AGRICULTURAL RESEARCH COMMUNICATION CENTRE

www.arccjournals.com / indianjournals.comAgric. Sci. Digest., 33 (2) : 109 - 113, 2013

MICRO PROPAGATION OF CARRIZO (CITRUS CARRIZO)THROUGH MATURE BUD CULTURE

J. Kanwar, S. Godara, , M.K. Kaul and A.K. Srivastava

Departmant of Horticulture,Agricultural Research Station, Sriganganagar - 335 001, India

Received: 27-06-2012 Accepted: 26-04-2013

ABSTRACTThe influence of the basal medium and different plant growth regulators on micropropagation of

nodal explants from mature trees Carrizo cultivars were investigated. Although the basal medium didnot affect. several combinations of 6-benzyl amino purine (BAP) and gibberellic acid (GA3) wereused to optimise the proliferation phase. The number of shoots was dependent on the BAP and GA3concentrations and the best results were obtained with BAP 1 mg /L + GA3 1 mg/L. Explants lengthwas shorter with the higher BAP concentrations and, shoot length was greater with increasing levelsof GA3. The best results for productivity (number of shoots 7.9 and the average shoot length 3.0 cm)were obtained with 1 mg/L BAP and 1 mg/L GA3, although explants with narrow leaves were observed.The presence of BAP and GA3 in the proliferation medium was essential for the explants multiplicationbut BAP had a greater influence in shoot multiplication and GA3› influence in elongation of shoots.

Keywords: Adult explants, Basal medium, Plant growth regulators, Proliferation, Elongation

INTRODUCTIONThe field grown tree buds are not easy to

multiply in culture because they have high rate ofcontaminations in in vitro growth. The harsh tissuesterilization not only damages the growing regionsof the buds but also affect their overall growthpotential. Objective of this study was to estimatethe efficiency of in vitro system for multiplication offield grown tree. For clean tissue growth there isculture initiation, subculturing, shoot growth andtheir proliferation. In citrus ,the infection by virusesand related pathogens have received attention dueto decline of citrus trees and it was mainly attributedto viruses (Tristeza, Psorosis, Greening Ring spotvirus, Exocortis, Gummosis, Xyloporosis, Bacteriaetc.), fungi (Phytophtora) and Bacterial canker.Besides other factors like rootstock incompatibility,poor management, malnutrition, marginal soils andirrigation water also affect citrus production. Tristezavirus has wiped out the citrus industry in manycountries. Therefore, In vitro Citrus budwood culturerequire proper nutritional medium for obtainingshoots (Sas-Sert Kaya and Cinae, 1999), Theseinvestigations were undertaken to determine theresponse of di fferent concentrations andcombinations of growth regulators on bud culturesof Carrizo.

MATERIALS AND METHODSAn experiment was conducted at

Biotechnology Lab, Department of Horticulture,Agricultural Research Station, Sriganganagar,SKRAU, Bikaner in the year 2011-12. The fieldgrown 10 year old Carrizo plant was used in thisstudy. Stem buds taken from nucellar seedlings wereused as explants because they are true to type anduniform in growth. To surface sterilize, explants werewashed under running tap water. The buds werewashed with detergent (Teepol), pre-treated with 0.2% (w/v) Bavistin (a systematic fungicide of BASF,India Ltd., Mumbai) for 10 minutes followed by threetime washing with double distilled water. In laminarair flow cabinet, first these were quick rinsed with70 per cent ethanol followed by treatment of HgCl2@ 0.1 per cent and NaOCl @ 0.5 per cent atdifferential time exposure for explants followed by3-4 washings with autoclaved double distilled water.The stem sections were cut from both ends that wasexpose to sterilizing chemicals leaving 1 cm stemexplants with bud in the centre. These explants wereplaced in the horizontal position on the surface ofMS medium (Murashige and Skoog, 1962). Theadditions were different concentration of BAP (0,0.5,1.0 and 2 mg / l) + GA3 (0, 0.5, 1.0 and 2 mg/l)

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+ 500 mg /L of malt extract powder (Table 1).Sucrose was 3% and the pH of medium wasadjusted as 5.8. It was solidified with 1% agar.Explants incubated jam bottles were kept inincubation chamber under the controlledenvironment of temperature (25± 2 0C) and light (13hd-1 illumination of 30 to 40 m mol M-2 S-1 spectralof Flux Photon SFP) and 60-70 per cent relativehumidity (RH). After 5-6 weeks of inoculation allresponding cultures were sub cultured on freshlyprepared medium composition. During sub culturing,dried leaves and weak shoots were removed.

