CH APTER 8

REFERENCES

Abdin MZ, Rehman RU, Israr M , Sirvastava PS, Bansal KC. Development o f

transgenic chicory (Cichorium intybus L.) in in vitro applications in crop improvement.

Edited by M ujeeb et al (Science publisher, Inc, USA). 2004; 285-296.

Abid M , Palms B, Derycke R, Tissier JP, Ram bour S. Transfom iation o f chicory

and expression o f the bacterial uidA and nptll genes in the transgenic regenerants. J. Ex.p. Bot. 1995; 46; 337-346.

Akhter S, Ahmad MZ, Singh A, Ahm ad I, Rahman M, A nw ar M, Jain GK, A hm ad FJ, Khar RK. Cancer Targeted M etallic Nanoparticle: Targeting Overview. Recent

Advancement and Toxicity Concern. Curr Pharm Des. 2011; 17; 1834-1850.Alam P, Abdin MZ. Over-expression o f HM G-CoA reductase and amoi'pha-4,11-

diene synthase genes in Artem isia annua L. and its influence on artemisinin content.

P lant Cell R eports. 2011; 30; 1919-1928.Anterola AM, Lewis NG. Trends in lignin modification: acomprehensive analysis

o f the effects o f genetic manipulations/mutations on ligniflcation and vascular integrity.

Photochemistry. 2002; 61; 221—294.Anwar M, W arsi MH, M allick N , Akhter S, Gahoi S, Jain GK, Talegaonlcar S,

Ahmad FJ, K har RK. Enhanced bioavailability o f nano-sized chitosan-atorvastatin

conjugate after oral administration to rats, Eur J Pham i Sci. 2011; 44; 241-249.

Aquil S, Husaini AM, Abdin MZ, Rather GM. Overexpression o f the HM G CoA

reductase gene leads to enhanced artemisinin biosynthesis in transgenic A rtem isia annua

plants. P lantaM ed. 2009; 75: 1453-1458.

Araceli A, Jose D. Antitumoral o f Pyrimidine derivatives o f sesquiterpen lactones.

J Pharm Pharm aceut Sci. 1999; 3; 108-112.Asad S, Mukhtar Z, N azir F, Hashmi JA, M ansoor S, Zafar Y, A rshad M. Silicon

carbide whisker-mediated embryogenic callus transform ation o f cotton {Gossypium

hirsutum L.) and regeneration o f salt tolerant plants. M ol BiotechnoL 2008; 40: 161-169.

A shraf M. The effect o f NaCl on water relations, chlorophyll and protein and

proline contents o f two cultivars o f blackgram (Vignamxmgo L.) Plant Soil.1989; 119:

205-210.Bach TJ. Some new aspects o f isoprenoid biosynthesis in plants-A

review. Lipids. 1995; 30: 191—202.

Department o f Biotechnology, Haradard University Page 162

Bais HP, Venkatesh RT, Chandrashekar A, Ravishanlcar GA. Agrobacterium rhizogenes-me.dr&tQd transformation of W itloof chicory- In vitro shoot regeneration and

induction o f flowering. Current Science. 2001; 80: 83.

Bailas N , Zakai N, Loyter A. Liposomes bearing a quaternary am m onium

detergent as an efficient vehicle for functional transfer o f TM V-RNA into plant

protoplasts. B iochim ica et Biophysica Acta (BBA)- Biom embranes. 1988; 939; 8-18.

Banerjee G, Car S, Craig S, Hodge B, W alton D. Alkaline peroxide pre-treatm ent

o f com stover; effects o f biomass, peroxide, and enzyme loadingand composition on yields o f glucose and xylose.Bioteclmology for Biofuels. 2011; 4: 16.

Baron C, Domke N, Beinhofer M, Hapfelm eier S. Elevated tem perature

differentially affects virulence, VirB protein accumulation, and T-pilus form ation in

different Agrobacterium tumefaciens and Agrobacterium vitis strains. J Bacteriol. 2001:

183; 6852-6861.Belletre A, Coullerot JP, Vasseur J. Effects o f glycerol on somatic embryogenesis

in Cichorium leaves. Plant Cell Rep. 1999; 19; 26—31.Bennici A, Schiff S, Mori B. M orphogenic effect o f colchicine in Cichorium

intyhus L. root explants cultured in vitro. Caryologia. 2006; 59: 284-290.

Berestovsky GN, Ternovsky VI, Kataev AA. Through pore diam eter in the cell

wall o f Chara coral Una. J. Exp. Bot. 2001; 52: 1173-1177.Berg, Sverre E, Skaug et al. Use o f nonviable particles comprising an internal

control (ic) nucleic acid. US20110065092A1, 2011.

Bernhardt R, Cresnar S, Bronislava L. Cytochrome p450 from rhizopus oryzae and

uses thereof. W O-042143A1, 2011.Bevan MW, Flavell RB, Chilton M-D. A chimeric antibiotic resistance gene as a

selectable marker for plant cell transformation. Nature. 1983; 304: 184—187.

Bhojwani SS, Razdan MK. Plant tissue culture: Theory and practice. 5‘ ed.

Elsevier science. 1996; 112-151.Binding H, Nehlis R , Kock R , Finger J. Comparative studies on protoplast

regeneration in herbaceous species o f the dicotyledoneae class. Z. Pfianzenphysiol. 1981;

101: 119-130.Bim boim HC, Doly J. A rapid alkaline extraction procedxire for screening

recombinant plasm id DNA. Nucleic Acids Res. 1979; 7: 1513—1523.

Department o f Biotechnology, Hamdard University Page 163

Bisht S, Bhakta G, M aitra S, M aitra A. pD NA loaded calcium phosphate

nanopaiticles: highly efficient non-viral vector for gene delivery. Int. J. Pham i. 2005; 288: 157-168.

Boxirgaud F., Hehn A., Larbat R., Doerper S., G ontier E., Kellner S., M atern U.

Biosynthesis o f coumarins in plants; a major pathw ay still to be unravelled for cytochrome P450 enzymes. Phytochemistry. Rev. 2006: 5; 293-308.

Boiiriquet R, Vasseur J. Croissance et bourgeonnem ent des tissus de feuilles

d ’endive en fonction de I’age et du lieu de prelevem ent des explantats. Bull. Soc. Bot. Fr. 1973; 120: 27-32.

Bradford MM. A rapid and sensitive m ethod for the quantitation o f microgram

quantities o f protein utilizing the principle o f protein-dye binding. Anal. Biochem. 1976; 72: 248-254.

Brown SA, Coumarins. In E.E. Comi, ed. The biochem istry o f plants. A

comprehensive Ti'eatise. Academic press, London. 1981; pp 269-300.Caboche M, Lurquin PF. Liposomes as earners for the transfer and expression o f

nucleic acids into higher plant protoplasts. M ethods in Enzymology. 1987; 148: 39-45.

Caboche M. Liposome-mediated transfer o f nucleic acids in plant protoplasts.

Physiologia Plantanuu. 1990; 79: 173—176.

Cao X, Deng W, Wei Y, Su W, Yang Y, W ei Y , Yu J, X u X, Encapsulation o f

plasmid DNA in calcium phosphate nanoparticles: stem cell uptake and gene transfer

efficiency. International Journal o f Nanomedicine. 2011; 6: 3335—3349.

Carpita N, Sabularse D, M ontezinos D, D elm er DP. D eterm ination o f the pore size

o f cell walls o f living plant cells. Science. 1979; 205: 1144—1447.

Casas AM, Kononowicz AK, Bressan RA, Hasegawa PM . Cereal transform ation

through particle bombardment. Plant Breed Rev. 1995; 13: 235-264.

Cha TS, Chen CF, Yee W, Aziz A , Loh SH. Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium -m ediated transform ation of

the unicellular green alga, Nannochloropsis sp. J M icrobiol M ethods. 2011; 84: 430-434.

Chandra S. Natural plant genetic engineer Agrobacterium rhizogenes'. role o f T-

DNA in plant secondary metabolism. Biotechnol Lett. 2012; 34: 407-15.

Department o f Biotechnology, Hamdard University Page 164

^M efm em ces

Chappell J, W olf F, Proulx J, Cuellar R, Saunders C. Is the Reaction Catalyzed by

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase a Rate-Lim iting Step for Isoprenoid

Biosynthesis in Plants? Plant Physiol. 1995; 109: 1337-1343.

Chappell J. Biochemistry and m olecular biology o f the isoprenoid biosynthetic

pathway in plants. Annu Rev Plant Physiol Plant M ol Biol. 1995; 46; 521—547.

Charrie 're F, Sotta B, Emile M, Gunther H. Induction o f adventitious shoots or somatic embryos on in vitro culhired zygotic embryos o f Helianthus annuus variation o f

endogenous hormone levels. Plant Physiol Biochem. 1999; 37: 751-757.

Chen R, Qian Y, Li R, Zhang Q, Liu D, W ang M, Xu Q. M ethazolam ide calcium

phosphate nanoparticles in an ocular delivery system. Yakugaku Zasshi. 2010; 130: 419- 424.

Chen XB, Xu CB, Luo YL, Li, BJ. Study on Effect o f K anam ycin on germ ination

o f Aptenia cordifolia. Seed. 2005; 24: 29-31.

Chen Z., Zhang L., Chen G. Carbon nanotube/poly (ethylene-co-vinyl acetate) composite electrode for capillary electrophoretic determination o f esculin and esculetin in

Cortex Fraxini. Electrophoresis. 2009; 30: 3419-3426.Cheng X, Kuhn L. Chemotherapy drug delivery from calcium phosphate

nanoparticles. Int J Nanomedicine. 2007; 2: 667-74.Choi D, W ard BL, Bostock RM. Differential induction and suppression o f potato

3 -hydroxy-3 -methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid. Plant Cell. 1992; 4: 1333—1344.

Chong J, Baltz R, Schmitt C, Beffa R, Fritig B, Saindrenan P. Down-regulation o f

a pathogen-responsive tobacco UDP-Gic: phenylpropanoid glucosyltransferase reduces

scopoletin glucoside accumulation, enhances oxidative stress, and w eakens virus

resistance. Plant Cell. 2002; 14; 1093-1107.Chowrira GM, Akella V, Fuerst PE, Lurquin PF. Transgenic grain legumes

obtained by in planta electroporation-mediated gene transfer. M ol Biotechnol. 1996; 5:

85-96.Chowrira GM, Akella V, Lurquin PF. Electroporation-m ediated gene transfer into

intact nodal meristems in planta. Generating transgenic plants w ithout in vitro tissue

culture. M ol Biotechn. 1995; 3:17-23.

m

Pepartment o f Biotechnology, Hamdard University Page 165

_________ M e f m e n c m

Cifuentes Z, Custardoy L, de la Fuente JM, M arquina C, Ibarra MR, Rubiales D,

Perez-de-Luque A. Absorption and translocation to the aerial part o f magnetic carbon-

coated nanoparticles through the root o f different crop plants. Journal o f Nanotechnology. 2010; 8: 26.

Corredor E, Testillano PS, Coronado MJ, Gonzalez-M elendi P, Fem andez-Pacheco

R, Marquina C, Ibarra MR, de la Fuente JM, Rubiales D, Perez-de-Luque A, Risueno

MC: Nanoparticle penetration and transport in living pum pkin plants: in situ subcellular identification. BM C Plant Biol. 2009; 9: 45.

Corsi K, Chellat F, Y ahia LH, Fernandes JC. M esenchym al stem cells, MG63 and

HEK293 transfection using chitosan-DNA nanoparticles. Biomaterials. 2003; 24; 1255— 1264.

Cowan AK, M oore-Gordon CS, Bertling I, W olstenholme BN. M etabolic control

o f avocado fruit growth. Plant Physiol. 1997; 114: 511—518.

Crepy L, Chupeau MC, Chupeau Y. The isolation and culture o f leaf protoplasts o f Cichoriiim intybus and their regeneration into plants. Z. Pflanzenphysiol. 1982; 107: 123—

131.Cronin MT, Miyada CG, Huang XC et al. Aixays o f nucleic acid probes for

analysing biotransformation genes. US7846659, 2010.Daffalla HH, A bdellatef E, Elhadi BA, Klialafalla M M . Effect o f Growth

Regulators on in vitro M orphogenic Response o f Boscia senegalensis (Pers.) Lam. Poir.

Using Mature Zygotic Embryos Explants. Biotechnol Res Int. 2011; 710-758.

Dafny-Yelin M, Levy A, Tzfira T. The ongoing saga o f Agrobacterium -host

interactions. Trends Plant Sci. 2008; 13: 102-105.

Dai S, Zheng P, M armey P, Zhang S, T ian W , et al. Comparative analysis o f transgenic rice plants obtained by Agrobacterium m ediated transform ation and particle

bombardment. M ol Breed. 2001; 7: 25-33.

Dainty J, Hope AB. Ionic relations o f cell o f Chara australis. Ion exchange in the

cell wall. Journal o f Biological Science. 1959; 12: 395—411.

Dan Y, Baxter A, Zhang S, Pantazis CJ, V eilleux RE. D evelopm ent o f efficient

plant regeneration and transformation system for impatiens using Agrobacterium tumefaciens and multiple bud cultures as explants. BM C Plant Biol. 2010; 9; 10; 165.

Department o f Biotechnology, Hamdard University Page 166

S le f& i& tc m

Dasgupta S, Bandyopadhyay A, Bose S. Reverse m icelle-m ediated synthesis o f

calcium phosphate nanocaixiers for controlled release of bovine serum albumin. Acta Biomater. 2009; 5: 3112-3121.

Datta SK, Soltanifar N, Domi G, Potrylcus I. Herbicide-resistant Indica rice plants

from IRRI breeding line 1R72 after PEG-mediated transform ation o f protoplasts. Plant mol bioL 1992; 20: 619-629.

Davis BD. intermediates in amino acid biosynthesis. Adv. EnzymoL 1955; 16: 247.

De la Riva GA, Cabi'era JG, Fadron RV, Pardo CA. Agrobacterium tumefaciens'. a

natural tool for plant transformation. Electron. J. Biotechnol. 1998; 1: 31.De Paepe S, De Buck K, Hoorelbeke J, N olf I. Peck, Depicker A. H igh frequency

of single-copy T-DNA transformants produced by floral dip in CRE-expressing

Arabidopsis plants. Plant J. 2009; 59: 517-527.

De R osa G, La Rotonda MI. N ano and m icrotechnologies for the delivery of

oligonucleotides with gene silencing properties. Molecules. 2009; 29: 2801-2823.

Decout E, Dubois T, Guedira M, Dubois J, Audran JC, V asseur J. Role of

temperature as a triggering signal for organogenesis or somatic embryogenesis in wounded leaves o f chicory cultured in vitro. J. Exp. Bot. 1994; 45: 1859—1865.

Deshayes A, Herrera-Estrella L, Caboche M. Liposom e-m ediated transform ation of

tobacco mesophyll protoplasts by Escherichia coli plasmid. EM BO J. 1985; 4: 2731—

2737.DeSmet PAGM. An introduction to herbal pharm acoepidemiology. Journal o f

Ethnopharmacology.1993; 38, 197—208.Dhar MK, Kaul S, Kour J. Towards the developm ent o f bettercrops by genetic

transformation using engineered plant chromosomes. Plant Cell Rep. 2011; 30: 799-806.Dillen W, De Clercq J, K apila J, Zambre M, M ontagu M V. The effect of

temperature on Agrobacterium tumefaciens-mediatQd gene transfer to plants. The plant

joumal. 1997; 12: 1459-1463.Do C.T., Pollet B., Thevenin J., Sibout R., Denoue D., Barriere Y., Lapierre C.,

Jouanin L. Both caffeoyl Coenzyme A 3-O-m ethyltransferase 1 and caffeic acid

Omethyltransferasel are involved in redundant ftinctions for lignin, flavonoids and

sinapoyl malate biosynthesis in Arabidopsis. Planta. 2007; 226: 1117-1129.

Department o f Biotechnology, Hamdard University Page 167

Siefjm em cc&

D obhal Sli, Pandey D, Kumar A, A graw al S. Studies on plant regeneration and transform ation efficiency o f Agrobacterium m ediated transform ation using neom ycin

phosphotransferase II {nptll) and glucuronidase (GUS) as a reporter gene. A frican Journal of Biotechnology. 2010; 9; 6853-6859.

D oran PM. Loss o f secreted antibody from transgenic p lan t tissue cultures due to surface adsorption. Trends Biotechnol. 2006; 24; 426-432.

D uan H, D ing X, Hong D, Zhou Ch, Lu L. Analele §tiintifice ale Universitatii, A lexandra loan Cuza, Secfiunea Genetica §i Biologic M oleculara, Tom IX. Effects o f

kanamycin on growth and developm ent o f Arabidopsis thaliana seedling and its root. 2008.

D ubey VS, Bhalla R, Luthra R. A n overview o f the non-m evalonate pathw ay for

terpenoid biosynthesis in plants. Journal o f Bioscience. 2003; 28: 637-646.

Duke JA. M edicinal Plants o f the Bible (Illustrated by Peggy K Duke) Out o f p rin t Trado-M edic Books Buffalo and NY. 1983. pp: 233.

D w ight T, Dennis B, Charisse MB. Tissue culture m ethod for transform ation o f

plant cells. 1995; EP0424047.Eung JP, Tae LH, Park EJ, Lim HT, M yung LJ. Establishm ent o f an efficient in

vitro plant regeneration system in chicory (Cichorium intybus L. var. sativus). A cta

Hortic. 1999; 483.- 367-370.

Evans E, Colem an JOD, Kearns A. Plant Cell W all and Gene D elivery in P lant

Cell Culture: the Basics, BIOS Scientific Publisher (Taylor & Francis Group), 2003, p.

37.

Fabricant DS, Farnsworth NR. The value o f plants used in traditional m edicine for

drug discoveiy. Environm ental Health Perspectives. 2001; 109: 69-75,

Foster JG, Kimberly A. Cassida, K enneth E. Turner. In vitro analysis o f the

anthelmintic activity o f forage chicory (Cichorium intybus L .) sesquiterpene lactones

against a predom inantly Haemonchus contortus egg population. V eterinary Parasitology.

2011; 180: 298-306.Fraley RT, Rogers SG, H orsch RB, Sanders PR, Flick JS , Adams SP, Bittner ML,

Brand LA, Fink CL, Fry JS, G alluppi GR, G oldberg SB, H offm ann NL, W oo SC. Expression o f bacterial genes in plant cells. Proc. Natl. Acad. Sci. USA. 1983; 80: 4803-

4807.

Departm ent o f Biotechnology, Ham dard U niversity Page 168

^ M e fm e n c e s

Fritig B , H eitz T, Legrand M. Antim icrobial proteins in induced plant defense. Curr. Opin. Im m unol. 1998; 10: 16-22.

Frulleux, W eyens G, Jgicobs M. Agrobacterium tumefaciens—mediata.ted

transform ation o f shoot-buds o f chicory. Fanny Plant Cell, T issue and O rgan Culture. 1997; 50: 107-112.

Fujino T, Itoh T. Changes in Pectin Structure during Epiderm al Cell E longation in

Pea (Pisum sativum ) and Its Im plications for Cell W all Architecture. Plant Cell Physiol. 1998; 39: 1315-1323.

G adgoli C, M ishra S.H. Antihepatotoxic activity o f Cichorium intybus. Journal o f Ethnopharm acology. 1997; 58: 131-134.

