enhanced shoot organogenesis in cassia angustifolia vahl. — a difficult-to-root drought resistant...

ORIGINAL ARTICLE

Enhanced shoot organogenesis in Cassia angustifoliaVahl. — a difficult-to-root drought resistant medicinal shrub

Shahina Parveen & Anwar Shahzad & Mohammad Anis

Received: 15 June 2011 /Accepted: 7 November 2011 /Published online: 13 December 2011# Society for Plant Biochemistry and Biotechnology 2011

Abstract In vitro regeneration was achieved through callusculture derived from cotyledon explants of Cassia angus-tifolia Vahl. on MS (Murashige and Skoog, 1962) medium.Calli were induced from cotyledon explants excised fromaseptic 14 days old seedlings on MS medium containing2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxy acetic acid) at different concentrationswith 3% sucrose and 0.8% agar. Optimal growth of calluswas obtained at 5.0 μM 2,4-D, which was proved to be thebest for shoot regeneration when sub cultured onto MSmedium supplemented with cytokinins either alone or incombination with an auxin. Maximum number of shoots(23.2±1.4) were produced at 5.0 μM 6-benzylaminopurine(BA) and 0.4 μM α-naphthalene acetic acid (NAA).Regenerated shoots produced prominent roots when trans-ferred to half strength MS medium supplemented with1.0 μM indole-3-butyric acid (IBA) and 5.0 μM phlor-oglucinol (PG). Rooted plantlets thus developed werehardened and successfully established in the soil. Thisprotocol yielded an average of 23 plants per cotyledonexplant over a period of 4 months.

Keywords Callus . Cotyledons . Histological evidences .

Indian senna . Organogenesis . Plant establishment

Abbreviations2,4-D 2,4-dichlorophenoxy acetic acid

2,4,5-T 2,4,5-trichlorophenoxy acetic acidBA 6-benzylaminopurineIBA Indole-3-butyric acidKn KinetinMS Murashige and Skoog’s mediumNAA α-naphthalene acetic acidPG PhloroglucinolPGR Plant growth regulator

Introduction

Cassia angustifolia Vahl. (Fabaceae) is an erect shrub andgrows to 1–2 m in height. It is one of the importantmedicinal plants, highly drought resistant and largelycultivated on marginal lands in some parts of India. Theleaves and pods contain an important alkaloid ‘sennosoid’which is administered in Ayurvedic and Unani system ofmedicine for the treatment of several diseases and valuedfor its laxative and cathartic properties (Anonymous 1962).

Conventionally the plant is propagated through seeds;however, low viability and poor seed germination restrictits propagation in natural conditions. In vitro regenerationprovides an alternative mean for large scale multiplicationand propagation of plants. Micropropagation with highmultiplication rates is an important asexual method that canbe used for the production of clonal plants; it also forms thebasis for the production of genetic variation or improve-ment of species by genetic transformation or somaclonalvariations. Large number of medicinal plants have beensuccessfully regenerated through micropropagation eitherdirectly (Raghu et al. 2006; Bouhouche and Ksiksi 2007;Parveen et al. 2010; Shahzad et al. 2011;) or indirectly viacallus formation (Reddy et al. 2001; Faisal and Anis 2005;

S. Parveen :A. Shahzad (*) :M. AnisPlant Biotechnology Laboratory, Department of Botany,Aligarh Muslim University,Aligarh (UP)-202 002, Indiae-mail: [email protected]

A. Shahzade-mail: [email protected]

J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):213–219DOI 10.1007/s13562-011-0094-x

Rout 2005; Khanna et al. 2006; Shahzad et al. 2006). As faras literature is concerned, there are only few reports on invitro regeneration of C. angustifolia using differentexplants, either through direct shoot regeneration or callusformation (Agrawal and Sardar 2003; 2006; 2007; Siddiqueand Anis 2007; Siddique et al. 2010; Parveen and Shahzad2011). Although the number of shoots produced was veryless in all these earlier reports. Therefore, in the presentstudy an attempt has been made to develop an improvedand efficient regeneration system for senna.

