Strength MS Salts Research Articles

MICORPROPAGATION OF VIBURNUM OPULUS (ROSEUM) BY USING SINGLE NODES

The present study was carried out to investigate the micropropagation of viburnum opulus (Roseum) by using single nodes with axillary buds excised from soft cuttings using full strength MS salts, 30g.l-1 sucrose, 7g.l-1 Agar and different concentrations of plant growth regulators in culture medium. Results of the experiment at initiation stage revealed that the culture of single nodes of viburnum on a medium containing 4 mg.l-1 BA gave the highest number of shoots (2.21 shoots/explant). Concerning the interaction, a nutrient medium containing (2 mg.l-1 BA+ 0.4 mg.l-1 IAA) gave the highest values of mean number of shoots, leaves and length of new shoots (1.61 shoots/explant, 2.01 leaves/explant, 1.61 cm respectively). At shoot multiplication stage, the addition of 2.0 mg.l-1 from both BA and kinetin gave the highest number of shoots per explant estimated at 2.65 and 3.31 shoots/explant respectively at root formation stage; the interaction treatment of full MS salt strength with the use of 0 .5 mg.l-1 NAA gave the highest rooting percentage reaching 85.5 %. The highest number of roots (18 roots/ explant) was recorded as well from the combined treatment of full MS salt strength with the use of 0.5 mg.l-1 NAA. While the longest roots (6.65 cm) were recorded from the interaction treatment of half salt strength and 0.50 mg.l-1 IBA..

Biomass and Flavonoid Production of Gynura procumbens Adventitious Roots Influenced by MS Salt Strength and Nitrogen Source in a Balloon-type Bubble Bioreactor

Biomass and Flavonoid Production of Gynura procumbens Adventitious Roots Influenced by MS Salt Strength and Nitrogen Source in a Balloon-type Bubble Bioreactor

Colchicine induced polyploidy in Common Ice plant Mesembryanthemum crystallinum L

The aim of this research was to obtain optimum concentration with mutation breeding techniques by using colchicine for polyploid induction in the common ice plant Mesembryanthemum crystallinum L. The common ice plant Mesembryanthemum crystallinum L wild type seed were soaked in five levels of colchicine concentration: 0% (control), 0.01%, 0.05%, 0.10% and 0.20% for 24 hours. Seeds were germinated in petri dish using growth medium with compound of 1.15 gr ml half strength MS salts, 0.5 ml B5 vitamins, 0.2% (w/v) Sucrose 1gr, 25 mg L−1 myo-inositol and Agar. After 15 days, seeds germinated and then the young plant were transferred to a fresh medium soil. The observation parameters consisted of leaf thickness, width of the leaf, and lenght of the leaf, calculated from 1st month until 8th month. The observational data obtained were analyzed in variance and if the treatment has significant effect, then the follow up test will be done with DMRT test at 95% confidence level. For polyploidy analysis was conducted using Flow Cytometry. The results show that colchicine can affect the vegetative growth and the number of chromosomes from Mesembryanthemum cristallinum L. plants. The colchicine treatment of 0.10% was the best treatment compared to other treatments in the parameters of the length, width and thickness of the leaves. Use of colchicine treatment of 0.10% changed the chromosome number of the Mesembryanthemum cristallinum L. plant from diploid (2n = 18) to Octaploid (8n = 108).

Micropropagation and Somatic Embryogenesis Induction of Gardenia jasminoides Plants

Gardenia jasminoides Ellis is an evergreen plant with fragrant creamy white flowers and glossy, darkgreen leaves, belongs to family Rubiaceae and member of the genus Gardenia. The current study aimed to afford a new technique for in vitro propagation and callus induction of G. jasminoides by using single nodes excised from soft cuttings using full strength MS salts supplemented with 30g/l sucrose, 4g/l gelrite and different concentrations of plant growth regulators. Generally, the current study showed that NAA and BA at 0.0 and 1.0 mg/l, respectively gave rise to the best results for initiation stage. Meanwhile, fortified the basal MS medium with BA and NAA at 2.0 and 0.250 mg/l, consecutively; resulted in the best results with respect to multiplication stage. Regarding rhizogenesis, the neoformed shoots produced from vegetative multiplication stage were rooted successfully upon cultured on MS medium supplemented with 1.0 mg/l IBA and NAA at either 0.250 or 0.500 mg/l, whereas the highest number of roots was recorded besides callus induction. Regarding embryonic callus induction, adding 2,4-D at 1.0 mg/l; enhanced embryonic callogenesis. Neoformed plantlets were acclimatized ex vitro and in vivo vigorously in mixture of perlite and peatmoss at (1:1, v/v), which resulted in the highest percentage value of survival / percent (100%) and successfully flowered and showed true-to-type plants ex vitro.