RESULTS AND DISCUSSIONThough Carrizo is propagated successfully

by seeds, the plant developed carries undesirabletraits like inborn virus diseases, soil borne fungi andno uniformity among plants. A lot of improvementin micro propagation of this woody plant tree hasnot yet attained the reputation of being modelsystems. However, the plant regeneration of a singlebud is dependent on high potential of bud sprouting.The potential of cell division and bud regenerationin in vitro conditions is low in Citrus species andcultivars due to browning and poor growth. Carrizobuds responded to BAP and GA3 medium. Nodalsegments needed longer culture period for shootregeneration as compared to bud culture. Thesubculture period was important. The growth wascomparatively faster in the second passage ascompared to initial culture. The bud sprouting andshoot development and proliferation were normaland stable indicating that hormonal balance presentwas optimal and addition of GA3 in the medium wasnecessary for shoot and elongation.

The efficacy of sterilizing agents wasadjudged in terms of maximum aseptic explants,which responded to sprouting. The highest frequencyof contamination free explants (80.00 per cent) wasrecorded when explants were treated with mercuricchloride (0.1 per cent) for 12 minutes followed byquick rinse in 70 per cent ethanol. The increase in

time exposure of both the surface sterilizing agentsdecreased contamination but it also reducedsprouting of explants. These results are in agreementwith the results of Kour et al. (2007) who reported inRough lemon that a treatment of 70 per cent ethanolfor 30 seconds followed by HgCl2 (0.1 per cent) for8 minutes was best surface sterilization treatment.

The similar findings are also reported byAltman et al. (1982); Al-Khayri and Al-Bahrany,(2001); Kotsias D and Roussous P A (2001),Sudhersan et al. (2001); Karwa, (2003); Altaf N(2006); Syamal et al. (2007) and Tornero et al(2010)

Number of days taken to bud break (days):Lower levels of BAP (0.0 - 1.0 mgl-1) and GA3 (0.0 -1.0 mgl-1) when added singly or in combination inMS basal medium reduced the time required to budbreak in Carrizo. When BAP and GA3 added singlyin MS medium, there was decrease in time requiredto bud break, as compared to control. High levels(2.0 mgl-1) of BAP and GA3 delayed bud break. Incombination of BAP and GA3, lower levels (0.5-1.0mgl-1) decreased the time taken in bud break andhigher levels (1.0-2.0 mgl-1) delayed the bud breakin Carrizo (Table 2). These results are in accordancewith Singh et al., 1994 who reported that the meanminimum number of days required to bud break wasdirectly dependent on citrus species and mediumcombination. They also reported that minimum daysto bud beak in Citrus reticulata and Citrus limonwere 17 and 18 days respectively, when explants ofboth species were cultured in MS medium modifiedby BAP 1.0 mgl-1 + kinetin 0.5 mgl-1+ NAA 0.5 mgl-1.These results were also similar with Altman et al.(1982) and Altaf N (2006).

Percent survival of explants: In Carrizo, whenBAP (0.0-2.0 mgl-1) was added singly in MS medium,significant increase in per cent survival of explantswas observed. Maximum (70 per cent) survival ofexplants was observed at 1.0 mgl-1 BAP. Furtherhigher level (2.0 mgl-1) decreased the per centsurvival. When various levels (0.0-2.0 mgl-1) of GA3›were added singly in MS basal medium, maximum(70 per cent) survival of explants was observed at1.0 mgl-1. It was appreciably reduced at 2.0 mgl-1

GA3.The maximum survival (80 per cent) of explantswas observed when BAP and GA3 were combinedtogether, both at 1.0 mgl-1 BAP + 1.0 mgl-1 GA3and 0.5 mgl-1 BAP + 1.0 mgl-1 GA3.

TABLE 1: Treatments details of experiment.

BAP (mg/L) GA3 › (mg/L)

0 00.5 0.51.0 1.02.0 2.0

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The higher levels (2.0 mgl-1) of both BAP andGA3 alone or in combination had negative effect inper cent survival of explants (Table 2). This may bedue to the endogenic levels of cytokinin in differentspecies. Parthasarathy and Nagaraju (1993)reported significant interaction between levels ofvalues for plantlet induction in citrus genotypes.Similar finding were also reported by Duran-Vila etal., 1989; Belouly, 1991; Baruah et al., 1996 andEl-Morsy and Millet, 1996. These results were alsosimilar with Altman et al. (1982) and Altaf N (2006).

Number of shoots: The effect of BAP and GA3either used alone or in combination increased the

shoot number. In Carrizo, addition of BAP singly(0.0-2.0 mgl-1) in the MS basal medium increasedthe number of shoots. The maximum (4.9) shootswere observed at 1.0 mgl-1 kinetin and furtherincrease in level (2.0 mgl-1) decreased it in MS basalmedium (Table 2). When GA3› added singly in MSbasal medium, the maximum (7.2) shoots observedon 1.0 mgl-1 level and numbers of shoots wereinhibited strongly at 2.0 mgl-1 level.