Materials and methods

Collection of explants

The mature and dried seeds of C. angustifolia obtainedfrom Prem Nursery and Seed Store, Dehradun, India wereused to raise aseptic seedlings. Seeds were washed underrunning tap water for 30 min to remove adherent particlesand then kept in 1% (w/v) bavistin (Carbendazim Powder)a broad spectrum fungicide, for 25–30 min followed bythorough washing with 5% (v/v) Teepol, a liquid detergent,by gentle shaking for 15 min. Further washing was doneunder laminar air flow hood with sterile double distilledwater (DDW) followed by a short treatment of 40 s with 70%(v/v) ethanol. Seeds were surface sterilized with freshlyprepared aqueous solution of 0.1% mercuric chloride (HgCl2)for 5–6 min and then finally rinsed 4–5 times with sterileDDW to remove excess of sterilants. Sterilized seeds wereinoculated on MS (Murashige and Skoog 1962) medium andplaced under controlled conditions for germination. Cotyle-donary leaves (CL) or cotyledons (1–1.5 cm) excised from 7,14 and 21 days old aseptic seedlings were used as explant todetermine the appropriate aged tissue for the maximizationof organogenic efficiency.

Culture medium and culture conditions

The MS medium supplemented with 3% sucrose and 0.8%agar (Hi media, India) was used throughout the experiments.The pH of the mediumwas adjusted to 5.8 using 1 NNaOH or1 NHCl prior to autoclaving at 121°C and 1.06 kgcm−2

pressure for 20 min. All the cultures were maintained at 24±2°C under 16 h photoperiod with a photosynthetic photonflux density (PPFD) of 50 μmol m−2 s−1 provided by coolwhite fluorescent tubes (40 W, Philips, India) and with 50–60% relative humidity.

Callus induction

Mature green cotyledons were excised from 7, 14 and21 days old aseptic seedlings and cultured on MS medium

supplemented with auxins; 2,4 dichlorophenoxy acetic acid(2,4-D) and 2,4,5 trichlorophenoxy acetic acid (2,4,5-T) atdifferent concentrations (1, 2.5, 5.0, 7.5 and 10 μM) toinduce organogenic calli. The percent response of explantsproducing callus and rate of callus growth was recordedafter 6 weeks of culture.

Shoot regeneration and proliferation

The green compact calli obtained from 14 days old cotyledonexplants at optimal concentration (5.0 μM) of 2, 4-D wereused for shoot regeneration when transferred to MS mediumcontaining cytokinins (BA and Kn) either alone or incombination with an auxin α-naphthalene acetic acid (NAA)at various concentrations (0.2, 0.4, and 0.6 μM). MS mediumwithout any plant growth regulator was used as control. Datafor percentage response, number of shoots per explant andshoot length were recorded after 6 weeks of culture.

In vitro rooting and acclimatization of regenerated plantlets

For in vitro root induction microshoots of appropriate size(3–4 cm) were isolated from the cultures and transferred torooting medium comprised of MS basal and half strengthMS without any plant growth regulator or supplementedwith indole-3-butyric acid (IBA) either alone or incombination with phloroglucinol (PG). Regenerated plant-lets were isolated from the culture tube and washed gentlyunder running tap water to remove any adherent gel,transplanted into plastic cups containing sterilized soilriteand covered with transparent polythene bags to retainmoisture content (>85%). Initially plantlets were irrigatedwith ¼ strength MS salt solutions (without vitamins) for2 weeks and thereafter with tap water. The polythene bagswere removed gradually starting after 2 weeks in order toacclimatize plants in controlled conditions. After 4 weeksplants were successfully transferred to earthen pots con-taining sterilized garden soil and maintained in greenhouseunder natural environment with 85% survival rate.

