1 Int.J.Curr.Biotechnol. Volume 4; Issue 3; Mar, 2016

Raghu Ellendula, Muralikrishna Narra, Srinivas Kota, Bharathkumar Kalva, Yashodhara Velivela, Pandarinath Savitikadiand Venkateswar Rao Allini,An efficient and high frequency regeneration protocol in two cultivars of Capsicum annuum. L cvs. G3 and G4, Int.J.Curr.Biotechnol., 2016, 4(3):1-8.

An efficient and high frequency regeneration protocol in two cultivars of Capsi-cum annuum. L cvs. G3 and G4

Raghu Ellendula, Muralikrishna Narra, Srinivas Kota, Bharathkumar Kalva, Yashodhara Velivela,Pandarinath Savitikadi and Venkateswar Rao Allini*.

Department of Biotechnology, Kakatiya University, Warangal-506009, India.

A R T I C L E I N F O A B S T R A C T

Article History:Received 10 February 2016Received in revised form 09 March20 16Accepted 15 March 2016Available online 30 March 2016Key words:BAP, TDZ, IAA, IBA, Callus, Mul-tiple shoots.

An efficient and high frequency protocol for regeneration and elongation was established intwo different cultivars of Capsicum annuum commonly known as pepper. Cotyledons from21 day old in vitro raised plantlets were selected as explants. The effect of two differentcytokinins BAP and TDZ in combination with a standard range of IAA were analyzed. Theregular combination of BAP and IAA was not shown to achieve satisfactory outcome in thesecultivars. In explants cultured on the above said combination callus proliferation was ob-served after 4-5 weeks of incubation. Whereas the explants of both cultivars cultured on MSmedia supplemented with TDZ and IAA were stimulated to induce multiple shoots. Maxi-mum number of multiple shoots were observed at 0.7 mg/L TDZ in combination with 0.2 IAAin G3 variety and in G4 the optimum concentration growth regulators was recorded as 0.5 mg/L and 0.2 mg/L IAA. Proliferated shoot buds were elongated on the same media without anyadditives. Shoots were excised and grown in rooting media provided with 0.1 mg/L IBA.Rooted plantlets were successfully hardened by using autoclave soil and manure in 1:1 pro-portion.

IntroductionCapsicum annuum. L commonly known as chilli plant isa dicotyledonous plant belongs to the genus Capsicumof the family Solanaceae. The genus Capsicum containsof about 25 wild and 5 domesticated species (Sanatombiand Sharma 2007). Capsicum is a commercially importantcrop plant and its uses are widely spread around theworld in several edible forms as it provides a rich sourcefor pungent flavor (Agarwal et al., 1988). The membersof Capsicum annuum are well known for their long timeusage in various cultures to treat a wide range ofafflictions including bronchitis, arthritis, diabetes andfatigue (Robinson and Maheswari 2013). It is widelygrown in South India for its pungent fruits, which areused both in green and ripened forms to impart pungencyto the food. This plant is well known for its pungentcharacter which is contributed by a naturally occurringlipophilic chemical capsaicin (Sanatombi and Sharma2006). In addition chilli contain numerous chemicalsincluding fatty oils, carotenoids, vitamins, proteins, fibers(Bosland and Votava 2000).

To fulfill the present market requirement and to usebiotechnology tools for crop improvement fasterpropagation strategies for mass multiplication areinvariably necessary. Conventional methods are tedious,utilize huge amounts of seeds and moreover they are

seasonal oriented (Sanatombi and Sharma 2006). Pepperseeds normally have short viability and they tend to losegermination capacity rapidly (Orliñska and Nowaczyk,2015). Some times because of low germination rate of theseeds and short span of viability seed basedmultiplication was facing difficulties (Rahul et al., 2015).As pepper plants lack natural vegetative mode ofpropagation, tissue culture methods provide an alternateapproach for the rapid multiplication of these plants(Rajan et al., 2010). As almost all the genetictransformation strategies are also involving the need oftissue culture for mass production of transformants, thistechnique has gained significance.

It is clear that, unlike many other members of the familySolanaceae , which show good rate of in vitroregeneration, the genus Capsicum is disobedient withregard to its in vitro regeneration (Sanatombi and Sharma2006). Tissue culture response in genus Capsicum differssignificantly with the type of explants and cultivar(Venkataiah et al., 2006; Kiramayee et al., 2008). Thegenotype also has a considerable influence onregeneration from various explants (Sanatombi and Sarma2006). In vitro regeneration is governed by many genesand it is cultivar dependent (Arroyo and Revilla 1991;Vinoth kumar et al., 2012; Orliñska and Nowaczykm 2015)and this makes it necessary to optimize in vitropropagation protocols for the specific cultivars.