Interaction effect of BAP and GA3 (0.0-2.0mgl-1) were strongly promote especially at 0.5, 1.0mgl-1 levels and maximum (7.9) shoots wereobserved at BAP 1.0 mgl-1 + GA3 1.0 mgl-1. This

FIG.1: Control FIG. 2: Effect of BAP 1.0 mg/L + GA3 1.0 mg/ L.

TABLE 2: Effect of BAP and GA 3 on in vitro growth of Citrus carrizo.

BAP + GA3 Number of days Per cent Length of Number of Number of(mg/L) taken to bud survival shoot shoot leaves

break(Days) (%) (cm)

0.0+ 0.0 18.7 40 1.7 2.2 3.10.0+ 0.5 18.5 50 2.0 5.9 4.80.0+ 1.0 18.3 70 2.5 7.2 5.20.0+ 2.0 18.5 60 2.1 5.7 5.00.5+ 0.0 18.5 60 2.3 3.5 4.50.5+ 0.5 18.4 70 2.2 5.4 4.90.5+ 1.0 18.2 80 2.6 7.4 5.60.5+ 2.0 17.9 60 2.9 7.3 5.41.0+ 0.0 18.3 70 2.4 4.9 5.21.0+ 0.5 17.6 70 2.7 6.6 5.71.0+ 1.0 17.1 80 3.0 7.9 6.11.0+ 2.0 17.2 70 3.0 7.7 5.92.0+ 0.0 18.5 60 2.3 4.4 4.22.0+ 0.5 17.9 70 2.6 6.2 5.22.0+ 1.0 17.7 70 2.7 7.3 5.72.0+ 2.0 17.3 60 2.8 7.6 5.8

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treatment was significantly superior over all thetreatments. Presence of both the hormones at 2.0mgl-1 each was strongly inhibitory for shoot numbers(Table 2 and Fig 1, 2). This may be due to cytokininsuppressed apical dominance and induced lateralbud formation. The rate of shoot multiplication wassignificantly affected by the concentration of BAP.Duran et al. (1992) also observed the important roleof BAP in shoot multiplication. These findings arealso supported by Otoni and Teixeira (1991) inSweet orange. According to them cytokinin promotesshoot proliferation by inducing cell division andenlargement. The similar results are also reportedby Bowman, 1994 in citrus rootstocks and Thakuret al. (2004) in Gerbera. These results were alsosimilar with Altman et al. (1982), Altaf N (2006)and Tornero et al (2010).

Length of shootBAP, GA3 added alone or in combination

produced variable influences on shoot length butgrowth was not pronounced. In Carrizo, increasedconcentration of BAP increased length of shoot.The maximum (2.4 cm) was recorded at 1.0 mgl-1 and further increase in concentration (2.0 mgl-1) decreased length of shoot. When various levelsof GA3 (0.0-2.0 mgl-1) were added singly in MSbasal medium, the effect on shoot length was lessthan that observed on GA3 (2.5 cm) length ofshoot was observed at 1.0 mgl-1. The higherlevels (2.0 mgl-1) GA3 drastically reduced lengthof shoot (2.1 cm). In combination of BAP (0.0-2.0 mgl-1) and GA3 (0.0-2.0 mgl-1), the maximum

length of shoot (3.0 cm) were observed at BAP1.0 mgl-1 + GA3 1.0 mgl-1 and BAP 1.0 mgl-1 +GA3 2.0 mgl-1 (Table 2 and Fig 1, 2). The resultsof present study are similar to that of Al-Bahrany(2002) who reported that in lime the length ofshoot increased in MS medium modified withBAP 0.5 mgl-1. Similarly Al-Khayri and Al-Bahrany (2001) observed 2.20 cm length ofshoot in MS medium modified by BAP 0.5 mgl-1. These results were also similar with Altman etal. (1982), Altaf N (2006) and Tornero et al(2010).Number of leaves

The effect of BAP and GA3 either used aloneor in combination increased the number of leaves.In Carrizo, addition of BAP singly (0.0-2.0 mgl-1) inthe MS basal medium increased the number ofleaves. The maximum (5.2) leaves were observed at1.0 mgl-1 BAP and further increase in level (2.0 mgl-1) decreased it in MS basal medium. When GA3›added singly in MS basal medium, the maximum(5.2) leaves observed on 1.0 mgl-1 level and numbersof leaves were inhibited strongly at 2.0 mgl-1 level.Interaction effect of BAP and GA3 (0.0-2.0 mgl-1)were strongly promotory especially at 0.5, 1.0 mgl-1levels and maximum (6.1) leaves were observed atBAP 1.0 mgl-1 + GA3 1.0 mgl-1. This treatment wassuperior over all the treatments. Presence of boththe hormones at 2.0 mgl-1 each, was stronglyinhibitory for leaves numbers. (Table 2 and Fig 1,2). These results were also similar with Altman et al.(1982), Altaf N (2006) and Tornero et al (2010).

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