Improving In Vitro Somatic Embryos Production of Medjool and Khalas Date Palm Cultivars via Modification of Ammonium and Potassium Nitrate

Date palm is one of the most economically powerful fruit tree grown in Egypt. Although Egypt tops the list of date's product in the world. Egyptian date palm industry suffers from many constraints. Low-quality varieties and limited conventional propagation methods are the main obstacles. Extension of date palm plantations in various areas of Egypt and replanting trees to compensate loss due to diseases or human factors are reduced expected to lack adequate planting materials. The knowledge of the interplay between some fundamental medium parameters allows not only the optimization of the micropropagation system but also gives models to investigate and rationalize the process of induction and development of somatic embryogenesis itself. In vitro growth and development of produced embryos and plantlets has been improved by using of full MS salt strength. MS medium modification of some nutrients concentrations, i.e. NH4NO3(1237.5 mg/l) which recorded (13.20 and 9.60 embryos/explant for Medjool and Khalas respectively) at the end of three subculture. KNO3 which gave a higher score of mature somatic embryos (14.40 and 10.00 embryo/culture for Medjool and Khalas respectively) was obtained with 1425 mg/l KNO3 after three subcultures as compared to those obtained from any other treatment combination. The morphological response of somatic embryos production is controlled by some internal factors that fall under the influence of the genetic make-up of the plant and specialized for each genotype that is responsible for the production of somatic embryos (Medjool and Khalas cultivars). The genetic make-up is a decisive factor during somatic embryos production. There are differences between the two cultivars named, Medjool and Khalas. The produced somatic embryos at germination stage were transferred from the two experiments namely ammonium nitrate and potassium nitrate to multiplication and rooting stages for more in vitro growth for 12 weeks at three re-cultures. Individual shoots were cultured on modified MS basal medium in addition to IBA (1.0 mg/l), sucrose (30 g/l) and solidified with phyto-agar (8.0 g/l) for more in vitro growth and development. In vitro plantlets were transferred to acclimatization stage in plastic pots 5*18 cm diameter containing peatmoss, perlite and washed sand at equal volume. In vitro date palm plantlets produced from rooting stage grow well in the greenhouse during acclimatization stage without morphological abnormality. The growth and development of Medjool cultivar were better than Khalas during in vitro culture and ex vitro acclimatization.

IN VITRO DIRECT AND INDIRECT PROPAGATION OF BLACKBERRY (RUBUS SP.)

In vitro direct and indirect propagation of blackberry (Rubus sp.) were studied. Blackberry explants are easily oxidized, so the uses of the running tap water for washing about one hour before sterilization allowed avoiding the oxidation. The disinfection process was represented using different concentrations of Clorox (10, 15, 20%) with exposure times (5 and 10 min.), the best treatment being achieved by the concentration of (10% for 5 min.) for shoot tips and (5% for 5min) for leaves disinfectant. A normal growth and development occurred only in the BA of the multiplication, but at Kin or TDZ decreased in the shoots growth was induced. Maximum volume of callus induction and callus % were produced at TDZ or 2mg/l NAA or 2,4-D each alone from leaf explants. Addition of 2.0mg/l TDZ into media increased the callus induction from in vitro leaves than ex vitro leaves and having the best response. The highest responses of organogenesis obtained from In-vitro leaves than ex vitro leaves. The increased rooting of shoots appeared when MS reduced to ½ salts strength and produced highest leaves number, plant length (cm) and roots number. While, MS reduced to ¼ salts strength resulted in tallest of roots (2.72 cm). The rooting of plants derived from ½ MS salt strength medium give highest leaves number, plant length, roots number and roots length than other strengths of MS after two months from acclimatization in greenhouses.

