Production of Leaf Curl Virus - Free Chilli by Meristem Tip Culture

  

Int ernat ional Journal of Pharmaceut ical Sciences Review and Research

Rishi kesh M eena, Kavi Gour* , Vidya Patni Plant pat hology, Tissue cult ure and Biot echnology Laborat ory, Depart ment of Bot any, Universit y of Rajast han, Jaipur, India. * Corresponding author’s E-mail: kavigour@gmail.com

  cult ure in comm ercial m icropropagation syst em is t o at t em pt for recovering virus -free plant s.

  Research Article

  Production of Leaf Curl Virus - Free Chilli by M eristem Tip Culture C

  M echanical inoculat ion w as done using t he leaf sap from nat urally infect ed and in vit ro grow n chilli plant s. Young leaves were homogenized in 0.02 M sodium phosphate buffer, pH.7.0 wit h m ort ar and pest le. The slurry t hus

  Virus detection and indexing of naturally infected and in vitro grown plants 2.2.1 Biological indexing

  The virus w as m aint ained on healt hy chilli var. Pusa jw ala by w hit e fly Bemisia tabaci inoculation at 3 t o 4 w eeks int erval. The experim ent al plant s used in t his invest igation w ere raised from seeds in pot s filled wit h loam y clay soil, sand and farm yard (1:1:1) m anure. From t hese plant s young leaves and m erist em tips w ere chosen for furt her det ection and t o produce virus-free plant s.

  M ATERIALS AND M ETHODS Collection and maintenance of viral culture Capsicum annuum var. Pusa jw ala show ing charact erist ic sym pt om s on leaves w ere collect ed from t he field st udy.

  disease in Capsicum annuum by PCR and in vit ro production of LCV free chilli plant s by m erist em t ip cult ure. This m ethod w ill help t o minimize virus infect ion and hence produce qualit y CLCV free chilli plant s. This is t he first report of leaf curl virus free plant product ion from m erist em tip of m at ure chilli plant s.

  15 In t he present paper, we report t he det ect ion of leaf curl

  C. annuum but no at t em pt s have been m ade t o eradicate LCV from chilli.

  M erist em t ip cult ure has been successful for Tobacco m osaic virus and or w it h Tom ato spot t ed w ilt virus or wit h bot h viruses in

  10-14

  9 The m ain advant age of using m erist em t ip

  Accepted on: 18-01-2014; Finalized on: 31-03-2014. ABSTRACT Chilli plant s infected w it h leaf curl virus (geminivirus) are charact erized by vein clearing, upw ard curling, deformat ion of leaves st unt ing of plant s and abscission of flow er buds. The virus w as ident ified as chilli leaf curl virus (CLCV) on t he basis of mechanical inoculation on Nicot iana tabacum and Chenopodium quinoa and by PCR. M S medium amended w ith BAP (3.0mg/ l) and IAA (3.0mg/ l) w as used for shoot proliferation. A maximum of 34.26±0.06 number of shoot s w as obt ained. Elongat ed shoot s w ere root ed on 1/ 2 M S supplement ed wit h IBA (2.0 mg/ l) and 2% act ivat ed charcoal. Regenerat ed plants gave negat ive result s for CLCV by PCR w it h specific primers. Virus free chilli plant s (80%) w ere obt ained from optimum size (0.5 mm) of merist em t ips. Virus indexing by PCR w as found t o be a reliable met hod for confirmat ion of CLC virus free nat ure of t he regenerat ed plant lets. This is t he first report of geminivirus-free chilli plant s product ion t hrough merist em tip culture.

  produce virus free plant s and in clonal m icro propagat ion in a w ide variet y of plant s How ever, knowledge of in vit ro grow t h and developm ent al responses of C. annuum L., is lim it ed.

  In 2007, during a survey of chilli varieties grow n in fields of Jodhpur and Jaipur (Rajast han, India) plant s w ere found infect ed w it h leaf curl disease. The virus induced leaf curl sym pt om s are characterized by vein clearing, upw ard curling, deform ation of leaves st unt ing of plant s and abscission of flow er buds. The w hole plant assum es a bushy appearance wit h st unted grow th. Few er flow ers and fruit s develop due t o disease. The plant s w ere t est ed for t he presence of leaf curl gem ini virus using PCR and w ere found t o be posit ive.

  4-7

  The PCR technique has been used by m any w orkers for det ection of virus in virus infect ed plant s.

  1-3

  Indonesia.

