In vitro clonal multiplication of an apple rootstock by culture of shoot apices and axillary buds

In vitro clonal multiplication of apple rootstock MM 111 using axillary buds and shoot apices were carried out. Vegetative axillary buds of the size of 0.2-2.0 cm and shoot apices measuring 4 mm in length were initiated to shoot proliferation on MS medium supplemented with BA (0.5 - 1.0 mgl(-1)), GA3(0.5 mgl(-1)), with or without IBA(0.05 - 0.1 mgl(-1)). Small size explants showed less phenol exudation and less contamination. Following establishment phase, the small shoots emerged from explants were subcultured on MS medium supplemented with different combinations and concentrations of growth regulators. BA (1.0 mgl(-1)) and GA3 (0.5 mgl(-1)) combination showed highest multiplication rate (1:5), andcl also produced longer shoots. Two step rooting was done by transferring microcuttings to auxin free solid medium after root initiation in dark on 1/2 strength MS liquid medium containing IBA (0.5 mgl(-1) ). Rooted plantlets were transferred to peat containing paper cups and resulting plants of MM 111 acclimated successfully for transfer to field.     

不同杂种优势类群玉米幼胚愈伤组织的诱导及植株再生特性的研究

以Reid、唐四平头和其它种质等3个杂种优势类群共19份玉米自交系为试验材料,以玉米幼胚作为外植体,研究了基因型、培养基和激素对玉米幼胚愈伤组织的诱导及植株再生的影响,结果表明供试材料均能进行愈伤组织的诱导,但是仅有12个自交系能再生植株。N6和改良N6培养基有助于提高愈伤组织的质量及其生长速度,2,4-D在愈伤组织的诱导中起着关键性作用。在诱导培养基中添加0.2mg／L的6-BA或KT会使胚性愈伤组织的诱导频率下降以及降低愈伤组织的质量。在胚状体诱导培养基中添加1mg／L的KT能促进绿苗的分化,但是浓度过高会使丛生苗分化过多。此外,通过对不同杂种优势类群自交系玉米幼胚培养特性的分析,发现在唐四平头类群的4个自交系中,黄早四的绿苗分化率仅为0.5％,其它3个自交系不能再生植株。但是,从Reid和其它种质类群的供试自交系中筛选出了胚性愈伤组织的诱导频率和绿苗分化率均较高的、适合于遗传转化的受体材料,如3189／4380、4380／陕综5、8103、先早17、18-599红、18-599白、501、178和冀53。     

Somatic embryogenesis in two Gossypium hirsutum genotypes on semi-solid versus liquid proliferation media

A comparison of semi-solid vs. liquid embryo proliferation media was made using two Gossypium hirsutum L. genotypes (Coker 312 and T25) and two callus initiation media. Sections of petioles from mature, flowering plants were cultured on two modified Murashige and Skoog media. Medium 1 included 4.0 mg l^-1 NAA and 1.0 mg l^-1 kinetin; medium 2 contained 0.1 mg l^-1 2,4-D and 0.1 mg l^-1 kinetin. After six weeks, callus was removed from each explant and divided in half. One callus portion was placed in liquid proliferation medium and the other on semi-solid (0.2% Gelrite) proliferation medium. Composition of proliferation medium was identical to that of initiation medium, except no growth regulators were added. Embryos were counted after eight weeks. The percentage of explants forming callus was influenced by genotype/initiation medium combination. Analysis of variance procedures revealed significant variability for callus initiation media, proliferation media (semi-solid or liquid), and an initiation medium x genotype interaction. Paired t-tests indicated that more embryos were produced in liquid proliferation medium (227.3 embryos/culture) than on semi-solid proliferation medium (134.6 embryos/culture).Abbreviations NAA naphtaleneacetic acid - 2,4-D 2,4-D dichlorophenoxyacetic acid     

In vitro clonal propagation of bird eye chilli (Capsicum frutescens Mill.)