Direct In Vitro Regeneration of Fig (Ficus carica L. CV. Benaty) from Apical and Lateral Buds

Benaty fig plant was successfully propagated by tissue culture technique and areproducible and reliable micropropagation protocol was developed through thisinvestigation. At initiation stage, healthy aseptic cultures from lateral and terminal budexplants of about 85% were achieved when the explants were treated with 0.15% HgCl 2for 7 minutes. A combined solution of ascorbic acid and citric acid (100 and 150 mgl -1 )was successfully used to overcome the problem of phenolic compounds exudation fromthe explants into the culture media. At shoot multiplication stage, the addition of 2.0 mgl -1 from both BA and kinetin gave the highest number of shoots per explant estimated at2.66 and 3.33 shoots/ explant respectively. As well as, activated charcoal promotedshoots multiplication in all multiplication parameters. The highest number of shoots perexplants (5 shoots/ explant) was obtained on MS medium containing 4.0 gl -1 AC. At rootformation stage, the interaction treatment of full MS salt strength with the use of 0.25mgl -1 NAA gave the highest rooting percentage reaching 87.5%. The highest number ofroots (18.5 roots/ explant) was recorded as well from the combined treatment of full MSsalt strength with the use of 0.25 mgl -1 NAA. While the longest roots (6.66 cm) wererecorded from the interaction treatment of half salt strength and 0.50 mgl -1 IBA. Theaddition of activated charcoal to the rooting culture medium decreased the rootingpercentage from 87.5% at control treatment to 50% and 62.5% when 2.0 and 4.0 gl -1 ACwere respectively added. The in vitro propagated fig plantlets were gradually moved in asuccessful way from lab to field conditions with 95% survival rate.

Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers

A reliable and reproducible protocol for in vitro regeneration has been developed for Alhagi maurorum, a rare and medicinally important plant of family fabaceae. MS medium with BAP (2.0 mg l−1) proved to be the best for shoot bud induction from nodal segments. The rate of shoot multiplication was found to be influenced by a number of factors, viz., media composition, plant growth regulator’s type and concentration, successive transfer of mother explant for different passage, culture vessels and gelling agents. Modified MS medium (modified having nitrates reduced to half) solidified with 0.14 % gelrite and containing BAP (0.5 mg l−1), IAA (0.1 mg l−1) and additives was found optimum for shoot multiplication which gave rise to maximum number of shoots (33.5 ± 3.43 per culture vessel). The in vitro regenerated shoots were rooted under both in vitro (on half strength MS salts with 1.0 mg l−1 IBA + 100 mg l−1 activated charcoal) as well as ex vitro (on sterile soilrite by treating shoot base with 250 mg l−1 each of IBA and NOA for 4 min in green house) conditions. Thereafter, the in vitro and ex vitro rooted plantlets were hardened under green house conditions with 70 and 90 % survival rate, respectively. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity of seven tissue cultured plantlets growing in green house condition with mother plant. The amplification products were monomorphic across all the seven micropropagated plants as well as mother plant produced by all SCoT, ISSR and RAPD primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for A. maurorum. To the best of our knowledge, this is the first report on micropropagation and genetic homogeneity assessment of A. maurorum, which can be applied for large scale multiplication of elite genotypes of A. maurorum.

High frequency multiplication of shoots using axillary buds for production of elite lines of <i>Stevia rebaudiana</i>

An efficient repeatable protocol has been developed for rapid clonal multiplication of an endemic, perennial herb Stevia rebaudiana using juvenile axillary buds from in vivo grown seedlings. MS basal medium supplemented with BAP (8.88 μM) and NAA (5.37 μM) was found to be the most suitable medium for initiation of shoots with 92% response. The repeated subculture resulted in high-frequency shoot multiplication (9.82 ± 1.93) at the average rate of 3-fold per subculture. The axenic multiple shoots were rooted ex-vitro on horticultural grate soilrite mix-peat:perlite:vermiculate (1:1:1 v/v) with half strength MS salts fortified with NAA (5.37 μM). The complete plantlets were successfully established in soil with 86% survival frequency. The statistical analysis of the data pertaining to multiple shoots and root formation revealed significant difference between and within the treatments.