Gaertig J, Thatcher TH, Gu L, G orovsky MA. Electroporation-m ediated replacement o f a positively and negatively selectable beta-tubulin gene in Tetrahym ena thermophila. P4roc Natl Acad Sci USA. 1994; 91: 4549-4553.

G aisser S, Heide L. Inliibition and regulation o f shikonin biosynthesis in

suspension cultures o f lithospermum. Phytochem istry. 1996; 41: 1065-1072.Galis I, Kakiuchi Y, Simek P, W abiko H. Phytochem istry. Agrohacterium

tumefaciens A K -6b gene m odulates phenolic com pound m etabolism in tobacco. 2004; 65:

169-79.

G arza RM , Tran PN, Hampton RY. Geranylgeranyl pyrophosphate is a potent

regulator o f H RD -dependent 3-H ydroxy-3-m ethylglutaryl-CoA reductase degradation in yeast. .1 Biol Chem. 2009; 284: 35368-35380.

G asser C, Fraley R. Genetic engineering plants for crop improvement. Science.

1989; 244: 1293-1299.

G elvin SB. V iral-m ediated plant transform ation gets a boost. Nature

Biotechnology. 2005; 23: 684-685.

G eorge J, Bais HP, Ravishankar GA. in vito propagation o f decalepis W & A an

endangered shrub through axillary bud culture. Ind. J. Exp. Biol. 1999; 37: 269-273.

G heysen G, V illarroel R, Van M ontagu M. Illegitim ate recom binationin plants: a

model for T-D N A integration. Genes Dev. 1991; 5: 287—297.

Girish K, Shankara Bhat S, Raveesha KA. In vitro screeningof system ic fungicides against Phom opsisazadirachtae, the incitant o f die-back disease o f neem. Archiv.

Phytopathol. Plant Protect. 2009; 42: 256—264.

Depai-tment o f Bioteclmology, Ham dard U niversity Page 169

^M e^fmemces

Gomez K.A. Statistical procedure for agricultural research with emphasis o f Rice

(Los Bans, Philippines International Rice Research Institute). 1976.

Gruenhagen JA, Lai ChY, Radu DR, V ictor S, L in Y, Y eung ES. Real-Tim e ATP

Imaging o f Tunable Release fi-om a M CM -41-type M esoporous Silica Nanosphere-Based

Delivery System. Appl. Spectrosc. 2005; 59: 424-431.

Gurel E, W ren MJ. In vitro developm ent from leaf explants o f sxigar beet (Beta

vulgaris L.) rhizogenesis and the effect o f sequential exposure to auxin and cytokinin. Ann Bot. 1995; 75: 31-38.

Gurib-Fakim A. M edicinal plants: traditions o f yesterday and drugs o f tomorrow. Mol Aspects Med. 2006; 27: 91-93.

Hackl EV, Kornilova SV, Blagoi YP. DNA structural transitions induced by

divalent metal ions in aqueous solutions. Int J Biol Macromol. 2005; 35: 175-191.Hacki EV, Yurij P. Blagoi. The effect o f temperatu^re on D N A structural transitions

under the action o f Cu2+ and Ca2+ ions in aqueous solutions. Biopolymers. 2005; 77: 315-324.

Hagiw ara K, Anastasia R, Nakata M, Sato T. Physicochem ical properties o f

pDNA/chitosan complexes as gene delivery systems. Curr Drug D iscov Technol. 2011; 8:

329-39.

Hain R, Stabel P, Czernilofsky AP, Steinbi HH, H errera-Estrella L. Uptake,

integration, expression and genetic transmission o f a selectable chimaeric gene by plant

protoplasts. Schell M olecular and General Genetics MGG. 1985; 199: 161-168.

H anda SS, Sharma A, Chakraborti KK. Natural products and plants as liver

protecting drugs. Fitoterapia. 1986; 57: 307-352.

Hao J, Han W, Huang S, Xue B, Deng X. M icrow ave- assisted extraction o f

artemisinin firom Artem isia annua L., Sep Purif TechnoL 2002; 28: 191-196.

Harbome. Phytochemical dictionary: H andbook ofbioactive compounds from

plants 2nd (Edn.). Taylorand Francis, London. 1999; 221-234.

H assan MA, Ahmed IS, Campbell P, Kondo T. Enhanced gene transfection using

calcium phosphate co-precipitates and low-intensity pulsed ultrasound. European Journal

o f Pharmaceutical Sciences. 2012; 4: 768-773.

Department o f Biotechnology, Plamdard University Page 170

M e f e m n c m

Hassan, HA, Yousef ML Am eliorating effect o f chicory (Cichorium intybus L.)-

supplemented diet against nitrosamine precursors-induced liver injury and oxidative

stress in male rats. Food Chem Toxicol. 2010; 48; 2163-2169.

Hayashimoto A, Li Z, Murai N . A Polyethylene G lycol-M ediated Protoplast

Transformation System for Production o f Fertile Transgenic R ice Plants. Plant Physiol. 1990; 93: 857-863.

Hazra BR, Sarkar S Bhattacharyya, Roy P. Tum our inhibitory activity o f chicory

root extract against Ehiiich ascites carcinoma in mice. Fitoterapia. 2002; 73: 730-733.

H em m erlin A, Hoeffler JF, M eyer O, Tritsch D, K agan I A , Grosdem ange-Billiard

C, Rohmer M , Bach TJ. Cross-talk betw een the cytosolic m evalonate and the plastidial

methylerythritoi phosphate pathways in Tobacco Bright Y ellow - 2 cells. J Biol Chem.

2003; 278: 26666-26676.H eirera Estrella L, De Block M, Messens E, H em alsteens J P, V an M ontagu M,

Schell J. Chimeric genes as dominant selectable markers in p lan t cells. EM BO Journal

1983; 2:987-995.Hey SJ, Powers SJ, Beale MH, Hawkins ND, W ard JL, H alford NG. Enhanced

seed phytosterol accumulation through expression o f a m odified HM G-CoA reductase.

Plant Biotechnol J. 2006; 4; 219-29.

Hiei Y, Komari T, Kubo T. Transform ation o f rice m ediated by Agrobacterium

tumefaciens P lant M ol Biol. 1997; 35: 205-218.

Hirakova N , Bhatt ID, Sakurai Y, Chang JI. A lkaloid production by somatic

embryo cultures o f Corydalis ambigua. Plant Biotechnol. 2004; 21: 361-366.

Hiscox JD, Israelstam GF. A m ethod for the extraction o f chlorophyll from leaf

tissue without maceration. Canadian Journal o f Botany. 1979; 57: 1332—1334.

Ho C, Kuo H. In vitro resveratrol-rich callus tissues derived from vitis thunbergii

Sieb.et Zucc and method for producing the same. US-0160560, 2008.

Ho CW, Chu T. Flavonoids-rich tissues from N eom arica gracilis and methods for

culturing the same. US patent No. 7824914. 2010,Ho CW, Hsien-Shen K. In vitro resveratrol-rich callus tissues derived from vitis

thunbergii Sieb.et Zucc and method for producing the same. US Patent A pplication No:

0160,560. 2008.

Department o f Biotechnology, Hamdard University Page 171

M e fm e m c e^

Hoflmarm-Tsay S, Ernst R Hoffmann F. Design, synthesis and application of

surface-active chemicals for the prom otion o f electrofiision o f plant protoplasts. Bioelectrochem Bioenerg, 1994; 34: 115-122.

Holmberg Cl, Roos PM, Lord JM , Eriksson JE, Sistonen L. Conventional and

novel PKC isoenzymes modify the heat-induced stress response but are not activated by heat shock. J. Cell. Sci. 1998; 111: 3357-3365.

Holm berget N, Biilow L. Improving stress tolerance in plants by gene transfer. Trends in Plant Science. 1998; 3: 61-66.

Hu Q, Zuo P, Shao B, et al. Admimstration o f nonviral gene vector encoding rat f?-

defensin-2 ameliorates chronic Pseudomonas aeruginosa lung infection in rats. Gene Med. 2010; 12: 276-286.

Hu T, M etz S, Chay C, Zhou HP, Biest N et al. Agrohacterm m m ediated large-

scale transform ation o f wheat {Triticum aestivum L.) using glyphosate selection. Plant

Cell Rep. 2003; 21: 1010-1019.

Huie CW. A review o f modern sample- preparation techniques for the extraction

and analysis o f medicinal plants, Anal. Bioanal. Chem. 2002; 373: 23-30.

Husaini AM, Abdin MZ. Development o f transgenic strawberry (Fragaria x

ananassa Duch.) plants tolerant to salt stress. Plant Sci. 2008; 174: 446-455.

Iba K. Acclimative response to tem perature stress in higher plants: approaches o f

gene engineering for temperature tolerance. Annu Rev Plant Biol. 2002; 53: 225-2245.

ICH Haromonised Tripartite Guideline, International Conference on

Harmonisation on Technical Requirements for Registration o f Pharm aceuticals for

Human Use, Q2A, Text on Validation o f A nalytical Procedures, Step 4 o f the ICH

process, 1994 and Q2B, Validation of Analytical Procedures: M ethodology, Step 4 o f the

ICH process, 1996, ICH Steering Committee.

Iqbal M M , Nazir F, A li S, A sif M A, Zafar Y , Iqbal J, A li GM. Over expression of

rice chitinase gene in transgenic peanut (Arachis hypogaea L.) im proves resistance

against leaf spot. M ol Biotechnol. 2012; 50: 129-136.

Ito H , Fukuda Y, M urata K, Kimura A. Transform ation o f intact yeast cells treated

with alkali cations. J Bacteriol. 1983; 153: 163-168.

Department o f Biotechnology, Hamdard University Page 172

S le f e m m c m

Jandera P, Veronika, Lucie R, Tom H, Baldri L, Gabriela S, K ellner V, H om a A.

EIP-HPLC analysis o f phenolic compounds and flavonoids in beverages and plant extracts

using a CoulArray detector. J. Sep. Sci. 2005; 28: 1005—1022.

Jha Z, Narayan Sharam Sh, Sharrna DK. D ifferential expression o f 3-hydroxy-3-

methylglutaryl-coenzyme A reductase of Andrographis paniculata in andrographolide accumulation. J. Chem. Pharm. Res. 2011; 3: 499-504.

Jiang Z, Berg H. Increase o f protoplast electrofiision supported by dextran

fractions. Bioelectrochem Bioenerg. 1995; 38: 383-387.

Jilek S, Ulrich M, M erkle HP, W alter E. Com position and surface charge o f DNA-

loaded microparticles determine m aturation and cytokine secretion in hum an dendritic

cells. Pharm Res. 2004; 21: 1240-1247.

.Jordan M, Wurm F. Transfection o f adherent and suspended cells by calcium phosphate. Methods. 2004; 33: 136—143.

.T0rgensen CB, Famm e P, Kxistensen HS, Larsen PS, M 0hlenberg F, Riisgard HU.

Transformation o f plants to introduce closely linked markers. 1996; EPOl 98288.

Jun L, Xuan-M ing L, Su-Yao X, Chuen-Yi T, Dong-Ying T L i-Jian Z.

Bioconjugated nanoparticle for DNA protection from ultrasound damage. A nal Sci.

2005; 21: 193-197.

Jun L, Xuan-M ing L, Su-Yao X, Chuen-Yi T, Dong-Ying T, Xuan-ming.

Preparation o f fluorescence starch-nanoparticle and its application as p lant transgenic

vehicle. J. Cent. South Univ. Technol. 2008; 15: 768-773.

Kai K ., Shimizu B., M izutani M., W atanabe K., Sakata K. Accum ulation o f

coumarins in Arabidopsis thaliana. Phytochemistry. 2006; 67: 379-386.

K akkar A, Verma VK. A grobacterium m ediated biotransformation. Journal o f

Applied Pharm aceutical Science. 2011; 01: 29-35.

K aram NS, Al-M ajathoub M. D irect shoot regeneration and m icrotuberization in

wild Cyclamen persicum M ill using seedling tissue. Sci Hort. 2000; 86: 235—246.

Kavi K ishor PB, Hong Z, Miao GH, H u CAA, Verm a DPS. Over-expression o f 1-

pyrroHne-5-carboxyiate synthetase increases proline production and confers

osmotolerance in transgenic plants. Plant Physiology. 1995; 108: 1387—1394.

K eck CM, Mtiller RH. Drug nanocrystals o f poorly soluble drugs produced by high

pressure homogenization. Eur. J. Pharm. Biopharm. 2006; 62: 3—16.

Department o f Biotechnology, Hamdard University Page 173

Kenel F, Eady C, Brinch S. Efficient Agrobacterium tumefaciens m ediated

transformation and regeneration o f garlic {Allium sativuni) im m ature le a f tissue. Plant Cell Rep. 2010; 29: 223-230.

Kester M, Heakal Y, Fox T, Sharma A, Robertson GP, M organ TT, A ltinogD lu El,

et al. Calcium Phosphate Nanocom posite Particles for In Vitro Im aging and Encapsulated Chemotherapeutic Dmg D elivery to Cancer Cells. Nano Lett. 2008; 12: 4116-4121.

Khan ST, Izum ikawa M, M otohashi K, M ukai A, Takagi M , Shin-Ya K.

Distribution of the 3 -hydroxyl-3-m ethylglutaryl coenzyme A reductase gene and

isoprenoid production in marine-derived Actinobacteria. FEM S M icrobiol Lett. 2010; 304: 89-96.

Kikkert JR, Vidal JR, Reisch BI. Stable Transform ation o f Plant Cells by Particle

Bombardment/Biolistics. M ethods in M olecular Biology. 1993; vol. 286. Transgenic

Plants; M ethods and Protocols. Edited by: L. Pena © Humana Press Inc., Totow a, NJ.

Kimbaris AC, Siatis NG, Daferera DJ, Tarantilis PA, Pappas CS, Polissiou MG.

Comparison o f Distillation and Ultrasound- Assisted Extraction m ethods for the isolation

o f sensitive Aroma compounds from Garlic {Allium Sativum), U ltrasonics Sonochem.

2006; 13: 54-60.

Kishinami I, W idholm JM. Auxotrophic Com plem entation in Intergeneric Hybrid

Cells obtained by Electrical and Dextran-Induced Protoplast Fusion. Plant Cell Physiol.

1987; 28: 211-218.

Klee H, Horsch R, Rogers S. A grobacterium -M ediated Plant Transform ation and

its Ftirther Applications to Plant Biology. A nnual Review o f P lant Physiology. 1987; 38:

467-486.

Koronfel M. Effects o f the Antibiotics Kanamycin, Cefotaxim e and Carbenicillin

on the Differentiation o f Flax Hypocotyls. Arab Journal o f Biotechnology. 1998; 1; 93-

98.

Kumari, Velayutham P, A nitha S. Comparative Study on Inulin and Esculin

Content o f in vitro and in vivo Plants o f Chicory (Cichorium intybus L. Cv. Lucknow

Local). Advances in Biological Research. 2007; 1: 22-25.

Lacroix B, Kozlovsky SV, Citovsky V. Recent patents on agrobacterium -m ediated gene and protein transfer, for research and biotechnology. Recent Pat D N A Gene Seq.

2008; 2: 69-81.

Department o f Biotechnology, Hamdard University Page 174

^M efm em ces

Lante A, Nardi T, Zocca F, Giacomini A, Corich V. M ultiple approaches to

evaluate red chicory extract antioxidant activity. Journal o f A gricultural and Food Chemistry. 2011; 59; 5318-5324.

Lazzeri PA. Stable transform ation o f barley via direct D N A uptake.

Electroporation- and PEG-mediated protoplast transformation. M ethods M ol Biol. 1995;

49:95-106.

Lebedev VG, Shestibratov KA, Shadrina TE, Bulatova IV, Abramochlcin DG,

Miroshnikov AI. Cotransformation o f aspen and birch with three T-D NA regions from

two different replicons in one Agrobacterium tumefaciens strain. Genetika. 2010; 46:

1458-1466.

Leon BR, Gonzalez BD, Galvan A, Fernandez E. Transgenic m icroalgae asgreen

cell-factories. Trends Bioteclinol. 2004; 22:45—52-

Levin R, Alper Y, Stav R, W atad A. Methods and apparatus for liquid m edia and

semi - automated micropropagation. Hort. Biotech. In Vitro Cult. Breed. 1999; 3: 659-

663.

Li C, Guo T, Zhou D, Hu Y, Zhou H, W ang S, C hen J, Zhang Z. A novel

glutathione modified chitosan conjugate for efficient gene delivery. J Control Release.

2011; 154: 177-188.

Li C-, Chen A., Chen X., Chen X., Hu Z. Separation and sim ultaneous

detem iination o f rutin, puerarin, daidzein, esculin and esculetin in m edicinal preparations

by non-aqueous capillary. J. Pharm. Biomed. Anal. 2005; 1: 125-31.

Li C., Chen A., Chen X., M a X., Chen X., Hu Z. N on-aqueous capillary

electrophoresis for separation and simultaneous determ ination o f fraxin, esculin and

esculetin in Cortex fraxini and its medicinal preparations. Biom ed. Chromatogr. 2005; 19:

696-702.

Li Q, Zhang Y, Wu B, Qu H. Identification o f indole alkaloids in Nauclea

officinalis using high-performance liquid chromatography coupled w ith ion trap and time-

of-flight m ass spectrometry. Eur J M ass Spectrom (Chichester, Eng). 2011; 17: 277-86.

Lichtenthaler HK, Rohmer M , Schwender J. Tw oindependent biochem ical

pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants.

Physiol Plant. 1997; 101: 643-652.

Department o f Biotechnology, Hamdard University Page 175

Lin C, Fugetsu B, Su Y, Watari F. Studies on toxicity o f m ulti-w alled carbon

nanotubes on arabidopsis T87 suspension cells. Nanotoxicology. 2008; 2: 28-32.

Linden L, Riikonen A. Effects o f 6-benzylaminopurine, thidiazuron and type o f

explant on in vitro shoot development o f Acer platanoides L Propag O m am Plants 2006;

6: 201-204.

Ling HQ, Kriseleit D, Ganal MW. Effect o f ticarcillin/potassium clavulanate on

callus growth and shoot regeneration in Agrobacterium m ediated transform ation o f

tomato (Lycopersiconesculentum Mill.). Plant Cell Reports. 1998; 17: 843—847*

Liu G, Dong J, W ang H, Hashi Y, Chen S. Characterization o f alkaloids in Sophora

flavescens Ait. by high-perform ance liquid chrom atography-electrospray ionization

tandem mass spectrometry. J Pharm Biom ed Anal. 2011; 54; 1065-1072.

Liu J, L iu XM , Xiao SY, Tong CY, Tang DY, Zhao LJ. B ioconjugated

Nanoparticle for DNA Protection from Ultrasound Damage. Anal. Sci. 2005; 21: 193-

197.Liu X, Qin D, Cui Y, et al. The effect o f calcium phosphate nanoparticles on

hormone production and apoptosis in human granulosa cells. Reprod B iol Endocrinol. 2010; 8: 32.

Liu Z, Zhu Z, Zhang H, Tan G, Chen X, Chai Y. Qualitative and quantitative

analysis o f Fructus Comi using ultrasound assisted m icrowave extraction and high

performance liquid chromatography coupled with diode array UV detection and tim e-of-

flightm ass spectrometry. J Pharm Biom ed Anal. 2011; 55: 557-62.

Loyter A, Scangos G, Juricek D, Keene D, Ruddle F. M echanism s o f D N A entry

into mam malian cells. II. Phagocytosis o f calcium phosphate DNA co-precipitate

visualized by electron microscopy. Exp Cell Res. 1982; 139: 223—234.

Lu CY. The use o f thidiazuron in tissue culture. In vitro cell. Dev. Biol. 1993; 29:

92-96.

Lu WX. Journal o f Southwest Agricultural University. 2001; 23: 130-133.

Lurqutn PF. Gene transfer by electroporation. M ol Biotechnol. 1997; 7: 5-35.

M acLachlan I, Palmer LR, Heyes J. M ethod o f inserting D N A into living cells.

US4394448, 1983.

M aitra A. Calcium phosphate nanoparticles; second generation non-viral vectors in

gene therapy. Expert Rev. MoL Diagnos. 2005; 5: 893-905.

pefjartm ent o f Biotechnology, Haradard University Page 176

M arcel B, Yuan Peng Z. Lipid-nucleic acid particles prepared v ia a hydrophobic

lipid-nucleic acid complex interm ediate and use for gene transfer. US5705385, 2008.

M argara J, Rancillac M. Recherches experim entales sur la neoform ation de

bourgeong inflorescentiels ou vegetatifs in vitro a partir d ’explantats d ’endive Cichorium

intybus L. II. Observations sur la vernalization preaiable de la racine. Ami. Physiol. Veg. 1966; 8:39-47.

M aroufi A, Karimi M, M ehdikhanlou Kh, Bockstaele EV, De Loose M.

Regeneration ability and genetic transform ation o f root type chicory {Cichorium intybus

var. sativum) African Journal o f Biotechnology. 2012; 11: 11874-11886.

M cCalla DR, Neish AC. M etabolism o f phenylpropanoid compounds in Salvia. II.

Biosynthesis o f phenolic cimiamic acids. Canad. J. Biochem. Physiol., 1959; 37: 531.

M enne E, Guggenbuhl E, Roberfroid M. Fn-type chicory inulin hydrolysate has a

prebiotic effect in humans. Am erican Society for N utritional Sciences. 2000; 22: 1197-

1199.

M eyer D, Stasse-W olthuis M. The bifidogenic effect o f inulin and oligofructose

and its consequences for gut health. Eur. J. Clin. Nutr. 2009; 63: 1277-1289.

M ineo L. Plant tissue culture techniques. Tested studies in laboratory teachings

Proc. ABLE, 1990; 11: 151-174.

M ohapatra DP, Gassara F, Brar SK. Nanoparticles production and Role in

Biotransformation. Journal o f N anoscience and N anotechnology. 2011; 11: 899-918.

M ostaghaci B, Hanifi A, Loretz B, Lehr CM. N ano-particulate calcium phosphate

as a gene delivery system. N on-Viral Gene Therapy. Prof. X ubo Yuan (Ed.). 2011; 577-

596.

M ukesh U, KuUcami V, Tushar R, M urthy RS. M ethotrexate loaded se lf stabilized

calcium phosphate nanoparticles: a novel inorganic carrier for intracellular drug delivery.

JB iom edN anoteclinol. 2009; 5: 99-105.M urashige T. Plant Propagation through Tissue Cul1:ures. Annu R ev Plant Physiol.

1974; 25: 135-166.

M urray HG, Thom pson WF. Rapid isolation o f h igh m olecular weight DNA.

Nucleilc Acids Res. 1980; 8: 4321-4325.

fiepartnient o f Biotechnology, Ham dard U niversity Page 177

M uthusamy VS, Anand S, Sangeetha KN, Sujatha S, Arun B, Lakshm i BS.

Tannins present in Cichorium intyhus enhance glucose uptake and inhibit adipogenesis in

3T3-L1 adipocytes through PTPIB inhibition. Chem Biol Interact. 2008; 174: 69-78.

N aaz F, Abdin MZ, Saleem J. Hepatoprotective effect o f Kasni against aflatoxin B

induced hepatic damage in mice. OPEM. 2006; 16: 1996-2001.

Nadkarni AK. Indian M ateria M edica popular Prakasam Pvt Ltd, Bombay. 1976.

N afis T, Akmal M, Ram M, A lam P, Ahlaw at S, M ohd A, A bdin MZ.

Enhancement o f Artemisinin content by constitutive expression o f the H M G -CoA

reductase gene in high-yielding strain o f A rtem isia annua L. Plant Biotechnol Rep. 2011;

5: 53-60.

N air R, Varghese SH, N air BG, M aekaw a T, Yoshida Y, K um ar DS.

Nanoparticulate material delivery to plants. Plant Science. 2010; 179: 154—163.

N ajmi K, Pillai tdC, Pal SN, A qil M , Sayeed A. H epatoprotective and behavioural

effects o f jigrine ingalactosamine induced hepatopathy in rats. Pharm aceutical Biology.

2010; 48: 764-769.

Nandagopal S, Ranjitha kumari BD. Adenine sulphate induced high frequency

shoot Organogenesis in callus and in vitro flowering o f Cichorium intybus Cv. Focus~a

potent medicinal plant. A cta Agriculturae Slovenica. 2006; 2: 415 — 425.

N anjo T, Kobayashi M, Y oshiba Y , W ada K, Tsukaya H, Kakaubari Y , Shinozaki

K. Biological functions o f proline inmorphogenesis and osmotolerance revealed in

antisense transgenicArabidopsis thaliana. Plant J. 1999; 18: 185-193.

N aqvi S, Maitra AN, A bdin M Z, A km al M d, A rora I, Sam im MD. Calcium

phosphate nanoparticle m ediated genetic transform ation in plants. Journal o f M aterial

Chemistry. 2012; 22: 3500-3507.

Narita JO, Gruissem W. Tom ato H ydroxym ethylglutaryl-CoA Reductase Is

Required Early in Fruit Developm ent but not during Ripening. The Plant Cell, 1989; I:

181-190.

Navarro E, Baun A, Behra R, H artm ann N. Environm ental behavioxir and

ecotoxicity o f engineered nanoparticles to algae, plants, and fungi. Eco-toxicol. 2008; 17:

372-386.N ess J, Minshull J. Biotransform ation using genetically m odified Candida. W O-

008231A2, 2011.

Department o f Biotechnology, Ham dard University Page 178

N euhaus G, Spangenberg G, M ittelsten Scheid O, Schweiger HG. Transgenic

rape seed plants obtained by the m icroinjection o f DNA into m icrospore-derived

embryoids. TAG Theoretical and Applied Genetics. 1987; 75; 30-36.

N eum ann S, Kovtun A, D ietzel ID, Epple M, H eum ann R. The use o f size-defined

DNA-functionalized calcium phosphate nanoparticles to m inim ise intracellular calcium

disturbance during transfection. Biomaterials. 2009; 30; 6794—6802.

Niidom e T, Huang L. Gene therapy progress and prospects: nonviral vectors. Gene

Ther. 2002; 9: 1647--1652.

Niness, K.R. Inulin and Oligofructose W hat Are They J. Nutr. 1999; 129; 1402-

1406.

Oard JH. Physical methods for the transformation o f plant cells. Bioteclinol Adv.

1991; 9; 1-11.

Ohadi Rafsanjani MS, Alvari A, Samim M, Abdin M Z, Hejazi M A. A pplication o f

Novel Nanotechnology Strategies in Plant Biotransformation: A Contem porary

Overview. Recent Pat Biotechnol, 2012; 6: 69-79.

Ohadi Rafsanjani MS, Alvari A, Anis M, Abdin MZ, H ejazi MA. In vitro

Propagation o f Cichorium intybus L. and Quantification o f Enhanced Secondary

Metabolite (Esculin). Recent Patents on Biotechnology. 2011; 5; 227-234.

O lson M.M., Roseland C.R. Induction o f the coumarins scopoletin and ayapin in

sunflower by insect feeding stress and effects o f coumarins on the feeding o f sunflow er

beetle (Coleoptera, Chrysomelidae). Environ. Entomol. 1991; 20: 1166-1172.

O 'Neill C, Horvath GV, Horvath E, Dix PJ, M edgyesy P. Chloroplast

transformation in plants: polyethylene glycol (PEG) treatm ent o f protoplasts is an

alternative to biolistic delivery systems. The Plant Journal. 1993; 3; 729-738.O rrantia E, Chang PL. Intracellular distribution o f DNA internalized through

calcium phosphate precipitation. Bxp Cell Res. 1990; 190; 170—174. >

Ozbas-Turan S, Aral C, Kabasakal L, K eyer-U ysal M , Akbuga J. Co-encapsulation

o f two plasm ids in chitosan m icrospheres as a non-viral gene delivery vehicle. J P hann

Pharm Sci. 2003; 6; 27-32.Pan X, Niu G, Liu H. M icrowave- assisted extraction o f tea polyphenols and tea

caffeine from green tea leaves, Chem Eng Proc. 2003; 42: 129-133.

P ^ a r tm e n t o f Biotechnology, Hamdard University Page 179

3 le ^ m s n c e &

Park EJ, Lim HT. Establishment o f an efficient in vitro plant regeneration system

in chicory (Cichorhim intybus L. var sativiis). Acta H ort 1999; 483: 367-370.

Pasupathy K, Lin S, Hu Q, Luo H, Ke PC. D irect plant gene delivery w ith a

poly(amidoamine) dendrimer. Biotechnol. J. 2008; 3; 1078-1082.

Pena L, Cervera J, Juarez J, N ararro A, Pina .TA, N avarro L. Genetic transform ation

o f lime (Citus aurantifolia Swing.): factors affecting transform ation and regeneration.

Plant Cell Rep. 1997; 16: 731-737.

Pem iiakova NV, Shumnyi VK, Delneko EV. A grobacterium -m ediated

transform ation of plants: transfer o f vector DNA fragments in the plant genome.

Genetika. 2009; 45: 305-317.

Petersen M, Strack D, M atem U. Biosynthesis o f phenylpropanoids and related

compounds. In M W ink, ed. Biochem istry o f Plant Secondary M etabolism. A cadem ic

Press, Sheffield, 1999, pp 151-185.

Philip F, Raj K, Channa B. Cationic lipids for intracellular delivery o f biologically

active molecules. US5264618, 1993.

Potrykus I. Gene Transfer to Plants: Assessm ent o f Published Approaches and

Results. Annu Rev Plant Physiol Plant M ol Biol. 1991; 42: 205-225.

P rud’homme G, Draghia-Akii R, W ang Q. Plasmid-based gene therapy of diabetes

mellitus. Gene Ther. 2007; 14: 553—564.Rabinow BE. Nanosuspensions in drug delivery. Nat. Rev. 2004; 3: 785—796.

Radu DR, Lai Ch, Jeftinija K, Rowe EW , Jeftinija S, L in VSY. A Polyam idoam ine

Dendrimer-Capped Mesoporous Silica Nanosphere-Based Gene Transfection Reagent.

Am. Chem. Soc. 2004; 126: 13216-13217.

R afla A, H Sehrish. Photochem istry o f light harvesting pigm ents and som e

biochemical changes under aluminium stress. Pak. J. Bot. 2008; 40: 779-784.

Rakoczy-troj anowska M. Alternative methods o f p lant transform ation —a short

review. Cellular & molecular biology letter. 2002; 7: 849—858.Ram M , Khan MA, Jha P, K han S, K iran U, et al. HM G-CoA reductase limits

artemisinin biosynthesis and accum ulation in Artem isia annua L. plants. A cta

Physiologiae Plantarum. 2010; 32: 859-866.

Department o f Biotechnology, Hamdard U niversity Page 180

M e fm e m c e s

R anjitha Kumari BD, V elayutham P, A nitha S. A Comparitive Stxidy on Inulin and Esculin Content o f In vitro and In vivo Plants o f Chicory {Cichorium intybus L. Cv.

Lucknow Local). Advances in B iological Research. 2007; 22-25.

Rao Q, Baklish A, Kiani S, Shahzad K. The m yth o f plant transform ation. Bioteclmol. Adv. 2009; 27: 753-763.

Rasm ussen MK, Zamaratskaiab, Ekstrand B, In vivo effect o f dried chicory root

{Cichorium intybus L.) on xenobiotica m etabolising cytochrome P450 enzymes in porcine liver. Toxicol Lett. 2011; 200: 88-91.

Rastogi RP and M ehrotra BN. Com pendium o f Indian M edicinal Plants Rastogi,

RP (Eds) M entha piperita against the pathogenic bacteria was Orient Longm an Ltd, Madras, 1994; pp: 74.

Re EB, Jones D, Learned RM. Co-expression o f native and introduced genes

reveals cryptic regulation o f H M G Co A reductase expression in Arabidopsis. Plant J. 1995; 7; 771-84.

Rehm an RU, Israr M, Srivastava PS. et.al. In vitro regeneration o f w itloof chichory

{Cichorium intybus L.) from leaf explants and accumulation o f esculin. In vitro Cell D ev

Biol Plant. 2003; 39: 142-146.

Reich TJ, Iyer VN, M iki BL. Efficient transform ation o f alfalfa protoplasts by the

intranuclear microinjection o f Ti plasmids. Nature Biotechnology. 1986; 4: 1001-1004.

Reid W , Zhang Q, Sekimoto H. Influence o f mem brane surface charge on nutrient

uptake by plants. Developments in Plant and Soil Sciences. 2002; 92: 198-199.

Reynolds EC, W ong A. Effect o f adsorbed protein on hydroxyapatite zeta potential

and Streptococcus mutans adherence. Infect Immun. 1983; 39: 1285-1290.

Ribas AF, Dechamp E, Cham pion A , Bertrand B, Combes MC, Verdeil JL,

Lapeyre F, Lashermes P, Etienne H. Agrobacterium -m ediated genetic transform ation of

Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures.

BMC Plant Biol. 2011; I I : 92-99.

R iva GA, Gonzalez-Cabrera J, Vazquez-Padron R, Ayra-Pardo C. Agrohacterium

tumefaciens: a natural tool for plant transform ation. J Bioteclmol. 1998; 315: 118-133.

Roberfroid, M. Functional effects o f food components and the gastrointestinal

system: chicory fructooligosaccharides- Nutr. Rev. 1996; 54: 38-42.

pepartm ent o f Biotechnology, Hamdaxd U niversity Page 181

fM e fm e m c m

Robins RI. Secondary products from cultured cells and organs: M olecular and

cellular approaches. In: Dixon R.A. and R.A Gonzales (Eds.). Plant Cell Culture, IRL

Press, Oxford, NY and Tokyo. Sugar J. 1994; 96; 1150-1156.

Rout SR, Behera B, M aiti TK, M ohapatra S. M ultifunctional magnetic calcium

phosphate nanoparticles for targeted platin delivery. D alton Trans. 2012; 41: 10777-

10783.

Roy I, M itra S, M aitra A, M ozum dar S. Calcium phosphate nanoparticles as novel

non-viral vectors for targeted gene delivery. Int J Pliarm. 2003; 250: 25-33.

Russell DW, Knight JS, W ilson TM. Pea seedling H M G-CoA reductases:

regulation o f activity in vitro by phosphorylation and Ca "* , and posttranslational control

in vivo by phytochrome and isoprenoid hormones. In: Current Topics in Plant

Biochemistry and Physiology. Columbia; U niversity o f Missouri. 1985. pp. 191—206.

Rusu MA, Tamas M, Puica C. et.al. The hepatoprotective action o f ten herbal

extracts in CC14 intoxicated liver. Phytother Res. 2005; 19: 744-749.

Ruzin SE, M cCarthy SC. The effect o f chemical facilitators on the frequency of

electro ftision o f tobacco mesophyll protoplast. Plant Cell Rep. 1986; 5: 342-345.Sailaja M, Tarakeswari M, Sujatha M. Stable genetic transform ation o f castor

(Ricinus communis L.) via particle gun-m ediated gene transfer using embryo axes from

mature seeds. Plant Cell Rep. 2008; 27; 1509-1519.

Saksi N , Dubois J, M illicamps JL, V asseur J. Regeneration de plantes de chicoree

w itloof cv. Zoom a partir de protoplastes: influence de la nutrition glucidique et azotee.

C. R. Acad. Sci- Paris. 1986; 302: 165-170.Sala, A.V. Indian M edicinal Plants: A Com pendium o f 500 Species, I'"* ed; Origent

Longmen; Chennai, 1994.

Samuel JK, Burroughs F, D ixit S, Zettler MW. Patent no. 20090104700. M ethods

for transferring molecular substances into plant cells. 2009.

Sasidharan S, Chen Y, Saravanan D, Sundram KM^ Yoga Latha L. Extraction,

isolation and characterization o f bioactive compounds from plants' extracts. A ir J Tradit

Complement A ltem Med. 2011; 8: 1-10.

Sawahel W, Cove D. Gene transfer strategies in plants. B iotechnol adv. 1992; 10:

393-412.

Department o f Biotechnology, Hamdard University Page 182

Sawahel WA. The production o f transgenic potato plants expressing hum an alpha-

interferon using lipofectin-m ediated transformation. Cell M ol Biol Lett. 2002; 7: 19-29.

Schaller H. The role o f sterols in plant growth and developm ent. Progress in L ipid Research. 2003; 42; 163-175.

Scheller H. The role o f sterols in plant growth and development. Prog Lipid Res.

2003; 42: 163-175.

Schmidt, Hartley T, et al. A ssem bly o f aqueous-cored calcium phosphate

nanoparticles for drug delivery. Chemistry o f materials. 2004; 24: 4942-4947.

Semple SC, K lim uk SK, H arasym T et al. Lipid-encapsulated polyanionic nucleic

acid. US8021686, 2011.

Seo JK, Lee HG, K im KH. Systemic gene delivery into soybean by simple rub-

inoculation with plasm id DNA o f a Soybean mosaic virus-based vector. A rch Virol.

2009; 154: 87-99.

Seong J, Yoon H, Sik H, Hee D, Seop I ET AL. M ethod for preparing ortho

dihydroxyiso flavones using a biotransform ation system. U S20110033904A1, 2011.

Shestibratov, Konstantin Aleksandrovich (Puschino, RU), Dolgov, Sergey

Vladimirovich. Method for producing a transgenic plant w ith the aid o f Agrobacterium

tumefaciens. US Patent Application No. 0124835, 2007.

Shihua Sh, X iujun Z, Y iming G, Yuxiang X. M icroinjection o f genes into in vitro

ovaries o f maize and identification o f transformed plants. A cta Botaiiica Sinica. 2001; 43:

1055-1057.

Shimabayashi S, Hashimoto N, K awam ura H, Uno T. Li: M ineral Scale Form ation

and Inhibition; Amjad Z, Ed. Plenum Press: N ew York, 1995; pp. 14-15.

Shrawat AK, Good AG. A grobacterium tum efaciens-m ediated genetic

transformation o f cereals using im mature embryos. M ethods M ol Biol. 2011; 710: 355-

372.

Signoretto C, M archi A, Bertoncelli A, Burlacchini G, Tessarolo F, Caola I,

Pezzati E , et al. Effects o f mushroom and chicory extracts on the physiology and shape o f

Prevote 11a intermedia, a periodontopathogenic bacterium. J Biom ed Biotechnol. 2011;

635348.

Department o f Biotechnology, Hamdard U niversity Page 183

^ M e fm e n c e s

Singer SD, Hily JM, Cox KD. Analysis o f the enhancer-blocking function o f the

TBS elem ent from Petunia hybrida in transgenic Arabidopsis thaliana and N icotiana

tabacum. Plant Cell Rep. 2011; 30: 2013-25.