An efficient and highly reproducible protocol for micropropagation of bird eye chilli Capsicum frutescens was attempted. Murashige and Skoog (MS) medium containing 0.5-3.0 mgl(-1) of 6-benzyladenine (BA), 2-isopentenyl adenine (2iP), kinetin and 0.5-2.0 mg l(-1) of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) along with 1 gl(-1) activated charcoal (AC) were used for shoot regeneration from both shoot tip and nodal explants. Shoot tip explants (100%) grew well on medium containing 1 mgl(-1) of kinetin and 1 mgl(-1) of IBA. Shoot proliferation (1-3) from nodal explants was effective on this medium. The regenerated shoots with 4-7 nodes had further growth upon sub-culturing onto kinetin (1 mgl(-1)) and IBA (1 mgl(-1)) and rooted simultaneously. The rooted plants were transferred to pots after hardening under controlled conditions. The survival percentage in pots was 80-90%.     

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 3l balloon type bubble bioreactor (BTBB) containing 2l 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 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. 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.     

Somatic Embryogenesis and Plant Regeneration from Papaya Suspension Cultures

Compact embryogenetic calli were obtained from explants on P3 medium after 4 weeks of culture and high-frequency somatic embryogenesis occurred after these calli were transferred into suspension culture. Experimental data showed that low level (0.2%W/V) of activated charcoal had beneficial effects on somatic embryogenesis. Abundant calli on P4 medium however, showed no embryogenesis. On the other hand, callus induction and somatic embryogenesis varied with different rarities of exptants. The efficiency of somatic embryogenesis was much higher, if roots were used as explants, whereas stems were more suitable for callus formation Mature somatic embryos with cotyledons were cultured on MS medium containing different plant hormones. The optimum medium for germination and growth of entire plantlet was Mso medium. The somatic embryos on MS2, MS and MS3 media germinated rapidly, but formed excessive callus from the surface of germinating embryos.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

油松体细胞无性系的建立

以油松（Ｐｉｎｕｓｔａｂｕｌａｅｆｏｒｍｉｓ）种胚为外植体,通过器官发生途径建立体细胞无性系。以ＭＳ为基本培养基,在芽的诱导和增殖培养基中,附加植物激素,以６ＢＡ＋ＫＴ和ＮＡＡ效果最好,两者的配比为５－１０：１,６ＢＡ和ＫＴ的浓度分别均不超过１ｍｇ／Ｌ。同时根据培养物的发育状态交替使用活性炭,则对芽的增殖有明显的促进作用。选择发育状态较幼嫩的刚抽茎的外植体,诱导生根,在１／２ＭＳ＋ＫＴ０．１＋ＩＢＡ０．１＋ＮＡＡ０．１ｍｇ／Ｌ的培养基中,生根率达３２．％。体细胞胚胎发生途径建立细胞无性系也已取得成功,筛选出可继代培养的胚性胚柄团无性系,在胚轴和子叶上诱导出成熟的体细胞胚并获得完整小植株。     

Induction of somatic embryogenesis in Pinus roxburghii Sarg.

Embryogenic calli were initiated from embryonic explants of Pinus roxburghii using female gametophytes containing immature pre-cotyledonary embryos. Zygotic embryos were collected at different developmental stages and cultured on various media. Initiation of embryogenic calli was achieved in pre-cotyledonary zygotic embryos of a 0.1-mm to 1.2-mm embryonal head on Douglus fir cotyledon revised medium (DCR) medium supplemented with 2,4-D or NAA and BA. Embryogenic callus development was initiated from the suspensor region of immature embryos. The method of immature embryo culture was significant as rapid embryogenic callus development occurred in megagametophytes where the suspensor was stretched onto the medium from the cut micropylar end. Sixty embryogenic lines were established from 2500 explants cultured during one season. A pro-embryo with six to eight meristematic cells and suspensor of six to ten long, vacuolated cells dominated the early phase of the callus development. Cleavage polyembryony occurred in proliferating callus, constituting a method of multiplication of these somatic embryos. Somatic embryos developed to stage-I and stage-II embryos on DCR medium supplemented with 5 μM 2,4-D or 10 μM NAA. Received: 30 June 1999 / Revision received: 15 November 1999 / Accepted: 3 December 1999     

Indirect somatic embryogenesis and plant regeneration from leaf and internode explants of Paulownia elongata

