Full-strength Murashige And Skoog Research Articles

An improved micropropagation of Spilanthes acmella L. through transverse thin cell layer culture

An efficient and improved in vitro propagation system for Spilanthes acmella L. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration from tTCL nodal segments was affected by concentrations of plant growth regulators and orientation of the explant. MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium with 5.0 mg dm−3 BAP was optimal for shoot regeneration. Upon this medium, the explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (about 97%), and the highest number of shoots (31.5) per explant. The intact node (1.0–1.5 cm) cultured on the same medium had significantly lower shoot multiplication ability with only 4.5 shoots per responsive explant. As compared to BAP alone, the combination of BAP and Kin or NAA did not have positive effects on shoot multiplication from tTCL nodal segments. Rooting of shoots was achieved on growth regulator free full-strength MS medium. Plantlets were transplanted into soil with 90–100% survival rate.

Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species

This article demonstrates the plantlet regeneration from alginate-encapsulated shoot tips of Spilanthes acmella. Shoot tip explants excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation for encapsulation of shoot tips was achieved using 3% sodium alginate and 100 mM calcium chloride. Maximum percent response for the conversion of encapsulated shoot tips into plantlets was obtained on growth regulator-free full-strength liquid MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium. The addition of MS nutrients in alginate matrix was found to have pronounced effect on shoot and root emergence from alginate beads. Encapsulated shoot tips could be stored at low temperature (4°C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully. The present synthetic seed technology could be useful in large-scale propagation as well as short-term conservation and germplasm distribution and exchange of Spilanthes acmella.

Direct shoot organogenesis from in vitro-derived mature leaf explants of pistachio

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl−1 IAA and 2 mgl−1 BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl−1 BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl−1 of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species.

In vitro regeneration of Leucaena leucocephala by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 100 mg dm−3 glutamine, 20.9 µM N6-benzylamino-purine (BAP) and 5.37 µM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm−3 myoinositol, 14.76 µM indole-3-butyric acid (IBA) and 0.23 µM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 40.28 µM NAA and 12.24 µM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 15.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 µM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 10.0 µM BAP, 2.5 to 5.0 µM IBA and 0.5 mM spermidine.

Micropropagation of Searsia dentata

A method for in vitro regeneration of Searsia dentata from nodal and shoot tip explants derived from mature trees is outlined. Nodal explants produced multiple shoots from the axis when cultured on Murashige and Skoog (MS) medium containing 3% sucrose supplemented with 0, 5, 7.5, 10, or 12.5 μM N6-benzyladenine (BA). An average of 5.3 shoots was obtained from nodal explants on 10 μM BA. For shoot tip explants, however, supplementation of α-naphthaleneacetic acid (NAA) with BA favored a caulogenic response. A maximum of 6.1 shoots were produced per shoot tip explant on MS containing 7.5 μM BA plus 5.0 μM NAA. The in vitro-regenerated shoots produced roots when transferred to full-strength MS medium containing 3% sucrose and 10 μM indole-3-butyric acid (IBA). The developed plantlets were transferred initially to a mist house. After an initial acclimatization period of 3–4 mo, plantlets were shifted to the greenhouse where they thrived for 9 mo. The standardized protocol for mass propagation of S. dentata should eliminate the dependence on natural stands of plants for traditional medicinal purposes, and will also serve as a means of conservation as the species is heavily overexploited.