Singh S, Bhardwaj P, Singh V, Aggarwal S, M andal UK. Synthesis o f

nanocrystalline calcium phosphate in microem ulsion—effect o f nature o f surfactants. J

Colloid Interface Sci. 2008; 319: 322-329.

Southern EM. Detection o f specific sequences am ong D N A fragments separated by

gel electrophoresis. Journal o f M olecular Biology, 1975; 98: 503—517.

Sporlein B, Streubel M, D ahlfeld G, W esthoff P, Koop HU. PEG -m ediated plastid

transform ation a new system for transient gene expression assays in chloroplasts. Theor.

Appl. Genet. 1991; 82: 717-722.

Stam M, Joseph NM , Kooter MJAN. The Silence o f Genes in Transgenic Plants.

Annals o f Botany. 1997; 79: 3-12.

Stanic G, .furisic B, Brkic D. HPLC Analysis o f Esculin and Fraxin in Horse- Chestnut Bark (Aesculushippo castanum L.). Croatica Chem ica Acta. 1999; 72: 827-834.

Stermer BA, Bianchini GM, K orth KL. Regulation o f H M G CoA reductase activity

in plants. J Lipid Res. 1994; 35: 1133—1140.

Subramanyam K, Subram anyam K, Sailaja KV, Srinivasulu M , Lakshm idevi K.

H ighly efficient Agrobacterium-mediated transform ation o f banana cv. Rasthali (AAB)

via sonication and vacuum infiltration. Plant Cell Rep. 2011; 30: 425-436.

Sultana S, Perwaiz S, Iqbal M, A thar M. Crude extracts o f hepatoprotective plants.

Solarium nigrum and Cichorium intybus inhibit free radical-m ediated D NA dam age. J

Ethnopharmacol. 1995; 45: 189-92.

Sumi Y. An enhancer o f tissue plasm inogen activator produced by heat processing

Bacillus natto culture solution; useful as pharm aceutical or food stuff for preventing

thrombotic diseases. JP 2008106064, 2008.

Swapan KD, Karabi D, N ouchine S, Gunter D, Ingo P. Herbicide resistant Indica

rice plants from IRRI breeding lin.e IR72 after PEG-m ediated transform ation o f

protoplasts. Plant M olecular Biology. 1992; 20: 619-629.

Swapna P, Subrahmanya RS, Sathyanarayana DN. W ater-soluble conductive blends o f polyaniline and poly (vinyl alcohol) synthesized by two em ulsion pathways.

Journal o f Applied Polym er Science. 2005; 2: 531—943.

Department o f Biotechnology, Hamdard University Page 184

TA Yu, Yeh ShD, Yang JSh. Com parison o f tlie effects o f kanam ycin and

geneticin on regeneration o f papaya from root tissue. Plant Cell, Tissue and O rgan

Culture. 2003; 74: 169-178.

Tadashi M, Hitoshi W, M ayum i O, Kiyoshi H, H ironori U. A m ethod o f plant

tissue culture which comprises culturing a tissue or an organ o f a plant, a part o f the same

or cultured cells in a m edium containing a culture filtrate and/or an extract o f a

photosynthetic procaryotic microorganism . 1993; Patent no. 5300128.

Tadashi M, Hitoshi W, M ayum i O, Kiyoshi H, Hironori U. Plant tissue culture

process. 1990; EF 0380692.

Takahashi S, Kuzuyama T, Seto H. Purification, characterization, and cloning o f a eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme involved in

biosynthesis o f terpenoids. J Bacteriol. 1999; 181; 1256-63.Tamm a RV, M iller GC. H igh performance liquid chromatographic analysis o f

coumarin and flavonoids from sections o f tridentate o f Artemesia. J. Chromatography.

1985; 322: 266-269.Tan X, Lin Ch, Fugetsu B. Studies on toxicity o f m ulti-w alled carbon nanotubes on

suspension rice cells. Carbon. 2009; 15: 3479—3487.

Taylor NJ, Fauquet CM. M icroparticle Bom bardm ent as a Tool in Plant Science

and Agricultural Biotechnology. DNA and Cell Biology. 2002; 21: 963-977.

T H E USE O F T H ID IA Z U R O N IN TISSU E C U L T U R E 1

The W ealth o f India, vol. Ill, Publication & Inform ation Directorate, Council o f

Scientific and Industrial Research, N ew Delhi. 1992, p. 555.

Tholi D, Sohrabi R, Huh J, Lee S. The biochem istry o f homoterpenes — Com m on

constituents o f floral and herbivore-induced plant volatile bouquets. Phytochem istry.

2011 ;72 :1635-1646 .

Tiwari KN, Sharma NC, Tiwari V, Singh BD. M icropropagation o f Centella

asiatica (L.) a valuable medicinal herb. Plant Cell Tissue and Organ Culture. 2000; 63:

179-183.

To KY, Cheng MC, Chen LF, Chen SC. Introduction and expression o f foreign

DNA in isolated spinach chloroplasts by electroporation. Plant J. 1996; 10: 737-743.

Pepartm ent o f Biotechnology, Hamdard University P age 185

Toponi M. Action combinee de la kinetine et de I’acide indolyiacetique sur la

neoform ation d ’organes par des fragments de feuilles d ’endive {Cichorium intyhus L.)

cultives in vitro. C. R. Acad. Sci. Paris. 1963; 257: 3030-3033.

Tom ey, Brian G, Trewyn, V ictor SY, Lin & Kan W ang. M esoporous silica

nanoparticles deliver DNA and chemicals into plants. Fran9ois N ature Nanotechnology. 2007; 108: 295-300.

Tousch D, Lajoix AD, Hosy E, Azay-M ilhau J, Ferrare K, Jahannault C, Cros G,

Petit P. Chicoric acid, a new compound able to enhance insulin release and glucose

uptake. B iochem Biophys Res Commun. 2008; 377: 131-5.

Tow ler MJ, Weathers P.T. Evidence o f artemisinin production from IPP stemm ing

from both the mevalonate and the non-mevalonate pathways. Plant Cell Rep. 2007; 26:

2129-2136.

Trigiano RN, Gray DJ. Plant tissue culture concepts and laboratory exercises, 2^

ed. 2000; CRC Press.

Truong-Le VL, W alsh SM, Schwabert E, Mao HQ, Guggino W B, August JT,

Leong KW. Gene transfer by DNA-Gelatin nanospheres. Arch Biochem Biophys. 1999;

361: 47-56.Tzfira T, Citovsky V. The Agrobacterium-Plant Cell Interaction. Taking Biology

Lessons from a Bug. Plant Physiology. 2003; 133: 943-947.

Varotto S, Lucchin M, Parrin P. Immature embryos culture in Italian red Chicory

(Cichorium intybus). Plant Cell Tiss O rg Cult. 2000; 62: 15-11,Varotto S, Lucchin M, Parrini P. Plant regeneration from protoplasts o f Italian red

chicory (Cichorium intybus L.). J. Genet. Breed. 1997; 51: 17—22.

Vasseur J. Action de I’acide indolyl-acetique, de la kinetine et de I’hydrazide

maleique sur la neoform ation des bourgeons et la synthese d ’A RN obsevees au cours de

la culture in vitro de fragments de feuilles etiolees d ’endive. C. R. Acad. Sci. Paris. 1979;

189: 93-96.V asseur J. Etude du bourgeonnem ent de fragments de feuilles etiolees d ’Endive. II.

Formation des bourgeons en fonction de caracteristiques biochim iques. Rev. Gen. Bot.

1979; 86: 113-190.V asudevan DM, Sree kumari S. Text Book o f B iochem istry for M edical Students

2nd Ed. 1995; Jaypee Brothers M edical Publisher (P) Ltd, N ew D elhi, India.

Department o f Biotechnology, Hamdard University Page 186

Velayutham P, Kumari R, Baskaran P. A n efficient in vitro plant regeneration

system for Cichorium intyhus L.—an im portant medicinal plant. Journal o f Agricultural Technology. 2006; 2: 287-298.

V enkatesw aran PS, M iilman 1, Blum berg BS. Effect of an extract from

Phyllanthusniruri on hepatitis B and w oodchuck hepatitis viruses: In vitro and in vivo studies. Proc. Natl. Acad. Sci. 1987; 84: 274-278.

Verma P, M athur AK. Agrobacterium tumefaciens m ediated transgenic plant

production via direct shoot bud organogenesis from pre-plasm olyzed leaf explants o f

Catharanthiis roseus. Biotechnol Lett. 2011; 33: 1053-1060.

Verm eulen A, Vaucheret H, Pautot V, Chupeaii Y. Agrobacterium m ediated

transfer o f a m utant Arabidopsis acetolactate synthase gene confers resistance to

chlorsulfuron in chicory {Cichorium intyhus L.) Plant Cell Reports. 1992; 11: 243-247.

V idyashankar S, Patki PS. Liv.52 attenuate copper induced toxicity by inhibiting

glutathione depletion and increased antioxidant enzyme activity in HepG2 cells. Food and

chemical toxicology. 2010; 48: 1863-8.

V ieira AL, Camilo CM. Agrobacterium tumefasciens m ediated transform ation of

the aquatic fungus Blastocladiella emersonii. Fungal Genet Biol. 2011; 48: 806-811.

Vogt T. Phenylpropanoid Biosynthesis. Mol. Plant. 2010; 3; 2-20.

W agner GM, Eneva T. Plant regeneration from Cichorium intyhus L. var. sativum

leaf midrib explants induced by ancim idol supplem entedculture medium.

Landbauforschung-Volkem'ode (FAL), Germany. 1998; 48: 53—55.

W agner GM, Eneva T. Positive effect o f cefotaxime on plant regeneration from

Cichorium intybus L. leaf material. Land bauforsch Volk. 1996; 46: 166-168.

W ang W, Tang J, W ang S, Zhou L, Hu Z. M ethod developm ent for the

determination o f coumarin compounds by capillary electrophoresis vi^ith indirect laser-

induced fluorescence detection. J Chrom atogr A. 2007; 1: 108-114.

W ang ZX, Yi ZL. Progress in Biotechnology. 2003; 23: 9-13.

W echuck JB , Ozruer A, Goins W F, W olfe D, Oligino T, G iorioso JC, A taai MM.

Effect o f temperatxire, m edium composition, and cell passage on production o f herpes-

based viral vectors. Biotechnol Bioeng. 2002; 5: 112-119.

W einstein LH, Porter CA, Laurencot HJ. Quinic acid as a precursor in aromatic

biosynthesis in the rose. Contrib. Boyce Thom pson Inst., 1959; 20: 121.

Department o f Biotechnology, Hamdard U niversity Page 187

^M efm em ces

W einstein LH, Porter CA, Laurencot HJ. Role o f quiaic acid in the aromatic

biosynthesis in higher plants Contrib. Boyce Thompson Inst. 1961; 21: 201.

W heeler JJ, Hope M, Cullis PR, Bally MB. Methods for encapsulating plasm ids in lipid bilayers. US5981501, 2003.

W ieber A, Selzer T, Kreuter J. Characterisation and stability studies o f a

hydrophilic decapeptide in different adjuvant dm g delivery systems: a comparative study

of PLGA nanoparticles versus chitosan-dextran sulphate m icroparticles versus DOTAP- liposomes. In t J Pharm. 2011; 421: 151-159.

W iele T, Boon N, Possemiers S, Jacobs H, Verstraete W. Prebiotic effects o f

chicory inulin in the simulator o f the human intestinal m icrobial ecosystem. FEMS M icrobiology Ecology. 2004; 51: 143—153.

W iesm an Z, Dom NB, Sharvit E, Grinberg S, Linder C, Heldman E, Zaccai M.

Novel cationic vesicle platform derived from vem onia oil for efficient delivery o f DNA

through plant cuticle membranes. J BiotechnoL 2007; 130: 85-94.

W u L, Zhang J, W atanabe W. Physical and chemical stability o f

drug nanoparticles. Adv Drug Deliv Rev. 2011; 63: 456-469.Wu Y, Kuzma J, M are' chal E, G raeff R, Lee HC, Foster R, Chua NH. abscisic

acid signaling through cyclic ADP—Ribose in plants. Science. 278; 2126-2130.

v^wv^.biotechnology4u.com

www.molecular-plant-biotechnology.info

Yang GD, Zhu Z, Li YE, Zhu ZJ. Journal o f Agricultural Biotechnology. 2002; 10:

127-132.Yang Z, Park H, Lacy GH, Cram er CL. D ifferential activation of potato 3-

hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen

challenge. Plant Cell. 1991; 3: 394-405.

Yaseen khan M, Aliabas S, K um ar V, Rajkum ar S. R ecent advances in medicinal

plant biotechnology. Indian Journal o f Biotechnology. 2009; 8: 9-22.

Y asseen MY, Splittstoesser W E. Somatic embryo genesis in w itloo f chicory

through leaf suspension culture. Plant Cell Rep. 1995; 14; 804-806.

Y asseen YM, Splittstoesser W E. Regeneration o f plants from stored w itloof

chicory for selection o f desirable storage traits. P lant growth reg. Soc. America. 1991; 1:

41-45.

Department o f Bioteclmology, Hamdard University Page 188

S le f,m e m c e 3

Yu TA, Y eh SD, Cheng YH, Y ang JS. Efficient rooting for establishm ent o f

papaya plantlets by micropropagation. Plant Cell Tissue and O rgan Culture. 2000; 61: 29- 35.

Y ucesan B, Turker AU, Gurel E. TDZ-induced high frequency plant regeneration

through multiple shoot formation in w itloof chicory {Cichorium intybus L.). Plant Cell Tiss Organ Cult. 2007; 91: 243-250.

Zayeda RM, W ink B, El-Shamy H. Z. In vitro O rganogenesis and Alkaloid

accumulation in D atura innoxia. Naturforsch. 2006; 61: 560-564.

Zhang BH, Liu F, Liu ZH, W ang HM , Yao ChB. Effects o f kanam ycin on tissue

culture and somatic embryogenesis in cotton. Plant Growth Regulation. 2001; 33: 137—

149.

Zhang F, Chen B, Xiao S, Y ao S. Optimization and Com parison o f different

extraction tecliniques for Sanguinarine and Chelerythrine in fruits o f M acleaya Cordata

(wild) R. B. Sep. Purif. Technol. 2005; 42: 283-290.

Zhang L, Hong F, Lu S, Liu C. Effect o f nano-T i02 on strength o f naturally aged

seeds and grow th o f Spinach. Biological Trace Elem ent Research. 2005; 105: 83-91.

Zhang Y, Nowak G, Reed DW , Covello PS. The production o f artem isinin

precursors in tobacco. Plant Biotechnol J. 2011; 9: 445-54.

Zhao GZ, Li J, Qin S, Huang HY, Zhu WY, X u LH, Li WJ. Streptomyces

artemisiae sp. nov., isolated from surface-sterilized tissue o f Artem isia annua L. Int. J.

Syst. Evol. Microbiol. 2010; 60: 27-32.

Department o f Biotechnology, Hamdard University Page 189

Recent Paients on Biotechnology 2012, 6, 69-79 69

Application of Novel Nanotecheology Strategies in P lant Biotransforma- tioiiJ A Contem porary OverviewMehrnaz S. Ohadi Rafsanjani’, Amene Alvari', M. Samim^, M. Amin Hejazi^ and M.Z. Abdin'*

Dept. o f Biotechnology, Faculty o f Science, Jamia Flamdard, New Delhi-62, India; ^Dept. o f Chemistry, Faculty o f Sci­ence, Jarnia Hamdard, New Delhi-62, India; ^Dept. o f Agriculture Biotechnology Research Institute o f Iran (ABRII),

Tabriz, Iran

Received: October 1 7, 2011 Revised: December 17, 2011 .Accepted: Jamiary 03, 2012Abstract; During the past epoch we liave gone through the remarkable progress in plant gene traiisfoniiation technology. The production of transgenic plants is considered as a valuable tool in plant research and the technology is eKtensively applied in phytomedicines and agricultural research. Gene transformation in plants is normally carried out by Agrobacterium species, application of some chemicals and physical techniques (electroporation, microprojectile, etc.). Now a days with better efficacy and reproducibility, novel technologies for the direct gene ti'ansfer like liposome, positively charged liposome (lipofectin) and naiioparticle based delivery systems are used for genetic transformation of plants. In this review, we have enlightened the novel nanotechnologies like liposome, Carbon nano-tube and nanoparticles with their current status and future prospects in transgenic plant development. Moreover, we have also highlighted the limitations of conventional techniques of gene transfer. Furtlrermore, we have tried to postulate innovative ideas on the footprints of established nanotechnology and chemical based strategy with improved efficacy, reproducibility and accuracy along with less time consumption.

Keywords; Biotransformation, Nanotechnology, Liposome, Lipofectin, Nanoparticle.

IN TROD UCTION

Plant tissue culture techtiique is an essential tool in agricultural and medicinal plant research. It relies on maintaifiing plattt cells in controlled aseptic condition on a suitable nutrient medium. The culture can be sustained as a mass o f undifferentiated cells for an extended period of time or regenerated into whole plants. For the production o f secondary plant metabolites and regeneration of plant with improved nutritional quality, higher yields and tolerance against biotic and abiotic stresses, new er genes need to be introduced in the preexisting plants with norm al physio and phytochemical properties [1-3]. T ill now tissue culture is grown at a mature level in each respective area o f gene transfer like protoplast fusion [2, 4-6], physical method (electroporation, particle bom bardm ent, micro-injection, etc.), direct gene transfer techniqites (protoplast fusion, transfection), gene transfer using plasma membrane destabilizing/precipitating agents (chemicals that facilitate the gene transfer through the cell membrane, for example, polyethylene glycol 6000, polyvinyl alcohol, NaNOa, DEAE, Dextran, DMSO) [7-14] and natural means o f gene transfer &uch as the use o f Agrobacterium species [15 , 16].

Although these conventional m ethods are effective in gene transfer, but their effectiveness and efficiency are com­promised over the rapidity as the application o f excessive energy in physical m ethods as w ell as the excess chemicals utilized in chemical m ethods damage the DNA and cells [6 ],

Encapsulation o f gene into biocom patible carriers is ex­pected to enhance the biotransformation process and related outcomes by providing protection to the cell, gene and en­zymes.

‘Address Coriespending to this Author at the Dept, of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi-62, India;Tel: +91-9818462060; E-mail: mzabdin@rediffmail.com

Based on this, currently new era o f novel nanotechnology based carriers like liposome, m odified positively charged liposome, inorganic nanoparticles, carbon nano-tnbe and Quantum dot etc. were successfully adopted as main or adju­vant technologies in genetic transformation. N um erous reports published in recent tim es are indicative o f significant contributions o f these novel technologies in gene transfer to the plant tissues and their culture techniques. In this review, we are discussing in detail about the liposomes and nanopar- dcles as novel nanotechnology based biotransformation sys­tems for gene deliveiy in plants. M oreover, wc will also describe in brief about com m only used gene transfer approaches and their limitations. A dditionally, we w ill try to postulate innovative ideas on the footprints o f estabhshed nanotechnology and chemical based strategy for im proved efficiency, reproducibility and accuracy o f bioiransforma- tion.