Propagação e conservação in vitro de vetiver

O vetiver (Chrysopogon zizanioides) é um capim perene com características físico-químicas e agronômicas importantes que o torna uma espécie destaque. De suas raízes é extraído um óleo essencial utilizado amplamente na produção de perfumes e devido à sua baixa volatilidade, como fixador de odor. Esse trabalho teve como objetivo desenvolver um protocolo de propagação e de conservação in vitro de vetiver. Para o ensaio de multiplicação in vitro testaram-se combinações dos reguladores de crescimento BAP e ANA. Na aclimatização foram testados substratos contendo pó de coco e/ou vermiculita suplementado com calcário, fertilizante NPK (3-12-6) e concentrações dos sais do meio MS. Nos ensaios de conservação in vitro foram avaliadas as temperaturas de 18 e 25°C, reguladores osmóticos (sacarose, manitol e sorbitol), o inibidor de crescimento ABA e diferentes concentrações dos sais MS. Verificou-se que a micropropagação de vetiver UFS-VET003 é promovida em meio MS líquido acrescido de 3,33 mg L-1 de BAP, sendo a etapa de enraizamento das brotações proporcionada em meio MS básico, também líquido. Para aclimatização recomenda-se o substrato PBC. A conservação in vitro é possível em meio MS semissólido com 25% dos sais MS e temperatura de 18°C por um período de 270 dias.

둥근마(Dioscorea opposita Thunb.)의 기내증식을 위한 배지조건

Abstract - This study was carried out to determine the effect of medium solidity, salt strength, sugar and nitrogen sources, and pH levels on in vitro multiplication of pathogen-free yam ( Dioscorea opposita Thunb.). Liquid medium was more effective in the growth of plant height, fresh weight, and formation of microbulb than the solid medium. Optimal condition for plant fresh weight, growth, and multiplication axillary bud was in 1MS salt strength with 60 g · L -1 sucrose and half strength of KNO 3. Optimal condition for microbulb formation was ½ MS salt strength supplemented with glucose 60 g · L -1 and half strength of KNO 3 . The number of leaves and nodes were sharply increased from 2 to 5 weeks, whereas plant fresh weight was steadily increased from 3 to 11 w eeks after inoculation. Microbulbs were formed at 2 weeks after inoculation and continuously increased until 12 weeks. Key words - Axillary bud, Explant, Nodal stem, Salt strength, Tuberization * 교신저자(E-mail): skwon@andong.ac.kr

MICROPROPAGATION OF GARDENIA Gardenia jasminoides

The present study was carried out to study the micropropagation of Gardenia (Gardenia jasminoides) by using single nodes excised from soft cuttings using half strength MS salts, 30gl-1 sucrose, 7gl-1 Agar and different concentrations of plant growth regulators in culture medium. Results of the experiment at initiation stage revealed that the culture of single nodes of Gardenia on a medium containing 5mgl-1BA gave the highest number of shoots (2.2 shoots/explant). Concerning the interaction, a nutrient medium containing (2 mgl-1 BA+ 0.4 mgl-1 IAA) gave the highest values of average number of shoots and leaves and length of new shoots (1.6 shoots/explant, 2 leaves/explant, 1.6cm respectively). At vegetative multiplication stage, the results showed that the medium supplemented by 2 mgl-1 BA gave the highest values of average number of shoots and leaves and length of new shoots (2.20 shoots/explant, 3.20 leaves/explant, 1.80cm respectively), whereas the medium supplemented by 0.3 mgl-1 IAA gave the highest number of shoots and length of new shoots. In order to promote the shoots produced at vegetative multiplication stage to root, it was noticed that the treatment of 8mgl-1NAA gave the highest percentage of rooting (90%) and the medium supplemented by 12mgl-1 IAA gave (100%) rooting. Plantlets obtained were transferred to pots and acclimatized with 95 % success.

In vitro micro propagation of “sand pear” Pyrus pyrifolia (Burm. f.) Nakai

This study was undertaken to standardize protocols for in vitro explant establishment, shoot multiplication and in vitro rooting for Pyrus pyrifolia, growing in the Kashmir valley. The explant used was nodal cuttings of 2–3cm size with one or two axillary buds. The highest explant establishment (over 90%) was observed during spring while the lowest (60%) explant establishment frequency was observed during winter season. The shoots sprouted from the axillary buds were excised and subcultured on MS basal medium. The highest shoot multiplication rate (130 shoots from a single nodal cutting) was obtained on MS medium supplemented with BAP 2.2 mg·L−1 and kinetin 1 mg·L−1 after four weeks. The shoots were inoculated for rooting in root induction medium of half strength MS salts supplemented with plant growth regulators like IBA, IAA, NAA, and 2, 4-D. However, the best rooting response (8.50 roots per shoot) was obtained in combination of hormones (IBA + NAA) 0.25 mg·L−1 each. The rooted micro shoots were acclimatized under decreasing humidity regime (95% RH to 75% RH over a period of 5±1 weeks). The highest ex vitro plantlet survival percentage (56.50%) of Pyrus pyrifolia was observed on sand + soil (1:1 v/v).

In vitro propagation of Dahlia variabilis

Several experiments were carried out to propagate Dahlia variabilis throughout tissue culture during period of 2004 to 2005. In tissue culture lab.- Horticulture- College of Agriculture- University of Baghdad. The studies included the effects of type of explants, plant growth regulators, adenine sulphate and seed extraction of Johnson grass. The results indicated that 100% response was achieved when apical shoots and single nods explants were cultured on free MS medium. The highest number of shoots/explant (4.00) were achieved when shoots were cultured on MS medium supplemented with 2µM of both BA and IAA. Adding of 60 mg/l adenine sulphate to the medium increased shoots number to 6.80 with 7.27 cm length. The addition of 300 ml/l of alcoholic extract from immature seeds of Johnson grass increased shoot number ( 2.50 shoots/explants), while modifying the medium with 200 ml/l of water extract of mature seeds of Johnson grass was superior on shoots length (7.76 cm). The highest root number/explant (5.00) and length (5.00 cm) were achieved when MS medium supplemented with 3 µM IBA. Shoots cultured on half MS salt strength supplemented with 45 g/l sucrose and 3 µM of IBA gave the highest number and length of roots (8.00 roots/explants, 10.40 cm). A 100% success of acclimatized of plantlets was achieved when they were planted in 1:1 peatmoss and loam under controlled humidity.

IN VITRO PROPAGATION OF POTENTILLA FULGENS HOOK (BAJRADANTI) – A HIGH VALUE MEDICINAL HERB FOR COMMERCIAL CULTIVATION

A rapid in vitro clonal propagation protocol is standardized for Potentilla fulgens, a high value medicinal herb of Indian Himalayas. The best promising results were obtained by culturing the tender petiole segments for adventitious shoot proliferation in Murashige and Skoog basal medium supplemented with a combination of benzyl adenine (BA, 1.5 mg/l) and indole acetic acid (IAA, 0.5 mg/l). The best rooting (92.3%) was obtained while culturing the in vitro raised shoots in MS medium modified with IAA (1.5 mg/l). Rooted plantlets were hardened in sterilized peat moss in plastic pots. Spray of quarter strength MS salts solution on alternate days is found reducing the mortality of plants considerably during the hardening process. As a result it is possible to obtain 30-35 rooted plant from a single explant within three months, and hence is useful for commercial cultivation.

MICROPROPAGATION OF GARDENIA Gardenia jasminoidesBY USING SINGLE NODES

The present study was carried out to study the micropropagation of Gardenia (Gardenia jasminoides) by using single nodes excised from soft cuttings using half strength MS salts, 30gl-1 sucrose, 7gl-1 Agar and different concentrations of plant growth regulators in culture medium. Results of the experiment at initiation stage revealed that the culture of single nodes of Gardenia on a medium containing 5mgl-1BA gave the highest number of shoots (2.2 shoots/explant). Concerning the interaction, a nutrient medium containing (2 mgl-1 BA+ 0.4 mgl-1 IAA) gave the highest values of average number of shoots and leaves and length of new shoots (1.6 shoots/explant, 2 leaves/explant, 1.6cm respectively). At vegetative multiplication stage, the results showed that the medium supplemented by 2 mgl-1 BA gave the highest values of average number of shoots and leaves and length of new shoots (2.20 shoots/explant, 3.20 leaves/explant, 1.80cm respectively), whereas the medium supplemented by 0.3 mgl-1 IAA gave the highest number of shoots and length of new shoots. In order to promote the shoots produced at vegetative multiplication stage to root, it was noticed that the treatment of 8mgl-1NAA gave the highest percentage of rooting (90%) and the medium supplemented by 12mgl-1 IAA gave (100%) rooting. Plantlets obtained were transferred to pots and acclimatized with 95 % success.