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*Corresponding author.Email address: vrao_allini@yahoo.com

ISSN: 2321 - 8371

Abbreviations:BAP – 6-Benzyl Amino Purine,TDZ – ThidiazuranIAA- Indole 3-AceticacidIBA – Indole 3-Butyricacid

Volume 4; Issue 3; Mar, 2016 Int.J.Curr.Biotechnol. 2

Plate – 1(a-g): Regeneration studies in cotyledonary explants of C. annuum (cv. G3)

a. In vitro germination of seedlings. b. Initiation of shoot buds from cut ends of explants on regeneration medium supplemented with 0.7 mg/L TDZ and 0.2 mg/L IAA.c & d. Proliferation of multiple shoots on same medium.e. Elongation of shoots.f. Rooting of single elongated shoot on medium containing 0.1 mg/L IBAg. Hardened plants shifted to Greenhouse

3 Int.J.Curr.Biotechnol. Volume 4; Issue 3; Mar, 2016

Plate - 2(a-g): In vitro regeneration of cotyledonary explants in C. annuum (cv. G4 ).

a. Adventitious shoot buds induction from cotyledonary leaf on regeneration medium containing 0.5 mg/L TDZ and 0.2 mg/L IAA.b & c. Proliferation of shoot buds.d. Elongation of multiple shoots on same regeneration mediume. Rhizogenesis induced on medium containing 0.1 mg/L IBAf & g. Acclimatization and Hardened plantlets

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_______________________________________________________________________________________________ S.no Growth regulators (mg/L) response in response in IAA BAP cv.G3 cv.G4

1. 0.20 0.10 NR NR 2. 0.20 0.20 NR NR 3. 0.20 0.30 NR NR 4. 0.20 0.40 Callus Callus 5. 0.20 0.50 Callus Callus 6. 0.20 0.60 Callus Callus 7. 0.20 0.70 Callus Callus 8. 0.20 0.80 Callus Callus 9. 0.20 0.90 Callus Callus 10. 0.20 1.0 1.4±0.213 1.6±0.322

Table - 1: Effect of IAA with BAP hormonal combination on multiple shoot induction in C. annuum (cv. G3 and G4)

NR; No Response, Data was taken as mean value of 25 replicates for each treatment.

_______________________________________________________________________________

S.no Growth regulators (mg/L) % of response Mean No. of shoots ± standard error

IAA TDZ

1. 0.20 0.10 68 4.1±0.439 2. 0.20 0.20 72 5.4±0.482 3. 0.20 0.30 76 5.3±0.492 4. 0.20 0.40 80 6.5±0.519 5. 0.20 0.50 80 6.2±0.532 6. 0.20 0.60 75 7.5±0.507 7. 0.20 0.70 80 8.6±0.528 8. 0.20 0.80 76 6.8±0.493 9. 0.20 0.90 70 6.2±0.507 10. 0.20 1.0 75 5.2±0.493 _________________________________________________________________________

Table - 2: Effect of IAA with TDZ hormonal combination on multiple shoot induction in C.annuum (cv. G3)

Data was taken as mean value of 25 replicates for each treatment.

_______________________________________________________________________________

S.no Growth regulators (mg/L) % of response Mean No. of shoots ± standard error

IAA TDZ

1. 0.20 0.20 65 4.3±0.409 2. 0.20 0.20 76 3.2±0.382 3. 0.20 0.20 70 5.4±0.492 4. 0.20 0.20 80 7.1±0.519 5. 0.20 0.20 80 8.5±0.532 6. 0.20 0.20 75 6.3±0.507 7. 0.20 0.20 72 5.1±0.528 8. 0.20 0.20 76 6.2±0.483 9. 0.20 0.20 70 8.4±0.507 10. 0.20 0.20 75 6.1±0.493 _________________________________________________________________________________

Table - 3: Effect of plant growth regulators on multiple shoot induction in C. annuum(cv G4)

Data was taken as mean value of 25 replicates for each treatment.

