Induction of somatic embryogenesis in lotus (Nelumbo nucifera Geartn.)

Callus induction and somatic embryogenesis of lotus (Nelumbo nucifera Gaertn.) cv. Satabankacha were studied. Callus was initiated by culturing bud, cotyledon, and young leaf explants on Murashige and Skoog (MS) (1962) medium containing a combination of 0, 4, 8 and 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 μM 6-furfuryl amino purine (kinetin) or substituting 0, 0.5 and 1 μM benzyladenine (BA) for kinetin. Bud explants cultured on medium containing 4 μM 2,4-D and 1 μM BA gave the best callus growth. For somatic embryogenesis, the calli initiated on MS medium containing a combination of 4, 6, 8 and 10 μM 2,4-D and 1 μM BA and subsequently transferred to media containing 2–4 μM 2,4-D and 0 or 0.5 μM BA produced the most somatic embryos. When cultures were 12-week-old, callus produced on medium with 6 μM 2,4-D and 1 μM BA showed the best growth for somatic embryo regeneration. When transferred to a medium with 2 μM 2,4-D and 0.5 μM BA somatic embryos were produced from 33% of the calli. Embryos developed to the stage proembryo within 4 weeks and formed globular, heart, torpedo and mature embryos within 16 weeks.     

Callus and axillary-bud culture of Vitis vinifera ‘Sylvaner Riesling’

Healthy growth of serially subcultured callus of the grape Vitis vinifera cultivar ‘Sylvaner’ was obtained by incubation at 30° C in continuous light in a defined culture medium containing 2% w/v sucrose, 1.0 mg l^?1 1-naphthaleneacetic acid (NAA) and 0.2 mg l^?1 kinetin (K). Organogenesis was not induced in this callus by alteration in the absolute or relative levels of NAA and K.Continued shoot initiation was obtained by culture of axillary buds in a medium containing 10^?5 M Benzyladenine (BA). Plantlets could be generated from these shoot buds by transfer to media containing 10^?7 M BA or lacking a cytokinin.     

Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

Somatic embryogenesis in a broad spectrum of grape genotypes

Somatic embryogenesis is the preferred method for cell-to-plant regeneration of grapevine. In this study, we tested the embryogenic capacity of anther-derived calli from 59 grape genotypes, representing a diverse group of Vitis vinifera and hybrid varieties, and hybrids and accessions of non-vinifera Vitis species. Most genotypes were tested on two types of media: MST1 medium, which contained plant growth regulators (PGRs) 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), and MSE medium, which contained 2,4-D and 6-benzylaminopurine (BAP). Twenty-four of the grape genotypes produced embryogenic callus on one or both of these media, eighteen of which have not been reported to form somatic embryos before. The results also suggested that the various PGR combinations are differentially effective at inducing somatic embryos in various classes of grape genotypes. For example, seven of the eight V. vinifera conv. occidentalis varieties brought forth somatic embryos on MSE medium, and three out of four American Vitis genotypes produced somatic embryos on MST1 medium. We could not observe any apparent association between frequency of callus formation and embryogenic capacity of the anthers.     

Somatic embryogenesis in Cymbopogon pendulus and evaluation of clonal fidelity of regenerants using ISSR marker

An efficient plant propagation system through somatic embryogenesis was established in Cymbopogon pendulus, an aromatic grass followed by analysis of genetic status of regenerants using ISSR markers. Optimum embryogenic callus induction was observed on MS basal medium supplemented with 13.57 μM 2,4-dicholorophenoxyacetic acid (2,4-D) with 8.88 μM N⁶-benzyladenine (BA). Subsequent culturing of embryogenic calli on MS medium containing 4.52 μM 2,4-D and 8.88–13.32 μM BA gave maximum number of somatic embryos. Addition of coconut water (CW) promoted induction, growth and differentiation of callus and somatic embryogenesis. Further development of embryos into plantlets was achieved on MS medium supplemented with lower concentration of biotin and calcium pantothenate (CaP) along with BA (4.44–13.32 μM) and kinetin (2.32–4.65 μM). The root meristems were established on half strength MS medium containing 2% sucrose and 2.46–9.84 μM Indole3-butyric acid (IBA) and successfully established in soil with 77.8% survival rate in field condition. Thirteen randomly selected regenerated clones were screened using six ISSR primers. Nine clones produced similar monomorphic amplification profiles while remaining clones showed minor variation with absence of certain parental bands and appearance of unique band. Majority of the regenerants maintained genetic fidelity with the generation of few variants as evidenced from similarity matrix estimates using Nei Li's coefficient of similarity data.     