Data collection and statistical analysis

The data for percentage response, rate of callus, number ofshoots per explant and shoot length were recorded after6 weeks of culture, whereas the data for rooting experimentswere recorded after 4 weeks of culture. All the experimentswere conducted with ten replicates per treatment and repeatedthrice. One replicate represents one explant per culturetube. The data were analyzed statistically through one wayANOVA using SPSS ver.10 (SPSS Inc., Chicago, USA).The significance of difference among means was carriedout using Tukey’s multiple range test at 5% level ofsignificance (P≤0.05).

214 J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):213–219

Histological studies

For histological examination, regenerative tissues at differ-ent developmental stages were fixed in FAA [formalin,glacial acetic acid, ethanol (70%) in the ratio of 5:5:90(v/v)] for 24 h and then kept in 70% ethanol. Thetissues were dehydrated through graded ethanol-xylolseries and then embedded in paraffin wax as describedby Johansen (1940). The embedded tissues were cut witha Spencer 820 rotary microtome (American OpticalCorporation, Buffalo, NY, USA) at 10 μm thickness andserial sections were mounted on glass slides. Afterdewaxing in xylol-ethanol series sections were stainedwith safranin and fast green followed by mounting inCanada balsam and observed under Olympus CompoundMicroscope (Olympus CH 20i) and photographs were shotwith Cannon Power Shot 640.

Results and discussion

Callus induction

Mature green cotyledon explants were excised from 7, 14and 21 days old aseptic seedlings and inoculated on callusinduction medium comprising of MS basal mediumsupplemented with two auxins (2,4-D and 2,4,5-T) atvarying levels of concentration whereas MS basal mediumwithout any plant growth regulator was used as control. Thedata for percent response of explants producing callus andrate of callus growth is depicted in Table 1. Explants failed

to respond on control and died within 2 weeks of culture.As it is evident from the table, all the treatments showedresponse to callus initiation but with different rates depend-ing on the concentrations of hormones as well as the age ofthe explants. The optimal growth of callus with highregeneration potential was obtained at 5.0 μM of 2,4-D in14 days old seedling explant. However at the sameconcentration 7 & 21 days old explant showed poorresponse. The earliest visible sign of callus growth wasnoted within 8 days of culture, the callus cells were firstinitiated at the cut ends of the explant and then extended tocover all over the explant. Initially calli were light green todark green in colour which later on converted into darkbrown. The increase in callus forming efficiency of theexplant was observed with the increase in concentration of2,4-D to 5.0 μM (optimal concentration) with 84.7%regeneration percentage in the 14 days old explants andbeyond this concentration, a decline was observed (Table 1).While calli induced at different concentrations of 2,4,5-Twere friable and loose as compared to that of 2,4-D andfurthermore the callus formation efficiency of the explantwas lower at 2,4,5-T. A growth regulator like 2,4-D helps toinduce embryogenic callus in a number of plant species likeAcacia sps (Ortiz et al. 2000) and Azadirachta indica(Akula et al. 2003). However, in the present study, 2,4-Dinduced only organogenic calli without any sign ofembryogenesis and these calli later on produced multipleshoot buds which transformed into healthy shoots on theregeneration medium comprising of cytokinin and auxin.Our results are in consonance with the earlier report ofShahzad et al. (2009) in Sansevieria cylindrica.

