
5
1.0 mg/l BAP and 50mg/l Ads (CH) was used for routine macro salt solution. Hardened plantlets were transferred to
sub culturing of multiple shoots at an interval of every 3 9 inch size poly bags containing soil: sand: farm yard manure
wks. By this procedure 5 sub-cultures have already been in 1:1:1 for acclimatization to ex-vitro environment and
carried out. The rate of shoot formation per culture watered with tap water daily. No mortality of plants was
th
increased 8-10 folds after 5 subculture. In vitro observed though the growth rate was slow.
Table: 3: Effect of different auxins and their concentrations on rooting of in vitro regenerated shoots of Eleocarpus sphaericus after 30
days. S. No. ½ strength MS medium
+PGR (mg/l) Rooting % Average number of
roots per shoot
(Mean ± SE)
Average length of
roots(cm)
(Mean ± SE) IBA NAA
1. - - - - -
2. 0.2 - 20 2.0 ± 0.21 1.2 ± 0.13
3. 0.5 - 60 2.8 ± 0.13 1.8 + 0.13
4. 1.0 - 80 3.4 ± 0.16 2.7 ± 0.15
5. 0.2 - - -
6. 0.5 40 1.6 ± 0.16 1.3 ± 0.15
7. 1.0 60 2.2 ± 0.13 1.9 ± 0.10
176 The Indian Forester [February