Recurrent somatic embryogenesis and plant regeneration in Angelica glauca Edgew., a critically endangered medicinal plant of the Western Himalaya

Summary

Secondary somatic embryogenesis and plant regeneration from seedling explants of Angelica glauca, an endangered medicinal plant of the Himalaya, is reported for the first time. Callus was obtained from all the explants tested in the present study (i.e., epicotyls, hypocotyls, and cotyledonary nodes). The highest frequency of callus formation (95.8%) was observed using epicotyl explants on 4.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), whereas 70.8% of hypocotyl explants, and 58.3% of cotyledonary nodes produced callus. One-hundred percent embryogenic callus was induced from epicotyl explants in 2.0 µM 6-benzyladenine (BA) and 2.0 µM μnaphthaleneacetic acid (NAA), together with the maximum number of somatic embryos (34.2 embryos per explant). Cotyledonary nodes did not produce somatic embryos. Histological studies confirmed the induction of somatic embryogenesis. Somatic embryos germinated into plantlets upon transfer to half-strength Murashige and Skoog (MS) medium without added plant growth regulators. We observed 85% survival of these plantlets under field conditions. The development of secondary embryos was also observed when primary embryos were sub-cultured on full-strength MS medium containing 2.0 µM NAA plus 2.0 µM BA. This system of recurrent somatic embryogenesis provides a route for gene transfer and also for the large-scale production of this critically endangered medicinal plant.     

Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.)

The effect of sucrose concentration on callus induction followed by differentiation of embryogenic callus derived from petal explants of four carnation cultivars (Nelson, Sagres, Spirit and Impulse) was investigated. Embryogenic calli were produced on Murashige and Skoog [Murashige, T., Skoog, F.A., 1962. Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 154, 73–479] basal medium (MS) culture medium containing six concentrations of sucrose (3, 6, 9, 12, 15 and 18%, w/v) all supplemented with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 μM 6-benzyladenine (BA). Maximum frequency of embryogenic callus was obtained from the media containing 9 and 12% sucrose. Somatic embryos were induced on a hormone-free MS media containing the seven concentrations of sucrose. Development of somatic embryos was enhanced by increasing sucrose concentration from 1.5 to 12%, while it was reduced in higher concentrations of 15 and 18%. However, normal embryos were not developed in the media containing 1.5 and 3% sucrose. Ninety-five percent of somatic embryos were regenerated to form the entire plantlets when they transferred onto the half-strength hormone-free MS culture medium containing 3% sucrose. Plantlets were also continued to grow normally under greenhouse condition.     

Direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis (Girard) Kuntze

The potentialities of direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis var. Golden Diamond invitro were investigated. Young whole leaf and cut leaf explants when cultured on MS basal medium supplemented with each of the growth regulators N⁶-benzyladenine (BA) (0.44–2.2 μM) or thidiazuron (TDZ) (4.54 μM) alone or in combination with a fixed concentration of α-naphthalene acetic acid (NAA) (1.07 μM) produced somatic embryos directly. More than 90% of the leaf explants produced white, globular somatic embryos on BA (2.2 μM) and NAA (1.07 μM) supplemented MS basal medium within 1 week of inoculation. Most of the embryos matured further and converted after 8 weeks of culture on the same medium. Histological observation showed that the somatic embryos originated from single cells of epidermal layer of leaf. Histological evidence of formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. After conversion the plantlets were further placed on MS medium containing 0.44 μM BA and 4.5 μM IBA for better shoot and root growth. About 90% of the plantlets transferred to the mixture of soil:perlite:vermiculite (1:1:1) in small plastic pots acclimatized successfully. Of these 85.5% plants survived after transferring into earthen pots containing a mixture of soil, coarse sand and cattle manure (1:1:1) under greenhouse or shady open condition.     

