EVALUATION THE EFFECTIVE FACTORS ON SHOOT PROLIFERATION OF WASHINGTON NAVEL ORANGE IN TISSUE CULTURE MEDIUM
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Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231– 6345 (Online)
An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm
2015 Vol. 5 (1) January-March, pp. 244-248/Jahromi and Dardan
Research Article
EVALUATION THE EFFECTIVE FACTORS ON SHOOT
PROLIFERATION OF WASHINGTON NAVEL ORANGE IN TISSUE
CULTURE MEDIUM
*Abdolhossein Aboutalebi Jahromi and Mahsa Dardan
Department of Horticulture, Jahrom Branch, Islamic Azad University, Jahrom, Iran
*Author for Correspondence
ABSTRACT
The present study was conducted to evaluate the effective factors on prosperity percent of shoot
proliferation of Washington navel orange in vitro culture by using young flashes having bud in
completely randomized design. For this purpose were selected young shoots having bud with length of
1.5 cm as explants and following disinfection by solution of sodium hypo chloride 1% and washing by
distilled water were cultured in MS medium with 30% sucrose and 7.5 gl -1 Agar supplemented with BA
(0.5, 1, 1.5, 2, 2.5 and 3 mgl-1) and NAA (0, 0.5, 0.75, 1, 1.25 and 1.5 mgl-1) and were placed in
conditions of 16 hrs light and 8 hrs dark for 4-6 weeks. In the end of the experiment, were measured shoot
number, shoot length, shoot quality, leaf number and the dropped leaf number. Based on the obtained
results, the highest number and quality of the produced shoot was relative to BA 2 mgl-1 + NAA 0.5 mgl-
1
. The greatest shoot length obtained from medium containing 1 mgl -1 BA + 1.5 mgl-1 NAA. The highest
leaf number and the dropped leaf were observed in BA 2 mgl-1 + NAA 0.5 mgl-1 and BA 1.5 mgl-1 + NAA
0.5 mgl-1.
Keywords: Sweet Orange, In Vitro Culture, Proliferation, Benzyl-adenine, Naphthalene Acetic Acid
INTRODUCTION
Some citrus cultivars such as Washington navel sweet orange have abiotic pollen, which produce
parthenocarpy fruits without pollination. Tissue culture in citrus is used in order to evaluate rootstocks,
economical production as well as to eliminate virus and virus-like pathogens. In order to eliminate virus
and virus-like pathogens is utilized embryo culture, ovule culture and shoot tip grafting in vitro
conditions. At present, virus and virus-like diseases is the most dangerous problem for citriculture in all
citrus growing regions that leading to sever yield reduction and dead of plants. Now, to remove these
pathogens in herbaceous plants is used meristem tip culture and thermotherapy (Cambra et al., 2000).
Rapid cloning of superior genotypes via in vitro adventitious shoot proliferation is used for many fruit
trees. The production of in vitro plants directly from shoot tips proliferation, nodal stem segments,
epicotyl and root segments is also reported in sweet orange, citron and lime, mandarin and Citrus mitis
(Paudyl and Haq, 2000).
Karimi et al., (2012) in order to study the regeneration condition of Mazandaran native sweet orange,
cultured epi- and hypo-cotyl in MS medium supplemented with BA (0, 0.5, 1, 2 and 3 mgl-1) and IBA (0,
0.2, 0.5 and 1 mgl-1 and reported that the best results was relative to BA 1 mgl-1 without IBA. The greatest
regenerated plantlets also obtained from above medium. The maximum enhancement of stem length in
regenerated plantlets was observed in medium containing 2 mgl-1 BA and 0.2 mgl-1 IBA. In relation to
explants type, epicotyls were better than hypocotyls in all treatments. Habibi and Amiri (2013) in
evaluation the physiologic reaction of two citrus rootstocks to salinity in vitro condition utilized MS
medium containing 8.9 µm BA and 0.5 µm NAA. Paudyl and Haq (2000) to determine the rate of shoot
proliferation of Pummelo (Citrus grandis L. Osbeck) used MS medium supplemented with various
concentrations of BA and TDZ, singly or in combination with NAA. The response of explants to all
concentrations of TDZ was very poor. The most adventitious shoots per explant (average 5.2) were
obtained on medium supplemented with 1.8 μM BA. NAA was superior to indole butyric acid (IBA) for
in vitro root induction. Over 75% of the shoots developed roots when transferred to half-strength MS
medium with 1.3, 2.7, or 5.4 μM NAA. Duran-Vila et al., (1989) in Morphogenesis and tissue cultures of
