Somatic embryogenesis from mature zygotic embryos of Distylium chinense (Fr.) Diels and assessment of genetic fidelity of regenerated plants by SRAP markers

The objective was to establish an efficient regeneration protocol for Distylium chinense based on somatic embryogenesis and evaluate the genetic stability of plants regenerated in vitro. To induce callus mature zygotic embryos were cultured on Murashige and Skoog’s (MS) medium that was supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N⁶-benzyladenine (BA). After 20 days, the highest rate of callus formation (88.9 %) occurred on MS medium supplemented with 0.5 mg l^?1 2,4-D and 0.1 mg l^?1 BA. It was observed that light-yellow, compact, dry, nodular embryogenic calli had formed. These calli were then subcultured on fresh MS medium supplemented with 0.1 mg l^?1 BA and 0.5 mg l^?1 α-naphthaleneacetic acid (NAA) for proliferation for an additional 30 days. To induce somatic embryos and plant regeneration, the embryogenic callus was transferred to fresh MS medium that was supplemented with different concentrations of BA and NAA. After 30 days, 0.5 mg l^?1 BA in combination with 0.5 mg l^?1 NAA produced the best result in terms of somatic embryogenesis (%), shoot differentiation (%), number of shoots per callus and shoot length. Next, the plantlets were transferred to the field for 5 weeks and a 95 % survival rate was observed. The sequence-related amplified polymorphism markers confirmed genetic stability of plants regenerated in vitro. To our knowledge, this is the first report that describes a plant regeneration protocol for D. chinense via somatic embryogenesis to be used for germplasm conservation and commercial cultivation.     

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl^?1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA₃), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO₄·7H₂O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.     

Somatic embryogenesis and adventitious shoot formation in Burma reed (<Emphasis Type="Italic">Neyraudia arundinacea</Emphasis> Henr.)

Burma reed (Neyraudia arundinacea Henr.) is a C₄ grass native to Southeast Asia and Indomalaya that grows quickly, exhibits strong resistance to environmental stresses, and is extremely adaptable. It can be widely utilized as a bioenergy crop for biomass conversion. In vitro multiple shoots were first established from axillary buds and then subcultured on propagation medium containing 10 μM 6-benzylaminopurine (BA) and 2.0 μM naphthaleneacetic acid (NAA). Multishoot clumps were used as explants to induce somatic embryogenesis and adventitious shoot formation. The results showed that auxin 2,4-dichlorophenoxyacetic acid or NAA play a key role for the induction of somatic embryogenesis and adventitious shoot formation, whereas cytokinin BA or kineatin enhance shoot proliferation and plant regeneration from callus and somatic embryos. Efficient somatic embryogenesis, mass propagation, and plant regeneration systems in Burma reed were established.     

Regeneration of daylily (<Emphasis Type="Italic">Hemerocallis</Emphasis>) from young leaf segments

Calli were initiated from leaf segments (~0.5 × 0.5 cm) of daylily incubated on Murashige and Skoog (MS) (1962) medium supplemented with 2,4-dichlorophenoxyacetic acid (2.4-D), and either benzyladenine (BA) or thidiazuron (TDZ). The highest frequency of callus induction was observed on medium with 6.79 μM 2,4-D plus either 4.55 or 6.81 μm TDZ. A period of callus maintenance on medium containing 5.37 μM naphthaleneacetic acid (NAA) plus 2.22 or 4.44 μM BA was necessary following induction to improve the quality of the callus, and significantly increase the frequency of embryogenic-like callus formation and shoot regeneration once calli were transferred to light. Over 70% of the regenerated shoots produced roots on ½ strength MS medium lacking plant growth regulators. The regenerated plantlets were successfully transferred into soil and acclimatized in growth rooms. This is the first report showing that leaf segments can be used for daylily regeneration.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

Effect of plant growth regulator combination and culture period on in vitro regeneration of spinach (Spinacia oleracea L.)

