Standardization of an Efficient Protocol for Sterilization and Me
Journal of Plant Biochemistry & Physiology

Journal of Plant Biochemistry & Physiology
Open Access

ISSN: 2329-9029

+44 1478 350008

Research Article - (2016) Volume 0, Issue 0

Standardization of an Efficient Protocol for Sterilization and Media for Direct Shoot Regeneration from Nodal Segments of Medicinal Herb Piper longum Linn

Ruby Tiwari*
Department of Biotechnology, IMS Engineering College, India
*Corresponding Author: Ruby Tiwari, Department of Biotechnology, IMS Engineering College, NH-24, Near Dasna, Ghaziabad, UP, India, Tel: +919451218865 Email:


In the present research it was aimed to the development and standardization of an effective sterilization protocol and media for shoot regeneration technique for a medicinal herb Piper longum linn. Nodal explants from juvenile plants were cultured on Murashige and Skoog’s (MS) media with an array of different combinations of Cytokinins and Auxins. Among the various protocols tested for sterilization, the one containing 200 mg activated charcoal in 250 ml autoclaved water has found to be the best, for nodal segments and cultured in antibiotic containing media. Maximum growth were obtained from the media containing (MSL7) BAP(5 mg/l) NAA(0.5 mg/l) and (MSL10) BAP(3 mg/l)+IAA (0.5 mg/l) along with 15 mg Kanamycin. The findings of this study have to be used for development of an effective protocol for the sterilization and establishment of shoots from nodal explants. Best shoot regeneration was observed from nodal explants was used for further studies and micro shoots were transferred for rooting in IBA containing media and then go for acclimatization and hardening.

Keywords: Piper longum linn; Explants; Shoot regeneration; Medicinal herb; Micro shoots


Piper longum linn (Piperaceae) is the accepted source of the drugs Pippali and Pippalimulam throughout the country. Pippali is the dried ripe fruits; Pippalimulam is the roots of this plant [1,2]. Piper longum is a medicinal plant, it is found almost all over India. It is slender aromatic climber with perennial woody stems. Common name of Piper longum is Pippali, Indian long pepper and papal. It is widely used in ayurvedic and unani systems of medicine particularly in diseases of respiratory tract egcough, bronchitis, asthma, etc. The herb has nerve depressant and antagonistic effects on electro-shock and chemo-shock seizures [3].

The plant is a dioecious slender aromatic climber with perennial woody roots, or a perennial creeping under shrub. Branchlets erect, glabrous with swollen nodes; roots clasping at nodes, which help to get attached to the host trees; leaves alternate, ovate, cordate, apex acute to acuminate, margin entire, glabrous. The male and female plants are morphologically very similar till the formation of spikes. Male spikes greenish-yellow, fleshy, cylindrical, with minute flowers, and female spikes, known as long pepper are shorter and thicker than the male spikes. Fruit spikes cylindrical, oblong, berries red or black when ripe, globose with aromatic odour and pungent taste [2,4-6]. It is used to treat flatulence, gout, laryngitis, and paralysis and used to treat abdominal tumors and gastric ulcers, improves the digestive system and also enhances the immune system. It is a native of North East India. It occurs in the hotter parts of India, from central Himmalayas to Assam, Magadhi, Kana, ushana (Sanskrit) [4]. Long pepper is also known and popular in parts of Africa, mainly in the Islamic regions of North and East Africa. It is used to treat flatulence, gout, laryngitis, and paralysis and used to treat abdominal tumors and gastric ulcers, improves the digestive system and also enhances the immune system. The fruits are used as spice and also in pickles. They have a pungent taste and cause salivation and numbness of the mouth [7]. It cures cough, dyspnoea, ascites, leprosy, diabetes, piles, colic indigestion, anemia, thirst and dispels cardiac and spleen disorders, chronic fever and loss of appetite. The drug is used in Ayurvedic treatment for abdorminal tumours and distention, to improve the digestive fire, kapha disorders, flatulence, gout, laryngitis, paralysis, rheumatic pain, sciatica, worms, and for the immune system. It is used in manufacturing cold relief balm, pain balm, joint care balm, and in heart and stress care and cough syrups [2].

Plant cell can differentiate into a whole plant, a process called regeneration, through addition of plant hormones in culture medium. This ability is known as totipotency. Regeneration of a whole plant from a single cell allows researchers to recover whole plants that have a copy of the transgene in every cell. The objectives of this study were to investigate the best surface sterilization method and standardize the media for direct shoot regeneration from nodal explants of Piper longum linn. And to remove the bacterial contamination produced after culture initiation by using antibiotics. Then root induction was done after shoot formation and finally plants go to lab to land.

