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

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1 Agric. Sci. Digest., 33 (2) : 09-3, 203 AGRICULTURAL RESEARCH COMMUNICATION CENTRE / indianjournals.com 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 , India Received: Accepted: ABSTRACT The 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 did not affect. several combinations of 6-benzyl amino purine (BAP) and gibberellic acid (GA 3 ) were used to optimise the proliferation phase. The number of shoots was dependent on the BAP and GA 3 concentrations and the best results were obtained with BAP mg /L + GA 3 mg/l. Explants length was shorter with the higher BAP concentrations and, shoot length was greater with increasing levels of GA 3. The best results for productivity (number of shoots 7.9 and the average shoot length 3.0 cm) were obtained with mg/l BAP and mg/l GA 3, although explants with narrow leaves were observed. The presence of BAP and GA 3 in the proliferation medium was essential for the explants multiplication but BAP had a greater influence in shoot multiplication and GA 3 influence in elongation of shoots. Keywords: Adult explants, Basal medium, Plant growth regulators, Proliferation, Elongation INTRODUCTION The field grown tree buds are not easy to multiply in culture because they have high rate of contaminations in in vitro growth. The harsh tissue sterilization not only damages the growing regions of the buds but also affect their overall growth potential. Objective of this study was to estimate the efficiency of in vitro system for multiplication of field grown tree. For clean tissue growth there is culture initiation, subculturing, shoot growth and their proliferation. In citrus,the infection by viruses and related pathogens have received attention due to decline of citrus trees and it was mainly attributed to viruses (Tristeza, Psorosis, Greening Ring spot virus, Exocortis, Gummosis, Xyloporosis, Bacteria etc.), fungi (Phytophtora) and Bacterial canker. Besides other factors like rootstock incompatibility, poor management, malnutrition, marginal soils and irrigation water also affect citrus production. Tristeza virus has wiped out the citrus industry in many countries. Therefore, In vitro Citrus budwood culture require proper nutritional medium for obtaining shoots (Sas-Sert Kaya and Cinae, 999), These investigations were undertaken to determine the response of di fferent concentrations and combinations of growth regulators on bud cultures of Carrizo. MATERIALS AND METHODS An experiment was conducted at Biotechnology Lab, Department of Horticulture, Agricultural Research Station, Sriganganagar, SKRAU, Bikaner in the year The field grown 0 year old Carrizo plant was used in this study. Stem buds taken from nucellar seedlings were used as explants because they are true to type and uniform in growth. To surface sterilize, explants were washed under running tap water. The buds were washed with detergent (Teepol), pre-treated with 0.2 % (w/v) Bavistin (a systematic fungicide of BASF, India Ltd., Mumbai) for 0 minutes followed by three time washing with double distilled water. In laminar air flow cabinet, first these were quick rinsed with per cent ethanol followed by treatment of HgCl 0. per cent and per cent at differential time exposure for explants followed by 3-4 washings with autoclaved double distilled water.the stem sections were cut from both ends that was expose to sterilizing chemicals leaving cm stem explants with bud in the centre. These explants were placed in the horizontal position on the surface of MS medium (Murashige and Skoog, 962). The additions were different concentration of BAP (0,,.0 and 2 mg / l) + GA 3 (0,,.0 and 2 mg/l)