Several plant growth regulators BA, TDZ, 2,4- and Kn were tested alone or in combination for their capacity to induce indirect somatic embryogenesis from leaf and internode explants of Paulownia elongata. Calli were produced when leaf explants were cultured on Murashige and Skoog (MS) medium containing 3 % sucrose, 0.4 % phytagel, 4 mg l^-1 TDZ and 0.1 mg l^-1 Kn after 3 weeks and the initiation rate was 54.1%. After subculturing on the same medium, embryos at various developmental stages (globular, heart and torpedo shaped) were transferred for maturation onto MS medium supplemented with 3 % sucrose, 0.4 % phytagel, 0.1 mg l^-1 TDZ, 1 mg l^-1 Kn and 2 mM glutamine. An average of 50.7 somatic embryos were obtained from 100 mg of embryogenic callus after 4 weeks at high frequency (64.7 %). Afterward, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination (80 %) and development into plantlets. Plantlets were transferred to pots with a mixture of peat and perlite in a 3:1 ratio and showed a survival rate of 70–80 %. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in the greenhouse.     

Indirect somatic embryogenesis and plant regeneration from leaf and internode explants of Paulownia elongata

Several plant growth regulators BA, TDZ, 2,4- and Kn were tested alone or in combination for their capacity to induce indirect somatic embryogenesis from leaf and internode explants of Paulownia elongata. Calli were produced when leaf explants were cultured on Murashige and Skoog (MS) medium containing 3 % sucrose, 0.4 % phytagel, 4 mg l^-1 TDZ and 0.1 mg l^-1 Kn after 3 weeks and the initiation rate was 54.1%. After subculturing on the same medium, embryos at various developmental stages (globular, heart and torpedo shaped) were transferred for maturation onto MS medium supplemented with 3 % sucrose, 0.4 % phytagel, 0.1 mg l^-1 TDZ, 1 mg l^-1 Kn and 2 mM glutamine. An average of 50.7 somatic embryos were obtained from 100 mg of embryogenic callus after 4 weeks at high frequency (64.7 %). Afterward, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination (80 %) and development into plantlets. Plantlets were transferred to pots with a mixture of peat and perlite in a 3:1 ratio and showed a survival rate of 70–80 %. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in the greenhouse.     

Optimization of somatic embryogenesis protocol in Lycopersicon esculentum L. using plant growth regulators and seaweed extracts

In the present study a simple and efficient somatic embryogenesis system was developed from leaf explants of Lycopersicon esculentum L. The protocol has been developed by using plant growth regulators and seaweed extracts a natural biostimulant. The leaf sections were initially cultured on to leaf embryogenic callus induction medium fortified with various concentration and combinations of 2,4-dichlorophenoxy acetic acid (0.2–1.0 mg L^?1), picloram (0.2–1.0 mg L^?1), and kinetin (0.1–0.5 mg L^?1). The best responding concentration in induction of friable embryogenic callus was tested for the proliferation. The friable cultures were detached from the mother culture and inoculated in three different media supplemented with plant growth regulators, plus 0–25 % Caulerpa scalpelliformis or 0–25 % Gracilaria corticata extracts for embryo development. A twofold increase in maturation and germination of somatic embryos was observed in the media containing seaweed extracts (MSMG2 and MSMG3) than the control (MSMG1). The plantlets transferred from plant growth chamber to greenhouse conditions exhibited higher survival rate (90 %) than directly shifted plantlets.     

Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.     

植物生长调节剂对南方红豆杉愈伤组织培养和紫杉醇合成的影响

通过不同种类和水平植物生长调节剂对南方红豆杉(Taxus chinensisvar.mairei)愈伤组织诱导、生长和紫杉醇合成能力影响的研究发现:诱导培养初期,以无植物生长调节剂的MS为基本培养基,在附加不同植物生长调节剂组合作用下愈伤组织产生的时间和生长、在相同植物生长调节剂组合作用下不同外植体愈伤组织的产生时间和生长均表现出较显著差异,2,4-D/NAA高于0.4时,不利于南方红豆杉愈伤组织的诱导。转换到附加不同植物生长调节剂组合的B5培养基上后,随培养继代次数的增加,生长差异逐渐缩小,直至不显著,表明参考不同文献报道最优配方所设计的各植物生长调节剂组合对南方红豆杉愈伤组织的生长均较适宜,有利南方红豆杉愈伤组织生长的植物生长调节剂优化组合没有唯一性。但不同调节剂组合作用下的同源愈伤组织中、相同调节剂作用下不同源愈伤组织中紫杉醇含量均存在着极显著差异,适当水平(2 mg/L)的2,4-D单用,或与适当水平的KT、6-BA、KT GA配合使用,对南方红豆杉愈伤组织紫杉醇的合成较有利,NAA则不太有利,幼茎和叶愈伤组织产紫杉醇的水平较其它愈伤组织为高。     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Putrescine-enhanced somatic embryos and plant numbers from elite oat (Avena spp. L.) and reciprocal crosses