The objective of this study was to develop an efficient system for the regeneration of spinach plants (Spinacia oleracea L.) by investigating the factors influencing callus and shoot induction. All plant growth regulator (PGR) combinations tested induced callus with high frequency (73–100 %), and the combination of 5 μM α-naphthaleneacetic acid (NAA), 10 μM 6-benzyladenine (BA) and 0.1 μM gibberellic acid (GA₃) had the most significant effect on callus growth in term of weight (120.98 ± 22.56 mg). A high auxin-containing medium induced competent callus for shoot formation, while high cytokinin-containing media enhanced callus growth and made callus incompetent for shoot regeneration. Longer periods of callus induction in a high auxin-containing medium were required to form competent callus and led to a high regeneration capacity. The PGR combination shift from a high auxin to cytokinin ratio (ACR) to a low ACR resulted in highly efficient regeneration. Among the regeneration systems tested, the combination of 10 μM NAA and 0.3 μM GA₃ for callus induction for 6 weeks followed by 2 μM NAA and 5 μM BA resulted in the highest plant regeneration frequency (83.33 ± 6.43 %) and the highest number of plantlets per explant (7.93 ± 1.24). Somatic embryos at cotyledonary stage and plantlets were transferred to PGR-free medium to establish whole plants. Regenerated female plants grew well to maturity in the greenhouse (77.17 ± 9.80 %) and produced seeds (175.21 ± 28.01 firm seeds per plant).     

Somatic embryogenesis and plant regeneration through cell suspension in diploid (AB) banana cultivar Elakki Bale (syn Neypoovan)

An efficient protocol was developed using cell suspensions for somatic embryogenesis and plantlet regeneration in a most popular diploid AB banana (M.accuminata X M.bulbisiana hybrid) cv. Elakki Bale (syn Neypoovan) known for its taste and keeping quality in southern India. Floral primodia from position 8–16 of male inflorescence which were more responsive for embryogenesis were used as explants for the embryogenic callus production in MS media supplemented with different concentration of 2,4-D. A concentration of 18.1 μM 2, 4-D produced maximum embryogenic calli in 1 % of the explants inoculated. Embryogenic calli on repeated sub culturing on MA2 media produced good embryogenic cell suspensions (ECS). Microscopic examination of ECS showed globular, smaller with dense cytoplasm filled with starchy granules characteristic of embryogenic cells. Highest number of somatic embryos (189) was produced on modified MA3 media. A germination percentage of 31 % were observed in BAP 22.19 μM concentration. Regenerated plants with normal shoot and root were hardened in soilrite. Direct somatic embryogenesis and plant regeneration was also noticed in embryogenic calli which did not pass through the ECS stage. The protocol optimized for somatic embryogenesis through cell suspension and also direct embryogenesis leading to plantlet regeneration can be used for the micropropagation and genetic manipulation.     

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.     

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.     

In vitro shoot growth, direct organogenesis and somatic embryogenesis promoted by silver nitrate in Coffea dewevrei

Direct differentiation of shoot buds in Coffea dewevrei was evident from the seedling shoots with collar region and also from collar region end of hypocotyl segments in presence of 40 μM AgNO₃, 8.88 μM of BA and 2.85 μM of IAA. Apart from this, shoot end of hypocotyl explants mainly supported yellow friable callus or somatic embryos. Subsequent transfer to the same medium induced secondary somatic embryogenesis. The collar region of the hypocotyl explants not only showed direct organogenesis by producing 1–3 shoots per explant and also able to produce globular somatic embryos and embryogenic yellow friable callus. Similarly direct somatic embryogenesis along with yellow friable embryogenic callus formation on 1/2 strength MS medium comprising 1.47 μM IAA, 2.22 μM BA and 40 μM AgNO₃ was noticed from cut portion of in vitro leaf and stalk of regenerated plants. The microshoots rooted well upon subculturing onto the same medium in 6 weeks and showed 60 % survival in green house and resumed growth upon hardening.     

