Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L^?1 BA and 0.2 mg L^?1 NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L^?1 BA and 0.5 mg L^?1 NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot–root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.     

Cloning adult trees of Arbutus unedo L. through somatic embryogenesis

Arbutus unedo L. is a perennial tree, native in the Mediterranean area, and tolerant to stress conditions. Due to its economic potential, there is an increasing demanding for plants by producers and farmers. In order to offer cloned material with assured quality, several micropropagation protocols have been developed, including somatic embryogenesis induction. However, little is known about this process on strawberry tree and a great deal of work is still necessary to successfully clone in A. unedo through somatic embryogenesis. Thus, the main goals of this work were: (i) to test the effect of the genotype on somatic embryogenesis induction, (ii) to analyse the role of adult and young materials on induction, and (iii) to perform a comparative histological analysis between somatic embryos and their zygotic counterparts. Somatic embryogenesis was induced on apical expanded leaves from in vitro shoots of several genotypes in Andersson medium with 3% sucrose and different concentrations of BAP (2.0 mg L^?1) and NAA (0.5, 1.0, 2.0, 5.0, 10.0, 15.0 and 20.0 mg L^?1). Embryogenesis induction rates ranged from 0 to 94.5%. Higher induction rates were achieved on the medium with 2 mg L^?1 BAP and 2 mg L^?1 NAA, and are genotype dependent. After a 3-month induction period, the highest somatic embryogenesis induction rate was 94.5% on genotype AU4. Embryos at different developmental stages were found, as well as abnormal somatic embryos. SEM images showed different anomalies being the most common embryos displaying more than two cotyledons or fused embryos. Embryo germination was not genotype dependent and the maximum embryo conversion rate achieved was 73.5%. However, only 39.21% of the embryos were able to grow into plantlets which displayed a normal chromosome number (2n?=?26). Histological analysis showed differences in the cell organization between somatic and zygotic embryos, as well as several morphological anomalies. Overall, the developed somatic induction protocol proved to be very efficient, with high induction rates achieved, both from seedling and adult material, but its genotype dependent. However, embryo conversion still needs to be improved, in order to fully seize the potential of this micropropagation technique.

     

Biochemical status of in vitro regenerated Lilium bosniacum and Lilium cattaniae plantlets

Lilium cattaniae (Vis.) Vis. and Lilium bosniacum (G. Beck) Beck ex Fritsch, endemic species of Balkan Dinaric Alps, were micropropagated from seeds collected from their natural habitats. The relationship between peroxidase activity, photosynthetic status and differentiation of Lilium cattaniae and L. bosniacum in vitro was investigated. Peroxidase activity recorded for somatic embryos of Lilium cattaniae obtained on Murashige and Skoog (MS) medium containing 9.05 mM 2.4-dichlorophenoxyacetic acid (2.4-D) and 4.44 mM N6-bezyladenin (BA), was about two times higher than for any other treatment. Photosynthetic status of plantlets obtained through regeneration was explant-specific and generally higher for plantlets regenerated from basal leaf explants than from bulb explants. The accumulation of anthocyanin was detected in some regenerated shoots and more often in plantlets obtained through regeneration from bulb explants. High frequency of somatic embryo formation was recorded for L. cattaniae on MS medium containing 9.05 mM 2.4-D and 4.44 mM BA. The peroxidase activity for L. bosniacum plantlets obtained through regeneration showed treatment-specific elevations. We consider that basal leaf parts are suitable for successful regeneration of these two lilies and that elevation in peroxidase activity is a good indicator of somatic embryogenesis in both lilies.     

Somatic embryogenesis and plant regeneration from immature zygotic embryo cultures of mountain ash (<Emphasis Type="Italic">Sorbus pohuashanensis</Emphasis>)

Plant regeneration through somatic embryogenesis was achieved using immature zygotic embryos (ZE) of Sorbus pohuashanensis as explants. Over 50% of immature ZEs from immature seed collected at 30 days after pollination produced direct somatic embryos (SEs) on Murashige and Skoog (MS) medium supplemented with 0–0.44 μM 6-benzyladenine (BA) in combination with 5.73 μM naphthaleneacetic acid (NAA) or with 0.91–2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) alone. Fourteen to 23 SEs per explant were regenerated on MS medium supplemented with BA 0.44 μM in combination with NAA 5.73 μM. SE formation decreased when sucrose concentrations were higher than 40 g L⁻¹. Repetitive embryogenesis occurred following culture on solid MS medium containing 12 μM abscisic acid, 75 g L⁻¹ polyethylene glycol, and 20 g L⁻¹ sucrose at 25 ± 1°C under a 16-h photoperiod with a light intensity of 40 μmol m⁻² s⁻¹. Over 40% of the mature SEs germinated on solid MS medium under light condition described previously. Up to 40% of the regenerated plantlets were successfully acclimatized under greenhouse conditions. Plantlets derived from SEs grew vigorously with similar morphology as those germinated from ZEs. Histological studies of explants at various developmental stages of somatic embryogenesis revealed that SEs passed through globular, heart, torpedo, and mature stages. Similar to ZE suspensors, similar structures of SE degenerated in later stages of embryo development. ZE and SE are a effective means of regenerating tissue culture plantlets for S. pohuashanesis.     