In Vitro Propagation of "Jarilla" (Larrea divaricata CAV.) and Secondary Metabolite Production

An efficient protocol for the in vitro germination and propagation of Larrea divaricata CAV. (Jarilla) was established. To determine the effect of different growth regulators on the growth rates and phenol production, apical-node microshoots from in vitro germinated plantlets were incubated on the following media: 1) full-strength MS (Murashige and Skoog) salt medium with different ratios of alpha-naphthaleneacetic acid (NAA) and N(6)-benzyladenine (BA); 2) after pre-treatment with indolebutyric acid (IBA), transfer to MS medium of different inorganic salt strengths; and 3) full-strength MS salt medium with different ratios of sucrose and IBA. Successful microshoot rooting percentages were achieved by the second and third strategies, the highest being 87.5-100%. The maximum principal shoot length and node number obtained by the second strategy corresponded to the plantlets previously induced with 50 microM IBA, and grown on half- or full-strength MS salt media (7.03+/-0.93 and 9.86+/-1.07 cm, respectively) while in the third strategy the most efficient micropropagation medium was full-strength MS salt medium supplemented with 7.5 microM IBA: 3% (w/v) sucrose (7.05+/-1.08 and 7.0+/-1.51 cm, respectively). The phenol concentration was determined by analytical HPLC. The highest content of nordihydroguiaretic acid (NDGA) accumulated in microplants of L. divaricata cultivated on half-strength MS salt medium (35.90+/-3.82 mg/g DW). Reducing the MS medium salt concentration by half, in the absence of IBA, it resulted in a higher NDGA production. NDGA production was not sensitive to the variation of IBA concentration. The medium supplemented with 5% (w/v) sucrose and 2.5 microM IBA induced not only a higher NDGA production but also a higher quercetin production.

Micropropagation of Pongamia pinnata through enhanced axillary branching

A complete protocol is presented for the first time for the micropropagation of Pongamia pinnata, a biofuel tree, using cotyledonary nodes derived from axenic seedlings. Multiple shoots were induced in vitro from nodal segments through forced axillary branching. Murashige and Skoog (MS) medium supplemented with 7.5 μM benzylaminopurine (BAP) induced up to 6.8 shoots per node with an average shoot length of 0.67 cm in 12 d. Incorporation of 2.5 μM gibberellic acid (GA3) in the medium during the first subculture after establishment and initiation of shoot buds significantly improved the shoot elongation. Single use of GA3 during the first subculture eliminated the need for prolonged culturing on BAP medium. Further use of GA3 in the medium was not useful. Shoot culture was established for at least two subcultures without loss of vigor by repeatedly subculturing the original cotyledonary node on shoot multiplication medium followed by shoot elongation medium after each harvest of the newly formed shoots. Thus, from a single cotyledonary node, about 16–18 shoots were obtained in 60 d. Shoots formed in vitro were rooted on full-strength MS medium supplemented with 1.0 μM indole butyric acid (IBA). Plantlets were successfully acclimated, established in soil, and transferred to the nursery.

Effect of nitrogen salts on the growth of Ceratonia siliqua L. Shoot cultures

Effects of reduced nitrogen salt nutriton on the growth, lenticel hypertrophy and anthocyanin accumulation of carob (Ceratonia siliqua L.) shoot cultures were investigated in conditions of light and darkness. Growth of shoot cultures was not significantly affected until nitrogen salts were reduced to less than ? of full-strength MS (Murashige and Skoog, 1962) values. Cultures in darkness were less affected and their main shoots even increased in length. Appearance of hypertrophied lenticels in light decreased, while in darkness they were absent in all treatments. Reduced nitrogen salt nutrition strongly affected anthocyanin accumulation of shoots and leaves, which greatly increased in both light and darkness. .

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N6-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.

Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides

Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l −1 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l −1 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l −1 gibberellic acid (GA 3). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3 l balloon type bubble bioreactor (BTBB) containing 2 l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3 l BTBB containing 2 l MS medium with 4 mg l −1 GA 3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials.