Table 1 Effect of auxins on callus induction through cotyledon explants of C. angustifolia

PGR (μM) 7 days old explant 14 days old explant 21 days old explant

2,4-D 2,4,5-T % Response Rate of callus growth % Response Rate of callus growth % Response Rate of callus growth

– – 0 NR 0 NR 0 NR

1.0 – 27.3±1.8ab – 33.0±2.9cd + 24.7±2.9bc –

2.5 – 30.0±2.3ab + 40.3±3.2c + + 28.7±3.5abc –

5.0 – 36.0±2.3a + + 84.7±2.6a + + + + 34.7±2.6ab +

7.5 – 31.0±2.0ab + 59.0±3.8b + + 39.7±3.2a + +

10.0 – 25.3±2.4ab + 37.7±3.2cd + 29.3±1.8abc +

– 1.0 22.0±4.2ab – 35.7±3.5cd + 20.0±2.3c –

– 2.5 32.0±5.3ab + 60.0±5.1b + + + 24.7±2.9bc +

– 5.0 27.0±4.7ab + 27.7±4.3cd + 35.3±2.9ab + +

– 7.5 21.3±4.7ab + 22.0±4.2d + 28.3±4.2abc –

– 10.0 14.7±4.7bc – 21.3±2.0d + 24.7±2.9bc –

Data recorded after 6 weeks.

Value represents Mean ± SE of three repeated experiments with 10 replicates each. Means followed by the same letter within columns are notsignificantly different (P≤0.05) using Tukey’s test.

NR No Response; -, very poor; +, poor; ++, moderate; +++, good, ++++, excellent.

J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):213–219 215

In general, a combination of 2,4-D along with acytokinin has been used for the induction of somaticembryogenesis in many legumes (Mc Kently 1991; Loiseauet al. 1995; Venkatachalam et al. 1997) as well as for theinduction of organogenic calli in several plants likeSolanum nigrum (Shahzad et al. 1999), Leucaena leucoce-phala (Saafi and Borthakur 2002) and Clitoria ternatea(Shahzad et al. 2007). Cotyledon explants have also beenproved superior over other explants in several earlierstudies conducted for organogenesis using different plantgrowth regulators (Saafi and Borthakur 2002; Shahzad etal. 2006).

Shoot regeneration from the callus

The calli raised on optimal medium (MS + 5.0 μM of 2,4-D)from 14 days old explants were selected for organogenesisand transferred to shoot regeneration medium comprised ofcytokinins (BA and Kn) either alone or in combinationof an auxin (NAA) at different concentrations. Shootregeneration from the calli was determined by the typeof growth regulator, its concentration and combination.The dark brown and compact callus when transferred toshoot regeneration medium, turned green when smallnodular structures came out and started to initiate shootbud primordial within 1 week of transfer in most of thecultures (Fig. 1a). Differentiated shoot buds started toemerge out after 2 weeks leading to healthy shootdevelopment. Data for percent response, mean number ofshoots per explant and mean shoot length are given inTable 2. Between two cytokinins tested for shoot regen-eration, BAwas found to induce more number of shoots ascompared to Kn. MS medium supplemented with 5.0 μMof BA produced a maximum number (10.7±1.0) of shootswith 91.0% regeneration percentage (Fig. 1b). Superiorityof BA for shoot regeneration may be due to the ability ofplant tissue to metabolize natural hormones more readilythan artificial growth regulators or due to the ability of BAto induce production of natural hormones such as zeatinwithin the tissue and thus work through natural hormonesystem (Sharma and Wakhlu 2003).

The optimal concentration of BA and Kn (5.0 μM) wasalso tested with various concentrations of an auxin (NAA)to evaluate the response of cytokinin—auxin interaction.Addition of NAA enhanced the rate of shoot buds multipli-cation and proliferation at an optimal concentration (0.4 μM)in both BA and Kn supplemented media. The MS mediumcontaining BA (5.0 μM) + NAA (0.4 μM) considerablyenhanced the regeneration efficiency of explant with theproduction of a maximum of 23.2±1.4 shoots/explant andmean shoot length of 5.0±0.3 cm (Fig. 1c and d). However,addition of 0.2 μM and 0.6 μM of NAA to the mediumcontaining optimal concentration (5.0 μM) of BA did not

show any pronounced increase in the number of shoots perexplant and percent response. In medium comprised ofMS + BA (5.0 μM) + NAA (0.2 μM), the number of shootswas 11.4±1.4 with a percent response of 90.5±2.6 ascompared to MS + BA (5.0 μM) wherein 10.7±1.0 shoots/explant were produced with 91.0±2.6% response (Table 2).While further increasing the concentration of NAA (0.6 μM)from optimal level (0.4 μM) resulted in heavy growth ofcallus which adversely affected the percent response and alsoretarded the growth and development of new shoots in BAsupplemented medium. The addition of NAA in Kn(5.0 μM) supplemented medium did not deliver any goodresponse as compared to single treatment and only slightincrease in the number of shoots/explant (6.8±0.2) wasobserved in Kn (5.0 μM) + NAA (0.4 μM).

Thus, the findings suggest that the critical concentrationof hormonal supplements played a vital role for thesignificant enhancement in shoot organogenesis and differ-entiation. Existing reports also suggested that auxins atlower concentrations along with cytokinins have a criticalrole in plant regeneration and proliferation as in severalsystems like Coleus forskohlii (Reddy et al. 2001), Eleusineindica (Yemets et al. 2003) and Sansevieria cylindrica(Shahzad et al. 2009). Earlier report on C. angustifolia(Agrawal and Sardar 2006) showed only 12.0±1.0 shootsfrom cotyledon derived callus on MS medium containingBA (5.0 μM) + NAA (0.5 μM), our results are much betterthan the previous report as more number (23.2±1.4) ofshoots were produced.

In vitro rooting and plant establishment

Microshoots measuring 3–4 cm were excised from thecultures and transferred to rooting medium comprised ofMS basal and half strength MS with or without any plantgrowth regulator (PGR). MS basal medium devoid of anyPGR (control) did not induce rooting in the microshootswhile half strength MS medium induced rooting but at verylow frequency (11.0%). When half strength MS mediumwas supplemented with different concentrations of IBA,59.0% shoots induced roots with 3.2±0.5 roots per shoot at1.0 μM of IBA (Table 3). Our results are in accordancewith earlier reports on many plant species where IBA hasbeen used as the most suitable auxin for rooting (Raghu etal. 2006; Shahzad et al. 2007 and Parveen and Shahzad2010). Addition of phloroglucinol (PG) along with optimalconcentration (1.0 μM) of IBA enhanced rooting percent-age as well as number of roots per shoot (Fig. 1e). Thus,maximum number (4.8±0.4) of roots per shoot wereobtained on half strength MS + IBA (1.0 μM) + PG(5.0 μM) in 82.0% shoots. Phloroglucinol, a phenoliccompound is responsible for the suppression of peroxidaseactivity in the culture and thus protects the endogenous


auxin from peroxidase-catalysed oxidation which facilitateshealthy root formation (De Klerk et al. 1999).

Acclimatization of regenerated plantlets is the most crucialand important phase of plant regeneration system. Regener-ated plantlets with well developed root and shoot system weresuccessfully transferred to plastic pots containing sterilizedsoilrite and hardened off under controlled conditions (Fig. 1f).Plantlets were covered with polythene bags and initiallyirrigated with ¼ strength MS salt solutions (without

vitamins) for 2 weeks and then with tap water. After 4 weekspolythene bags were removed and plants were transferred toearthen pots containing sterilized garden soil and maintainedin green house with 85% survival rate.

Histological observations

The histological analysis of callus tissue at variousdevelopmental stages revealed the pattern of organogenesis

Fig. 1 In vitro regeneration of C. angustifolia via callus formationthrough cotyledon explants excised from 14 days old seedlings aInduction of shoot buds from the cotyledon derived callus on shootregeneration medium. b Development of shoots from the callus on MS +BA (5.0 μM). c–d Multiplication and proliferation of shoots on MS +BA (5.0 μM) + NAA (0.4 μM). e In vitro root induction in half strength

MS + IBA (1.0 μM) + PG (5.0 μM). f An acclimatized plantlet insoilrite. g Organization of meristematic zones (meristemoids) in thecallus tissue (arrow head) which later transformed into shoot buds. Bar:120 μm. h Development of a shoot bud at the surface of callus tissueshowing apical dome (ap) surrounded by well organized leaf primordia(lp). Bar: 50 μm


via callus formation in C. angustifolia. The histologicalsections showed the organization of several meristematiczones (meristemoids) within the callus tissue. Thesemeristemoids were constituted of densely cytoplasmic,isodiametric cells with prominent nuclei. Majority ofmeristemoids developed at the peripheral region of the calli(Fig. 1g). Our findings are similar to the earlier report ofSaravitz et al. (1993). The peripheral meristemoids fre-quently developed into shoot buds which later transformedinto healthy shoots (Fig. 1h). However the meristemoidswhich are deep seated into the callus tissue remainsuppressed and showed slower growth and development.The application of hormones within MS medium lead the

parenchyma cells to dedifferentiate, subsequently forming ameristemoid and finally an organ (Thorpe 1980). Thecombining effect of cytokinins (BA) and auxin (NAA)enhanced shoot regeneration from organogenic calli as ithas been evident in several earlier studies (Gonzalez-Benitoand Alderson 1990; Jethwani and Kothari 1996).

In conclusion the present study provides a much better andreproducible plant regeneration protocol for mass multiplica-tion of C. angustifolia from cotyledon derived callus. Thesignificance of the work lies in the production of regener-ative callus, which would be a good target for co-cultivationwith Agrobacterium to achieve genetic transformation of thisspecies.

Table 2 Effect of various plantgrowth regulators on shootorganogenesis from cotyledonderived callus of C. angustifolia


Value represents Mean ± SE ofthree repeated experimentswith ten replicates each. Meansfollowed by the sameletter within columns are notsignificantly different(P≤0.05) using Tukey’s test.

PGR (μM) % Response Mean number of shoots/explant Mean shoot length (cm)

BA Kn NAA

– – – 0 0 0

1.0 – – 70.7±2.3ef 3.1±0.6de 1.4±0.3fg

2.5 – – 77.3±2.6cd 5.5±0.8cd 2.1±0.3def

5.0 – – 91.0±2.6ab 10.7±1.0b 4.0±0.2b

10.0 – – 84.7±2.9bc 4.9±0.6cd 2.6±0.3cd

– 1.0 – 55.3±2.6h 2.6±0.2de 1.2±0.1g

– 2.5 – 62.0±2.3fgh 3.2±0.4de 1.7±0.3efg

– 5.0 – 66.7±3.5gh 4.3±0.6de 2.6±0.3cd

– 10.0 – 61.7±3.5gh 2.2±0.8e 2.2±0.3def

5.0 – 0.2 90.5±2.3ab 11.4±1.4b 4.1±0.2b

5.0 – 0.4 96.3±1.5a 23.2±1.4a 5.0±0.3a

5.0 – 0.6 79.3±1.8cd 9.2±0.6b 2.7±0.2cd

– 5.0 0.2 67.3±3.1efg 4.4±0.6cd 2.7±0.2cd

– 5.0 0.4 74.3±3.5de 6.8±0.2c 3.0±0.1cd

– 5.0 0.6 67.0±2.9efg 3.8±0.4de 2.5±0.3cde

Table 3 Effect of various plant growth regulators on in vitro root development in microshoots of C. angustifolia

PGR (μM) % Response Mean number of roots/shoot Mean root length (cm)