Somatic embryogenesis and plant regeneration of <Emphasis Type="Italic">Lilium ledebourii</Emphasis> (Baker) Boiss., an endangered species

A somatic embryogenesis (SE) protocol was established for the regeneration of Lilium ledebourii (Baker) Boiss. whole plants using new vegetative bulblet microscales and transverse thin cell layers (tTCLs) of young bulblet roots as the explant sources. Bulblets were induced from bulb scale explants cultured for at least 3 months in the dark on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar, and different concentrations of α-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and thidiazuron. Embryo-like structures were obtained from tTCL explants of 3-month-old bulblets (excised from bulb scale explants) following culture on solid MS medium containing 3% sucrose and various concentrations of NAA and BA for 3 months in the dark. Both the explant source and the type of plant growth regulators affected the differentiation of somatic embryos. The highest percentage (65.55%) of embryogenesis was obtained from bulblet microscale tTCLs cultured on solid MS medium containing 0.54 μM NAA and 0.44 μM BA. Plants with normal shoots and roots were obtained following a 3-month culture of embryos on growth regulator-free MS medium at 25 ± 1°C under a 16/8-h light/dark photoperiod (light intensity 40 μmol m⁻² s⁻¹, cool-white fluorescent light). The plants were successfully acclimatized in the growth chamber.     

Somatic embryogenesis to overcome low seed viability and conserve wild banana (Ensete superbum (Roxb.) Cheesman)

The seed viability, ex vitro germination, and percentage of in vitro zygotic embryo germination were found to be very low in Ensete superbum (Roxb.) Cheesman. Only 33.33% of seeds were viable, and the ex vitro germination percentage was only 5%, while the percentage of in vitro zygotic embryo germination was 33%. Somatic embryogenesis experiments produced competent callus on Murashige and Skoog (MS) medium supplemented with 2.5 mg L⁻¹ 2,4-D and 3 mg L⁻¹ BAP from inflorescence explants. The embryogenic callus produced the maximum number of somatic embryos on MS basal medium kept in a dark chamber for 15 wk. Half-strength MS medium supplemented with 500 mg L⁻¹ glutamine was optimal for somatic embryo germination and development of plantlets. Regenerated plants had 80 to 90% survival rate. Therefore, somatic embryogenesis can be considered as an efficient method to overcome a drastic reduction in population and to achieve germplasm conservation.

     

Large-scale somatic embryogenesis and regeneration of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10⁴) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA₃), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.     

Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

A rapid system for micropropagation of <Emphasis Type="Italic">Swertia chirata</Emphasis> Buch-Ham. ex Wall.: an endangered medicinal herb via direct somatic embryogenesis

An improved method of direct somatic embryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness to induce somatic embryos. Leaf explants with abaxial side in the medium produced maximum number of somatic embryos. This system omits the callus stage and thus reduces the process of SE in S. chirata by 35–45 days. Embryos at different stages of development were observed. Maturation of heart stage embryos were observed on Murashige and Skoog (MS) medium containing 1 mg L⁻¹ 2,4-D. Upon transfer to the germination medium, they were converted to cotyledonary stage and then plantlets of 33% and 68% of them were converted to cotyledonary stage and then plantlets on MS medium supplemented with 0.05 and 0.1 mg L^-1 GA₃ respectively. The 2,4-D alone at 1.0 or 1.5 mg L⁻¹ was found to be better for embryogenic tissue initiation than 2,4-D in combination with indole-3-acetic acid or α-naphthalene acetic acid. For further embryo development, 2,4-D was combined with cytokinins such as 6-benzylaminopurine (BAP) and kinetin or plant growth regulator free medium or medium with 50% reduced concentration of the same hormone while subculturing. Mean germination and percentage of survival were maximum in the medium containing 1.0 mg L⁻¹ 2,4-D in combination with 0.1 mg L⁻¹ BAP. Regenerated plantlets were morphologically and genetically identical. This method offers a vast scope for the clonal propagation of endangered plants.     