A new medium formulation for in vitro rooting of carob tree based on leaf macronutrients concentrations

Experiments were performed to optimize the macronutrients concentrations for in vitro rooting of Ceratonia siliqua micropropagated shoots. Several dilutions of Murashige and Skoog (MS) medium were tested: full-strength MS, half-strength MS (1/2MS), and 1/2MS + full N. The frequency of in vitro rooting was enhanced when the 1/2MS was used (50 % rooted shoots). Mature leaves from 20 - 30 year-old carob trees and from 2 year-old micropropagated plants were collected and the concentrations of macronutrients (N, P, K, Ca, Mg) assessed. Based on the mineral composition of the leaves a new medium was formulated and compared with the previous ones showing an increment of the rooting frequency to 80 %. Moreover, shoots rooted in the new medium did not show symptoms of apical necrosis that occurred in the other tested media.

Liquid culture system for shoot multiplication and secoiridoid production in micropropagated plants of Centaurium erythraea Rafn

An efficient shoot multiplication method from shoot tips of Centaurium erythraea using liquid Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (0.1 mg l −1) and 6-benzylaminopurine (BAP) (1.0 mg l −1) was developed. Under these conditions, almost 60 microshoots per explant were produced within 4 weeks. This is three times more as compared to shoots obtained on agar-solidified medium. Shoots taken from liquid culture were rooted with frequency 85% and 77% on hormone-free full-strength and half-strength MS medium, respectively within 6 and 4 weeks. The plantlets were transferred into soil and they survived acclimatization with 90% success, producing healthy plants, morphologically similar to plants derived from shoots grown on solid culture during their multiplication stage. The shoots of 10-week-old micropropagated plants of C. erythraea accumulated secoiridoid glucosides (gentiopicroside, swertiamarin and sweroside) up to 149 mg g −1 dry weight. The value was significantly higher than those achieved for other tested plant materials, such as shoot cultures and aerial parts of wild-grown plants. It is hoped, that the system using liquid shoot culture could be useful for large-scale micropropagation of C. erythraea plants with high production of pharmacologically important products.

Toward Introgression of Ornamental Cucurbita Germplasm: Generation of C. maxima ×pepo hybrids

Cucurbita maxima and C. pepo are difficult to hybridize, and it was our objective to generate F1 hybrids between ornamental cultivars of the two species. C. maxima `Lakota' and C. pepo `Jack O'Lantern'; and `OZ'; were selected as parents. `Lakota' (L) is an heirloom, hubbard-type cultivar producing pear-shaped, red-orange fruit with dark green mottling, `Jack O'Lantern'; (J) is an open-pollinated Halloween-type pumpkin cultivar and `OZ' is a Halloween-type hybrid. Sixteen plants of each cultivar were greenhouse-grown in a CRB design during the period July-Sept. 2003. Interspecific crosses were made in both directions, with intraspecific crosses (J × O) and selfs (L) serving as controls. Fruits were harvested about 20 d after pollination. Embryos were excised under aseptic conditions and grown on either full strength Murashige and Skoog (MS) media with 6% sucrose (S6), full strength MS media with 6% maltose (M6), or half strength MS media with 3% sucrose (S3). Fruit set was generally greater in the intraspecific crosses (33%) and selfs (67%) than in the interspecific crosses (15 %), with the notable exception of the interspecific combination L × J (85% fruit set). Embryos of interspecific and control crosses were about 1.5mm and >1cm long, respectively. Hypocotyl and root growth 10 d after plating was better on S3 (3.2 and 1.7 cm) than on S6 (1.6 and 0.25 cm) or M6 (0.35 and 0 cm), and a greater number of functional hybrids were obtained from embryos grown on S3 (6 plants) than on S6 (2 plants) or M6 (2 plants). The interspecific plants were backcrossed to one of the parents and novel combinations of shape, color and variegation in hybrid fruit were observed.

Micropropagation and hairy root culture of Solanum Laciniatum Ait.