Miscanthus×giganteus plant regeneration: effect of callus types,ages and culture methods on regeneration competence

The perennial rhizomatous grass, Miscanthus×giganteus is an ideal biomass crop due to its rapid vegetative growth and high biomass yield potential. As a naturally occurring sterile hybrid, M. ×giganteus must be propagated vegetatively by mechanically divided rhizomes or from micropropagated plantlets. Plant regeneration through somatic embryogenesis is a viable approach to achieve large‐scale production of plantlets in tissue culture. Effect of the callus types, ages and culture methods on the regeneration competence was studied to improve regeneration efficiency and shorten the period of tissue culture in M. ×giganteus. Shoot‐forming calli having a yellow or white compact callus with light‐green shoot‐like structures showed the highest regeneration frequency. Percentage of shoot‐forming callus induction from immature inflorescence explants was 41% on callus induction medium containing 13.6 μM 2,4‐d and 0.44 μM benzyladenine (BA). The use of a regeneration medium containing 1.3 μM NAA and 22 μM BA was effective at shortening the incubation period required for plantlet regeneration, with 69% of total regenerated plantlets obtained within 1 month of incubation on regeneration medium. Embryogenic‐like callus morphotype could maintain regeneration competency for up to 1 year as suspension cultures. Field grown regenerated plants showed normal phenotypic development with DNA content and plant heights comparable to rhizome propagated plants. Winter survival rates of the regenerated plants planted in 2006 and 2007 at the University of Illinois South Farm, Urbana‐Champaign, Illinois, were 78% and 56%, respectively.     

Organogenesis and somatic embryogenesis in Foeniculum vulgare: histological observations of developing embryogenic callus

Different NAA plus kinetin or BA combinations were tested on Francia Pernod fennel seedlings for callus induction and plant regeneration. Callogenesis from hypocotyls was obtained in all auxin/cytokinin-containing media. The organogenic response was observed especially in presence of NAA plus kinetin. The highest frequency of shoot regeneration was found when the auxin and kinetin were used at a 1:1 ratio. Moreover, a prolonged culture period increased shoot formation. Somatic embryogenesis was tested on several fennel populations. The results gave evidence of the genotypic importance. Two different protocols were used for somatic embryo induction. Using the first protocol among the different fennel genotypes tested, only Francia Pernod showed embryogenic capacity. In this case, from a primary non-embryogenic callus cultured for 12 months in presence of 2,4-D, an embryogenic secondary callus was produced. When transferred to the medium without 2,4-D (agarized or liquid), this gave embryogenic plants in high frequency. As far as the second embryogenic method is concerned, secondary embryogenic callus developed only in the presence of 2,4-D plus kinetin in Francia Pernod genotype. Thereafter, the replacement of those growth regulators by GA₃ into the medium greatly increased the somatic embryo development, especially in `Francia Pernod', but also in `Aboca erbe' callus, a population with a very poor embryogenic capacity. In Francia Pernod, the primary and secondary (embryogenic) calli showed different morphological and histological responses, either when the secondary callus was induced by 2,4-D alone or by 2,4-D plus kinetin. Ontogenetic processes leading to somatic embryo formation are described in this context. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration through somatic embryogenesis of a medicinal plant, <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr.

Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8 μM 6-benzylaminopurine (BA) and 2.0–8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryogenic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.     

尾巨桉胚状体诱导及愈伤组织差异研究

以尾巨桉优良无性系无菌苗茎段为外植体,通过对多种不同浓度生长调节剂组合的优化,进行胚状体诱导研究;并对胚性与非胚性愈伤组织进行形态解剖学观察、相关生理指标检测以及相关基因荧光定量PCR分析,以揭示尾巨桉胚性愈伤组织非胚性化发生的机理,为建立尾巨桉体细胞胚胎再生体系提供参考。结果表明:(1)胚性愈伤组织在MS+0.1mg/L NAA+0.01mg/L TDZ培养基中诱导得到胚状体,外植体经过0.5mol/L蔗糖处理12h有助于胚性愈伤组织产生胚状体,胚状体最高发生率为16.7%。(2)尾巨桉胚性与非胚性愈伤组织石蜡切片观察发现,两者的细胞形态特征存在明显的差异,胚性愈伤组织细胞体积小,排列紧密,表现出典型的胚性细胞特征,而非胚性细胞比较大,排列疏松,细胞呈不规则形状。(3)生理生化指标检测结果表明,非胚性愈伤组织中蛋白质含量、SOD、PPO及CAT活性均显著低于胚性愈伤组织,非胚性愈伤组织中木质素、可溶性糖含量以及PAL和POD活性要高于胚性愈伤组织,二者的反肉桂酸4-单加氧酶基因、淀粉磷酸化酶基因、谷胱甘肽硫转移酶基因、葡萄糖-1-磷酸腺苷酸转移酶基因、葡萄糖六磷酸异构酶基因、分支酸合酶基因以及苯丙氨酸解氨酶基因表达差异也达到显著水平。     