© Copyright 2014 | Centre for Info Bio Technology (CIBTech) 244Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231– 6345 (Online) An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm 2015 Vol. 5 (1) January-March, pp. 244-248/Jahromi and Dardan Research Article three citrus species reported that The optimal concentrations of NAA to induce root formation on stem segments were 10 mg l-1 for sweet orange and lime, and 3 mg l-1 for citron. The optimal BA concentration for shoot and bud proliferation was 3 mg l-1 for sweet orange and citron, and 1 mgl-1 for lime. Callus initiation was accomplished in a culture medium containing 10 mgl-1 NAA and 0.25 mgl-1 BA. Tao et al., (2002) observed a multiplication rate of 5–7 shoots from shoot tip culture on MS medium with 0.89 μM BA. Roots developed when regenerated shoots were cultured on MS medium with 9.84 μM IBA and 5.37 μM NAA. MATERIALS AND METHODS Plant sample was supplied from a Washington navel sweet orange in the garden in Jahrom city. To prepare explants, young 20-cm shoots were cut and transferred to tissue culture laboratory. Following elimination of the leaves, small shoots having bud with 1-1.5 cm length were cut by scissors and were disinfected by sodium hypochlorite 1% for 30 min and then were washed 3 fold by distilled water. To establish and proliferation shoot of Washington navel orange was used Murashige and Skoog medium (MS) containing 30 gl-1 sucrose and 7.5 gl-1 agar. Benzyl-adenine (BA) in the concentrations of 0.5, 1, 1.5, 2, 2.5 and 3 mgl-1 and Naphthalene acetic acid (NAA) in the concentrations of 0, 0.5, 0.75, 1, 1.25 and 1.5 mgl-1 were added to the medium and the characteristics such as shoot number and length, leaf number and its dropping as well as quality of shoot growth were recorded. After culturing explants in the perti dishes containing medium, were transferred them to culture room and were kept them for 4-6 weeks. This study was done in completely randomized design with 3 replications. Data was analyzed by using SAS software and the means were compared by Duncan’s multiple range test (DMRT). RESULTS AND DISCUSSION The results of anova table (table 1) showed that single effects of BA and NAA as well as interaction effect of BA × NAA were significant (p
Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231– 6345 (Online)
An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm
2015 Vol. 5 (1) January-March, pp. 244-248/Jahromi and Dardan
Research Article
Table 2: Mean comparison of interaction between BA × NAA on shoot number
NAA (mgl-1) 0 0.5 0.75 1 1.25 1.5
BA (mgl-1)
0.5 1.30jk 1.41jk 1.67jk 2.00ijk 1.00k 0.95k
defg fgh ghi
1 4.35 3.31 3.00 2.00ijk 1.65jk 1.00k
hij c c
1.5 2.29 7.73 7.36 5.71d 4.03efg 2.36hij
2 3.69efg 12.42a 7.35c 5.70d 5.70d 4.65de
defg b c
2.5 4.37 10.00 7.71 5.69d 5.04de 4.01efg
c defg defg
3 7.35 4.35 4.35 4.00efg 3.67efg 3.36fgh
Means having similar letters have not significant difference together (pIndian Journal of Fundamental and Applied Life Sciences ISSN: 2231– 6345 (Online)
An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm
2015 Vol. 5 (1) January-March, pp. 244-248/Jahromi and Dardan
Research Article
In relation to leaf dropping, the highest and the lowest leaf dropping was observed in medium containing
1.5 mgl-1 BA + 0.5 or 0.75 mgl-1 NAA (0.60 and 0.65 respectively) and 0.5 mgl-1 BA + all concentrations
of NAA (without dropping) respectively (Table 5).
Table 5: Mean comparison of interaction between BA × NAA on leaf dropping
NAA (mgl-1) 0 0.5 0.75 1 1.25 1.5
BA (mgl-1)
0.5 0.0c 0.0c 0.0c 0.0c 0.0c 0.0c
b b b b
1 1.0 1.0 1.0 1.0 1.0b 1.0b
b a a b
1.5 1.0 1.60 1.65 1.0 1.0b 1.0b
b b b b
2 1.0 1.0 1.0 1.0 1.0b 1.0b
b b b b
2.5 1.0 1.0 1.0 1.0 1.0b 1.0b
b b b b
3 1.0 1.0 1.0 1.0 1.0b 1.0b
Means having similar letters have not significant difference together (pIndian Journal of Fundamental and Applied Life Sciences ISSN: 2231– 6345 (Online) An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm 2015 Vol. 5 (1) January-March, pp. 244-248/Jahromi and Dardan Research Article Habibi F and Amiri MA (2013). Evaluation the physiological responses of two citrus rootstocks against salinity stress in vitro culture. Journal of Horticultural Sciences 27(3) 350-357 (In Persian). Jain A, Kantia A and Kothari SL (2001). De novo differentiation of shoot buds from leaf-callus of Dianthus caryophyllus L. and control of hyperhydricity. Scientia Horticulturae 87 319-326. Karimi MR, Dehestani Kalagar A and Jannatabadi AA (2012). Improving tissue culture and direct regeneration of Siavorz sweet orange in order to gene transfer. M.Sc Thesis of Agricultural Engineering Trend of Agricultural Biotechnology, Islamic Azad University of Sabzevar (In Persian). Khoskhui M (1998). Techniques of Plant Tissue Culture for Horticultural Plants (Gardening) (Shiraz University Press) (In Persian). Lesani H and Mojtahedi M (2000). Basic of Plant Physiology (Tehran University Press) (In Persian). Paudyl KP and Haq N (2000). In vitro propagation of pummelo (Citrus grandis L. Osbeck). In vitro Cellular & Developmental Biology - Plant 36(6) 511-516. Shahsavar A (2004). Comparison of different citrus rootstocks for micro-grafting. Journal of Iranian Horticultural Sciences and Techniques 5(2) 109-116 (In Persian). Shahsavar A and Khoshkhoi M (1994). The effect of several variables on shoot tip grafting of mandarin onto citrange. Iranian Agricultural Research. Tao H, Shaolin P, Gaofeng D, Lanying Z and Gengguang L (2002). Plant regeneration from leaf- derived callus in Citrus grandis (Pummelo): Effects of auxins in callus induction medium. Plant Cell, Tissue and Organ Culture 69(2) 141-146. © Copyright 2014 | Centre for Info Bio Technology (CIBTech) 248
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