Int ernat ional Journal of Pharmaceut ical Sciences Review and Research
67
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
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.
Keywords: Chilli leaf curl virus, Geminivirus, In vit ro production, PCR, Virus free chilli.
INTRODUCTION
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.1
Several viruses have been report ed t o affect chilli such as Cucumber mosaic virus (CM V), Pepper vein banding virus, Pepper vein mott le virus, Potato virusy (PVY), Tobacco et ch virus and Tobacco mosaic virus (TM V).
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, Chilli leaf curl multan virus and Pepper yellow leaf curl Indonesian virus w ere report ed from Pakist an and Indonesia.1-3 The PCR technique has been used by m any w orkers for det ection of virus in virus infect ed plant s.4-7 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.8
Apical m erist em cult ure has been successfully used t o 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.9 The m ain advant age of using m erist em t ip cult ure in comm ercial m icropropagation syst em is t o at t em pt for recovering virus -free plant s.10-14 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 C. annuum but no at t em pt s have been m ade t o eradicate LCV from chilli.15
In t he present paper, we report t he det ect ion of leaf curl 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.
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. 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.
Virus detection and indexing of naturally infected and in vitro grown plants 2.2.1 Biological indexing
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
Production of Leaf Curl Virus - Free Chilli by M eristem Tip Culture
C
obt ained w as squeezed t hrough double layered m uslin clot h. Nicot iana t abacum (syst emic host ) Chenopodium quinoa (local lesion host ) w ere inoculat ed w it h sap t o t est t he presence of virus.
PCR Detection
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.
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.
Name
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
In vitro production of chilli (Capsicum annuum)
Source of explants 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. 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.
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).
Figure 1: PCR detection of chilli leaf curl virus by coat Prot ein gene am plificat ion Posit ion of sample
RESULTS AND DISCUSSION
Virus indexing
11-Int ernat ional Journal of Pharmaceut ical Sciences Review and Research Delhi is caused by some ot her virus. (Figure 1)
M eristem culture and shoot multiplication
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
PGR’s mg/ l No. of shoots buds per explant
M ean ±SE
Figure 2: Production of leaf curl virus free plant s of chilli using m erist em t ips. production of leaf curl virus free chilli plant s
0.185) w ere m axim um (Table 3). Root s w ere thick w ith w hit e root hairs and healt hy in nat ure (Figure 2, E) Plantlet s w it h 6-7 leaves and w ell developed root syst em w ere rem oved and transferred t o pot s cont aining soil rit e. These pot s w ere kept in grow t h cham ber for 15 days at characterization of an isolat e of Begom ovirus in Pakist an and found t hat isolat e w as not relat ed t o t he t w o t om ato isolat es of Begom ovirus from Pakist an.2
The nut rient m edium generally used t o init iat e cult ures is com posed of basal salt s (m ajor and minor element s) and organic supplem ent s. Besides t he vit am ins, grow th regulat ors also play a vit al role in grow t h of m erist em . As m erist em s are incapable of synt hesizing t he grow th horm ones ot her t han auxins,ext ernal supply t hrough m edium becomes absolut ely essential.18
In contrast t o above m entioned result s som e researchers observed t hat t he com binat ion of BAP and IAA on M S-m ediuS-m favoured S-mult iple shoot bud induction in
Capsicum annuum (Sobhakum ari and Lalit hakum ari,
2003) and in Salvia nemorosa L. by Skaia and Nska (2004).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.21 In m ost species IBA has also been found t o be 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
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 due t o cell injury during explant s excision.26,27
Regenerated plant s w ere longer, fruit s were healt hy, t issue cult ure raised plant s. This proposal can be used for t he production of CLCV-free plant s of chilli.
Int ernat ional Journal of Pharmaceut ical Sciences Review and Research
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