  Chilli leaf curl multan virus and Pepper yellow leaf curl Indonesian virus w ere report ed from Pakist an and

  In India, Tomato Leaf Curl New Delhi Virus (ToLCNDV) has recent ly been show n t o be associated wit h chilli leaf curl disease. Several ot her begomovirus species associat ed w it h chilli leaf curl such as Cot ton leaf curl mult an virus,

  Cucumber mosaic virus (CM V), Pepper vein banding virus, Pepper vein mott le virus , Potato virus y (PVY), Tobacco et ch virus and Tobacco mosaic virus (TM V).

  hilli (Capsicum annuum L.) an im port ant spice, is grow n w idely in t he t ropics and sem i t ropics t hroughout t he w orld but t he productivity is very low . One of t he m ain reasons for t he low product ivit y is t he susceptibilit y t o various pest and diseases including viral diseases which not only reduce yield but also t he qualit y.

  INTRODUCTION

  Keywords: Chilli leaf curl virus, Geminivirus, In vit ro production, PCR, Virus free chilli.

1 Several viruses have been report ed t o affect chilli such as

8 Apical m erist em cult ure has been successfully used t o

  

Int ernat ional Journal of Pharmaceut ical Sciences Review and Research

  In vitro production of chilli (Capsicum annuum) Source of explants

  t abacum produced leaf curl sym pt om on t he leaves in 11-

  On m echanical inoculation from nat urally leaf curl infect ed chilli and in vit ro grow n plant s, Nicotiana

  RESULTS AND DISCUSSION Virus indexing

  Prot ein gene am plificat ion Posit ion of sample

  Figure 1: PCR detection of chilli leaf curl virus by coat

  1. M arker (1 kb); 2. Infect ed leaf (Jaipur); 3. Healt hy leaf of Chilli; 4. Infected leaf Jodhpur; 5. Infect ed leaf (New Delhi).

  The cult ures w ere regularly subcult ured on fresh m edium aft er 4 w eeks. M ult iple shoot s obtained w ere lat er t ransferred t o elongation medium supplem ent ed w ith different grow t h horm ones. Elongat ed and healt hy shoot s w ere t ransferred t o ½ M S m edium supplement ed w ith IBA (2m g/ l) w it h activat ed charcoal (0.2%) for rooting. Regenerated plant let s w ere t hen t ransferred t o pot s cont aining st erilized soil and verm iculit e (3:1) in an insect proof net house. They w ere rout inely checked for presence of virus.

  Shoot t ips w ere w ashed w it h 2% liquid detergent solut ion (Extran, E. M erck- a comm ercial grade det ergent ) for 5 m inutes under running t ap w ater and imm ersed in 1.0% Sodium hypochlorit e solut ion for 2-3 minut es follow ed by t hree w ashings w it h st erile dist illed w at er. Shoot t ip m erist em s, 0.25–1.0 mm in size w ere excised asept ically under st ereomicroscope and w ere kept in lam inar flow clean bench. The excision w as perform ed in a st erile glass pet ridish, lined w it h st erile m oist filt er paper t o avoid desiccation of t he sm all explant. M S-m edium supplement ed wit h various concent rations and com binat ions of auxins (1-5 m g/ l) and cyt okinins (1-5 m g/ l) w as prepared. The pH of t he m edium w as adjust ed t o 5.8 and aut oclaved at 15 psi for 20 m inutes. These w ere planted wit h t he cut -end slight ly em bedded in t he m edium. All the cult ures were incubat ed at 26±2°C and exposed t o 16 hr phot operiod illum inated by fluorescent light of about 1500-2500 lux light int ensit y. Relat ive hum idit y of 55±5% w as m aint ained in t he cult ure room.

  Establishment of aseptic cultures

  Shoot t ips (2-3mm long) w it h t w o leaf prim ordia w ere chosen from chilli var. Pusa jw ala field grow n infect ed plant s.

  PCR product w as separat ed by electrophoresis on a 1.0% agarose gel at 80 V. The gel w as st ained in et hidium bromide (1mg/ ml) and visualized in transillum inat or. An aliquot (500 ng) of 1kb DNA ladder (M .B.I. ferm ent as, USA) w as used as a m olecular w eight m arker. The PCR product w as cut from t he gel and isolat ed from gel elut ion kit (Prom ega inc. USA) in 30 m lof aut oclaved double dist illed wat er.

  obt ained w as squeezed t hrough double layered m uslin clot h. Nicot iana t abacum (syst emic host ) Chenopodium

  10X PCR react ion buffer, 0.5 m lof Taq DNA polym erase and aut oclaved double dist illed w at er t o m ake up t he volume. The t herm o cyclic programm e w as st art ed at 90°C for 5 m inut es follow ed by 35 cycles of denat urat ion at 90°C for 1 m inut e annealing at 42°C for 1 m inute and synt hesis at 72°C for 2-3 minut es. The final ext ension w as at 72° C for 10 m inutes.