In vitro direct regeneration in mint from different explants on half strength MS medium.

In vitro shoot regeneration of Mentha piperita L. was investigated using shoot meristems, node, internode and petiole explants. Shoot regeneration was achieved on ½ strength MS salts and vitamins supplemented with various concentrations of BAP, Kin alone or with NAA. Shoot meristems and nodal segments showed variation in the frequency of shoot regeneration with single to multiple shoots. However, internode and petiole explants were not potent and failed to regenerate shoots at some of the tested plant growth regulators concentrations. Variable frequency of rooting was recorded at four different concentrations of auxins. The rooted plants were successfully acclimatized to in vivo conditions in the greenhouse. Key words: Mentha piperita, plant hormone, organogenesis, shooting.

Direct Shoot Bud Formation and Tuberization from Aseptically Cultured Root Tubers of Calla Lily (Zantedeschia aethiopica L)

We describe here the development of a micropropagation protocol for mass multiplication of Zantedeschia aethiopica by using root tubers as explant. The surface sterilized root tubers produced five to six shoot-buds on semi-solid Murashige and Skoog’s (MS) medium with 10.0 mg l−1 of 6-benzylaminopurine (BAP) and additives (50.0 mg l−1 of ascorbic acid; 25.0 mg l−1 each of adenine sulphate, L-arginine and citric acid). The cultures were multiplied by sub-culture of individual shoot bud produced in vitro and clumps of shoot buds generated in vitro in cultures on MS medium containing 3.0 mg l−1 of BAP and additives. Further multiplication of propagules was achieved through tuber formation along with amplifying shoots on MS medium with 5.0 mg l−1 of BAP. The micropropagated shoots were rooted both in vitro as well as ex vitro. Cent percent of the cloned shoots rooted in vitro within 15–18 days on hormone-free 1/2 strength MS salts with 200.0 mg l−1 of activated charcoal. Alternatively 95–100% shoots rooted ex vitro under greenhouse conditions on soilrite after pulse-treatment with 500.0 mg l−1 of Indole-3-butyric acid (IBA) or β-naphthoxyacetic acid (NOA) for 300 sec. The cloned plants were hardened in the greenhouse. The hardened plants were transplanted to soil for further acclimatization.

In vitro direct regeneration in mint from different explants on half strength MS medium.

In vitro shoot regeneration of Mentha piperita L. was investigated using shoot meristems, node, internode and petiole explants. Shoot regeneration was achieved on 1⁄2 strength MS salts and vitamins supplemented with various concentrations of BAP, Kin alone or with NAA. Shoot meristems and nodal segments showed variation in the frequency of shoot regeneration with single to multiple shoots. However, internode and petiole explants were not potent and failed to regenerate shoots at some of the tested plant growth regulators concentrations. Variable frequency of rooting was recorded at four different concentrations of auxins. The rooted plants were successfully acclimatized to in vivo conditions in the greenhouse.

Nutrient factors affecting in vitro cultivation of Stevia rebaudiana

The present work was carried out to display in details the whole protocol for Stevia rebaudiana propagation through tissue culture techniques to produce and introduce Stevia rebaudiana plants as a new sweetener crop to Egyptian agriculture. In order to maximize plant propagation efficiently via direct organogenesis, it is important to study the influence of plant nutrient medium and its components at different levels. The study included the effect of MS salt strength and major elements containing medium at different levels on the in vitro growth and development of Stevia rebaudiana The best nutrient medium was MS at full salt strength or modified MS major elements at these levels (NH4NO3 at 1237.5 mg/l, KNO3 at 950 mg /l, MgSO4 at 185 mg/l, CaCl2.2H2O at 440 Mg/l and KH2PO4 at 85 mg/l) and minor elements supplemented with 30 g/l sucrose, 2 mg/l glycine, 0.5 mg/l pyrodoxine, 0.5 mg/l nicotinic acid, 0.1 mg /l thiamine and solidified with 1.5 g/l agar + 1.5 g/l gelrite. The ex vitro plantlets were successfully acclimatized in planting medium containing peat, sand and vermoculite at equal volume. Hardening plants were cultivated under Sadat soil conditions (10 volume sand + 3 volume loam) which achieved good growth and these plants used as a source of vegetative propagation after 50 days by using terminal cuttings.