Materials And Methods

Media preparation

MS Murashige and Skoog medium with 10 different combinations of Auxin and Cytokinin were analyzed for shoot formation Table 1. After preparation of the medium, and pH was maintained at 5.8-5.9 then Clarigel was used as a gelling agent. Media was dispensed into small culture bottles; the same were autoclaved for 20 min. at 15 psi and 121°C and stored at 23±1°C for 2 days. MS medium with auxin and cytokinin were analysed for bacterial contamination was prepared by adding the antibiotics in the same medium by filter sterilization method under the laminar air flow. And for root induction different rooting media (Table 2) was prepared.

MS Media PGR (mg/l)
Media code BAP AS NAA IAA
MSL1 2   0.5  
MSL2 3   1  
MSL3 2   1.5  
MSL4 4   0.5  
MSL5 4   1  
MSL6 4   1.5  
MSL7 5   0.5  
MSL8 5   1  
MSL9 5   1.5  
MSL10 3     0.5

Table 1: Different media composition for shoot regeneration.

Media Code Sucrose PGRs (mg/l) ml/l from stock
  MS   IBA
MSR-1a 3%(30gms/L) 0.25-0.5
MSR-2a 3%(30gms/L) 0.5-1

Table 2: Different media composition for root formation.

Plant materials

The Piper longum linn (Figure 1) nodal explants were collected from the wild habitats and make into herbarium sheets finally identified with the help of recognized scientist of National herbaria.


Figure 1: Graphical representation of Plant growth.

Explants surface sterilization

Freshly harvested nodal segments were cleaned and dead parts separated, that were washed with the running tap water for 10 minutes to remove the dust or sand particles. These were then surface sterilized by using two different methods. In the first method, all explants were treated with Labolene (10 ml) and Savlon (5 ml) for 15 min. then 1% Bavistin (a brood spectrum systemic fungicide containing 50% WP carbendazim) for 25 mins. And then washed with 70% Ethanol for 1 min. Then inside the laminar airflow treat with 0.15% HgCl2 for 8 min and then finally washed thrice with autoclaved water.

In the second method, first treated the explants with 10 ml Labolene and 5 ml Savlon in 250 mg water for 15 min. and washed with 1% Bavistin (a brood spectrum systemic fungicide containing 50% WP carbendazim) for 25 min. Then inside the laminar air flow treated with 0.15% HgCl2 for 5 min. and 100% Ethanol for 30 seconds only, then treated with 200 mg Activated Charcoal in 250 ml of autoclaved water (chilled) for 25 min with gentle shaking under sterile conditions. After that, the explants were washed three times with autoclaved water. Now the explants were ready for culture initiation.

Initiation of explants

Inside the laminar air flow, the nodal explants were cut at both ends to remove the undesirable/dead portions after surface sterilization and subsequently all explants were then transferred aseptically in the bottle containing media then cultures were stored under controlled environment of growth room, and later were exposed to fluorescent tube lights, with a photoperiod of 16 hour in every 24 hour. Cultures were observed regularly to observe their response to tissue culture. Data were recorded after every 10th day for four weeks.

Shoot induction

Nodal segments are grown on the medium containing different composition of cytokinin and auxin. And after that proliferated shoots were sub cultured for every 15 days for further mass multiplication.

Root induction and acclimatization

After successful shoot induction, when multiple shoots were generated then regenerated shoots were separated and transferred into MS medium containing IBA (In dole 3 butyric acid) for rooting. Acclimatization of rooted plantlets was achieved by transferring them into pots containing garden soil and sand under shade house [8] conditions where approximately 90% seeding survive.

Results And Discussion

Effect of sterilization method

The surface sterilization was optimized that prevented the blackening of tissues. The sterilization with 200 mg activated charcoal proved a little bit successful procedure as maximum explants were undamaged and responded to tissue culture medium which grow well and only 25% of them became infected, because plants produce a huge variety of secondary compounds as natural protection against microbial and insect attack [9] and activated charcoal plays a important role (for sterilization) in plant growth (Figure 1) at the time of tissue culture. On the other hand, treatment with 70% ethanol in 250 ml water failed due to contamination of 90% explants. But after using antibiotics in culture medium by filter sterilization, only 5% contamination occurs in our second sterilization procedure. So 200 activated charcoal proved the most successful sterilization procedure in the medium containing 15 mg Kanamycin (antibiotic), as all the explants safely grow on that medium.


Figure 1: Stepwise Regeneration of nodal explants of Piper longum linn.

Shoot induction

Auxins and Cytokinins are major growth regulators that have showing great results of cell division, shoot induction and regeneration. And Auxin-Cytokinin interactions are usually considered to be the most important for regulating growth and organized development in plant tissue and organ growth [10]. In the present study maximum growth were obtained from the media containing (MSL7) BAP (5 mg/l) NAA (0.5 mg/l) and (MSL10) BAP (3 mg/l)+IAA (0.5 mg/l) along with 15 mg Kanamycin (Table 3) Overall Plant Growth progress (Table 4).

Media code Media composition Treatments Shoot inductionpercentage Plant growth size in 1month
MSL7 MS+BAP(5)+NAA(0.5)+Kanamycin 30 92% 4-5 cm
MSL10 MS+BAP(3)+IAA(0.5)+Kanamycin 30 85% 5 cm

Table 3: Effect of different media composition on Shoot formation or plant growth.

S.No. Plant Week-1 Week-2 Week-3 Week-4
No. of Leaf Height No. of Leaf Height No. of Leaf Height No. of Leaf Height
1 1 1 1 cm 3 1.5cm 4 2.5cm 5 4cm
2 2 1 1cm 3 2cm 4 3 cm 5 4cm
3 3 2 1cm 4 2.5cm 5 3.5cm 6 5cm
4 4 1 1cm 3 2cm 3 2.5cm 4 4cm
5 5 2 1cm 4 2cm 5 3.5cm 5 4cm

Table 4: Effect of different media composition on shoots formation or plant growth.

Root induction and acclimatization

Auxins are major growth regulators that have showing great results of root formation. In the present study maximum rooting (70%) were obtained from the media containing IBA 0.5-1 mg/l in Table 5.

S.No. Media No. of cultures Survivability Sprouting Root induction
1 MSR1a 15 12 positive MSR 2a show 70% rooting
2 MSR2a 15 13

Table 5: Rooting effects on plants.


in vitro regeneration of plants has gained significance during recent years as this technique can be used for the rapid multiplication and ex situ conservation of some plants having threatened aspects. Selected media with different combinations of Auxins and Cytokinins were analyzed to determine the best media composition for shoot induction in P. longum Linn. In the present study, in vitro regeneration of P. longum was reported wherein nodes of in mother plant were utilized as initial explants for multiple shoot formation, sterilization of explants, followed by rooting of individual microshoots and successful transplantation of plantlets. The findings of this study have resulted in development of an effective protocol for sterilization and establishment of shoot formation from nodal explants. When the regenerated shoots become 6-7 cm long, they are transferred to MS medium containing rooting hormones for rooting. And after that rooted plantlets are transferred to greenhouse conditions. This method of micro propagation can be used for the development large scale plantlets, establishment of nurseries of P. longum and also be used for the ayurvedic medicinal purposes.


Author is grateful to the Mr. Santosh K. Mishra, Assistant Professor, Department of Biotechnology, IMS Engineering College Ghaziabad, UP, for providing lab facilities & library consultation. And very special thanks to Dr.C.S.Rana, Research Scientist, Dabur India Limited, Ghaziabad; who is the real source of inspiration and motivation for author during her research work.


  1. Sivarajan VV,Balachandran(1994)Ayurvedi c drugs and their plant sources. Oxford and IBH publishing Co., Pvt. Ltd, New Delhi, IndiaPp: 374-376.
  2. Manoj P, Soniya EV, Banerjee NS, Ravichandran P(2004) Recent studies on well-known spice. Piper longumlinn, Natural product Radiance 3: 222-227.
  3. Banerjee S, Mallick MA, Genetic Transformation & Gus Gene Expression in Piper longum. International Journal of Science and ResearchPp: 2319-7064.
  4. Sumy O, Ved DK, Krishnan R (1999) Tropical Indian Medicinal Plants propagation methods.FRLHT, Bangalore, IndiaPp:268-269.
  5. Banerjee NS, Manoj P, Das MR (1999) Male sex associated RAPD markers in Piper longumlinn. Current Science77:693-695.
  6. Viswanathan TV (1995) Medicinal and Aromatic plants. KL Chadha and R Gupta Eds, Malhotra Publishing House, New Delhi Pp: 373-383.
  7. Neelam, krishnaswamy Kamala (2001) Nutritive value of Pepper. InPepper market review, National institute of Nutrition, Hyderabad Pp: 1-5.
  8. Sandeep S, Purohit VK, Prasad P,Nautiyal AR(2015) Micropropagation of ValerianaWallichii DC.Indian Valerian through nodes, Indian journal of Biotechnology 14: 127-130.
  9. Ruby Tiwari, Rana CS(2015) Plant secondary metabolites. International Journal of Engineering Research and General Science 3: 661-670.
  10. Gaspar T, Kevers C, Penel C, Greppin H, Reid DM,et al. (1996) Plant hormones and Plant Growth regulators in Plant tissue vitro Cellular & Developmental biology-Plant 32: 272-289.
Citation: Tiwari R (2016) Standardization of an Efficient Protocol for Sterilization and Media for Direct Shoot Regeneration from Nodal Segments of Medicinal Herb Piper longum Linn. J Plant Biochem Physiol 4:163.

Copyright: © 2016 Tiwari R. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.