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Some of the reports were already available with regardsto the tissue culture of Capsicum annuum. But most ofthem were selected shoot tip and nodal segments onlyas their explants (Christopher and Rajam 1994; Venkataiahet al., 2006; Sanatombi and Sharma 2006; Ju Yeon Songet al., 2010; Mechuselie Kehie 2011; Otroshy et al., 2011;Philip Robinson and Maheswari 2013; Rahul et al., 2015).Stunted growth, rosette leaves and abnormal shoots arethe major problems in elongation of regenerated shootsof Capsicum (Steinitz et al., 1999; Vinoth Kumar et al.,2012). Therefore, the present experiment was undertakento establish a efficient protocol for rapid in vitroregeneration and elongation in two important chilli peppercultivars G3 and G4 by using cotyledonary explants fromin vitro-germinated seedlings.

Materials and MethodsTwo different and commercially important cultivars ofCapsicum annuum (G3 and G4) were selected for theregeneration studies. Seeds of both cultivars G3 and G4were collected from Agricultural Research Station (ARS)LAM, Guntur, A.P. The collected seeds were firstsubjected for germination test and those which shownhigh germination rate were used for establishment ofaseptic plants.

Establishment of in vitro cultures:Seeds of both cultivars were soaked overnight in distilledwater. These seeds were surface sterilized with 0.1%Mercuric chloride (HgCl2) for 3 minutes followed byrinsing with sterile distilled water for 4-5 times. Later theseeds were dried and inoculated on Murashige and Skoog(MS) (Murashige and Skoog 1962) basal mediumsupplemented with 2% sucrose, 100 mg/L myoinositol.The pH of the media was adjusted to 5.75 by using 1 NNaOH or 1 N HCl. Media was solidified with 0.8% ofAgar Agar (Himedia). All media used for regenerationstudies were autoclaved at 15 lbs pressure for 15 minutes.All the materials used in culture work were made freefrom microbes by various approaches like autoclaving,flame sterilization, and wiping with 70% ethanol etc.Flasks inoculated with seeds were incubated in tissueculture room.

Regeneration studies were conducted by culturing the21 days old cotyledon explants of Capsicum annuum L.cvs G3 and G4. Young and healthy cotyledons wereaseptically trimmed from the seedlings. Explants wereprepared by removing basal and tip portions with sterilescalpel and inoculated on to regeneration media. Twodifferent cytokinins Benzyl amino purine (BAP- 0.1, 0.2,0.3…1.0 mg/L) and Thidiazuran (TDZ- 0.1, 0.2, 0.3….1.0mg/L) separately in combination with stable range of 0.2mg/L Indole acetic acid (IAA) were applied to evaluatetheir effect on shoot bud induction. Cultured explants

were incubated in culture room illuminated with coolfluorescent lamps at 25±2ºC. Multiple shoot buds fromcut edges of explants were induced in clumps after fourweeks of culture. Regenerated shoots were cultured onsame medium for further elongation.

Rooting and acclimatization:Elongated shoots of both cultivars were excised andcultured on rooting medium containing half strength MSbasal salts fortified with various concentrations (0.1, 0.2,0.3, 0.4 0.5 mg/L) of Indole butyricacid (IBA) andincubated in culture room at same conditions specifiedabove. Rooted shoots were excised carefully from themedium and washed thoroughly with sterile distilled waterand planted in a plastic cup containing autoclaved soil:manure (1:1). Acclimatized plant is covered with plasticbag to maintain the relative humidity. Plants weremaintained in green house and watered daily. After 10days of shifting into plastic cups, humidity was graduallydecreased by increasing the size of holes in the plasticbags and the bags were finally removed. Four week-oldhardened plants were then transferred to bigger earthenpots (Plate-2 g).

Results and DiscussionSeeds were germinated after one week of culturing onMS basal salts supplemented with 2% sucrose andexpanded cotyledons were developed after three weekswhich were used as explants (Plate - 1 a). Two cytokininswere applied separately and in combination with standardrange of auxin. These two combinations were tested withten treatments varying with respect to the concentrationof cytokinin. Each and every treatment is tested withfifteen replicates.