In vitro culture of cotyledon tissue of Castanea sativa Mill

Establishment of callus tissue culture from cotyledon fragments of Castanea sativa Mill. is described. The explants were grown on various combinations of auxins (IAA, IBA, NAA, 2,4-D) and cytokinins (K, BA) to induce callus and regeneration of organs. The best growth occurred on basal medium with 2,4-D (1 and 10 mg/l) plus both K or BA (0.5 mg/l). Root regeneration was achieved with both IBA and NAA (10 mg/l) supplemented with K or BA.     

Somatic embryogenesis and plant recovery from callus of ‘Nabali’ Olive (Olea europea L.)

Regeneration via somatic embryogenesis from callus was studied in ‘Nabali’ olive (Olea europea L.). Among different explant sources (leaf blades, leaf petioles, hypocotyls of germinated seeds and roots of germinated seeds), roots gave the highest (46%) callus induction. Somatic embryogenesis was induced from root callus on embryogenesis induction medium (EIM) containing 5.0 μM 2,4-D, 0.5 μM kinetin and 5.0 μM NAA in darkness. Embryo regeneration was studied by transferring the callus from EIM to embryogenesis expression medium (EEM) containing different concentrations (0.0, 5.0, 10.0 and 15.0 μM) of 2-isopentenyladenine (2iP), BA, thiadiazuron (TDZ), zeatin or kinetin. Among the tested concentrations, 2iP at 10.0 μM outperformed the other growth regulators. 2,4-D at 5.0 μM in the EIM was satisfactory for embryogenesis induction. Sucrose at 0.2 M evoked higher embryogenesis than any other concentration of fructose and glucose in EIM, while sorbitol and mannitol at 0.1, 0.2 or 0.3 M reduced embryogenesis significantly and inhibited it totally at 0.4 M. Somatic embryos were rooted by transferring them to hormone-free medium (HFM). About 85% of embryos converted to rooted plantlets, 5% showed secondary embryogenesis and 10% were not developed and died. Rooted plantlets gave 95% survival when acclimatized ex vitro. Acclimatized plantlets developed into whole plants in the greenhouse and they were phenotypically similar.     

In vitro shoot regeneration from immature seeds of Epimedium alpinum induced by thidiazuron and CPPU

In vitro propagation of Epimedium alpinum L. was carried out using immature seed explants. The effects of various concentrations of thidiazuron (TDZ) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), on the induction of organogenic callus, were evaluated. Organogenesis occurred most efficiently when explants were transiently exposed (48 h) to 20 μM CPPU or 80 μM TDZ followed by culture on hormone-free woody-plant medium (WPM). Organogenic callus consisting of white, compact clumps of tissue proliferated slowly on hormone-free WPM. To promote adventitious shoot induction, the effects of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) were investigated. The highest per cent shoot regeneration, 66.7% of explants, and the maximum mean number of shoots, 2.6 per explant, were obtained on WPM containing 1.1 μM 2,4-D and 22 μM BA. Shoots were rooted on hormone-free WPM and well-developed plantlets were successfully transferred to soil.     

In vitro somatic embryogenesis from Mangifera indica L. callus

The nucellus and globular adventitious proembryos were removed from 2-month-old fruits of mango (Mangifera indica L.) cultivars ‘Ono’ and ‘Chino’, and were cultured on sterile, solid Murashige and Skoog (MS) medium that had been modified as follows: half-strength major salts and chelated iron; 20% (v/v) coconut water (CW); 6% sucrose; 100 mg l^?1 ascorbic acid and 400 mg l^?1 glutamine. Embryogenic explants were sub-cultured after 4–6 weeks in liquid modified MS medium containing 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) instead of CW. Rapidly growing cultures were established and were sub-cultured monthly. Somatic embryogenesis was induced following sub-culture from MS medium with 2,4-D to MS without growth regulators and with or without activated charcoal (0.5%). Germination of somatic embryos appeared to be enhanced by 1 mg l^?1 benzyladenine (BA); however, most of the germinating embryos became embryogenic.     

Factors affecting shoot development in apically dominant Acer cultivars in vitro.