2 0 AGRICULTURAL SCIENCE DIGEST mg /L of malt extract powder (Table ). Sucrose was 3% and the ph of medium was adjusted as 5.8. It was solidified with % agar. Explants incubated jam bottles were kept in incubation chamber under the controlled environment of temperature (25± 2 0 C) and light (3 hd - illumination of 30 to 40 m mol M -2 S - spectral of Flux Photon SFP) and - per cent relative humidity (RH). After 5-6 weeks of inoculation all responding cultures were sub cultured on freshly prepared medium composition. During sub culturing, dried leaves and weak shoots were removed. RESULTS AND DISCUSSION Though Carrizo is propagated successfully by seeds, the plant developed carries undesirable traits like inborn virus diseases, soil borne fungi and no uniformity among plants. A lot of improvement in micro propagation of this woody plant tree has not yet attained the reputation of being model systems. However, the plant regeneration of a single bud is dependent on high potential of bud sprouting. The potential of cell division and bud regeneration in in vitro conditions is low in Citrus species and cultivars due to browning and poor growth. Carrizo buds responded to BAP and GA 3 medium. Nodal segments needed longer culture period for shoot regeneration as compared to bud culture. The subculture period was important. The growth was comparatively faster in the second passage as compared to initial culture. The bud sprouting and shoot development and proliferation were normal and stable indicating that hormonal balance present was optimal and addition of GA 3 in the medium was necessary for shoot and elongation. The efficacy of sterilizing agents was adjudged in terms of maximum aseptic explants, which responded to sprouting. The highest frequency of contamination free explants (80 per cent) was recorded when explants were treated with mercuric chloride (0. per cent) for 2 minutes followed by quick rinse in per cent ethanol. The increase in BAP (mg/l) TABLE : Treatments details of experiment. GA 3 (mg/l) time exposure of both the surface sterilizing agents decreased contamination but it also reduced sprouting of explants. These results are in agreement with the results of Kour et al. (2007) who reported in Rough lemon that a treatment of per cent ethanol for 30 seconds followed by HgCl 2 (0. per cent) for 8 minutes was best surface sterilization treatment. The similar findings are also reported by Altman et al. (982); Al-Khayri and Al-Bahrany, (200); Kotsias D and Roussous P A (200), Sudhersan et al. (200); Karwa, (2003); Altaf N (2006); Syamal et al. (2007) and Tornero et al (200) Number of days taken to bud break (days): Lower levels of BAP ( -.0 mgl - ) and GA 3 ( -.0 mgl - ) when added singly or in combination in MS basal medium reduced the time required to bud break in Carrizo. When BAP and GA 3 added singly in MS medium, there was decrease in time required to bud break, as compared to control. High levels ( mgl - ) of BAP and GA 3 delayed bud break. In combination of BAP and GA 3, lower levels (-.0 mgl - ) decreased the time taken in bud break and higher levels (.0- mgl - ) delayed the bud break in Carrizo (Table 2). These results are in accordance with Singh et al., 994 who reported that the mean minimum number of days required to bud break was directly dependent on citrus species and medium combination. They also reported that minimum days to bud beak in Citrus reticulata and Citrus limon were 7 and 8 days respectively, when explants of both species were cultured in MS medium modified by BAP.0 mgl - + kinetin mgl - + NAA mgl -.These results were also similar with Altman et al. (982) and Altaf N (2006). Percent survival of explants: In Carrizo, when BAP (- mgl - ) was added singly in MS medium, significant increase in per cent survival of explants was observed. Maximum ( per cent) survival of explants was observed at.0 mgl - BAP. Further higher level ( mgl - ) decreased the per cent survival. When various levels (- mgl - ) of GA 3 were added singly in MS basal medium, maximum ( per cent) survival of explants was observed at.0 mgl -. It was appreciably reduced at mgl - GA 3.The maximum survival (80 per cent) of explants was observed when BAP and GA 3 were combined together, both at.0 mgl - BAP +.0 mgl - GA 3 and mgl - BAP +.0 mgl - GA 3.

3 Vol. 33, No. 2, FIG.: Control FIG. 2: Effect of BAP.0 mg/l + GA3.0 mg/ L. The higher levels ( mgl-) of both BAP and GA3 alone or in combination had negative effect in per cent survival of explants (Table 2). This may be due to the endogenic levels of cytokinin in different species. Parthasarathy and Nagaraju (993) reported significant interaction between levels of values for plantlet induction in citrus genotypes. Similar finding were also reported by Duran-Vila et al., 989; Belouly, 99; Baruah et al., 996 and El-Morsy and Millet, 996. These results were also similar with Altman et al. (982) and Altaf N (2006). shoot number. In Carrizo, addition of BAP singly (- mgl-) in the MS basal medium increased the number of shoots. The maximum (4.9) shoots were observed at.0 mgl- kinetin and further increase in level ( mgl-) decreased it in MS basal medium (Table 2). When GA3 added singly in MS basal medium, the maximum (7.2) shoots observed on.0 mgl- level and numbers of shoots were inhibited strongly at mgl- level. Interaction effect of BAP and GA3 (- mgl-) were strongly promote especially at,.0 mgl - levels and maximum (7.9) shoots were observed at BAP.0 mgl- + GA3.0 mgl-. This Number of shoots: The effect of BAP and GA3 either used alone or in combination increased the TABLE 2: Effect of BAP and GA 3 on in vitro growth of Citrus carrizo. BAP + GA3 (mg/l) Number of days taken to bud break(days) Per cent survival (%) Length of shoot (cm) Number of shoot Number of leaves