Somatic embryogenesis and plant regeneration from leaf and petiole explants of <Emphasis Type="Italic">Campanula punctata</Emphasis> Lam. var. <Emphasis Type="Italic">rubriflora</Emphasis> Makino

A simple and efficient protocol was developed for somatic embryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L⁻¹ 6-benzyladenine (BA). On this medium, a mean number of 19.5 and 31.2 SE were developed per leaf and petiole explants, respectively. Embryos were induced both light and dark conditions but culturing the explants 2 weeks in the dark followed by 3 weeks under light resulted in high frequency of embryo formation. Globular embryos germinated best on MS medium supplemented with 0.3% (w/v) activated charcoal (AC) and 1.0 mg L⁻¹ GA₃. The germinated plantlets grew further on MS medium containing 0.3% AC. Plantlets were successfully acclimatized in the greenhouse with 94% survival rate. This is the first report on induction of somatic embryogenesis in this genus and also has implications for genetic transformation, and mass clonal propagation.     

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1^−l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1⁻¹) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N⁶-benzyladenine (BAP, 0.75 mg 1^−l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1^−l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.     

In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.

     

Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos

Summary For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl⁻¹ (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl⁻¹ (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.     

Somatic embryogenesis and plant regeneration in two wild cotton species belong to G genome

The present work describes the plant regeneration via somatic embryogenesis in two wild cotton species belonging to G genome: Gossypium nelsonii Fryx and Gossypium australe F Muell. The role of plant hormones and carbohydrates was also evaluated for somatic embryogenesis and somatic embryo development. Normal plants were obtained from G. nelsonii Fryx; abnormal plants and somatic embryos were obtained from G. australe F Muell. The best medium for callus induction for these G genome wild cotton species was MSB₅ supplemented with 0.1 mg L⁻¹ KT and 0.1 mg L⁻¹ 2,4-D. For embryogenic callus proliferation, the best medium used was MSB₅ supplemented with 0.2 mg L⁻¹ KT and 0.5 mg L⁻¹ IBA. The medium MSB₅ supplemented with 0.15 mg L⁻¹ KT and 0.5 mg L⁻¹ NAA was used successfully for root initiation and plant growth. In addition, adding CuSO₄ and AgNO₃ in the callus-inducing and proliferation medium resulted in a number of somatic embryos. Glucose and maltose, the carbon sources in somatic culture, were used for callus induction, but maltose worked even better than glucose for proliferation of embryogenic callus and development of somatic embryos.     

Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshurica Rupr.)

Using mature cotyledonary explants of Fraxinus mandshurica, an efficient plant regeneration system was developed via somatic embryogenesis. More than 67 % of mature cotyledons of zygotic embryos yielded 23–159 somatic embryos (SEs) per explant when incubated on medium consisting of half-strength Murashige and Skoog (MS) salts and vitamins (MS1/2) supplemented with 8.88 μM 6-benzyladenine (BA), 26.84 μM naphthaleneacetic acid (NAA), 75 g L^?1 sucrose, and 400 mg L^?1 casein hydrolysate (CH). Approximately, 82 % of induced SEs were observed on browning cotyledonary explants. Histological studies of cotyledon explants at various stages of somatic embryogenesis revealed that the SEs originated from single epidermal cells and developed to the globular, heart, torpedo, and cotyledonary stage embryos. Secondary somatic embryos (SSEs) formed on the surface of radicle tips of the SEs. Addition of low concentrations of NAA and 200–400 mg L^?1 CH to MS1/2 medium increased SSE induction. Cotyledonary SSEs were cultured on MS1/2 medium with 10 mM abscisic acid in the presence of light to promote maturation, and >92 % of mature SSEs were able to germinate with normal shoots. After 8 weeks in culture in the presence of light on medium with one-third of the MS macroelements as well as 0.06 μM NAA, >94 % of the germinated SSEs converted into plantlets. Plantlets acclimatized successfully to ex vitro conditions and developed normal phenotypes under field conditions.     