Summary

Axillary shoot cultures of both Acer saccharinum L. ‘Pyramidale’ and A. platanoides L. ‘Crimson King’ displayed strong apical dominance and prolific basal callus in vitro, which was not conducive to rapid multiplication and rooting. Basal callus was reduced in ‘Pyramidale’ by replacing 6-benzylaminopurine (BAP-5μM) with zeatin (5μM), but this also reduced axillary shoot growth. The addition of 2,3,5-triiodobenzoic acid (1-20 μM) altered callus development and promoted a concentration-dependent increase in axillary shoot growth. Supplementing medium with thidiazuron (0.005 and 0.05 μM) in addition to BAP (1 μM) enhanced shoot growth, especially with nodal shoot sections, and increased subsequent rooting. Although thidiazuron also increased basal callus, this correlated with better shoot growth in ‘Crimson King’. Selection of apical buds from ‘Crimsom King’ stockplants was essential for the establishment of sustainable cultures; axillary bud-derived expiants quickly died. Once shoots of ‘Pyramidale’ and ‘Crimson King’ had elongated, they could be readily rooted in vitro, and plantlets were successfully weaned under high humidity ‘dry fog’.     

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.     

Propagation of Lilium hybrids. II. Production of plantlets from bulb-scale callus cultures for increased propagation rates

A method has been developed for the large-scale propagation of lilies from callus cultures. Bulb scales, being available at all times, are an ideal source of explants. Callus was induced throughout the annual cycle of all the 12 Oriental hybrid cultivars tested, by treating the bulb scales with a combination of 5 μM BA and 5 μM 2,4-D. Once initiated, this callus grew vigorously on media lacking exogenous growth substances. Callus induction by BA and 2,4-D, and the subsequent growth of such callus in the absence of exogenous growth substances, occurs within a broad range of cultivars and species within the genus. The callus could be maintained on a medium lacking ammonium salts, but the maximum growth rate was obtained at 10 mM NH₄NO₃. At low (0.2 mM) or zero levels of NH₄NO₃, light as compared with darkness was inhibitory to growth.The callus maintained its capacity for organogenesis. Maximum production of plantlets was obtained in continuous light on agar medium with 0.5 μM NAA.Potentially, the successive use of liquid and agar cultures can produce 6 × 10¹² plants per year from 1 g (dry wt.) of callus. Plantlets derived from callus were consistently diploid and could be readily transferred to soil.     

Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

An efficient indirect somatic embryogenesis and Agrobacterium-mediated transformation protocol for Limonium sinense has been established, wherein neomycin phosphotransferase II (npt II) and β-glucuronidase (GUS) genes were used as selectable and screenable markers, respectively. The efficiency of plantlet regeneration from transformed tissue was compared between direct embryogenesis from leaf and indirect embryogenesis from callus. Embryogenic callus (EC) was initiated from leaf explants on MS medium supplemented with 6.7 μM 2,4-D and 2.22 μM BA. The somatic embryos were induced, matured, and germinated when ECs were transferred onto MS medium supplemented with 4.44 μM BA and 1.07 μM NAA. Agrobacterium tumefaciens strain LBA 4404 containing the vector pBI121 was used for the transformation. Transient GUS expression frequency was evaluated and putative transgenic plants were successfully grown on culture medium in presence of kanamycin (80–100 mg L^?1). PCR analysis of putative transgenic plants confirmed the presence of GUS and nptII genes. The transformation efficiency obtained through indirect embryogenesis from calluses (4%) was much higher than through direct embryogenesis from leaf explants (0.9%).     