C O N V E N T IO N A L B IO T R A N S F O R M A T IO N N O L O G IE S IN PLA N TS

T E C H -

Plant biotransformation vectors and m ethodologies have been im proved with the time to increase the effectiveness o f biotransfonnation and achieve stable expression o f trans­genes, Currently available biotransform ation m ethods can be divided into two inain groups: indirect and direct biotrans­form ation method. The indirect m ethod is based on the in ­troduction of a plasm id-canying gene construct into the tar­get cell [15, 16], The m ost com m only applied indirect m ethod is Agrobacterium tumefaciens or Agrobacterium rhizogenes based gene transfer in w hich the xmlque natural ability o f Agrobacterium is utilized to precisely transfer spe­cific DNA sequences to cells [17-27]. Im portant events o f T-DNA transfer and components involved in the Agrobacte­rium mediated gene transfer are outlined in Fig. (1). Agro- bacteriiim-haseA DNA transform ation offers m any unique advantages: (1) precise integration o f DNA sequences with

1872-2083/12 SIOO.OO+.OO © 2012 Bentham Science Publishers

70 Recent Patents on Biotechnology 2012, Vol. 6, No. I Ohadi et al.

V in ilen te pvoteisi P In iU C e l l

Irian-iporter C'lianiiel

jVucleiis

M tto c lio n rtr iii

Bactei’in! C lii’omciQinesChloroplnst

Fig. (1). Diagrammatic presentation of Agrohacterium mediated gene transfer in higher plants.

defined etid, (2) transfer o f desired DNA along with the marker gene, (3) high frequency o f stable and intact gene transfer, (4) low rate o f transgene silencing, and (5) the abil­ity to transfer long stretches of T-D NA (> 150 kb) [20].

Although, Agrobacterium m ediated gene transfer is a commonly used technique with uniqueness, but it has several drawbacks [18. 20, 22-25], These include;

a) Hmitation to carry limited size base pair (<500kb) [25 ],b) chances o f transgene silencing,c) poor gene transfer efficiency,

To improve the efficiency o f Agrohacterium m ediated gene transfer, newer modifications in indirect methods (e.g. application o f acetosyringone) have been applied [20], Fur­ther advances were made while developing transgenic plants devoid o f antibiotic rnarker genes, co-transformation o f m ul­tiple T-DNAs, site-specific recom bination strategies and de­ployment of Ac/Ds-based transposition tliat helped in the elimi- natioti of marker genes in transgenic plants [20,22,25-27].

PHYSICAL M ETH O D S

Electroporation of In ta c t P lan t C ell and Tissue

Protocols for the electroporation o f cell suspensions have been worked out for many species, e.g. tobacco, rice and wheat. So far, however the best results have been obtained for maize [28]. The efficiency of electroporation was found to be relatively high in this case and 90 transgenic plants were regenerated from 1440 embryos (6.25%) and 31 plants from 55 calli (54.6%) in a study conducted by To et al. [29], which is fully comparable with the best results obtained for this species after micro bom bardm ent [28, 29-30]. Similar fesults were obtained for maize by other investigator on 445

transgenic cell lines selected from 24m l packed cell volume (PCV) [31]. Modifications in electroporation such as post pulse addition o f ascorbic acid or ascorbate could signifi­cantly increase the efficiency o f process without having any negative influence on cell viability [32], Although electropo­ration seems to be a simple and effective method, but its ap ­plication is lim ited to only few species. In addition, application o f electric current dam ages the gene leading to m isleading codons and w rong translational end product [2 9 ,3 3 ,3 4 ],

MicroinjectionIn case of microinjection, D N A is directly injected into

plant protoplasts or cells (specifically into the micleus or cytoplasm) using fine tip o f 0 .5-1,0 m icrometre diam eter made up of glass needle or micropipette. This method o f gene transfer is used to introduce D N A into large cells such as oocytes and the early em biyonic stage [34, 35-38],

Silicon C arb ide-M ediated T ran sfo rm atio n (SC M T )

In this technique, silicon carbide fibres are simply added to a suspension, containing both plant tissue and the plasm id DNA, It is then rigorously mixed so that DNA-coated, fibres penetrate the cell wall [34], A lthough SCM T approach is easy, fast and economical m ethod that is applicable to vari­ous plants but this methodology have disadvantages like low transformation efficiency, damage to cells that negatively influence their further regeneration capability, and the neces­sity o f taking extraordinarily rigorous precautions during kib w ork as breathing the fibres in, especially asbestos ones, can lead to serious sicknesses [34, 39, 40], M oreover, the effi­ciency o f SCMT is affected by various variables like fibre size, vortexing, shape o f the vessels used, plant m aterial and

Application o f Novel Nanotechnology Strategies in Plant Biotransformation Recent Patents on Biotechnology 2012, Vol. 6, No, I 71

the characteristics o f the plant cells (particularly the thick­ness o f the cell wall) [41],

T em perature M ediated Gene T ran sfe r

Elevation in temperature enhances the gene transfer that is successfolly adopted and conformed to animal cell trans­fection process [42|. It seems from the literature that at tem ­perature above 37"C, the gene transfer tendency gets in­creased and further rise in ambient temperature to 43°C with certain period o f time provided greater transient transfection. This is confirmed by work on interleukin-2 and swine growth hormone expressions using indirect FiLISA [43, 44],

Based on the above concept Dillen et al. in the year 1997 worked on the effect o f temperature on Agrobacterium tiiine- faciem-mediiiied gene transfer in plant [45], They reported the effect of temperature (15°C-29°C) in biotransformation process with A. tuinefaciens involved in co-cultivation of Phaseolus acutifolhis and further in Nicotiana tabacum biotTcinsformation [45], In both the situations, iiTespective of the type of helper plasmid, the level o f transient uidA expres­sion decreased notably when the temperature was raised above 22°C, further lowered dow n in temperature at 27°C and was undetectable at 29°C [45, 45]. Coiiamonly used con­ventional method o f biotransformation discussed here is suminarized in Table I along w ith their advantage, mecha­nism of gene transfer and limitations.

NEED O F N A N O T E C H N O L O G Y A PPR O A C H

Particles/globules having the size range of 1-lOOOnm are considered as nanoparticle or nano vesicle nanocarriers [47, 48], The small size with unique surface and bulk properties have made them versatile carriers for bioactives and genes.

Direct and indirect physical biotransfom iation processes commonly suffer from instability o f gene in the environ­ment, deletion of gene due to the high energy impact, poor penetration and reproducibility. Literature revealed that the process o f nanotechnology based gene transfer in plant seems to be slower but nanotechnology is expected to pro­vide numerous benefits when utilized in biotransfomiation;

a) comparatively faster mode o f gene transfer than the Agrobacterium based biotransfonnation,

b) DNA is protected due to encapsulation and effectively transformed in controlled manner,

c) the probability o f energy induced DNA damage related gene mutation (commonly seen in case o f physical methods) is very much reduced, and

d) nanotechnology approaches are also easily amalgamated with physical methods leading to effective and improved gene transfer [48],

Till date in plant biotransformation process, liposome, modified liposome, lipofectin, nanoparticles, carbon nano- lube, and quantum dots have been reported as gene carrier [48], Although, very few nanotechnology based methods have been reported so far in biotransfoxniation, but they seems to be effective in terms o f time consumption and DNA protection during transfonnation process. Furthennore, use

of nano-formulations encapsulated gene with other physical methods like electroporation, microinjection and m icropro- jectile bom bardm ent technique may enhances the gene trans­fer efficacy with integrity o f DNA. In plants, liposomes and nanoparticles were successfully adopted as an adjuvant w ith physical methods as common nano-approaches. Likewise, with some strategic modification in nano-approaches, they can directly be adopted as the sole biotransfom iation method,

Liposom e M ediated G ene Transsfer

Liposomes are colloidal, vesicular structures consisting of one or nrore lipid bilayers that sruTound an equal num bers o f aqueous compartments [48-49], Due to the presence o f aqueous core and lipophilic vesicle structure, the plasmid/ gene is expected to be encapsulated in aqueous environment. Liposomal gene transfer technique has been successfully tried and adopted in gene delivery as the novel technique [50],

Gene loaded liposomes provide numerous advantages;

1 ) providing stability to gene,

2) diminish or reduce the deletion in the DNA, while used along with the physical techniques,

3) control release pattern o f gene delivery,

4) surface modification o f liposome (positively charged) potentiate the penetration within a cell,

5) show higher degree o f reproducibility,

6 ) applicable to a wide range o f cell types, and

7) free from cellular toxicity.

In this technique, the m echanism o f D NA entry through the protoplast seems to be mediated by endocytosis process o f liposome by the cells [48], which may involve the follow­ing steps:

1 ) adhesion o f the liposomes to the protoplast surface,

2 ) fusion o f liposomes at the site o f adhesion,

3) formation o f endosome w ithin the cytoplasm, and

4) release o f plasmids inside the cell.

The liposomal technique has been successfully used to deliver DNA into the protoplasts o f a num ber o f p lant spe­cies (e.g. tobacco, petunia, carrot, etc.) [50]. Possible m echa­nism o f gene transfer in plant by liposome is illustrated in Fig. (2).

First time, in the year 1985 Deshayes et al. developed positively charged liposomes bearing EschericM a coll plas­mid (pLGV23neo) carrying a kanaiiiycin resistance gene, used in transgenic tobacco development. Protoplasts isolated from their leaves were resistant to 1 0 0 m icrogram s/m l ka- namycin [50],

Later in the year 1987, Caboche et al. prepared plasmid vector pUCSCaM VCAT with CA T gene (chloram phenicol acetyl transferase) encapsulated unilam ellar liposom al vesicle, and successfully tried to introduce gene in tobacco protoplast. They also reported that introduction o f

Table t. Summarized Features, Mechanisms of Gene Transfer and Limitations of Conventional Biotrarisforination Methods

72 Recent Falenis on Biotechnology 2012, Vol. 6, No. 1 Ohadi et al.

Methods Features Mechani,siri Limitations

Physical Methods

Elactropofation

Electrical impulse (1.2kVolt/s) through the suspension of protoplast in the solution con­

taining DNA resulting in gene transfer.

The process is rapid and sjjecialized vcctors are not needed.

Useful for high-efticiency transient expression of foreign genes,

In plants, sometime, selectable marker is not needed.

Poorly understood, but probably the electrical impulse enhance the pore size for passive diffusion. In addi­

tion, development of elcctricai giadient potentiate.s the iiitra cellu­

lar transport of DNA.

Frequency of stable trausfortuation is low (0.001 or less). Nonnally the

transformed DNA i.s cKtensivcIy reananged leading to mitotic and

meiotie instability.Can be only used with protoplasts.

Probability of deletion is high.Application is limited to only few

species.

Microprojectile/ Panicle bombardment/

Biolistic

DNA coated gold particles are bombarded over the cells, tissues or whole plants.

More stable transfomiation possibility. Vectors are not required

Applicable to wide range of plants. Applicable with other methods that are needed

for tissue culture.Highly efficient.

Forced bombardment of small gold particles directly introduce gene into the cells through reversible

penetration. Fomiation of passive diffusion pores is also probably the

reason.

The transformed DNA is highly rearranged leading to mitotic and

meiotie instability.Amenable tissue cul­

ture/regeneration systems caniTot completely he eliminated.

The transformed cells survival rate is decreased because of high mem­

brane damage.

Micro injectio u

The f)NA is directly injected into plant proto­plasts or cells using fine tipped glass needle or

niioropipette.Suitable for gene introduction in large cells.

Effective gene transfer to the nucleus is easy.

Direct introduction of gene by pene­trating the cellular membrane,

Not suitable for small cells or tis- ,sues.

Infrequent gene transfer method in plant biotransformation.

Efficiency is low.Suitable for large animal cells.

Temperature mediated gene transfer

Under the influence of temperature protoplasts fuse together or gene transformed into the cells.

Method is economical and easy to adopt.

At elevated temperature and enhanced randomization, formation

of pores due to the lipid fluidization.

Only used for experimeiital pur­pose.

Infi'equent use.

Only bo adopted with protoplast. Slow and less efficient process.

With the intention for rapid biotransformation, enhanced tem­perature damage the nucleic acid

and cells.

Co-pracipitationDivalent metals (Ca, Mg) and DNA

co-precipitates when formed at cell surfaces, they can be taken up by cells.

-

Wall-less cells are amenable to biotransformation through co-

precipitation.Mitotic and meiotie instability may

occur.

Micromanipulation

Single-cell technique.

Features similar to biolistic method.An excellent tool for specific transient expres­

sion studies.

Similar to biolistic method.

Little use for stable transformation of plants.

Other limitations similar to biolistic method

Chemical Method

Protoplast fiision/gene ti-ansfer is carried out by fusagens/plasma membrane destabilizing/ pre­

cipitating agents (eg. PEG, PVA, Dextran, etc.).

Process is rapid- and highly efficient

Fusion of protop last/cells under the influence of their charge interaction.

Surfactant like behavior enhances the cellular permeation.

Damage of cellular components and gene itseif

Only is applicable in case of proto­plasts. The regeneration of trans­

formed protoplasts is veiy difficult, which lowers (he transfomtacion

frequency.

Application of Novel Nanotechnology Strategies in Plant liiotramformation Recent Patents on Biotechnology 2012, Vol. 6, No, I 73

(Tiibie 1) coutd....

Methods Features Mechanism Limitations

Biological Methods

Agrobactcrium mediateU Iraitsfannation

Natural ability oi' Agvohacterium is utilized in which Ti plasmid of the liacteria containing gene of interest transforni the plant.

Simple technique.

The transformed cells are fairly stable for mi­totic and nieiotic process.

Large loading capacity in teniis of foreign DNA size.

Easy to adopt in large number of plant species,

Adaptable to different types of cell, tissues, cultures and non-culture techniques.

Biotransformation occur in follow­ing steps:

1) precisc integration of DNA se­quences with defined end, 2) trans­fer o f desired DNA along with the selection marker, 3) high frequency of stable and intact gene transfer, 4) low rate of transgene silencing, 5) The ability to transfer long stretches ofT-DNA,

Host range is limited; to dicot plants.

With vulnerable plants, accessible culture systems must be adapted.

Chances of tran.sgetie silencing.

Poor gene transfer efficiency.

Virus mediated transformation

Plant’s virus ability to express foreign gene is utilized.

Potentially useful for transient and whole-plant expression.

Independent of host range limitation.

Host cell mediated endocytosis. Moreover, lysogenesis process by

virus cannot be ruled out.

Non selectivity.

Slow process.

Low DNA size carrying capacity.

Endosome fomiation leading to endocytosis

Fig. (2 ). Illustration of possible means of gene transfer in plants by liposomes indicating the probable steps.

74 Recent Patents on Biotechnology 2012, Vol. 6, No. I O/tiU/i e( id.

polylysine into the media increases the penetration o f encap­sulated gene conjugate, that is probably due to the introdiic- tion of positive charge over the liposome surface that can be easily adsorbed over the negatively charged cellular mem­brane surface. In addition, the reports also disclosed that the CAT gene expression efficiency was decreased [51].

The reasons behind such responses were not explored by Caboche et a i, but the probable reasons behind decreased CAT gene expression possibly are as follows;

a) entire vesicular liposomal form may enter info the cell due to endocytosis process,

b) inside the cytoplasm, liposome release the encapsulated gene not at a time but in controlled manner, which is a property o f vesicular system, and

c) due to the fusion process, the surface adheres liposome fused with the membrane and released the core content.

Caboche e/ al further worked on tlie liposomal mediated gene transfer that was corroborative to their earlier work[52]. Balias et al. formulated tobacco mosaic vims RNA {TMV RNA) encapsulated liposome that was made up o f phospholipid (Phosphatidylcholine) and cholesterol [53]. The developed liposomes were further surface fnnctionaiized by introducing hydroxy] group over liposomal lipid layer by using a quaternary ammonium detergent, di-isobutyl cresoxyethoxyethyl dimethyl benzylaramonium (DEBDA [OT-r]). This enables the functional transfer o f TISW RNA within small span o f time (48h) into tobacco and petunia protoplasts [53].

They proposed two alternative modes thorough which the ti'ansfomiation process o c c u r r e d ; d u e to the presence o f cationic detergent, the liposomal surface gets positive charge. Incubation of positively charged liposomes witb plant protoplasts resulted in transfer o f the enclosed RNA. Second, biotransfomiation may also be enhanced by complex formation between the surfactant present over the surface o f liposome and externally added TMV-RNA. The result fur­ther supported by the experimental outcome that showed liposomes lacking the quaternaiy ammonium detergent failed to transfer TMV-RNA into tobacco and petunia protoplasts[53].

Recently, Caboche worked on liposome mediated trans­fer o f nucleic acids in plant protoplasts and reported the spontaneous interaction between liposome and surface o f protoplast, Caboche developed and characterized stable transfomiants expressing kanamycin resistance gene by hy­bridization technique and progeny analysis [52].

IVfodification in Liposom al Technique

Wiesnian et al., in the year 2007 synthesized novel cati­onic amphiphilic compounds from vemonia oil (a natural epoxidized triglyceride) and further utilized the same in the preparation o f liposomes that were able to encapsulate DNA[54], The developed liposomes have positive chai'ge on vesi­cle surface as well as excellent stability due to charge equi­librium provided by ampiphilic compound [54].

The ampiphilic compound based liposome was character­ized for surface morphology and vesicular size by TEM , SEM, AFM and confocal laser systems that showed the spherical shape with diameters between 40 and 110 nm [54], Liposomes were able to encapsulate a condensed 5.2kb plasmid (p.rD328) and showed interaction and crossed cuti­cle membrane in intact form. In addition, deliveiy rate of encapsulated radio-labelled 2,4-D (2,4-dichloropenoxy acetic acid) obtained with these vesicles was greater as compared to liposomes prepared from dipalmiloyl phosphatidyl choline (DPPC) and the control (un-encapsulated 2,4-D). The ob­tained result clearly indicates the significance of positive charge over the liposomal surface introduced by ampiphilic compound used in the preparation

N anoparticle

Nanoparticles are now established under the new era of medicine called as nanornedicine. Particularly in oncology, Doxil® and Abrexan® are commercially established nanomedicines for the treatment of breast cancer. In addi­tion, different nanoparticulate delivery carriers like polym ­eric nanoparticle, metallic nanoparticle and quantum dots have also been successfully adopted by scientists in gene therapy as a gene carrier [47, 55-56].

In plant biotechnology, nanoparticle as a gene carrier in biotransformation process could protect DNA damage from ultra sound as reported by Liu et al., with liposomal form, thus it is easily coupled w ith physical method of gene trans­fer. Liu et al. in their study used green fluorescence protein (GFP) tagged with nanoparticle to demonstrate its effective­ness and reported that nanoparticle alone goes into the cyto­plasm as seen in green cytoplasmic content [57], W hile coupled with physical method o f gene transfer (rdtrasound), it exhibited dramatically increased fluorescence, indicating the pIRGFP plasmid DNA-PLL-StNP complexes were trans­ferred into COS’’ cells mediated by ultrasound [57]. They successflilly developed surface-functionalized silicananoparticles resembling to honey comb mesoporous form with the pore size of 3nm that can transport DNA into iso­lated plant cells and intact leaves. They also developed capped and uncapped gold nanoparticles and reported that uncapped particles released the chemicals and triggered gene expression in the plant under controlled-release condition. Working in the same area, Jun et a l, (2005) developed starch nanoparticles by water/oil micro-emulsion technique [58], The developed nanoparticles were coated w ith poly-Z,- lysine to induce negative surface charge and their surfaces were coupled with fluorescent molecule Ru(bpy)3^^ 6 H2 0 . It has been found that plasmid DNA coupled with nanoparticle successfully transformed the plant suspension cells of Dio- screa zigiberensis with significant reduction in the incuba­tion time for transformation even under the influence o f ul­trasound and Dnase I [58], They also tried to explore the influence o f particle size on the biotransformation. Fluores­cence starch-nanoparticles with the size 50-100 nm effec­tively crossed the cell wall, plasma membrane and nuclear membrane. This nano-biomaterial can efficiently solve the problem that exterior genes cannot traverse the plant cellTT7o1T

Applkation o f Novel Nanotechnology Strategies in Plant Biotransfonnmion Becmt Patents on Biotechnology 2012, Vol. 6, No, I 75

Carbon Naiiotubes

Carbon nanotubes have fascinated scientists with their extraordinary properties. These nano-materials have become increasingly popular in various fields simply because o f their small size and amazing optical, electric and magnetic proper­ties when used alone or with additions o f metals [47]. Re­cently, it has been reported that rice cells suspension (Oiyza .sativa L.) cultured with multiwall carbon nanotubes (MWCNTs) increased reactive oxygen species (ROS) and decreased cell viability [59], As reported in case of carbon nanotube exposure on cell cuUru’e, it seems that cell wall undergoes hyposensitive response due to ROS defence re­sponse [59].