  The PCR react ion mixt ure of 50 m lconsist ed of 1 m lof t ot al DNA (about 200 ng), 2 m lof 10 mM dNTP 1 mleach of forw ard and reverse prim ers (about 1 ng/ ml), 5 m l of

  10 5´ TG/ TAATTCGT/ ATAC / ACGAG/ TTC ATA3´

  0.75 Coat protein gene AVR

  9 5´ATGTCG/ CAAGCGAG / CCTAGCC/ AGATAT3´

  Name of the primer Sequence Expected product (Kb) Used for amplification of AVF

  The PCR w as carried out t o amplify t he genom e of t he virus associated w it h chilli leaf curl disease. Thus, a pair of prim ers (AVF9 & AVR10) over t he put at ive coat prot ein gene w as effective in det ecting t he virus by polym erase chain reaction. The specific prim ers AVF9 and AVR10 designed at IARI, New Delhi w ere used in t he present invest igat ion for com paring t he put ative coat prot ein gene. Sequence available for present st udy and from t he gene bank w as very useful in diagnosis of 750bp fragm ent s. These specific prim ers are valuable m olecular t ools in t he diagnosis of chilli leaf curl virus. Tw o prim ers (AVF9, AVR10) of following sequences w ere used.

  The leaves of chilli infect ed w ith leaf curl disease used in present st udy w ere obt ained from field sam ples from Jodhpur, Jaipur and New Delhi. Tot al DNA w as isolat ed from t he infected and healt hy leaves using DNA easy m ini kit (Qaigen Inc. USA). About 50 m g of leaves w ere ground in liquid nitrogen and processed as described in Qaigen DNA m anual. The t ot al DNA w as elut ed w it h 100 m l aut oclaved double dist illed w at er.

  PCR Detection

  quinoa (local lesion host ) w ere inoculat ed w it h sap t o t est t he presence of virus.

  13 days and on Chenopodium quinoa chlorotic local

• 0.5 - 4.82 ± 0.33
• 1.0 - 5.22 ± 0.11
• 2.0 - 8.15± 0.21
• 3.0 -
• 4.0 - 15.22 ± 0.45

  3.0 3.0 - 34.26 ± 0.06

  production of leaf curl virus free chilli plant s

  Table 2: The effect of size of m erist em t ip in t he

  Sm all size merist em s (0.2-0.3m m) t aken in t his st udy t ransform ed int o callus and were hence not test ed for t he presence or absence of virus. The 0.5 mm long m erist em produced only shoot s and 80% plant s regenerat ed from t hese m erist em s t est ed negative for leaf curl virus as indexed by PCR. As t he size of m erist em increased, t he percent age of obt aining t he virus free plant s decreased and w it h 0.7-1.0 mm long merist em , no virus –free plant s w ere obt ained. (Table 2)

  Effect of meristem size on its establishment and virus elimination

  Production of leaf curl virus free plant s of chilli using m erist em t ips.

  Figure 2:

• 5.0 - 6.80± 0.54

  34

  27

  80

  0.7 Shoot s

  0.5

  4.5 2.5 - 16.4 ± 0.336

  0.5 Shoot s

  3.0 2.0 - 21.0 ± 0.427

  2.0 1.5 - 23.4 ± 0.220

  1.0 1.0 - 5.4 ± 0.401

  0.5 0.5 - 9.0 ± 0.336

  21.11 ± 0.10

  IAA BAP Kn 0.5 - - 21.58± 0.29 1.0 - - 25.81 ± 0.37 2.0 - - 31.33 ± 0.15 3.0 - - 32.22 ± 0.18 4.0 - - 30.20 ± 0.18 5.0 - - 25.01± 0.22

  PGR’s mg/ l No. of shoots buds per explant M ean ±SE

  form at ion from shoot t ip explant of Capsicum annuum L on M S-m edium

  Table 1: Effect of Plant Grow t h Regulat ors (PGR) on shoot

  Shoot t ips of Capsicum annuum L. w ere isolat ed asept ically and cult ured on M S-m edium supplem ent ed w it h cyt okinins and auxins for init iating veget at ive grow th and inducing a m axim um num ber of plant let s. Therefore, in t he present st udy experim ent s w ere aim ed at obt aining m ultiple shoot proliferat ion in capsicum annuum L. In t he present st udy, M S-medium fort ified w ith a com binat ion of BAP (3 mg/ l) and IAA (3 m g/ l) produced m axim um num ber of (34.26±0.06) m ultiple shoot (Table.1, Fig.2 A- D).