An efficient system for in vitro micropropagation of Solanum laciniatum Ait. has been established. Shoot induction on leaf explants was most successful on Murashige and Skoog (MS) medium supplemented with 10 μM N6-benzyladenine (BA) and 1 μM α-naphthaleneacetic acid (NAA). BA (13 μM) was optimal for further shoot multiplication, and rooting of separated shoots was achieved on medium without plant growth regulators. At each subculture, 20–25 shoots were obtained on each explant, from which six to eight were suitable for separation and further rooting. Leaf explants grown in vitro were successfully infected by Agrobacterium rhizogenes ATCC 15834. The established hairy root culture was, on the basis of dry weight, more productive when grown on half-strength MS medium than on full-strength MS (3% sucrose) and full-strength MS (6% sucrose) medium. The amount of solasodine-containing glycoalkaloids in hairy roots as measured by a colorimetric method was 0.3–1% of dry weight, which is higher than in the shoot culture (0.5% of dry weight) and lower than in leaves of in vivo-grown plants (1.1–1.4% of dry weight). The amount of solasodine-containing glycoalkaloids in leaves of in vivo-grown plants of S. laciniatum was similar to the related species Solanum aviculare Forst. Both species are morphologically similar, therefore we effectively distinguished them by flow cytometry. The genome size of S. laciniatum was determined as 4.03 pg and the genome size of S. aviculare as 1.69 pg.

Effect of medium salt concentration on differentiation and maturation of somatic embryos of cassava (Manihot esculenta Crantz).

Culture of cassava somatic embryos on media with an altered macro- and micro-nutrient salt concentration affected embryo development and germination capability. In the tests, quarter-, half-, full- or double-strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half- or full-strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full-strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half- and full-strength MS medium yielded 8.3 and 8.6 germinants g(-1) NEC tissue, respectively. For this important but often disregarded culture factor, either half- or full-strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination.

Factors Affecting In Vitro Multiplication of Date Palm

Rapid method of in vitro multiplication of date palm was developed. Shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg dm-3 dimethylaminopurine (2iP) + 1 mg dm-3 naphthalene acetic acid (NAA). Shoot buds were proliferated from white nodular cultures on hormone free medium. Shoot bud proliferation strongly enhanced when cultured on MS-medium contained 3 mg dm-3 2iP + 0.5 mg dm-3 NAA. Culturing on full-strength MS medium showed higher multiplication rate compared with half-strength MS medium. Among four concentrations of sucrose used, 30 g dm-3 speeded up the bud proliferation more than 10, 20 and 40 g dm-3. However, the largest shoot buds were observed with 40 g dm-3 sucrose. Solidification of culture media by 1.75 g dm-3Phytagel showed the highest proliferation rate, but the largest buds were observed with 1 g dm-3Phytagel.

Micropropagation of Echinacea angustifolia, E. pallida, and E. purpurea from Stem and Seed Explants

Micropropagation of three Echinacea species, E. angustifolia DC., E. pallida Nutt., and E. purpurea Moench., was investigated as a potential means of germplasm preservation of species faced with overcollection in the wild and rapid clonal propagation of elite individuals with unique medicinal or ornamental properties. Very high contamination rates occurred with shoot-tip explants but not with nodal segments. Contamination rates for seed explants were inversely related to the number of seedcoat layers removed, ranging from 100% contamination from intact seeds to near 0% contamination from excised embryos. Dormancy of seed explants was also eliminated when the pericarp and integument were removed. Addition of benzyladenine (BA) to the culture medium induced shoot multiplication and inhibited root formation in all three species. Shoot multiplication rates were low (1-3 shoots per culture) when seed explants were placed on a medium with BA levels low enough to avoid adventitious shoot formation (0.45 μm). Shoot count was higher on half-strength Murashige and Skoog (MS) minerals, while leaf size was greater on full-strength MS minerals. Cultures did not perform well in Woody Plant Medium. Reducing subculture frequency from 4 to 2 weeks increased shoot multiplication from 1.4 to 1.8 shoots per subculture and total shoots produced per subculture after 12 weeks from 2.8 to 23.9. Rooting occurred readily on shoots isolated from E. purpurea cultures and was not promoted by addition of IBA to the rooting medium. Rooting was low and nil on shoots from cultures of E. angustifolia and E. pallida, respectively. Methods described in this study allow rapid multiplication of three Echinacea species and subsequent rooting of E. purpurea. Future improvements in root induction treatments will allow these methods to be used effectively for micropropagation and maintenance of disease-free germplasm of Echinacea species. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); 1H-indole-3-butyric acid (IBA).