Summary Mature embryos from hulled, regenerable GP-1 (A. sativa L.), hull-less, recalcitrant Tibor (A. nuda L.) and reciprocal crosses were cultured in vitro on a putrescine- (Put) containing medium. Hormone-free Murashige and Skoog medium (MS-0) or shoot proliferation medium (SPM) [2.0 mgl⁻¹ (9.0 μM) 2,4-dichlorophenoxyacetic acid (2,4-D)], with and without 0.5 mM Put or 1 mM Put, were tested for effects on somatic embryogenesis and plant regeneration. Put/SPM (0.5mM) was the best medium for both somatic embryos (SEs) and plant numbers per gram of callus, regardless of genotype. This effect was most evident in Tibor, which produced no somatic embryos or plants on SPM, a previously published regeneration medium, and in Tibor ×GP-1, which produced reduced numbers of SE and plants on the remaining media. The number of SEs per gram of callus for GP-1 and GP-1× Tibor showed little significant differences between the different media. Put treatments produced plants from the four genotypes but the regeneration efficiency on Put-containing medium was similar or even better than on SPM for explants containing maternal GP-1 germplasm. This suggests that Put-containing MS-0 medium can be used for testing regeneration of other oat lines. In addition, SPM containing 0.5 mM Put can be used to induce significant regeneration of plants from normally recalcitrant genotypes. This improvement greatly increases the number of potential germplasms for further transformation efforts.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis in elm

We show that isolated zygotic embryos of Ulmus minor and U. glabra can produce embryogenic cultures provided they are isolated from immature seeds before storage proteins begin to accumulate. Rates of somatic embryogenesis were highest among zygotic embryos collected 6 weeks post-anthesis when they were at the midcotyledonary stage, were about 5 mm long and had a fresh weight of approx. 10 mg. At this time, induction was even possible in Murashige and Skoog basal medium with no plant growth regulators, but addition of 2,4-dichlorophenoxyacetic acid was necessary at earlier stages of zygotic development. In medium supplemented with benzyladenine (BA) only, no embryogenic induction was observed. The formation of callus was an essential step not only for the induction of embryogenic masses, but also for the maintenance of embryogenic competence through successive subculture of callus on induction media supplemented with 0.1 mg l(-1) BA. Nine embryogenic U. minor lines and 24 U. glabra lines have been maintained in this way for 3 years. However, conversion into plantlets has occurred only rarely.     

Somatic embryogenesis of two new <Emphasis Type="Italic">Panax ginseng</Emphasis> cultivars,Yun-Poong and Chun-Poong

Somatic embryogenesis from single cells is important for normal plant regeneration of ginseng. Cotyledon explants from zygotic embryos of two new ginseng cultivars, Chun-Poong and Yun-Poong, produced somatic embryos on Murashige and Skoog (MS) basal medium and MS medium containing growth regulators. The highest frequency of single somatic embryo formation was obtained when cotyledon explants were excised from premature (cultured for 1 day) zygotic embryos (about 6 mm in length) of both cvs. Chun-Poong and Yun-Poong and then cultured on MS medium supplemented with 7% sucrose. The frequency of single somatic embryo formation was strongly enhanced when Chun-Poong cotyledons were subjected to plasmolysis with 0.1–0.5 M sucrose for 24 h and Yun-Poong cotyledons to plasmolysis with 1.0 M sucrose for 24 h and then cultured on MS medium with 2,4-D.     

Recurrent somatic embryogenesis and plant regeneration from seedlings of <Emphasis Type="Italic">Hepatica nobilis</Emphasis> Schreb.

A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.