MS 0 0 0

½ MS 11.0±2.1ef 1.3±0.2cd 1.2±0.2cd

½ MS + IBA (0.5 μM) 26.0±2.6def 2.1±0.2bc 1.6±0.4bc

½ MS + IBA (1.0 μM) 59.0±7.8abc 3.2±0.5ab 2.7±0.4b

½ MS + IBA (2.0 μM) 34.3±3.5cde 2.0±0.3bc 1.1±0.2cd

½ MS + IBA (1.0 μM) + PG (2.5 μM) 62.7±10.1ab 3.6±0.5ab 3.1±0.4ab

½ MS + IBA (1.0 μM) + PG (5.0 μM) 82.0±6.4a 4.8±0.4a 4.3±0.5a

½ MS + IBA (1.0 μM) + PG (10.0 μM) 62.3±4.5abc 3.8±0.5ab 3.5±0.4ab


Value represents Mean ± SE of three repeated experiments with ten replicates each. Means followed by the same letter within columns are notsignificantly different (P≤0.05) using Tukey’s test.


Acknowledgements The author Anwar Shahzad gratefully ac-knowledge the financial support promoted by the Department ofScience and Technology (DST), UP-CST (vide no. CST/D 3836)and UGC (vide no. 39-369/2010(SR)), Government of India, NewDelhi. The author Shahina Parveen also acknowledged theresearch assistance provided by UGC under the scheme ofMaulana Azad National Fellowship for the award of SeniorResearch Fellowship (SRF).

References

Agrawal V, Sardar PR (2003) In vitro organogenesis and histomor-phological investigations in senna (Cassia angustifolia)—amedicinally valuable shrub. Physiol Mol Biol Plant 9:131–140

Agrawal V, Sardar PR (2006) In vitro propagation of Cassiaangustifolia through leaflet and cotyledon derived calli. BiolPlant 50:118–122

Agrawal V, Sardar PR (2007) In vitro regeneration through somaticembryogenesis and organogenesis using cotyledons of Cassiaangustifolia Vahl. In Vitro Cell Dev Biol Plant 43:585–592

Akula C, Akula A, Drew R (2003) Somatic embryogenesis in clonalneem. Azadirachta indica A. Juss. and analysis for in vitroazadirachtin production. In Vitro Cell Dev Biol Plant 39:304–310

Anonymous (1962) The wealth of India: a dictionary of Indian rawmaterials and industrial products. CSIR, New Delhi, pp 354–363

Bouhouche N, Ksiksi T (2007) An efficient in vitro plant regenerationsystem for the medicinal plant Teucrium stocksianum Boiss. PlantBiotech Rep 1:179–184

De Klerk GJ, Vander Krieken W, de Jong JC (1999) The formation ofadventitious roots: new concepts, new possibilities. In Vitro CellDev Biol Plant 35:189–199

Faisal M, Anis M (2005) An efficient in vitro method for masspropagation of Tylophora indica. Biol Plant 49:257–260

Gonzalez-Benito E, Alderson PG (1990) Regeneration from Alstroemeriacallus. Acta Hort 280:135–138

Jethwani V, Kothari S (1996) Phenylacetic acid induced organogenesisin cultured leaf segments of Dianthus chinensis. Plant Cell Rep15:869–872

Johansen DA (1940) Plant microtechnique. Mc Graw Hill, NewYork-USA, pp 126–154

Khanna PK, Ahuja A, Sharad A, Ram G, Koul K, Kaul MK (2006)Regeneration via organogenesis in callus cultures of Argyrolobiumroseum. Biol Plant 50:417–420

Loiseau J, Marche C, Denuff YL (1995) Effects of auxins, cytokinins,carbohydrates and amino acids on somatic embryo inductionfrom shoot apices of pea. Plant Cell Tiss Org Cult 41:267–275

McKently AH (1991) Direct somatic embryogenesis from axes ofmature peanut embryos. In Vitro Cell Dev Biol Plant 27:197–200

Murashige T, Skoog F (1962) A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiol Plant 15:473–497

Ortiz BOC, Reyes ME, Balch EPM (2000) Somatic embryogenesisand plant regeneration in Acacia farnesiana and A. schaffneri. InVitro Cell Dev Biol Plant 36:268–272