4 2 AGRICULTURAL SCIENCE DIGEST treatment was significantly superior over all the treatments. Presence of both the hormones at mgl - each was strongly inhibitory for shoot numbers (Table 2 and Fig, 2). This may be due to cytokinin suppressed apical dominance and induced lateral bud formation. The rate of shoot multiplication was significantly affected by the concentration of BAP. Duran et al. (992) also observed the important role of BAP in shoot multiplication. These findings are also supported by Otoni and Teixeira (99) in Sweet orange. According to them cytokinin promotes shoot proliferation by inducing cell division and enlargement. The similar results are also reported by Bowman, 994 in citrus rootstocks and Thakur et al. (2004) in Gerbera. These results were also similar with Altman et al. (982), Altaf N (2006) and Tornero et al (200). Length of shoot BAP, GA 3 added alone or in combination produced variable influences on shoot length but growth was not pronounced. In Carrizo, increased concentration of BAP increased length of shoot. The maximum (2.4 cm) was recorded at.0 mgl - and further increase in concentration ( mgl - ) decreased length of shoot. When various levels of GA 3 (- mgl - ) were added singly in MS basal medium, the effect on shoot length was less than that observed on GA 3 (2.5 cm) length of shoot was observed at.0 mgl -. The higher levels ( mgl - ) GA 3 drastically reduced length of shoot (2. cm). In combination of BAP (- mgl - ) and GA 3 (- mgl - ), the maximum length of shoot (3.0 cm) were observed at BAP.0 mgl - + GA 3.0 mgl - and BAP.0 mgl - + GA 3 mgl - (Table 2 and Fig, 2). The results of present study are similar to that of Al-Bahrany (2002) who reported that in lime the length of shoot increased in MS medium modified with BAP mgl -. Similarly Al-Khayri and Al- Bahrany (200) observed 2.20 cm length of shoot in MS medium modified by BAP mgl -. These results were also similar with Altman et al. (982), Altaf N (2006) and Tornero et al (200). Number of leaves The effect of BAP and GA 3 either used alone or in combination increased the number of leaves. In Carrizo, addition of BAP singly (- mgl - ) in the MS basal medium increased the number of leaves. The maximum () leaves were observed at.0 mgl - BAP and further increase in level ( mgl - ) decreased it in MS basal medium. When GA 3 added singly in MS basal medium, the maximum () leaves observed on.0 mgl - level and numbers of leaves were inhibited strongly at mgl - level. Interaction effect of BAP and GA 3 (- mgl - ) were strongly promotory especially at,.0 mgl - levels and maximum (6.) leaves were observed at BAP.0 mgl - + GA 3.0 mgl -. This treatment was superior over all the treatments. Presence of both the hormones at mgl - each, was strongly inhibitory for leaves numbers. (Table 2 and Fig, 2). These results were also similar with Altman et al. (982), Altaf N (2006) and Tornero et al (200). REFERENCES Al-Bahrany, A.M. (2002). Effect of photohormones on in vitro shoot multiplication and rooting of lime (Citrus aurentifolia Christm.) Swing. Scientia Horticulture 95 : Al-Khayri, J.M. and Al-Bahrany, A.M. (200). In vitro micro-propagation of Citrus aurantifolia. Curr. Sci. 8, Altaf, N (2006) In vitro bud culture of kinnow tree. Pak. J. Bot., 38: Altman, A. and Goren, R. (978). Development of citrus bud explants in culture. J. Am. Soc. Horti. Sci. 03 : Baruah, A., Nagaraju, V. and Parthasarathy, V. A. (996). Micro-propagation of three endangenered citrus species-2, rooting ex vitro, Ann. of Plant Physiol. 0: Belouly, N. (99). Plant regeneration from callus cultures of three citrus rootstocks. Plant Cell Tissue Org Cult 24: Bowman, K.D. (994). Micro-propagation of smooth flat Seville and Yuma citrus rootstocks. Fla State Hort Soc 07 : 5-8. Duran-Vila, N., Chambra, M., Medina, V., Ortega, C. and Navarro, L. (989). Growth and morphogenesis of citrus tissue cultures infected with citrus tristeza virus and citrus infectious variegation virus. Phytopath 79 : Duran-Vila N., Gogorcena, Y., Ortega, V., Ortiz, J. and Navarro, L. (992). Morphogenesis and tissue culture of sweet orange (Citrus sinensis (L) Osb.) : Effect of temperature and photosynthetic radiation. Plant cell Tiss Org Cult 29 : -8.

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