Highly efficient plant regeneration via somatic embryogenesis from cell suspension cultures of Boesenbergia rotunda

Boesenbergia rotunda is a perennial ginger species rich in flavonoids, flavones, and cyclohexenyl chalcone derivatives. Several of these secondary metabolites have shown promising antiviral and anticancer activities, and thus, it is important to optimize methods for robust production of clonal materials. In this study, cell suspensions were established and their growth capacities were evaluated in liquid media supplemented with varying growth regulator compositions. The highest settled cell volume of 6.1?±?0.3 ml with a specific growth rate of 0.0892?±?0.0035 was achieved by maintaining cells in Murashige and Skoog liquid media supplemented with 1.0 mg L^?1 of 2,4-dichlorophenoxyacetic acid and 0.5 mg L^?1 6-benzyladenine, representing a 12-fold increase in cell volume during the culture period. A somatic embryogenesis rate of 1,433.33?±?387.84 somatic embryos per milliliter of settled cells was achieved with an inoculation cell density of 50 μl settled cell volume and on growth regulator-free agar plates. Around half (53.5?±?7.9%) of the somatic embryos germinated into complete plantlets on media supplemented with 3 mg L^?1 6-benzyladenine and 1 mg L^?1 α-naphthaleneacetic acid. The plantlets were successfully transferred to soil and grown in the greenhouse. Phytochemical profiling via high-performance liquid chromatography analysis revealed that regenerated plantlets retained the capacity to produce and accumulate bioactive compounds. Hence, this protocol will be helpful for metabolic engineering and functional studies of genes and enzymes involved in the biosynthetic pathway of valuable compounds in B. rotunda.     

Direct somatic embryogenesis and plant regeneration from immature explants of chickpea

A protocol for plant regeneration via somatic embryogenesis was developed in two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri. Somatic embryos were induced from immature cotyledons on Murashige and Skoog’s (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), α-naphthaleneacetic acid (NAA) and picloram alone or in combination with 0.5 — 2.0 mg dm⁻³ N⁶-benzylaminopurine (BA) or kinetin (KIN). NAA was better for somatic embryo induction compared to other auxins. The well formed, cotyledonary shaped embryos germinated into plantlets with 36.6 % frequency on MS medium supplemented with 2.0 mg dm⁻³ BA + 0.5 mg dm⁻³ abscisic acid (ABA). The frequency of embryogenesis and plantlet regeneration was higher in cv. ICCV-10 as compared to cv. Annigeri. Regenerated plants were transferred to soil (40 % survival) and grown to maturity. Histological studies of explants at various developmental stages of somatic embryogenesis reveled that somatic embryos developed directly from the cotyledon cells and they were single cell origin.     

Plant regeneration,developmental pattern and genetic fidelity of somatic embryogenesis derived Musa spp.

Multiplication of banana cvs. Grand Naine (Musa AAA, Cavendish-sub group) and Rasthali (Musa AAB, Silk-sub group) were attempted through somatic embryogenesis. The influence of position of male flower buds, amino acid supplements in the induction of somatic embryogenesis and field performance of embryogenic cell suspension (ECS) derived banana plants were studied. Differentiated immature male flower buds positioned at 6–8?th bract whorl as explants showed better callus induction and somatic embryogenesis. Supplementation with glutamine at 400?mg?L^?1 along with 20:20?g?L^?1sucrose: maltose in maturation media induced a 10-fold increase in somatic embryo formation compared to control. Cotyledonary stage somatic embryos desiccated for 2?h showed higher germination compared to non-desiccated embryos. The plantlets generated were hardened, and the genetic fidelity of the plantlets was confirmed using ISSR marker. To check the field performance of ECS derived plants, plantlets were hardened and planted in the field along with meristem and sucker. During the field growth, these ECS derived plants were morphologically similar to those of control plants. In this experiment, it was observed that ECS derived banana plants displayed normal phenotype as that of plants grown from meristem and sucker. The protocol developed could be useful highly for large-scale micropropagation or genetic manipulation studies in these commercially important banana cultivars.     

Production of eleutherosides in in vitro regenerated embryos and plantlets of <Emphasis Type="BoldItalic">Eleutherococcus chiisanensis</Emphasis>

High frequency somatic embryogenesis of Eleutheorcoccus chiisanensis was achieved through suspension culture of embryogenic cells in hormone-free Murashige and Skoog liquid medium supplemented with 30 g sucrose l⁻¹. Cotyledonary somatic embryos were germinated and converted into plantlets using 20 μM gibberellic acid which were then grown in a 10 l airlift bioreactor. HPLC analysis revealed the accumulation of eleutheroside B, E and E1 in the embryos and plantlets. Thus mass production of embryos and plantlets of E. chiisanensis can be achieved in liquid cultures and the biomass produced may become an alternative source of eleutherosides.     

Direct somatic embryogenesis from leaves,cotyledons and hypocotyls of <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis>

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm⁻³ kinetin and 0.2, 0.5 mg dm⁻³ indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.