LipoFcctiii

Lipofectins are cationic liposomes capable o f gene trans­fer in plant cell line. Sawahel (2002) reported that lipofectin effectively transfuse the mesophyll protoplast of Nicotiana tabacum and N. phanbaginifolia [60, 61], They reported that the transfonnation efficacy of B-glucoronidase gene under the control o f CaMV-35S promoter was lower than the PEiG mediated gene transfer. Advantage with the use of lipofectin in biotransformation is similar to that of liposome with addi­tional advantage o f stably transformed plants with or without additional application of electi'oporation [61].

FUTURE PRO SPECTS

On ground of earlier reports that revealed the application of liposomal and nanoparticle based gene transfer, various mechanistic ideas can be postulated to improve the efficacy of this novel non viral gene protected biotransformation ap­proach- Adaptation o f physiochemical properties of nanocar­

riers, protoplast and plasm a membrane may significantly improve the liposomal and nanoparticle encapsulated gene into the plant cell and establishing these gene transfer m eth­ods as sole process rather than adjuvant [62], Some of the approaches to improve nanoparticles based gene delivery into plant cells are given below:

a) It is a well-known fact that protoplast and plasma m em ­brane negative surface charge and their surface potential vary from -lOmV to -30mV [62], This surface charge can be utilized for the enhanced adhesion of encapsu­lated DNA, if we introduce positive charge over their surface. This idea is successfully tried earlier by some investigators [54].

b) The pore size or cellular gap in protoplast is 0 .Inm - 5000nm [63]. fn addition the pore size in plasma rnein- brane also varies between Inm to 50nm [63]. Size based penetration enhancement o f nanomaterial is possible in this case as it is reported that the size o f such carriers varied from lOnm to lOOOnm [64].

c) Straylamine, triethylamine and natural biodegradable polymers like chitosan can easily functionalize liposo­mal or nanomaterial surface with positive charge [65].

Amalgamation of these mechanisms with nanoparticles is presented in simplified manner in Fig. (3). These scientific methodologies may open the way for further development o f efficient plant transformation. The recent patents and patent applications US patent application 20110065092 A1 (gene encapsulated non-viral particle) and US6534484 (gene en­capsulated liposome for bio transformation) signify the im ­portance and strength o f this novel technology in biotrans­formation. Some more patents assigned to nanotechnology based biotransformation are enlisted in Table 2.

CONCLUSION

In conclusion, efficient transformation o f plants with transgenes encapsulated in nanocarriers is relatively the new area in plant biotechnology. Till now, liposomes have been explored and other nanotechnologies (nanoparticle, carbon nano-tiibe, quantum dots, nanoemuision, etc.) will definitely be used as they were successfully explored in animal bio­technology. Although nanotechnology based biotransforma­

76 Recent Patents on Biotechnology 2012, Vol. 6, No. I

tion is relatively efficient, its combination with commonly applied approaches in transgenic plant development will surely improve in terms o f efficiency and productivity, and the chances o f transgene silencing can also be minimized. The mechanism of biotransformation in nanotechnology is still not clearly understood, but the possible role o f endocy- tosis process as well as transcellular and paracellular trans­port of the nanocarriers encapsulated transgene cannot be ruled out.

Ohadi et at.

' ' ' K ’h

MM

si

A

R

Fig. (4). Diagrams of nanotechnology based gene carriers studied in plant biotechnology (A. Liposome, B. Carbon nano-tube, C. Ncmopariicle).

Table 2. Summary of Patents Sanctioned in the Area of Biotransformation by Means of Conventional and Novel Nanoteclinology

Pafcnl No Title Publication Date Refcrcnce.?

Conventional biotramfnrmation method

W02011064259A1 Method for isolating an alkanol from an aqueous biotransformation mixture 2011-06-03 1661

W02011042143A1 Cytochrome p450 from rhizopus oiyzae and uses thereof 2011-04-14 [67]

W02011008231A3 Biotransformation using genetically modified Candida 2011-06-16 [6S]

DE102009051687A1 Verwendung von Alginit als Reaktionsadditiv bei Biotransformationen 2011-04-28 mUS20110033904A1 Method for preparing orthodihydroxyisoflavones asing a biotransfonnation system 2011-02-10 [70]

US7943356 Alcohol dehydrogenase for tlie stereoselective production of hydroxy compounds 2011-05-17 [71]

EP2366293A1 Taste and flavour modulation by biotransfonnation in raiifc products 2011-09-21 [72]

EP2348107A2 Method for preparing evolved micro-organisms, enabling the creation or modification of metabolic pathways

2011-07-27 [73]

US7846659 Arrays of nucleic acid probes for analysing biotransfonnation genes 2010-12-07 174]

Novel Nanotechnology based bio transformation

US7915450 Transfection reagents 2011-03-29 [75]

US2011006509 2A1 Use of nonviable particles comprising an internal control (IC) nucleic acid 2011-03-17 [76]

US8021686 Lipid-encapsulated poly-5nionic nucleic acid 2011-07-19 [77]

AppUcaiion o f Novel Nanotechnology Strategies in Plant Biotmnsfonnation

(Table 2) conlcl....

liecent Patents on Bioiechnology 2012, Vol 6, No. 1 77

Patent No Title I’ubliciUion Date Refftrcnces

Novel Nanotechnology based biotransfonnation

US4394448 Method of inserting DNA into living cells 1983-06-19 [78]

US5264618 Cationic lipids for intracellular delivery of biologically active molecules 1993-1 1-23 [79]

US.'i 976567 Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer

200B-11-02 [80]

US65344S4 Methods for encapsulating plasmids in lipid bilayers 2003-18-03 [SI]

CA274!918(A1) T-DNA/protein nano-complexes for plant ti-ansformation 2 0 1 1 - 1 2 -0 2 [82]

US20I 1203013 (Al) Delivering compositions of interest to plant cells 2011-08-18 [83]

W02011016053 (Al) DNA loaded supported gold nanoparticles, process for the preparation and use thereof 2 0 1 1 - 0 2 - 1 0 [84]

W02007050715 (A2) Compositions and methods for safe delivety of biologically-acti vc plant transforfflation agents using non-fibrous silicon carbide powder 2007-05-03 [85]

A C ™ O W L EI>G E M EN T

Centre for Transgenic Development, Dept, of Biotech­nology and University library, Jatnia Hamdard University, New Delhi'62 are conceded for providing the adequate fa­cilities for the completion o f the work. The authors are also thatikful to Dr. Saleem Javed, Associate professor in the De­partment of Biocheinistry, Jamia Hamdard for his critical evaluation of the manuscript.

CONFLICT O F IN T ER EST

Authors declare no conflict o f interest.

REFERENCES

[1] Ohadi Rafsanjani MS, Alvari A, Mohammad A, Abdin MZ, Hejazi MA. In vitro propagation of C, intybus L„ and quantification of en­hanced secondary metabolite (esculin). Recent Patent on Biotech­nology. 2011; 5: 227-234.

[2] Bhojwani SS, Razdan MK. Plant tissue culture: Theoiy and prac­tice. 5“' ed. Elsevier science. 1996,112-151,

[3] Dodds JH, Roberts LW. Experiment in plant tissue culture. 2'“' ed. International potato centre. 1990; 104-119,

[4] Murashige T, Plant Propagation through Tissue Cultures. Annual Review of Plant I^hy.siology. 1974; 25: 135-166.

[5] Husaini AM, Abdin MZ. Development of transgenic strawbeny {Fragaria x ananassa Duch.) plants tolerant to salt stress. Plant Science. 2008; 174:446-455.

[6] Potiykus I, Gene Transfer to Plants: As.sessment of Published Approaches and Results. Annual Review of Plant Physiology and Plant Molecular Biology. 1991; 42: 205-225.

[7] Datta SK, Datta K, Soltanifar N, Donn G, Potiykus I. Herbicide- resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts. Plant Molecular Biol­ogy, 1992:20:619-629.

[S] Hayashimoto A, Li Z, Murai N. A Polyethylene Glycol-MediatedProtoplast Transformation System for Producdon of Fertile Trans­genic Rice Plants. Plant Physiol. 1990; 93:857-863.

[9] O'Neill C, Horvath GV, Horvath Dix PJ, Medgyesy P. Chloro- plast tiunsfonnation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. The Plant Journal. 1993;3:729-738.

[10] Ito H, Fukuda Y, Murata K, Kimura A. Transfonnation of intact yeast cells treated with alkali cations. . J Bacteriol. 1983;153: 163- 168.

[11] Ruzin SE, McCarthy SC. The effect of chemical facilitators on the frequency of eieetrofusion of tobacco raesopliyll protoplast, Plant Cell Reports. 1986: 5; 342-345.

[12] Hoffmann-Tsay S, Ernst R Hoffmann F, Design, synthesis and application of surface-active oheiuicals for the promotion of elec­trofusion of plant protoplasts, Bioelectrocheinistiy and Bioenerget- ics.l994;34J15-l22,

[13] Jiang Z, Berg H, Increase of protoplast electrofusion supported by dextran fractions. Bioelectrocheiriistry and Bioenergeties. 1995; 38: 383-387.

[14] Kishinami 1, Widholm JM, Auxotrophic Complementation in In- tergeneric Hybrid Cells Obtained by Elechical and Dextran- Induced Protoplast Fusion. Plant Cell Physioll9S7; 28: 21 1-218.

[15] Vieira AL, Camilo CM. Agrobacterium tumq/ai-ciam-mediaXad transformation of the aquatic fungus Blastocladiella emersonii. Fungal Genet Biol. 2011; 48: 806-8011.

[16] Cha TS, Chen CF, Yee W, Aziz A, Loh SH. Cinnamic acid, cou-marin and vanillin: Alternative phenolic compounds for efficient Agi-ohacterium-msdiated transfonnation of the unicellular green alga, Nannochloropsis sp. J Microbiol Methods. 2011; 84: 430-434.

[17] Subraraanyam K, Subramanyam K., Sailaja KV, Srinivasulu M,Lakshmidevi K. Highly efficient Agrobacterium-mediated trans- fonuation ofbanana cv, Rasthali (AAB) via sonication and vaciumi infilh-ation. Plant Cell Rep. 2011; 30:425-436.

[18] Shrawat AK, Good AG. Agrobacteriuni tumefaciens-roediatedgenetic transformation of cereals using immature embiyos. Methods Mol Biol. 2011; 710: 355-372.

[19] Venna P, Mathur AIC. Agrohacterium ftwie/a'CKW-mediated trans­genic plant production via direct shoot bud organogenesis from pre-plasmolyzed leaf explants of Catharanthus rosexis. Biotechnol Lett. 2011; 33: 1053-1060.

[20] Tzfira T, Citovsky V. The Agrobacterium-Y\mt Cell Interaction. Taking Biology Lessons from a Bug. Plant Physiology. 2003; 133: 943-947,

[21] Permiakova NV, Shumnyi VK, Demeko EV, Agrobacterium- ‘mediated transformation of plants: transfer of vector DNA frag­ments in the plant genome. Genetika, 2009; 45: 305-317.

[22] Dafny-Yelin M, Levy A, Tzfira T. The ongoing saga of Agrohacte- rium-host interactions. Trends Plant Sci, 2008; 13: 102-105.

[23] Lacroix B, Kozlovsky SV, Citovsky V. Recent patents on Agrohac- teriiim-mcd\ated gene and protein transfer, for research and bio- teclmology. Recent Pat DNA Gene Seq, 2008; 2: 69-81.

[24] Klee H, Horscli R, Rogers S. Agrohacterium-Medmted Plant Trans- fonnation and its Further Applications to Plant Biology. Annual Review of Plant Physiology. 1987; 38: 467-486.

[25] Shestibratov, Konstantin Aleksandrovich (Puschino, RU), Dolgov, Sergey Vladimirovich, Method for producing a transgenic plant

78 Recent Patents on Biotechnology 2012, Vol, 6, No. 1 Ohadi et a i

with the aid of Agrobacterium thumefaciens, US Patent Application [49]20070124 835; 2007.

[20] Kai<kar A, Venna VK., Agrobacterium mediated biotransformatioii.JoiUTial of Applied F’haniiaceutica! Science. 2011; 29-35.

[27] www.biotecfmology4u.com [50][28] Lurquin PF. Gene transfer by electroporation. Mol Biotecliriol,

1997;7:5-35.[29] To ICY, Cheng MC. Chen LF, Chen SC. Introduction and expres- [51]

sion of foreign DNA in isolated spinach chloroplasts by electropo­ration. Plant J. 1996; 10: 737-743.

[30] Chowrira OM, Akclla V, Fuerst PE, Lurquin PF. Transgenic grain [52]legumes obtained by in planta electroporation-mediated gene trans­fer. Mol Biotechnol. 1996; 5: 85-96. [53]

[31] Chowrira GM, A!<ella V, Lurquin PF. Electroporation-mediatedgene transfer into intact nodal meristeins in planta. Generating transgenic plants without i/i vilio tissue culture. Mol Biotechn 1995;3:17-23. [54]

[32] Lazzeri PA. Stable tninsforination of barley via direct DNA uptake. Electropoi'ation- and PEG-mediated protoplast transfomiation.Methods Mol Biol, 1995; 49; 95-106.

[33] Gaertig .1, Thatcher TH, Gu L, Gorovsky MA. Electroporation- [55]mediated replacement of a positively and negatively selectable beta-tubuliii gene in Tetrahyinena thennophila. P4roc Nati AcadSci USA, 1994; 91; 4,549-4553. [56]

[34] RaJtoczy-tfojanovvska M. Alternative methods of plant ttansfoima- tiori -a short review. Cellular & molecular biology letter, 2002; 7;849-858. [57]

[35] Oard JFL Physical methods for the transformation of plant cells.Biotechnol Adv. 1991; 9: 1-11.

[36] Casas AM, Kononowicz AK., Bressan RA, Hasegawa PM. Cerealhansforination through particle bombardment. Plant Breed Rev, [58]1995; 13: 235-264.

[37] Neuhaus G, Spangenberg G, Mittelsten Scheid O, Schweiger HO.Transgenic rapeseed plants obtained by the inicroinjection of DNAinto microspore-derived embtyoids. TAG Theoretical and Applied [59]Genetics 1987;75:30-36,

[38] Holmberg N, Billow L. Improving stress tolerance in plants bygene fransfer. Trends in Plant Science. 1998; 3: 61-66. [60]

[39] Asad S, Mukhtar Z, Nazir F, Hashmi JA, Mansoor S, Zafar Y,Arshad M. Silicon carbide whisker-mediated einbiyogenie callus traiisfonnation of cotton (Gossypiinn hirsutum L„) and regeneration [61]of salt tolerant plants. Mol Biotechnol. 2008; 40; 161-9.

[40] Sailaja M, Tarakeswari M, Sujatha M. Stable genetic trans Forma­tion of castor (Rioinus communis L.) via particle gun-mediated [62]gene transfer using embryo axes from mature seeds. Plant Cell Rep.2008; 27:1509-1519,

[41] Asad Sh, Mukhtar Z, Nazir F, Hashmi JH, Mansoor Sh, Zafar Y, [63]'Arshad M. Silicon Carbide Wlrisicer-Mediated Erabryogenic Callus Transfortnation of Cotton (Gossypiura hirsutum L.) and Regenera­tion of Salt Tolerant Plants. Molecular Biotechnology, 2008; [64]40:161-169, [65]

[42] Wechuok JB, Ozuer A, Goins WF, Wolfe D, Oligino T, Glorioso JC, Ataai MM. Effect o f temperature, medium composition, andcell passage on production of heipes-based viral vectors. Biotech- [66]nolBioerig. 2002; 5:112-119.

[43] Kenel F, Eady C, Brinch S, Efficient Agrobactemm tnmefaciens- [67]tnediated transfonnation and regeneration of garlic {AH’mm sati­vum) ifflinatiire leaf tissue. Plant Cell Rep. 2010; 29: 223-230. [68]

[44] Baron C, Dornke N, Beinhofer M, Hapfelmeier S. Elevated tetn-peratiire differentially affects virulence, Vir.B protein accrmralation, [69]and T-pilus fonnation in different Agrobacterium tumefacieris and Agrobacterium vitis strains. J Bacteriol, 2001: 1 S3; 6852-6861. [70]

[45] Dillen W, De Clercq J, Kapila J, Zambre M, Montagu M V. Theeffect of temperature on Agrobacterium tumefaciem-msA\a.tsA gene [71 ]transfer to plants. The plant journal. 1997; 12; 1459-1463,

[46] Iba K. Acelimative response to temperature stress in higher plants: [72]approaches of gene engineering for temperature tolerance. AnnuRev Plant Biol, 2002; 53: 225-2245. [73]

[47] Akhter S, Ahmad MZ, Singh A, Ahmad I, Rahman M, Anwar M,Jain GK, Abroad FJ, Khar RK. Cancer Targeted Metallic Nanopar-dole: Targeting Overview, Recent Advanceinent and Toxicity Con- [74]cern. CurrPharmDes, 2011; 17: 1834-1850.

[48] Chen ZY, Liang K, Qiu RX, Luo LP, Ultrasound- and liposome [75]microbubble-mediated targeted gene transfer to cardiomyocytes in [76]vivo accornpanied by polyethylenimine. Ultrasound Med. 2011; 30:1247-1258.

Shirazi RS, Ewert KIC, Leal C, Majzoub RN, Bouxsein NF, Safinya CR. Synthesis and characterization of degradable iiiultivalent cati­onic lipids with disulfide-bond spacers for gene delivery. Biochim Biophys Acta. 20) 1; 1808:2156-2166.Deshayes A, Henera-Estrella L, Caboche M. Lipo.some-inediated traiisfonnation of tobacco mesophyll protoplasts by an Escherichia co« plasmid. EMBO J.1985; 4: 2731-2737.Caboche M, Lurquin PF. Liposomes as carriers for the transfer and expression of nucleic acids into higher plant protoplasts. Methods in Enzymology. 1987; 148: 39-45.Caboche M. Liposoine-mediated transfer of imcleic acids in plant protoplasts. Physiologia PUmtaruni. 1990; 79: 173-176.Balias N, Zakai N, Loyter A. Liposotnes bearing a quaternoiy ammonium detergent as an efficient vehicle for ftinctional transfer of TMV-RNA into plant protoplasts. Biochimica et Biophysica Acta(BBA)- Bioinembrane.s, 1988; 939: 8-18.Wiesman Z, Dora NB, Shaivit E, Grinberg S, Linder C, Heldinan E, Zaccai M. Novel cationic vesicle platfomi derived from vernonia oil for efficient delivery of DNA through plant cuticle membranes. .1 Biotechnol. 2007; 130: 85-94.De Rosa O, La Rotonda MI, Nano and inicrotechnologies fbt' the delivery of oligomicleotides with gene sileticing propertie.";. Mole­cules. 2009; 29: 2S01-2823.Trnoiig NP, Jia Z, Burgess M, Payne L. McMillan NA, Monteiro MJ. Snl[-Catiilyzed Dcgniduble Cationic Polymer for Release of DNA. Biomacromolecules, 2011 (in press).Jiin L, Xuan-Ming L, Su-Yao X, Chuen-Yi T, Dong-Ying T, Xuan- ming 1, Preparatton of fluorescence starch-nanoparticle and its ap­plication as plant transgenic vehicle. .1. Cent. South Univ. Technol, 2008; 15: 768-773.Inn LIU, Xuan-Ming LIU. Su-Yao XIAO, Chueii-Yi TONG, Dong-Ying TANG, Li-Jian ZHAO. Bioconjugated Nanoparticle for DNA Protection from Ultrasound Dainage. Anal. ,Sci.2005; 21: 193-197.Lin C, Fugetsu B, Su Y, Watari F. Studies on toxicity of multi­walled carbon nanotubes on Arabidopsis T87 suspension cells, J Hazard Mater. 2009; 30: 578-583.Sawahel WA. The production of transgenic potato plaut,s express­ing human alpha-interferon using lipofectin-mediated transforma­tion, Cell Mol Biol Lett. 2002; 7: 19-29.Mohapatra DP, Gassara F, Brar SIC. Nanoparticles roduction and Role in Biotranaforraation. Journal of Nanoscience and Nanotech­nology. 2011: 11; 899-918.Reid W, Zhang Q, Sekimoto H. Influence o f membrane surface charge on nutrient uptake by plants, Developments in Plant and Soil Sciences, 2002; 92: 198-199.Berestov,sky G N, Ternovsky V I, ICataev A A, Through pore diameter in the cell wall of Chara corallina, J. Exp, Bot. 2001; 52: 1173-1177.www.molecular-plant-biotechnology.info Li C, Guo T, Zhou D, Hu Y, Zhou li, Wang S, Chen J, Zhang Z. A novel glutathione tnodified chitosan conjugate for efficient gene delivery. J Control Release. 2011; 154: 177-188.W02011064259A1, Method for isolating an alkanol froiti an aque­ous biotransfomtation mixture. 2011-06-03 W02011042143A1. Cytochrome p450 from rhizopus oryzae and uses thereof. 2011-04-14.W02011008231A3, Bilotransfonnation using genetically modified

Candida. 2011-06-16,DE102009051687A1. Verwendung voii Alginit als Realctionsaddi- tiv bei Biotransformationen. 2011-04-28.US20110033904A1. Method for preparing orthodihyd.roxyisof!a- vones using a biotransfonnation system. 2011 -02-10.US7943356. Alcohol dehydrogenase for the stereoselective produc­tion of hydroxy compounds, 2011-05-17.EP2366293A1. Taste and flavour modulation by biotransfonnation in milk products. 2011 -09-21.EP2348107A2. Method for preparing evolved micro-organisms, enabling the creation or modification o f metabolic pathways. 2011- 07-27.US7846659. Airays of nucleic acid probes for analysing biotransfonnation genes. 2010-12-07.US7915450. Transfection reagents. 2011 -03-29.US20110065092A1. Use of nonviabie particles comprising an internal control (JC) nucleic acid. 2011 -03-17.

Application o f Novel Nanotechnology Strategies in Plant Uiotransformation Recent I’atents on Biotechnology 2012, Vol, 6, No. I 79

[77] US8021686. Lipid-encapsulated poly-5nionic nucleic acid. 2011- 07-19,

[78] US4394448. Method of inserting DNA into living cells. 1983-06-19.

[79] US526461S. Cationic lipids for intracellular delivety of biologi­cally active molecule,s. 1993-11-23.

[80] US5976567. Lipid-nucleic acid particles prepared via a hydropho­bic lipid-nucleic acid complex intennediate and use ft)r gene tran.s- fer. 2008-11-02

[81] US6534484, Methods for encapsrdating plasmids in lipid bilayer,?,2003-18-03,

[82] CA2741918 (Al) T-DNA/protein iiano-complexcs for plant ffansfonnation, 2011 -12-02 .

[83] US2011203013 (Al). Delivering compositions of interest to plant cells. 2011-08-18.

[84] W020ri0160.‘i3 (Al). DNA loaded supported gold nanoparticles, process for the preparation and use thereof. 2011 -02-10.

[85] W02007050715 (A2) CoiTipositions and methods for safe deliveiy of biologically-active plant tran.s formation agents using non-tlbrous silicon carbide powder. 2007-05-03,

Recant Patents on Biotechnology 2011, 5, 227-234 227

In Vitro Propagation of Cichorium intybus L» and Quantificatioo of Enha­nced Secondary Metabolite (EsciiUn)

Mehrnaz S. Ohadi Rafsanjani*, Ainene Alvari^” Anis Mohammad’, M.Z. Abdin’ and M.A. Hejazi^

'Department o f Biotechnology, Faculty o f Science, Jamia Hamdard University, New Delhi-62, India; ^Agriculture Bio- lechnology Research Institute o f Iran (ABRII) Tabriz, Iran

Received: July 05, 20IJ Rcviaed: July 25, 201J Accepted: August 10, 20! 1A bstract: In this report, rapid and effective slioot as well as root regeneration system through direct multiplication was successfully developed for Cichorium intybus L. Furthermore, the effect o f exogenous growth regulators (TDZ and lAA) at different concenb-atiojis on the regulation process o f the plant was also studied. Enhanced production o f esculin in d e­veloped C. iiuyhus L. was evaluated using leaf extract. Only on the expense of 20 days, regeneration was seen and very low dose o f TDZ was seen to be more effective. When 0.02mg/L o f TDZ was combined with 1.5mg/L o f lAA, nearly 100% of explants produced shoots with the highest number o f regenerated shoots (85.37). With further increase in con ' centration (>0.05rng/L), the number of shoots per explants get decreased. A lower NAA to IB A ratio (1 .Omg/L o f IFiA and 0.5mg/L o f NAA) seemed to be more effective for root generation and considered to be the most effective combination among the tried groups. IBA was more effective in root development than NAA, but both were comparatively effective.On quantitative analysis by RP-HPLC, the 76.23% of Esculin were found in leaf extract o f the in vitro developed C. inty­bus L. This amount was 26.77% higher than normal grown plants.

Keywords: Cichorium intybus L, TDZ, lAA, IBA, NAA, in-vitro propagation, Esculin extraction, HPLC.

IN T R O D U C T IO N

Plants are major sources o f natural products used as me­dicinal and pharmaceutical additives, pesticides, flavor and fragrance ingredients. The search for new plant derived chemicals for the welfare o f human being through medicinal atid agricultiu'al values should be a priority in current and future efforts toward sustainable conservation and rational utilization o f biodiversity. In the search of alternatives for production o f desirable medicinal compounds in higher amount from plants and regeneration/production of disease resistant herbal drugs, plant tissue culture technique, well known biotechnological approaches, are found to has poten­tial as a supplement to traditional agriculture in the industrial production. Plant tissue culture technique have been sug­gested as well established tool for the production o f useful secondai7 metabolites.

Recently assigned patents related to tissue culture are summarized in Table 1. US 20080160615 described about in-vitro propagation o f flavonoid-rich tissue from Neomarica gracilis having antitumor activity, which alters flavonoid content of TV. gracilis. US 20080160560 assigned for in vitro resveratrol-rich callus tissue development derived from Vitis thunbergii Sieb. et Zucc. induced in a tissue culture system containing one or more plant growth regulators; useftil for callus induction and producing resveratrol. US 20080152630 discloses the method for generating tissue’s cells in culture.

*Address con'espondence to this author at the Centre for Transgenic Plant Deveiopinent, Department of Biotechnology, Faculty of Science, Jainia Hamdard University, New Delhi'62, India; Tel: +91-981S462060;E-mail: mzabdin@rediffmail.com

JP2008106064 is u seM plant tissue culture concerning re­search outcome ascribed about enhanced production o f tissue plasminogen activator as secondary metabolite from Bacillus natto culture solution EP1962875A4. Obtaining pharmaceu­tical, nutraceutical, functional food, or cosmetic composition for treating inflammation or autoimmune diseases (e.g. asthma), by extrticting chicoi^ extract [1-5, 8 ],

These patents signify the potential and future directions in plant tissue culture and plant regeneration technology.

Thus, using this technology, therapeutically active bio­constituents as secondary metabolite can be produced under controlled and reproducible conditions, independent of geo­graphical and climatic factors. Cichorium intybus L., com ­monly known as chicory or kasni (in Hindi) is a medicinally important plant that belongs to the family Asteraceae. This herbal drug has been implemented in folk medicine in A f­rica, South Asia for several hundred years, Almost evety part o f this species has been used as ethnomedicines. The tuber­ous root of this plant contains medicinally important bioac­tives such as inulin, bitter sesquiterpene lactones, coumarins, flavonoids and vitamins [9]. In addition, chicory leaves con­tain esculin as dominant component which is a well-known natural UV protective agent and one of the phytomedicine used for the treatment o f various peripheral vascular disor­ders [10,11]. Furthermore, the plant is also used in the treatment o f AIDS, cancer, diabetes, dysmenor-rhoea, impotence, insomnia, splenitis and tachycardia [12]. Inulin obtained, from this plant is used to replace fat or sugar and to reduce the calories o f food. Antidiabetic activity is also

1872-2083/11 SIOO.OO+.OO © 2011 Bentham Science Publishers

Table 1. Some Examples of Patent Application in Plant Tissue Culture Applied to and Assigned by Different Goveniment

228 Recml Patents on Biotechnology 2011, Vol. 5, No. 3 0/uuli et iil.

PatentNumber

Inventor A.s.signee TitlePriority

ApplicationDate

PublicationDate References

US20080160615

Clu! T, Ho CTatung company

and Tatimg University

Flavoiioids-rich tis.sues from Neomaiica gracilis and methods for

culturing the same12/29/2006 7/3/2008 [1]

US20080160560

Ho C, Kuo HHo, Chin-Wen

Kuo, Hsieii-Shen

In vitro resvi>ralroI-rich callus tissues derived from vitis thimbergii

Sieb.et Zncc and method for producing the same

1/3/2007 7/3/2008 [2]

US200s0 i52630

Ghiis, Irene Schwartz, Aharon

Shinar, Doron Shirvaii, Mitchell

Ginis r,Schwartz A,Shinar D, Shirvan M

Method of generation and expansion oftissne-progenitor calls and mature tissue cells from intact hone marrow

or intact umhiliccd cord tissue

12/7/2006 6/26/2008 [31

WO2008068776

Deshpaude Mani- sha S ,Rao Sith-

amraju H, Wangi- kar Pralhad B,

Kuchroo Pushpa

Reliance life sci­ences Pvt Ltd

Three dimensional tissue equivalent using macromass cidture 12/g/2006 6/ 11/200s m

JP2008106064

Sumi Y Honda Trading K.K (Tokyo), Sumi Y

An enhancer of tissue plasminogen activatorproduced by heat process­ing Bacillus natto culture solution: useful as a pharmaceutical or food­stuff fir preventing thrombotic dis­

eases

9/28/2006 5/8/2008 [5]

CN101152580

LuH Lii HI, Wang X

A tissue-engineered skin manufactur­ing methodinvolving obtaining skin tissue, confecting anepidermal stem suspension with a specijiedconcen- tration, adding epidermal stem cells

into atissuc culture medium and carrying the cell viasubmerged cul­

ture

9/13/2007 4/2/2008 [6]

JP2008086509

Muiiakata M, Tajima K., Tsu-

chiya Y

Sun Medical KK (Morioka, Japan)

A composition for regenerating pcriodorrtal-tissiie (e.g., gum) com­prising a culture medium having

aceil suspension of human periodon­tal membranepositioned on the up­per region and a chemotaxissuh-

stance blended on the lower region of the medium

9/29/2006 4/17/2008 [7]

EPI962875A4

Christophe R ;

Barbara S; Ilic Nebojsa i;

Ilya I "

UNIV RUTGERS

United States of America

In vitro and in vivo anti­inflammatory effects of a sesquiter­pene lactone extract from chicory

(Cichoriiim Intybus L.)

2006-10-11 2011-01-05 [8]

reported [13] and an iniilin clearance test is used to measure glomerular filtration rate-GFR [14]. Recent pharmacological investigation o f the root extract o f this plant revealed immune modulator, anti tumor and anticancer properties [15, 16 ],

Recently, esculin has also been reported as having hepa- toprotection action against CCI4 induced hepatotoxicity [17]. Till this date, there is no established synthetic drag in the

market with dedicated hepatoprotective activity. So, there has been considerable interest in creating an economically feasible biotechnological approach using plant tissue culture for the production of hepatoprotective bioactives like inulin, bitter sesquiterpene lactones, coumarins, flavonoids, v ita­mins and esculin fom i C. intybus. So, the aim of this sttidy was to perform the in vitro propagation of C. intybus and

evaluation o f total hepatoprotective esculin content in in vi­tro derived C. intybus compared with nonnally grown plants.

M ATERIAI.S AND M ETH O D S

Plant M ateria ls

Well grown plant and seeds o f chicory {Cichoriiim inty­bus L. cv. Focus) were obtained from the herbal garden o f Jamia Hamdard, New Delhi, India.

Seeds S terilization and G erm ination

Seeds were washed in mnning tap water for 30 min and soaked in solution o f liquid detergent (1% v/v Teepol) for 10 min, washed with distilled water 2-3 times and taken to a laminar flow chamber where they were disinfected with 0 .1 % (w/v) aqueous mercuric chloride (HgCl2) solution for 3-5 min. Seeds were further rinsed 5-6 times in sterile dis­tilled water, soaked in 1% sodium hypochlorite solution for 5 minutes, rinsed with sterile distilled water 5-6 times to re­move the traces o f mercuric chloride and sodium hypochlo­rite and germinated aseptically on basal MS (Murashigue & Skoog, 1962) medium containing 3% w/v sucrose,0.7% agar [18], pH adjusted to 5.8 and autoclaved at 121“C for 25 min. The cultures were incubated in a growth chamber at 2 6 ± r c under 16/8 h (light/dark) photoperiod using white fluorescent light (150-200 j.iE nV" s " ) and 55-65% o f relative humidity.

In vitro P ropagation of C. intybus

Establishment Stage

Ten- days-old seedlings with 5-7mm leaves were trans­ferred to basal medium supplemented with Thidiazuron (TDZ) (0.05 mg/1) alone or in combinations with different concentrations of Indole acetic acid (lAA) (0.0, 0.3, 0.5, 1.0, 1.5, 2.0mg/l). The cultures were maintained at 26=tl°C and 16 h of photoperiod.

Multiplication StageWe studied the effect o f combination between lAA (0.0,

0.3, 0.5, 1.0, 1.5, 2.0mg/L) and TDZ (0.0, 0.002, 0.005, 0,008, 0.01, 0,02, l,0m g/L) using leaf explants (lamina). Table 2 indicates the following patterns; a) treatments in­volving no plant growth regulators (lAA and TDZ), b) only TDZ in varying concentrations without lAA, c) only lAA in varying concentration with no TDZ and d) both lAA and TDZ present in different varying concentrations. After four weeks shoot length and num ber o f leaves and shoots were recorded. The obtained plantlets were used as plant material for further subculturing. The effect o f TDZ and lAA and their different ratios on shoot induction, growth and esculin accumulation was determined by adding TDZ and lAA re­spectively. Furthermore, in similar way root induction was carried out by using Indole butyric acid (IBA) (0.1, 0,5, 1.0, 1.5mg/l) and a~Naphthalene acetic acid (NAA) (0,1, 0.5, l,0mg/l). Moreover, in the same experiment we carried out to find the effect o f combination on production o f shoots and regeneration capacity of explants. Different concenh'ations o f lAA (0.3mg/L to 2.0mg/L) with TDZ in the concentration o f (0.005mg/L to l.Omg/L) taking with each lamina explant is given in Table 2 and the propagation stages is given in Fig. (la , b and c).

Propagation o f Cichoriiim iniybm and Estimation ofEsciiUn

Adaptation and Acclimatization StageThe plantlets were removed from the culture vessels and

washed under tap water to remove agar from the roots. The washed plants were soaked for 1 0 min in 0 .2 % v/v H 2 O2 and cultured in small pots containing peat moss and transferred to green house. The cultivated plants were irrigated w ith water for next 2 0 days.

Esculin E x trac tion and A nalysis

Entire plants of C. intybus developed with tissue cultiu'e technique were collected and dried for 15 days in room tem ­perature before final drying in oven at 50"C for next 24 hours. The dried plants were powdered in electric grinder. For the preparation o f plant extract, five grams o f C. intybus powder placed in 100ml o f 70% v/v ethanol, in a soxhlet apparatus. The extract was filtered and concentrated under reduced pressure and at temperature of 4G°C on rotary evaporator. Appropriate weights o f the residue were pi'e- pared in methanol to obtain the various concentrations used for the experiment.

Q uantification o f Esculin by R P-H P L C M ethod

F,sculin was quantilled from the extract o f shoot parts o f in vitro propagated and harvested aerial parts of naturally grown C. intybus L, by an in-house developed RP-HPLC method. In brief, one month old well grown plants w ere taken for determination o f the esculin content and com pared its esculin content with naturally occurritig C. intybus L . Entire shoots o f C, intybus were dried for 15 days in room temperature before final drying in oven at 50“C for next 24 hours. The dried plants from both categories were pow dered in electric grinder. Five g o f dried powdered C. intybus was dissolved in 100ml of absolute ethanol. The mixtures w ere filtered through 0,45(am pore size filter paper before HPLC analyses were carried out. For RP-HPLC quantification, HPLC analysis was cari'ied out on a hchrosorb Cl 8 analyti­cal column (250mm X 4.6mni i.d., lOjum; Shiseido Fine chemicals, Japan). The mobile phase was consisted o f methanol and water (60:40 v/v) and pum ped at a flow rate o f 1 .Oml/min and the detection was carried out at 249 nm. The temperature in the laboratory was 25±2°C. One ml o f esculin solution was pipetted out from 15g/100ml of esculin solution which was diluted up to 1 0 ml with the mobile phase to be used as analyte. 20).il o f the sample w as injected for HPLC analysis.

Statistical AnalysisThe percentage o f response and different parameters re ­

lated to the growth were monitored. D ata o f three independ­ent experiments represented by 1 0 replicates from each ex­periment were subjected to statistical analysis (M eaniSE ) and New Duncan’s M ultiple Range Test [19J.