テッポウユリ‘ジョージア’のりん片培養によるカルス誘導とカルスからのウイルスフリー子球の生産

テッポウユリ'ジョージア'のカルス培養によるウイルスフリー個体作出を目的とし, カルス継代および植物体再生条件を検討し, 再生した子球のウイルス検定を行った.1. 'ジョージア'のカルスは5.0μM picloramを添加したMS培地で継代する間には器官の再分化は起らず, カルス増殖のみであった.しかし, 継代数を増加するにつれてその増殖率は低下した.2. 上述の培地で継代培養したカルスをMS培地と1/2MS培地(無機塩濃度が1/2に希釈され, 他の成分は同一濃度)に移植すると, シュートはMS培地よりも1/2MS培地で培養したカルスからよく形成された.長期間継代培養したカルスでも0.5μM NAAの単独添加培地ではシュートを形成したが, その率は継代数の増加につれて低下した.3. 再生したシュートから発達した子球を低温処理したあと6ケ月間圃場で栽培した.これらの植物体からのウイルス検出率はカルスの継代回数の多いカルスから得られた植物体で低く, 継代5回の子球のウイルス検出率は約31%となり, CMVは全く検出されなかった.4. 抗ウイルス剤であるDHTおよびribavirin(5, 50μM)を培地に添加してもカルスからのシュート形成には影響しなかった.それらの添加培地から再生した子球の培養終了時点でのウイルス検出率は, 無添加区より低くなった.特に50μM DHTの添加区では温室に移植した6ケ月後でも培養終了時のウイルス検出率とほぼ同等で, 19%であった.5. ウイルスに感染した'ジョージア'のりん片から誘導されたカルスを3回以上継代するか, あるいは50μM DHTを含む培地でカルスを培養して植物体を得る方法により, ウイルスフリー個体を効率的に生産できることが示唆された.

In vitro morphogenetic responses and plant regeneration from pepper (Capsicum annuum L. cv. Early California Wonder) seedling explants

Explants from 13-d old pepper (Capsicum annuum, L. cv. Early California Wonder) seedlings were cultured in Murashige and Skoog (MS) medium supplemented with different levels of 1-naphthalene acetic acid (NAA) and 6-benzylamino purine (BAP). Multiple shoot-buds proliferated from the cut surfaces of cotyledon, shoot-tip and hypocotyl explants in one month. The best NAA to BAP combinations (mg/l: mg/l) for multiple shoot-bud regeneration of the above three explant types were 0.1 ∶ 5.0, 0.0 ∶ 5.0, and 0.1 ∶ 10.0, respectively. Root explants did not express any new morphogenetic response in all hormonal combinations tested. Regenerated shoot-buds were excised from the explants and cultured in 1/2X or 1X MS medium supplemented with different levels of Indole-3-acetic acid (IAA) or NAA. When cultured in full strength MS medium with 0.5 mg/l IAA or 0.4 mg/l NAA, 70% of the buds rooted in one month. Plantlets were established successfully ex vitro under greenhouse mist and grown to maturity.

Micropropagation of Ulmus pumila L. from mature trees

Multiple shoots were induced from nodal segments of mature trees of Ulmus pumila L. on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). Further multiple shoots were obtained from nodal segments taken from in vitro proliferated shoots when cultured in MS medium containing 0.5 mg.l(-1) BA. Rooting of the shoots was achieved on half or full strength MS medium or in MS medium supplemented with 0.1 mg.l(-1) naphtaleneacetic acid (NAA). Rooted plantlets were able to resume independent growth after a short period of acclimatization.