  M eristem culture and shoot multiplication

  lesions appeared in 6-7 days.However t he in vit ro grown plant s, did not produce local lesions on C. quinoa. PCR w as found t o be a reliable met hod in com parision to biological indexing. Chilli leaf curl disease in Jaipur and Jodhpur is caused by gem inivirus because t hese sam ples gave an am plification of 0.75Kb fragm ent but t he sam ple collect ed from New Delhi fields did not show t he band so it can be concluded t hat leaf curl disease of chilli in New Delhi is caused by some ot her virus. (Figure 1)

  34

  Size of meristem (mm) Tissue differentiation No of shoots grown in vitro Virus indexing by PCR No of virus free plants % virus free plants 0.2-0.3 Callus - - -

0.5 Nil

  3.0 3.0 4.35 ± 0.60 (A) M erist em t ip in culture; (B) int iat ion of proliferat ion of shoot s; (C) Prolifirat ion of several shoot s; (D) Elongat ed virus free (t est ed) shoot s; (E) Root ing of virus free (t est ed) shoot in t he medium; (F) The chilli leaf curl virus-free plant w it h green and red fruit s.

  2.5 2.5 5.38 ± 0.45

  1.5 1.5 3.22 ± 0.88

  1.0 1.0 3.10 ± 0.72

  

Int ernat ional Journal of Pharmaceut ical Sciences Review and Research

  1.0 Shoot s

  34 Rooting, hardening and acclimatization

  Proliferat ed shoot s aft er 8 w eeks w ere t ransferred t o M S- m edium fort ified wit h IBA (2.0 mg/ l) and activat ed charcoal (0.2%).On t his m edium , root ing percent age (85%), num ber of root s (37-38) and root lengt h (10.6 ±

  2.0 2.0 4.16 ± 0.63

  

Int ernat ional Journal of Pharmaceut ical Sciences Review and Research

  part icle w it h it and all t he shoot s produced from t hese w ere found t o be virus infect ed. There are various explanat ions for virus eliminat ion during in vitro cult ure e.g. act ion of grow t h regulat ors part icularly cyt okinin phenol-am ines loss of enzym es necessary for viral replicat ion and viral RNA degradat ion due t o cell injury during explant s excision.

  19,20

  Similar effect of ½ M S m edia com bination w ith

  IBA w as observed by Rao et al. (2006) in t he sam e plant species.

  pot ent root auxin for in vit ro grow n shoot s and pot ent ialit y of act ivat ed charcoal on rooting w as assessed by several w orkers in many plant species.

  21-24

  In consonance t o t his, Kanyand et al. (1994) report ed successful t ransfer of in vitro regenerat ed plantlet s from flask t o field condit ion in Arachis hypogaea. Virus elim inat ion depends on various fact ors such as m erist em size, t he virus concerned, physiological condition of m ot her plant s and m erist em posit ion on it. The larger t he size of t he m erist em cult ured, t he great er is t he num ber of regenerat ed plant s, w hile t he num ber of virus-free plant let s obt ainable is inversely proport ional t o t he size of t he cult ured t ips.

  25 In our experim ent s, merist em tips of size 0.5 m m w ere

  found t o be opt im um for eliminat ing leaf curl virus from chilli. M erist em of 0.7-1.0 mm size carried t he virus

  26,27

  Capsicum annuum (Sobhakum ari and Lalit hakum ari,

  Regenerated plant s w ere longer, fruit s were healt hy, green and elongat ed. Seeds obt ained from in vit ro plant s w ere m ore in num ber t han t hat of in vivo plant s. Plant s regenerated from shoot t ip merist em (0.5 mm ) did not show t he 0.75 kb band hence t hey w ere gemini virus (LCV) free. This w as observed m orphologically and confirmed by PCR. (Figure 3) M erist em –tip cult ure t o produce leaf curl virus free plant s of chilli is an efficient t echnique and t he indexing by PCR is t im e saving and a reliable m et hod for cert ificat ion of chilli leaf curl free t issue cult ure raised plant s. This proposal can be used for t he production of CLCV-free plant s of chilli.

16 The prim ers have been used

17 Shih et al.. (2003) carried out m olecular

  1. Sample from leaf of chilli leaf curl virus infect ed plants grow ing in pots; 2. Sample from leaf of chilli plant s raised in vit ro; 3. St andard DNA of Gemini virus; 4. M arker (1 Kb) Figure 3: Det ect ion of virus free plant by PCR Posit ion of

  Sam ples

  Acknow ledgement: The first aut hor expresses his

  grat itude t o CSIR, New Delhi for providing financial support and t o IARI (New delhi, India) for providing facilities of PCR.

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  o

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  18 In contrast t o above m entioned result s som e researchers

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  6,7

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21 In m ost species IBA has also been found t o be

  

Int ernat ional Journal of Pharmaceut ical Sciences Review and Research

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