Callus induction and culture of Rosa

Leaf and stem explants of Rosa manetti Hort. and R. hybrida L. ‘Tropicana’ were placed on Murashige and Skoog (MS), Schenk and Hildebrandt (SH) and Gamborg and Wetter (1-B5C) media, containing growth regulators, casein hydrolysate (CH) or coconut milk (CM), to determine the optimum conditions for callus initiation and maintenance. The explants were cultured either in dark or in light (2 Klux 16 h/day) at 26±2°C. Friable, fast growing callus was evident after 3 weeks for both rose species on MS + 2.0 mg/l 2,4-D, 0.25 mg/l kinetin (K) and 2.0 g/l CH as well as on SH + 0.5 mg/l 2,4-D, 2.0 mg/l p-CPA and 0.1 mg/l K. Callus initiation occurred sooner on SH than on MS. However, during sub-culture of callus arising on SH, rapid oxidation often occurred, resulting in severe browning of the callus, which made it unsatisfactory for further experimental use. Callus initiation occurred faster in dark than in light, but deteriorated when continuously sub-cultured in the dark regardless of media. Although ‘Tropicana’ initiated callus sooner than R. manetti, very fast growing callus of the latter was obtained when leaf callus was transferred to MS + 2.0 mg/l NAA and 0.5 mg/l BA. Unsuccessful attempts to regenerate callus and induce adventitious shoots on the 2 explants are discussed.     

‘过山香’香蕉多芽体的诱导及其体细胞胚的发生

 以我国重要香蕉种质‘过山香’ (AAB) 为材料, 研究了香蕉多芽体诱导、多芽体茎尖薄切片即分生组织性的表层结构物( scalp) 愈伤组织诱导和体细胞胚发生的合适条件。结果表明, 该品种单个不定芽茎尖在P4培养基(含BAP 100 μmol·L ^{- 1}和IAA 1 μmol·L ^{- 1} ) 中继代5个周期后可诱导获得类似花椰菜结构的多芽体。从30 μmol· m ^{- 2} ·s^{- 1}光强, 光/暗周期(16 h /8 h) 培养的多芽体所获得的scalp在添加2,4-D 5μmol·L ^{- 1}和Zeatin 1 μmol·L ^{- 1}的愈伤组织诱导培养基中黑暗培养, 45 d后愈伤组织诱导率可达97.6%。诱导20 d后黄色分生小球体类结节状愈伤组织开始发生, 90～120 d后在分生小球体局部可获得白色或浅黄色松散的胚性愈伤组织(诱导率为17.5% ) 。从胚性愈伤组织诱导的体细胞胚在成熟培养基上培养60 d后体胚萌发率和植株转化率分别为14.5%和11.1%。     

Propagating palms in vitro with special emphasis on the date palm (Phoenix dactylifera L.)

Tissue-culture methods are described for the vegetative propagation of several palm species either through shoot tip culture or plantlet differentiation via embryogenic callus. The influence of explant size, medium composition and physical environment required for the establishment of palm shoot tips in vitro was determined. Date palm (Phoenix dactylifera L.) seedling shoot tips of various sizes were cultured in either liquid or agar modified Murashige and Skoog (MS) medium containing 0.0–1.0 mg 1^?1 α-naphthaleneacetic acid (NAA) and 0.0–15.0 mg 1^?1 benzyladenine or N⁶-(Δ²-isopentenyl) adenine (2iP) in order to enhance shoot growth and induce axillary budding. Satisfactory date palm shoot tip growth and proliferation was obtained from explants that were 3 mm in length, consisting of the apical meristem region and 2–5 adjacent leaf primordia. Optimum shoot tip development and axillary budding was obtained by initially establishing explants on an agar medium for 2 weeks, then transferring to a liquid medium. Shoot tips from several palm species were cultured on MS media containing 100 mg 1^?1 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg 1^?1 2iP and 3 g 1^?1 activated charcoal, or on MS medium containing 1 mg 1^?1 NAA and charcoal, to determine their morphogenetic responses in vitro. Shoot tips of Metroxylon sp., Phoenix canariensis Hort. ex. Chabaud., P. dactylifera ‘Khalasa’, ‘Thoory’ and ‘Zahidi’, and P. roebelenii O'Brien planted on medium with 2,4-D and 2iP initiated callus, asexual embryos and free-living plantlets after 4–8 months in culture. Shoot tips from Erythea edulis S. Wats., P. canariensis, P. dactylifera ‘Khalasa’, Thoory' and ‘Zahidi’, Washingtonia filifera Wendl. and W. robusta Wendl. cultured on medium containing NAA developed into plantlets with well-developed leaves and adventitious roots within 2–6 months from the time of planting. In some cases, cultured date palm shoot tips gave rise to axillary buds.