After 4-5 weeks of culture period varied of responseswere observed. Explants of both cultivars growing onMS medium with different levels of BAP in combinationwith constant range of IAA were shown mixed results.Among the tested ten treatments initial three were foundto be inefficient to induce response. In almost all remaining(except last one) treatments of this combination white togreen fragile callus formation from cut edges of explantswas observed clearly in both type of cultivars (Table 1).Well proliferated green callus was carefully cut into smallportions and sub cultured on MS media supplementedwith same combination of plant growth regulators.However the subsequent sub culturing of this calluscultures did not result any shoot bud proliferation. Themedia in replicates of last treatment which was addedwith 1.0 mg/L BAP and 0.2 mg/L IAA shown to haveability to induce shoot buds in the explants of bothcultivars but the efficiency was not appreciable (Table1).

________________________________________________________________________ S.No IBA Avg No. of roots Avg. length of roots (in cm.) (mg/L) G3 G4 G3 G4 _______________________________________________________________________

1. 0.1 8 7 6.5 6.8 2. 0.2 6 6 4.7 5.5 3. 0.3 5 6 4.3 5.2 4. 0.4 3 4 3.7 4.2 5. 0.5 4 3 3.3 3.1

Data was taken as mean value of 25 replicates for each treatment.

Table - 4: Effect of IBA in rhizogenesis from regenerated shoots of Capsicum annuum

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Results of shoot multiplication at different concentrationsof TDZ in combination with IAA were tabulated (Table2). First treatment of this combination was only confinedto callus proliferation. In early days of incubation whiteand fragile callus was proliferated from the cut edges ofexplants which covered the surface of explants as theincubation proceeds (Plate - 1b & c). Later tiny greenishshoot buds were developed from the callus (Plate – 1 d).When the TDZ concentration was increased in culturemedia it was clear that the gradual increasing of numberof shoots per explants was observed in both types ofcultivars. And a maximum of (8±0.528) shoots per explantswas recorded in culture medium containing 0.70 mg/LTDZ in combination with 0.2 mg/L IAA in cultivar G3(Table 2). Whereas in G4 cultivar the maximum numbershoots per explants (8±0.532) were observed in culturemedium having 0.50 mg/L TDZ in combination with 0.20mg/L IAA (Table 3). Initially callus proliferation wasobserved and it slowly produced shoot buds. (Plate – 2a, b & c). The number of shoots regenerated from singleexplants increased with increase of concentration of TDZin culture media. When the concentration of TDZ wasexceeded from 0.8mg/L there was a remarkable decreasein the number of regenerated shoots from the singleexplants of G3 variety. The same phenomenon wasobserved in G4 cultivar also when the concentration ofTDZ was exceeded 0.50 mg/L. This was indicating thatTDZ has no potential effect when it used higher thanoptimum concentration. Elongated shoots of bothcultivars (plate – 1 e & plate - 2d) were excised andsubjected to rooting. Among the five concentrations ofIBA tested 0.1 mg/L of IBA was shown to optimum forinduction of rhizogenesis in sub cultured shoots (Plate -1f & Plate - 2e). In vitro regenerated plants wereacclimatized by using 1:1 proportion of soil and manure(Plate - 1g & Plate - 2f).

The influence of plant growth regulators and theirinteraction with plant tissue culture have been discussedthoroughly by Thomas (Thomas G et. al 1987). Themeaningful combination of cytokinins with auxins playsimportant role for successful regeneration system. Whiledoing with tissue culture methods generally, acombination of a cytokinin and an auxin is often used toachieve high ratios of shoot induction (Ju Yeon Song2010). Therefore, the effect of combinations of IAA withBAP and TDZ were tested in present experiment. Bypromoting the cell division and cell elongation cytokininscontributes for plant tissue culture. Shoot proliferationwas the function of cytokinin but sustained andsuccessful growth ultimately depends upon thesynergistic balance between cytokinins and auxins(Thomas et al., 1987).

Thidiazuran a substituted phenylurea (N-phenyl-N-1,2,3-thiadiazol-5-ylurea) is used as a plant growth regulatorto stimulate high rate of shoot proliferation. It wascategorized as cytokinins due to induction ofmorphological responses which are very near to thosegiven by natural cytokinins (Ahmad et al., 2006 Khan etal., 2006; Ju Yeon Song et al., 2010). It has beenconsidered to be more potential than most of thecommonly used purine type cytokinins like BAP andKinetin (Huetteman and Preece, 1993). TDZ directlypromotes growth due to its own biological activities in afashion similar to that of an N- substituted cytokinin or itmay induce the synthesis and accumulation of anendogenous cytokinin (Emmanuel Youmbi et al., 2006;

Abdulrahman 2015). A synergistic effect of TDZ incombination with an auxin has been demonstrated invarious plants. Low levels of TDZ induce the shootproliferation and earlier reports indicating that TDZ mayalso have auxin like activity in root induction (Chin yi lu1992). It may promote callus proliferation or somaticembryogenesis at its higher concentrations (Stanislavaand Velichka 2015).

Even though the standard combination of BAP and IAAfound to be efficient to induce shoot bud proliferation inlarge varieties of species in earlier studies but in thepresent experiment this combination was not worked outto approach significant outcome. The other combinationtried TDZ and IAA was found to be more potent ininduction of shoot buds in both G3 and G4 cultivars.However, present results differ considerably with theearlier reports in Capsicum annuum, which used higherlevels of phytoharmones and obtained a comparativelylower frequency of shoots. Very few reports wereavailable regarding cotyledonary regeneration in pepper.Even though Vinoth et al. (2012) were tried with differentexplaants like hypocotyls, cotyledons and leaf discs onlyhypocotyls showed shoot induction while the rest ofexplants formed callus at the cut end of the explants orformation of rosette leaves. In earlier studies Ju yeon etal. (2010) observed the effect of TDZ alone and also incombination with NAA in two different cultivars ‘Hivitared’ and ‘Hivita yellow’ by using leaf as explants. In TDZand NAA combination they got similar response as weobtain in TDZ and IAA combination. But theconcentration of TDZ they used was comparativelyhigher (2.0 mg/L) than we tested (0.70 mg/L). Most of theearlier reports focused on pepper expressed that ananother medium is required for elongation whichsupplemented with higher dose of cytokinin or a newcytokinin other than used for regeneration or some timewithout any cytokinin (Agarwal et al., 1988; Venkataiahet al., 2006; Ahmad et al., 2006; Ju Yeon Song 2010; VinothKumar et al., 2012; Stanislava and Velichka 2015).Protocols developed by them were obviously includesan additional step. By culturing on same regenerationmedium we got successful elongation without any rosetteleaves and mal-shoot formation. Even though Kiranmayeeet al. (2008) were tried with TDZ and IAA combinationthey tested only two concentrations of TDZ (2.0 mg/Land 3.0 mg/L) which were very higher than we tried. Theydid not tried with lower concentrations. And the maximumnumber of shoot buds they reported was 5.8± 0.63 whichwas comparatively lower than we achieved (8.5±0.532) atone fourth concentration of TDZ they used. Howeverthis new approach ensures the reduced consumption ofTDZ, a cost effective plant growth regulator. The optimumlevel of these phytoharmones to induce maximum shootbuds in the explants of G3 cultivar was considered as0.70 mg/L TDZ and 0.20 mg/L IAA while that is for G4was 0.50 mg/L TDZ and 0.20 mg/L IAA. In the presentinvestigation it is clear that the genotype has a remarkableinfluence on response regards to tissue culture as thecultivar G3 produced maximum number of shoot buds at0.70 mg/L TDZ whereas cultivar G4 displayed similarresponse at comparatively lesser range of phytoharmonesi.e 0.50 mg/L of TDZ.

Rhizogenesis stage is also very important as properrooting and its development may directly influence thefurther growth of in vitro raised plantlets. 0.10 mg/L IBAwas studied as optimum level of concentration for rootinduction. There is no appreciable morphologicalresponse in rhizogenesis even though we increased IBA

7 Int.J.Curr.Biotechnol. Volume 4; Issue 3; Mar, 2016

concentration in rooting medium gradually. Plant tissueculture efforts are said to be fruitful when the tissuecultured plantlets survive in ex vitro conditions in thefield. Upon transferring of hardened plants into bigearthen pots, 75% of survival rate was observed in bothcultivars. Autoclaved soil and manure mixture wasconsidered as very convenient for acclimatization.

ConclusionThe well established protocol for in vitro propagation ofpepper plants through cotyledonary leaf culture wouldallow rapid production of plantlets with in short time.The developed protocol may be successfully used ingenetic transformation studies of Capsicum annuumcultivars G3 and G4.

AcknowledgmentsAuthors are greatly acknowledged to Prof. A.Sadanandam former dean, Dept. of Biotechnology,Kakatiya University for his kind advice andencouragement. This study was the part of UGC MajorResearch Project sanctioned to A.V. Rao to which we arevery grateful.

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