RESULTS AND DISCUSSIONIn-vitro development o f C. intybus has been reported

earlier by several scientists using somatic embryogenesis with explants including leaf, root, callus, suspension culture and different hormonal combination [20], B ut these works do not explain the prodviction o f esculin. Rehman et al. in the

Recent Patents on Biotechnology 20 i 1, Vol. 5, l\o. if 229

230 Recent Patents on Biotechnology 2011, Vol. 5, No. 3 O/iaili et al.

Table 2. Shoot Regeneration from Leaf Explants Cultured on MS M edium Containing D ifferent Concentrations of TDZ Alone or in Combinations with lA A (0.00 mg/L- 2.0 mg/Ij) for 4 Weeks

lAA (mg/L) TDZ (mg/L)% of Explants

Forming ShootsMean IViimJjer of

Shoots I’er Explants

0.00

0,00

0,00

0.00

0,30

0.30

0.30

0.30

0.50

0,50

0.000 5.2

0,002

0.005

0.008

39.7

54.4

0.000

0.002

0.005

0.01

0.000

0.002

69.7

4,1

76.8

9.3

48.2

5.9

76.5

01.13

10.1

15.8

14.5

09.4]

39.7

4i.6

21,2

07,24

58,70

0.50

0.50

1.0

0.01 51.09

0.02 79.26

0.002 100

31,70

62.50

73.10

1,0

1,0

1,0

1,5

1,5

1,5

0.01 91,08

0.02 100

0.05 85.43

0.002 1000.01 41.20

0.02 99.16

79.57

83.89

57.74

84,47

35,41

85.37

1,5 0.05 37.73 21,64

2,0 0.002 27.15 17.35

2.0 0.02 24.73 16.82

2.0

2,0

0.05 6.7

0,1 1.4

06.21

04.00

year 2003 reported the in-vitro regeneration o f C. intybus L. from leaf expiant and accumulation o f esculin. Report said the consumption o f high concentration of lAA along with specific growth regulator casein hydrolysate [lOOmg/L], Furthermore, the experimental duration seems to be higher [one month] and esculin content in fully grown regenerant was low [2 1 ],

We report a direct multiple shoot regeneration o f C. inty­bus L. from explants in just 20 days, with significant produc­tivity (100% of explants produced 73.10% o f shoots and 99.16% of explants produced 85.37 shoots) when cultured on media with 0.02mg/L TDZ and 1.5mg/L lAA (Table 2).

Our report showed remarkably higher productivity in tenns o f frequency and total weight than an earlier report [22], In earlier works, numerous approaches were adopted for enhanced shoot and secondary metabolite production like

pretreatment o f leaf explants with 330mM glycerol, applica­tion o f antibiotic cefotaxime (lOOmg/L) in the culture m e­dium [23] or antimitotic agent (colchicine at 0.001%) used alone or combined with kinetin, but in case o f C, intybus L, this is not common. Furthermore, there was no significant positive impact for shoot production o f this species in earlier reports [24], In our study, the effect o f TDZ and lAA on di­rect shoot regeneration and production of esculin as secon­dary plant metabolite were studied and the effect o f IBA and NAA on the root formation was also studied. In our study we regenerated the shoot parts within 2 0 days.

Effect of Different Concentration of TDZ and lAA (in Combination) on Shoot Production

When TDZ was used alone, fewer explants (39-69%) with lesser number o f shoots ranging from 10 to 14,5 were

Propagation o f Clchovium intyhus and Estimation o f EscuUn Recent Patents on Biotechnology 2011, Vol. 5, No. 3 231

a - - .. .. .. *l«r

4 .

P; -'

IP

Fig. (1). Photographs showing the different stages o f grown C. intybtis L. A) at the end o f 11‘'‘ day B ) at the end o f 16"' day and C) on the 25"' day showing the well-grown leaves in teim s o f number and length.

produced, while varying the concentration of TDZ (O.OOSmg /L to 1 .Omg/L) alone and in combination with lA A (0.3mg/L to 2.0nig/L) a varying number o f shoots per explants (4.0 to 85.37) were produced (Table 2).

When TDZ (0.005mg/L to 1.Omg/L) was combined with 0.3 to 2,0 mg/L of lAA, significant improvement in percent­age o f regenerated shoots and mean number o f shoot were achieved. It seemed that when O.Olmg/L or 0.02mg/L o f TDZ was combined with 1 .Omg/L of lAA, with a mean o f 79,57 and 83,89 shoots per explant, respectively, almost 100% of explants developed shoots. Combination o f TDZ with higher concentrations o f lA A was always more fruitful than the lower concentrations as seen in combination o f TDZ with 1,Omg/L o f lAA, which produced 83.89 shoots per ex­plant while at 0,3ing/L o f lA A induced 4L6 shoots per ex­plant. The highest number o f shoots and number of explants that developed the shoots w ere seen when 0.02mg/L o f TDZ was combined with L5mg/L of lA A forming 85.37 shoots per explant. As the concentration o f TDZ gone high (i.e., 0.5 mg/I or more) the number o f exphmts producing shoots and shoots per explant got decreased. Our data showed that, al­though TDZ produced some amounts o f shoots when used alone at low concenti'ations (0.005mg/L, O.Olmg/L, and 0.02 mg/L), its combinations with lA A was far more effective in shoot induction (Table 2). The result was in corroboration with earlier report as de-differentiation and re-differentiatioii o f plant cells is under the influence of exogenous chemical regulators and endogenous plant hormones in normal, direct and indirect regeneration [25]. Furthermore, in case of C. intybus, leaf lamina segments were more productive for cal­lus and shoot development than petiole segments because petiole cells depend on leaf cells to some extent for showing its totipotency [26, 27], Furthermore, the promoting effect o f

TDZ on in-vitro development has been reported earlier for many species [28],

Enhanced esculin accumulation on application o f TDZ along with growth regulator can be explained on the basis o f following possible reasons: a) in the presence o f growth regulators (here TDZ and lAA), nucleic acid synthesis get increased leading to enhanced translation b) Positive regula­tion o f biosynthetic pathway (shikimate pathway) may also be responsible for enhanced biosynthesis o f esculin c) in­creased lateral branching, number of leaves and its size are responsible for accumulating high esculin.

In case o f lamina explants, vascularization o f tissues, hormone synthesis and metabolite production are fast that increases the overall shoot formation ability [29-30], These findings are consistent with our data when TDZ was used alone or combined with lAA, higher concentrations of TD Z induced more shoot formation on the lamina explants.

E F F E C T O F D IF F E R E N T AUXINS O N R O O T IN ­D U C TIO N

Multiple shoots were cut out from the parent plants and cultured on MS media containing different NAA and IBA concentrations for rooting (Table 3). In teniis of root genera­tion, low ratio o f NAA to IBA (l.Onig/L o f IBA and 0,5mg/L o f NAA) seemed to be the most effective combination (Ta­ble 3). This treatment showed generation of 6.83±0.31 roots/ culture (mean±S.D) with 7.1±0.27cm (mean±S,D) increased concentration of NAA and IBA enhanced the num ber and length o f roots per culture (Table. 3). Results indicate that the combination between IBA (constant concentration) and low concentration o f N A A (0.5mg/L) enhanced the root formation. Nevertheless when concentration o f IBA and

2,12 Recent Patents on Biotechnology 2011, Vol. 5, No. 3 Ohndi lit a t

Table. 3. E ffect of D ifferent A uxins on R oo t Induction o f / / i vitro D erived L ea f E xplan ts of C. intybiis L. U n d er T otal D arkness in M S Solid M edium (H alf-S trength)

Auxins (mg/L)

NAA+IBA

0,1 + 0,1

Response (%) (Mcan± S.D)

51,42±6.19

Number of Root/Culture (Meiin ±S.D)

3.04±0.08

Root Length/ Culture (cm) (M eani S.D)

3.9±0.09

0.1+0.5

0.5+0.1

1.0+0.5

1.0+1.0

69.00+6.58

61,70±5.17

93.60+11.01

82.42+8.30

3.71+0.12

2.95+0.09

6.83+0.31

5.57+0.19

4.5+0.16

5.0+0.20

7.1+0,27

6.4+0.24

1.5+1.0 6S,48±7,42 3.31+0,14 4.2±0.13

NAA were high (1;1 ratio) root length and number o f roots were still high [5.57±0.19 roots/culture (m eaniS.D ) o f 6.4±0.24cm (mean±S.D).

The small C, intybus plantlets were successiully trans­ferred to sterilized soil and kept under favorable conditions for two weeks and transferred to room condition at low hu­midity. Approximately 89% o f the vshoots survived through the hardening process. Rooting o f the regenerated shoots was readily achieved when excised shoots were cultured on me­dium containing varying concentrations o f NAA and further IBA, It was clear that IBA was more effective in root devel­opment than NAA but both were comparatively effective. Auxins are considered obligatory for the maintenance o f meristematic tissue proficiency. W ith the increased concen­tration of auxins, potency o f root and plant development get enhanced but upto certain level. This finding is in corrobora­tion with earlier reports that showed excess o f auxin have inhibitory effect on lower plant part development including adventitious roots [31-33].

Q uantification o f Escuiiii by R P-H PL C M ethod

Esculin is poorly water soluble dmg but soluble in or­ganic solvent like methanol. The RP-HPLC method was op­timized with a view to develop a quantitative method for the estimation o f Esculin in C. intybus L. extract. The mobile phase was optimized as methanol-water (60;40%v/v) at a flow rate of Iml/min with the retention time o f Esculin 6.925 min and theoretical plate o f 20680, which shows good sensi­tivity of the developed method. The UV spectnim o f esculin showed the highest absorbance at 249nm in solution made up o f mobile phase. Quantification was achieved with UV detection at 287nm based on the peak area. Chromatograms obtained for the Esculin standard and extract revealed that they had similar pattern as shown in Fig, (2A and 2B). Line­arity was evaluated over working concentrations containing 1, 2, 4, 8, 10, 20 and 50(ig/ml o f Esculin, Peak area and con­centration were subjected to feast square linear regression analysis to calculate the calibration equation and con’elation coefficient. The regression equation was y= 22899x (where y is the response and x the am ount o f Esculin), Linearity was found over the concentration range 1 -50/ig/ml with a correla­tion, coefficient of 0.999. The linearity of the calibration

curve was validated by a high value correlation coefficient. DL (limit of detection) and QL (quantification limit) of the method were found to be 19.97ng/ml and 60.596ng/ml re­spectively as per the quantitative formula for LOD and LOQ given by ICH [34] (ICH Harmonized Tripartite Guideline) which indicate that the proposed method can be used for detection and c)uantification of Esculin in a very wide con­centration range. Our experimental outcome o f the culture technique showed that exogenous plant growth regulator (here in this case TDZ) and endogenous growth regulators (auxins) have proportional effect on plant development and secondary metabolite synthesis (here esculin from the leaf o f C. intybus L.), Quantification o f esculin indicated signifi­cantly high recovery/yield (76,23%) from our in-house tissue culture developed C. intybus L. as compared to the naturally occurring C. intybus L, (49,45%). Such response maybe due to nutritional differences {In-vitro m edia and field condition) and may even be due to different genotypes.

C O N C LU SIO N

In vitro propagation o f C. intybus and evaluation o f total hepatoprotective esculin content in in vitro cultured plants were quantified and compared with norm al grown plants and ITPLC quantification indicated significantly high recov­ery/yield (76.23%) from our in-house tissue culture devel­oped C. intybus L. as compared to the naturally occurring C. intybus L. (49.45%), In short span of time (20 days) 99.16% o f explants produced 85.37 shoots per leaf explants when cultured on media with 0.02mg/L TDZ and lA A (1.5mg/L). Approximately 79% of the shoots sui-vived through the hard­ening process. Our findings may be helpful in germplasm generation o f disease free C. intybus as well as to develop a large-scale production o f secondary metabolites, particularly esculin.

A C K N O W LED G M EN T

Centi-e for Transgenic plant development. Dept, o f B io­technology, Jamia Hamdai'd University, New Deihi-62 are conceded for providing the adequate facilities for this work.

C O N FL IC T O F IN T E R E ST

Authors declare no conflict o f interest.

Propagaiiort o f Cichorhtm miyhus and Estimation o f Esculin ilcceiit Pat{int% on Biotechnology 201), Voi 5, No. S 233

R e s t e n t iB n T i m e ft

H L

~ Z u . 1 ” [ . . J s ? . A _____1 ...........................1 1 ( 1 ' 1 1

. }.Q(3

2.B

Fig. (2). A) Chromatogram o f Esculin in the mobile phase; methanol and water in the ratio o f 66:34 (%v/v). B) Chromatogram of C. intylms L. extract in the mobile phase; m ethanol and water in the ratio of 66:34 (%v/v).

R EFEREN CES

[1]

[2]

[14]

Chu T, Ho C. Fiavoiioids-rich tissues from Neoraarica gracilis and methods for culturing tlie same, US 20080Ui0615 2008. [15]Ho C, Kuo H, In vitro resveratrol-rich callus tissues derived from vitis thunbergii Sieb.et Zuoc and method for producing the same. [16]US 20080160560 2008,

[3] Oinis IS, Aharon S, Doron S. Method of generation and expansionof tissue-progenitor cells and mature tissue cells from intact bone [17]marrow or intact umbilical cord tissue, US 20080152630 2008,

[4] Manisha D, Rao S, Sithainraju H, Wangikar PB. Three dimensionaltissue equivalent using macroraass culture. WO 2008068776 2008, [18]

[.5] Sami Y. An enhancer of tissue plasminogen activatorproduced byheat processing Bacillus natto culture solution; usefiil as a pharma­ceutical or foodstuff for preventing thrombotic diseases, JP 2008106064 2008, [19]

[(ij Lu H, A tissue-engineered skin manufacturing inethodinvolvingobtaining skin tissue, confecting anepidennal stem suspension with a specifiedconcentration, adding epidermal stem cells into atissue [20]culture medium and carrying the cell viasubraerged culture. CN 101152580 200S.

[7] Munalcata M, Tajiina K, Tsuchiya Y. A composition for regenerat- [21]ing periodontal-tissue (e.g., gum) comprising a culture mediumhaving acell suspension of human periodontal membranepositioned on the upper region and a chemotaxissubsfance blended on the lower region of the medium. JP 2008086509 2008, [22]

[8] Christophe R, Barbara S, Nebojsa 1, Ilya R./n vitro and in vivo anti- inflammatoiy effects of a sesquiterpene lactone extract from chic-oiy {Cichorium Mybus L.). EP1962875A4, 2011 [23]

[9] Varotto S, Lucchin M, Parrin P, Immature embiyos culture in Ital­ian red Chicory {Cichorium intybus). Plant Cell Tiss Org Cult 2000;62:75-77, [24]

[10] Nadkarni AK, (1976) Indian IWateria Medica popular Prakasam Pvt Ltd. Bombay,

[11] Rastogi RP and Mehrotra BN. Compendium of Indian Medicinal [25]Plants Rastogi, RP (Eds) Mentha piperita against the pathogenic bacteria was Orient Longman Ltd, Madras, 1994; pp:74, [26]

[12] Duke JA, Medicinal Plants of the Bible (Illustrated by Peggy K Duke) Out of print Trado-Medic Book,s Buffalo and NY, 1983, pp:233. [27]

[13] Niness KR, Inulin and Oligofructose What Are They J, Nutr. 1999;129: 1402S-1406S.

Vasudevan DM and Sreekumari S. Text Book of Biocheinistiy for Medical Students 2nd Ed. 1995; Jaypee Brothers Medical Publisher (P) Ltd, New Delhi, India.Araceli A, Jose D. Antiammral o f Pyrimidinederivative,s of sesquiterpen lactones, JPharm PharmaceutSci 1999; 3:1 08-112, Hazra BR, Sarkar S Bhattacharyya, P Roy. Tumour inhibitoiy activity of chicory root extract against Ehrlich ascites carcinoma in mice, Fitoterapia 2002; 73: 730-733,Rusu MA, Tamas M, Puica C. et.al. The hepatoprotective action of ten herbal extracts in CC14 intoxicated liver. Phytother Res 2005; 19; 744-749.Yucesan B, Turker AU, Gnrel E, TDZ-induced high frequency plant regeneration through multiple shoot fonnation in witloof chicoiy (Cichorium intybus L), Plant Cell Tiss Org Cult 2007; 91: 243-250.Goinez, K.A, and Gomez, K.A. (1976) Stati.stical procedure for agricultural research with emphasis of Rice (Los Bans, Philippine,? International Rice Research Institute).Park EJ, Lim HT, Establishment oi an efficient iti vitro plant re­generation system in chicoiy (Ciehoriuin intybus L. var sativus). Acta Hort 1999; 483:367-370,Rehman RU, Israr M, Srivastava PS. et.al. In vitro regeneration of witloof chichoiy (Cichorium intybus L.) from leaf explants and ac­cumulation of esculin. Tn vitro Ceil Dev Biol Plant 2003; 39:142- 146.Belletre A, Coullerot JP, Vasseur J. Effects of glycerol on somatic embryogenesis in Cichorium leaves. Plant Cell Rep 1999; 19: 26- 31.MixWagner G, Eneva T. Positive effect of cefotaxime on plant regeneration from Cichorium intybus L leaf inateiial. Land- bauforseh Volk 1996; 46: 166-168.Bennici A, Schiff S, Mori B, Moi-phogenic effect of colchicine in Cichorium intybus L root explants cultured in vitro. Caiyologia 2006; 59; 284-290,Trigiano RN, Gray DJ, Plant tissue culture concepts and laboratoiy exercises, 2"‘‘ ed, 2000; CRC Press.Karam NS, Al-Majathoub M. Direct shoot regeneration and micro- tuberization in wild Cyclamen persicuin Mill using seedling ti,ssue. Sci Hort 2000; 86:235-246.Park EJ, Lim HT. Establishment of an efficient in vitro plant re­generation system in chicory {Cichorium intybits L var sativus), Acta Hort 1999; 483:367-370.

234 Recent Patentn on Biotechnology 2011, Vo!, 5, No. J Ohadi el al.

f28J Linden L, Riikonen A. Effects of 6-benzylaminopurine, thidiazuronand tyjie of explant on in vitro shoot dovelopmeiit of Acer plata- noides L Propag Ornam Plants 2006; 6:201-204.

[29] Wang J, Bao MZ, Plant regeneration of pansy (Viola wittrockiaiia) ‘Caidie’ via petiole-derived callus. Sci Hort20Q7; 111:266-270,

[30] Jones MPA, Yi ZJ, Murch SJ, Saxena PK. Thidiazuronitiduoedregeiieration of Echinacea pwpurea L micropropagation in solid and liquid culture systems. Plant Cell Rep 2007; 26: 13-19.

[31] Waag .1, Bao MZ, Plant regeneration of pansy {Viola wittrockiana)‘Caidie’ via petiole-derived callus. Sci Hort2007; 111: 266-270,

[32] Cliarric're F, Sotta B, Emile M, Gunther H, Induction of adventi­tious shoots or somatic embryos on in vitro cultured zygotic

embryos of Helianthus anniius variation of endogenou.s lionnone levels. Plant Physiol Biochenil999; 37: 751-757,

[33] Cure] E, Wren M.I. In vitro development from leaf explant,'; of sugar beet (Bela vulgaris- L) rhizogenesis and the effect of sequen­tial exposure to auxin and cytokinin. Ann Bot 1995; 75: 31-38,

[34] ICH Haromoni.sed Tripartite Guideline, International Conference on Harmonisation on Technical Requirements for Registration of Pharmaceuticals for Human Use, Q2A, Text on Validation of Ana­lytical Procedures, Step 4 of the ICH process, 1994 and Q2B, Vali­dation of Analytical Procedures: Methodology, Step 4 of the ICH process, 1996, ICH Steering Committee.