Parveen S, Shahzad A (2010) TDZ-induced high frequency shootregeneration in Cassia sophera Linn. via cotyledonary nodeexplants. Physiol Mol Biol Plant 16(2):201–206

Parveen S, Shahzad A (2011) A micropropagation protocol for Cassiaangustifolia Vahl. from root explants. Acta Physiol Plant 33:789–796

Parveen S, Shahzad A, Saema S (2010) In vitro plant regenerationsystem for Cassia siamea Lam., a leguminous tree of economicimportance. Agrofor Syst 80:109–116

Raghu AV, Geetha SP, Martin G, Balachandran I, Ravindran PN(2006) Direct shoot organogenesis from leaf explants of Embeliaribes Burm. f.: a vulnerable medicinal plant. J For Res 11:57–60

Reddy PS, Rodrigues R, Rajasekharan R (2001) Shoot organogenesisand mass propagation of Coleus forskohlii from leaf derivedcallus. Plant Cell Tiss Org Cult 66:183–188

Rout GR (2005) In vitro somatic embryogenesis in callus cultures ofAzadirachta indica A. Juss,-a multi purpose tree. J For Res10:263–267

Saafi H, Borthakur D (2002) In vitro plantlet regeneration fromcotyledons of the tree-legume Leucaena leucocephala. PlantGrowth Regul 38:279–285

Saravitz CH, Blazich FA, Amerson HV (1993) Histology of in vitroadventitious bud development on cotyledons and hypocotyls ofFraser fir. J Am Soc Hort Sci 118:163–167

Shahzad A, Hasan H, Siddiqui SA (1999) Callus induction andregeneration in Solanum nigrum L. in vitro. Phytomorph 49:215–220

Shahzad A, Ahmad N, Anis M (2006) An improved method oforganogenesis from cotyledon callus of Acacia sinuata (Lour.)Merr. using Thidiazuron. J Plant Biotech 8:15–19

Shahzad A, Faisal M, Anis M (2007) Micropropagation through excisedroot culture of Clitoria ternatea and comparison between in vitroregenerated plants and seedlings. Ann Appl Biol 150:341–349

Shahzad A, Ahmad N, Rather MA, Husain MK, Anis M (2009) Improvedshoot regeneration system through leaf derived callus and noduleculture of Sansevieria cylindrica. Biol Plant 53:745–749

Shahzad A, Parveen S, Fatema M (2011) Development of aregeneration system via nodal segment culture in Veronicaanagallis-aquatica L.—an amphibious medicinal plant. J PlantInteract 6(1):61–68

Sharma RK, Wakhlu AK (2003) Regeneration of Heracleum candi-cans wall plants from callus cultures through organogenesis. JPlant Biochem Biotech 12:71–72

Siddique I, Anis M (2007) In vitro shoot multiplication and plantletregeneration from nodal explants of Cassia angustifolia (Vahl.): amedicinal plant. Acta Physiol Plant 29:233–238

Siddique I, Anis M, Aref IM (2010) In vitro adventitious shootregeneration via indirect organogenesis from petiole explants ofCassia angustifolia Vahl.—a potential medicinal plant. ApplBiochem Biotech 162:2067–2074

Thorpe TA (1980) Organogenesis in in vitro: structural, physiologicaland biochemical aspects. In: Vasil IK (ed) Perspectives in plantcell and tissue culture. Academic, New York, pp 71–111

Venkatachalam P, KaviKishore PB, Jayabalan N (1997) Highfrequency somatic embryogenesis and efficient plant regenerationfrom hypocotyls explants of groundnut (Arachis hypogea L).Curr Sci 72:271–275

Yemets AI, Klimkina LAA, Tarassenko LV, Blume YB (2003)Efficient callus formation and plant regeneration of goosegrass(Eleusine indica). Plant Cell Rep 21:503–510


enhanced shoot organogenesis in cassia angustifolia vahl. — a difficult-to-root drought resistant...

Documents