Volume 16(1), 220-224, 2012 JOURNAL of Horticulture, Forestry and Biotechnology www.journal-hfb.usab-tm.ro Establishment of in vitro multiplication biotechnology of the species Albizzia lebbeck L. Tudor Radu C.M. 1 * NRDIBH Stefăneşti Argeş *Corresponding author. Email: mihai_radutudor@yahoo.co.uk Abstract This paper aims to establish a in vitro biotechnology for Albizzia lebbeck species, because there was an increase in requests for such domestic plants and classic multiplying seed yield is below 50%, but also in the future to study if the concentration of substances used in pharmaceutical found in micro cuts are economically satisfactory. For this settled culture media for initiate cultures in vitro, multiplication and rooting. All utensils used in the in vitro multiplication were sterilized before each use and culture media were autoclaved. After each stage the plants were kept in growth chambers at a temperature of 240C, a 16-hour photoperiod and light intensity of 300 lux. The results obtained were good, so for initiation phase rates was 57 percent, for multiplication phase was obtained an average of 6 micro cuts / explant, and the rooting percentage was 84. The results recommend this technology for its use on an industrial scale. Key words in vitro culture, Albizzia lebbeck, initiation, multiplicatre, rooting In vitro cultivation of plant cells and tissues is a branch of biological sciences that provide modern research and production of planting material of high biological value. Over the years, this field has progressed rapidly, and can speak today of a set of techniques grouped under the name of plant biotechnology. Research in this paper, have used the results obtained to date using in vitro cultures to establish a rapid propagation biotechnology horticultural species poor in our country, but with market demand (Albizzia lebbeck). Concerns about biotechnology achievement of in vitro propagation of this species have been justified and that the importance of culture in terms of decoration, food, medicine, the inefficiency of traditional methods of propagation, the unsatisfactory results obtained in breeding by conventional methods. Materials and Methods In the first stage of culture in vitro was studied the influence of nutrient media composition on growth of Albizzia explants. Sterilization of instruments (tweezers, knives, cataract) was done in the oven at 120 C for two hours. The biological material consisted of meristems with 1-2 leaf primordial, which were collected in laminar air flow hood from terminal or axillary buds Disinfection of biological material was done in 94% ethyl alcohol for 10 minutes, after which he passed in calcium hypochlorite solution with a concentration of 6% for 20 minutes - the treatment of dormant buds. For alive buds and peaks in full growth, treatment was reduced by half (in time). Disinfection prior operation was washing the biological material with tap water plus a few drops of Domestos. Inoculii were puted in7 different variations of the composition of culture media in the macro, micronutrients, vitamins and growth regulators (Table 1). Before autoclaving the ph of the culture medium was adjusted to 5.6. Culture media were sterilized by autoclaving at a temperature of 1200C for 20 minutes. For explants growth phase were used tubes that were obturated with polyethylene. The growth chamber was provided optimal growth conditions of the explants (16 hours photoperiod, temperature range 21-250C). 220
Table 1 Composition of nutrient media used for growth of Albizzia explants Components (mg/l) V.1 V.2 V.3 V.4 V.5 V.6 V.7 Macroelements MS MS MS MS MS MS MS Microelementes MS MS MS MS MS MS MS Vitamine J J MS MS LS LS LS NaFeEDTA 32 32 32 32 32 32 32 BAP 1 0,5 1 0,5 1 0,5 0,7 AIA 0,2-0,2-0,2 - - Giberelic acid - 0,1-0,1-0,1 0,1 Naftilacetic acid - 1-1 - 1 1 Ascorbic acid - 5-5 - 5 5 Dextrose g/l 40 40 40 40 40 40 40 Agar g/l 7 7 7 7 7 7 7 Legend: MS = MURASHIGE - SKOOG (1962); J = JACQUIOT (1959) LS = LINSMAIER - SKOOG (1965) Before autoclaving the ph of the culture medium was adjusted to 5.6. Culture media were sterilized by autoclaving at a temperature of 1200C for 20 minutes. For explants growth phase were used tubes that were obturated with polyethylene. The growth chamber was provided optimal growth conditions of the explants (16 hours photoperiod, temperature range 21-250C). For the multiplication phase of in vitro propagation has been used as biological material, explants grown in the previous phase and passed on the following nutrient medium: - Mineral salts Murashige - Skoog (1962), vitamins Jacquiot (1959), 0.1 mg / l giberelic acid, 1 mg / l naftilacetic acid, 32 mg / l NaFeEDTA, 40 g / L glucose, 7 g / l agar. On this basic medium was tested citochininelor action BAP (2.0 mg / l), Kin (4.0 mg / l), 2iP (5.0 mg / l). Composition of nutrient media tested for multiplication faze of albizzia Components (mg/l) V.1 V.2 V.3 Macroelements MS MS MS Microelements MS MS MS Vitamins J J J naftilacetic Acid 1 1 1 Benzilaminopurine 2 - - Kinetin - 4-2iP - - 5 NaFeEDTA 32 32 32 Glucose 40.000 40.000 40.000 Agar 7.000 7.000 7.000 Legend: MS = Murashige - Skoog (1962); J = Jacquiot (1959); 2iP = N 6 - (2 - isopentyl) adenine. Albizzia multiplication phase was provided a 16-hour photoperiod, temperature 22-24 0 C and a light intensity of 3000 lux. Components (mg/l) Table2 To initiate research on in vitro rooting explants were taken from in vitro propagation phase. Composition of nutrient media tested for in vitro rooting of Albizzia lebbeck Variable factors A.1. A.2. A.3. A.4. Macroelements M&S 1/2 M&S 1/2 M&S 1/2 M&S 1/2 Microelements M&S 1/2 M&S 1/2 M&S 1/2 M&S1 /2 Vitamins LS LS LS LS NaFeEDTA 38,0 38,0 38,0 38,0 Dextrose 30.000 30.000 30.000 30.000 Agar 7.000 7.000 7.000 7.000 Giberelic acid 0,1 0,1 0,1 0,1 Indolilbutiric acid 0,2 0,4 0,6 0,8 Legend: LS = Linsmaier Skoog (1965) Vitamins LS (mg/l): Tiamine 0,4; Inozitol 100,0 Table 3 221
%explants grow For Albizzia the nutrient media were tested from the above table (Tab.2) and were provided the same aseptic conditions as in previous phases. During rooting, growth chamber, photoperiod was 16 hours, light intensity of 3000 lux and a temperature of 22-24 0 C. Results obtained Initiation phase Explants of Albizzia lebbeck growth was influenced by explant type and nutrient medium composition. Regarding the influence of explant type was found to have better behavior apical bud explants taken from the values recorded were between 8 and 57% depending on the composition of the nutrient medium. Explants taken from axillary buds were weaker behavior, increase the minimum explants 2% and maximum 43% of nutrient media tested, best results were obtained under the influence of phytohormones: 0,1 mg / l giberelic acid, 1 mg / l naftilacetic acid, 0.5 mg / l BAP maximum value of 57% was obtained by explants grown addition of these phytohormones in a basic culture media, salts Murashige - Skoog and vitamins formulated Jacquiot. Broths using a report that auxinic / citokinine of 0.2 mg / l indolilbutiric acid / 1 mg / l benzilaminopurină have ensured the growth of a smaller number of explants, explants grown 23% maximum is obtained by adding phytohormones those in rural basic Murashige - Skoog. Increasing concentrations benzilaminopurină from 0.5 mg / l (V.6) to 0.7 mg / l (V.7) led to higher rates of explants increased to 15 if the apical bud and axillary buds 8 for but the values are inferior to other alternatives. (Fig. 1). Albizzia explants showed no oxidative phenomena. 60 50 40 30 20 10 0 Muguri apicali Muguri axilari V.1 V.2 V.3 V.4 V.5 V.6 V.7 Culture media Fig. 1. The influence of culture media and explants tips over the grow of Albizzia explants Fig. 2. Albizzia lebbeck explant Multiplication phase The tested nutrient media for propagation of Albizzia lebbeck explants had good behavior. It should be noted that in all culture media variants appeared callus on explants base. The callus volum formed was bigger in the presence of kinetin and benzylaminopurine than with 2 ip. 222
% rooting Fig. 3 Citochine influence over callus appearance at albizia micro cuts Micro cuts formed on culture media V.1 and V.2 has presented callus not only at the base, but even on internodes parts. Regarding the process of organogenesis, only in variant 2iP were obtained micro cuts, propagation rate is 6 microlăstari / explant (Fig. 7). Fig.4. The influence of 2iP over the multiplication of Albizzia lebbeck explants Fig. 5.In vitro rooting of albizzia micro cuts Although microcuts elongation was lower as the callus was higher, however, Albizzia lebbeck microcuts have dimensions ranging from 1.5 to 4 cm. Rooting phase At Albizzia has been obtained in vitro rooting decreasing from 84% in 0.2 mg / l acid indolilbutiric variant from 40% in 0.8 mg / l acid indolilbutiric variant. Callus size formed at the base of micro cuts is increasing in the callus formed while auxine concentration is increasing. 90 80 70 60 50 40 30 20 10 0 0,2 mg/l 0,4mg/l 0,6mg/l 0,8mg/l IBA Fig. 6. The influence of AIA over Albizzia micro cuts rooting 223
Conclusions Best results of in vitro initiation phase were obtained under the influence of phytohormones: 0,1 mg / l giberelic acid, 1 mg / l naftilacetic acid, 0.5 mg / l BAP maximum value of 57% was obtained by explants grown addition of these phytohormones in a basic culture media, salts Murashige - Skoog and vitamins formulated Jacquiot. Regarding the process of multiplication, only in variant 2iP were obtained micro cuts, propagation rate is 6 micro cuts/ explant. At în vitro rooting phase had been obtained in vitro rooting 84% on variant with 0.2 mg / l acid indolilbutiric The results obtained in each phase recommende to use with success and at large scale this technology by businesses firms and individuals persons interested. Bibliography 2.Constantinescu, Gr., Haţieganu Elena 1983- Biologia moleculară a celulei vegetale. Editura medicală Bucureşti, 3.Danci M. 2007 Culturi in vitro si micropropagare Editura Eurobit Timisoara 4. Danci M, Danci Oana 2007- Gid practic de culturi in vitro Editura Eurobit Timisoara Dixon R.A. -1985- Plant cell culture a practical approach - Editura IRL Press 3. Eckart Haenchen - 2006 -Cultura trandafirilor Editura: M.A.S.T. 5. Enciclopedia Truffaut 2004 Gradini şi plante de interior Editura RAO 6.Gautheret R. J. - 1959 - La Culture des Tissus vegetaux. Masson et Cie, Paris; 7.Iliescu Ana Felicia 2002 Cultura arborilor şi arbuştilor ornamentali. Ed CERES, Bucureşti; 8.Stănică F 2004 Microînmulţirea plantelor horticole. Ed. INVEL Multimedia; 9.Teodorescu A., Marinescu L. 1999 Tehnici de culturi in vitro pentru înmulţirea şi ameliorarea plantelor. Ed. Tiparg. 1. Boxus P. 1995 - Biotehnologies Vegetales UNISAT Universite audiovisuelle francophone 224