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum,an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Induction of somatic embryos and plant regeneration was demonstrated for the first time in Alnus glutinosa. Somatic embryos were initiated from zygotic embryos collected 1–3 weeks post-anthesis (WPA), i.e., when they were at globular or early cotyledonary stage and were 0.5–1 mm in length. Induction frequency (16.6 %) and the mean number of somatic embryos (4.5 embryos/explant) were highest after culture of zygotic embryos, collected at 3 WPA, on Murashige and Skoog medium (MS) supplemented with 0.9-μM 2,4-dichlorophenoxyacetic acid and 2.22-μM benzyladenine (BA). No embryogenic induction was observed on medium with BA alone. Initial somatic embryos differentiated indirectly from callus tissue formed at the surface of the zygotic embryos. Embryogenic competence was maintained by secondary embryogenesis, which was affected by explant type, plant growth regulators and genotype. Secondary embryogenesis was induced by culture of small groups of whole somatic embryos or isolated cotyledon explants on medium consisting of MS medium (half-strength macronutrients) supplemented with 0.44-μM BA. Histological study of isolated cotyledon explants revealed that secondary embryos developed directly from differentiated embryogenic tissue on the surface of cotyledons. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were evident. For plantlet conversion, somatic embryos were transferred to maturation medium supplemented with 3 % maltose, followed by 6 weeks of culture in Woody Plant Medium supplemented with 0.44-μM BA and 0.46-μM Zeatin (Z). This novel protocol appears promising for mass propagation, conservation and genetic transformation of black alder.     

Plant regeneration from different explant types of Bituminaria bituminosa and furanocoumarin content along plant regeneration stages

The first protocol for in vitro plant regeneration from different explants of Bituminaria bituminosa, a pasture and medicinal species, has been established. Three explant types (petiole, leaflet and petiole-leaflet attachment “PLA”) cultured on media with different combinations of benzylaminopurine (BA; 5.0, 10.0 or 20.0 μM) and naphthalene acetic acid (NAA) or indole acetic acid (IAA; 0.5 or 5.0 μM) were tested for calli induction, and with 5 μM BA + 0.5 μM NAA or IAA for shoot development. The average number of shoots (≥5 mm) per callus depended on the explant type and the calli induction medium. The highest average number of shoots per callus was achieved by culturing leaflet and PLA explants on 5 μM IAA + 10 μM BA for calli induction and on 0.5 μM IAA + 5 μM BA for shoot development, and by culturing petiole explants on 0.5 μM NAA + 10 μM BA followed by a second culture on 0.5 μM NAA + 5 μM BA. The highest frequency of shoot rooting was achieved with 10.0 μM NAA and 1.0 μM gibberellic acid (GA₃). Rooted plants were acclimatised in a culture chamber, reaching 96 % survival. Acclimatised plants were transferred to a greenhouse and finally to the field, reaching 100 % survival. The furanocoumarin (FC) accumulation was evaluated in organogenic calli, in vitro shoots, ex vitro plants in the greenhouse and in ex vitro plants in the field (after 1 and 4 months of acclimatisation). The content of FCs depended on the plant material evaluated, being higher in ex vitro plants in the field (up to 9,824 μg g^?1 DW total FC) and lowest in organogenic calli (up to 50 μg g^?1 DW total FC). This effect may be due to cell organization, longer exposure to environmental factors and the developmental stage.     

High efficient somatic embryogenesis development from leaf cultures of <Emphasis Type="Italic">Citrullus colocynthis</Emphasis> (L.) Schrad for generating true type clones

We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5 mg L^?1 BAP + 1.0 mg L^?1 2,4-D which resulted in 25 % somatic embryos from 85 % nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5 mg L^?1 BAP + 0.5 mg L^?1 NAA from 55 % embryogenic globular callus i.e., 16 %. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot.