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.

     

Protocols for efficient repetitive and secondary somatic embryogenesis in Helianthus maximiliani (Schrader)

 Indirect somatic embryogenesis was induced on leaf explants of greenhouse-grown Helianthus maximiliani plants. Leaves of the regenerated plants were used as starting explants for the induction of direct somatic embryogenesis. Another cycle of somatic embryogenesis was induced on the leaves of regenerated plants. In both cases, leaf explants were cultured on media containing different auxin/cytokinin ratios. The auxin/cytokinin ratio had an influence on the intensity of embryo formation, germination and the capability to regenerate plants. Somatic embryogenesis was generally more intensive on the medium with lower concentrations of 6-benzylamino-purine. Further, the percentage of regenerated plants was higher when embryos were induced on high-cytokinin, low-auxin medium. Secondary somatic embryogenesis was induced on embryos by culture in liquid hormone-free medium. Similar to direct embryogenesis the efficiency of secondary embryogenesis depended on the medium used for the induction of the primary embryos. In contrast to the mostly low frequencies of conversion of secondary embryos into plants that has been observed in other species, the percentage of regenerated plants from secondary embryos of H. maximiliani was quite high, although slightly lower than that obtained in primary embryos. Received: 28 March 2000 / Revision received: 1 September 2000 / Accepted: 2 October 2000     

Plant regeneration via direct somatic embryogenesis in Panax japonicus

Panax japonicus is one of the important medicinal plants. Here, we established the protocol for plant regeneration of P. japonicus via direct somatic embryogenesis. Somatic embryos were directly obtained from the segments of zygotic embryos on MS medium with 4.4 μM 2,4-D. Thereafter, somatic embryos were produced by repetitive secondary somatic embryogenesis. The secondary somatic embryo formation was enhanced by plasmolyzing pretreatment (1.0 M mannitol for 10 h). Frequency of secondary somatic embryo formation from cotyledon segments was lowered by plasmolyzing pretreatment, but the number of somatic embryos per explants was greatly increased. Plasmolyzing pretreatment resulted in retardation of embryo growth and required subculture to fresh medium for further growth of embryos into cotyledonary stage. Without plasmolyzing pretreatment, cotyledonary embryos were obtained after 8 weeks of culture. All the cotyledonary somatic embryos germinated by 5 μM GA₃ treatment, but only 15.3% were germinated on hormone-free medium. After 2 months of culture on 1/2 strength WPM medium, plantlets produced flowers spontaneously. In the anthers of in vitro flowers, microsporogenesis occurred normally with low number of pollen grains.     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.Abbreviations BAP 6-benzylaminopurine - Kinetin 6-furfurylamino purine - 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.     

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.     

Somatic embryogenesis from cultured epicotyls and primary leaves of soybean [Glycine max (L.) Merrill]

Summary Regeneration of several varieties of soybean [Glycine max (L.) Merrill] by somatic embryogenesis from cultured epicotyls and primary leaves has been demonstrated. Somatic embryogenesis was induced from epicotyls and primary leaves when cotyledon halves with the intact zygotic embryo axes were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg 1⁻¹ (45.2 μM) 2,4-D. Stable, continuously proliferating globular embryo cultures (GEC) were established from small groups of somatic embryos on MS medium supplemented with 20 mg 1⁻¹ (90.5 μM) 2,4-dichlorophenoxyacetic acid (2,4-D). Rapid multiplication of shoot tips from germinating somatic embryos was achieved on Cheng’s basal medium (CBO) containing 2.5 mg 1⁻¹ (11.3 μM) 6-benzyladenine. Fertile plants were obtained from individual somatic embryos and in vitro propagated adventitious shoot bud cultures.     

Micropropagation of <Emphasis Type="Italic">Kalopanax pictus</Emphasis> tree via somatic embryogenesis

Summary Kalopanax pictus (Thunb.) Nakai is a tall tree, and its wood has been used in making furniture, while its stem bark is used for medicinal purposes. Here, we report on the micropropagation of Kalopanax pictus via somatic embryogenesis. Embryogenic callus was induced from immature zygotic embryos. The frequency embryogenic callus induction is influenced by days of seed harvest. Callus formation was primarily observed along the radicle tips of zygotic embryos incubated on Murashige and Skoog (MS) medium with 4.4 μM 2,4-dichlorophenoxyacctic acid (2,4-D). Somatic embryogenesis was observed following transfer of embryogenic callus to MS medium lacking 2,4-D. Somatic embryos at the cotyledonary stage were obtained after 6 wk following culture. Frequency of conversion of somatic embryos into plantlets was low (35%) on a hormone-free MS basal medium, but it increased to 61% when the medium was supplemented with 0.05% charcoal. Gibberellic acid (GA₃) treatment markedly enhanced the germination frequency of embryos up to 83%. All plantlets obtained showed 98% survival on moist peat soil (TKS2) artificial soil matrix. About 30 000 Kalopanax pictus plants were propagated via somatic embryogenesis and grown to 3-yr-old plants. These results indicate that production of woody medicinal Kalopanax pictus plantlets through somatic embryogenesis can be practically applicable for propagation.     

Direct somatic embroygenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.)

Direct regeneration of somatic embryos was obtained from immature zygotic embryos of Dalbergia latifolia. Immature embryos dissected from green pods 90 d after flowering gave the highest frequency of somatic embryo formation. Preculture on high 2,4-D medium for 4 weeks induced direct somatic embryogenesis, which was expressed during the second culture phase in the presence of low 2,4-D along with a high sucrose concentration. Embryos were separated and transferred to the maturation medium containing MS + 0.5–1.0 mg/L BAP, where embryos developed into plantlets. Somatic embryos failed to convert into complete plants without BAP treatment. This method of direct regeneration of somatic embryos without a callus phase has direct application for genetic manipulation studies.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - KIN Kinetin     

Somatic embryogenesis and plant regeneration from leaflets of peanut,Arachis hypogaea

Somatic embryos were induced on peanut (Arachis hypogaea) leaflets from aseptically germinated embryo axes. Leaflet size influenced percent somatic embryogenesis; 5–8 mm long cut leaflets were superior to 2–3 mm long uncut leaflets. Maximum embryogenesis of 14.6% was obtained after a 15 d incubation on induction medium (modified MS with B5 vitamins, 30 g/l sucrose, 4 g/l Gel-Gro, 40 mg/l 2,4-D +0.2 mg/l kinetin) followed by transfer to a secondary medium with 5 mg/l 2,4-D+0.2 mg/l kinetin. Primary somatic embryos were fused along the axes with no distinct cotyledons, but secondary embryos had single axes with two cotyledons. Other treatments had lower percent embryogenesis, no secondary embryogenesis, and embryos with single axes with two cotyledons. Some somatic embryos converted into normal plants capable of greenhouse survival.Abbreviations MS Murashige and Skoog (1962) medium - B5 Gamborg et al. (1968) B5 medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6benzylaminopurine - NAA 1-naphthaleneacetic acid     

Efficient somatic embryogenesis and plant regeneration from long-term cell suspension cultures of Medicago Truncatula cv. Jemalong

Summary Plants were suecessfully régenerated via somatic embryos from 3-yr-old cell suspension cultures of Medicago truncatula Gaertin. cv. Jemalong line M9-10a. The cultures were originally initiated from callus induced in well-expanded leaflets of 30 d in vitro-grown plants, Suspension cultures were established in stirred-liquid Murashige and Skoog (MS) basal salts and vitamins supplemented with 2.3 μM 2.4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin (Kin) and subeultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45 μM 2,4-D and 0.91 μM zeatin (Zea), during 1,2, and 3wk after subculture. Induced and non-induced cultures were transferred to solid embryo proliferation medium [EPM-MS basal salts and vitamins solidified with 0.2% (w/v) gelrite]. Somatic embryos developed until the late torpedo/dicotyledonary stages. We found that the best condition for the development of somatic embryos was achieved when suspension cultures were not subjected to the induction step. Induction of 1 and 2 wk led to a decrease in the recovery of somatic embryos and the 3-wk treatment resulted in no differentiation of somatic embryos. Plant regeneration was obtained in all conditions (except for 3wk induction) when embryos were transferred to an embryo conversion medium [ECM, similar to EPM but solidified with 0.7% (w/v) agar]. Embryo conversion rates were 54.5±1.6, 52.5±18.5, and 41.6±8.4% for 0, 1, and 2 wk induction treatments, respectively. These plants were successfully transferred to the greenhouse where they matured and produced seeds.     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

The histological analysis of indirect somatic embryogenesis on <Emphasis Type="Italic">Drosera spathulata</Emphasis> Labill

We studied indirect somatic embryogenesis in the callus tissue of Drosera spathulata Labill. originated from isolated leaves. Callogenesis was induced on MS medium (Murashige and Skoog 1962), supplemented with various concentrations of NAA and BA. Somatic embryos regenerated on half-strength MS medium supplemented with 20 μM of NAA or without growth regulators. The highest efficiency of somatic embryo production was achieved on hormone-free medium. Globular, heart-, torpedo- and cotyledonary-shaped embryos were observed in embryogenic clusters. Histological and scanning electron microscopy analysis verifies somatic embryogenesis. Regenerated plants were transferred to soil and were grown to maturity.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Somatic embryogenesis and plant regeneration from immature seeds of Magnolia obovata Thunberg

We have tested plantlet formation by somatic embryogenesis using immature seeds of Magnolia obovata. Seed collection date appeared to be critical for embryogenic cell induction. The optimal collection date was 3–4 weeks postanthesis. The embryogenic cells proliferated, formed somatic embryos, and were subsequently converted into normal plantlets under optimized culture conditions. Somatic embryo formation from the embryogenic calli was better on sucrose medium than on glucose medium. The optimum level of sucrose appeared to be 3% with an average of 28 somatic embryos per plate. About 25% of somatic embryos were converted into normal plantlets in 1/2 MS medium containing gibberellic acid (GA₃). During somatic embryo germination, secondary embryogenesis was frequently observed in the lower part of the hypocotyl or radicle ends of germinating somatic embryos. Finally, about 85% of converted plantlets survived in an artificial soil mixture, were transferred to a nursery, and have grown normally.     

Embryo production through somatic embryogenesis can be used to study cell differentiation in plants

Somatic embryogenesis is the process by which somatic cells, under induction conditions, generate embryogenic cells, which go through a series of morphological and biochemical changes that result in the formation of a somatic embryo. Somatic embryogenesis differs from zygotic embryogenesis in that it is observable, its various culture conditions can be controlled, and a lack of material is not a limiting factor for experimentation. These characteristics have converted somatic embryogenesis into a model system for the study of morphological, physiological, molecular and biochemical events occurring during the onset and development of embryogenesis in higher plants; it also has potential biotechnological applications. The focus of this review is on embryo development through somatic embryogenesis and especially the factors affecting cell and embryo differentiation.     

Influence of triacontanol on somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

Summary A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.     

松柏类植物体细胞胚胎发生的研究进展

松柏类植物的体细胞胚胎发生既是繁育的一种手段,又是研究胚胎发育过程中结构、生理和分子事件的一种重要的模式系统.整个体细胞胚胎发生过程主要包括3个步骤:胚性组织的诱导和增殖、体细胞胚的成熟以及体细胞胚的萌发和转换.过去为了提高胚胎发育过程所做的努力主要都集中在胚的成熟阶段,这是因为一直认为能否成功再生的关键在于胚发育成熟阶段的处理.然而,在过去几年里,结合生理生化以及分子生物学的研究发现,胚胎发生的早期阶段对于完成整个发育过程也是至关重要的,早期阶段培养条件的优化可以显著提高培养过程中体细胞胚的数量和质量.此外,萌发过程培养条件的调节对于提高成熟体细胞胚的萌发率和转换率也很重要.因此,这些新的研究成果对于改善松柏类植物体细胞胚胎发生中的胚的诱导率和转换率低的现象具有重要的意义.     

A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation

Somatic embryogenesis of the carrot (Daucus carota L.) depends on a set of factors, some of which accumulate in culture medium (conditioned medium, CM). When embryogenic cell clusters were transferred to an embryo-inducing medium, addition of CM derived from somatic embryo culture markedly stimulated somatic embryo formation. The active principles were purified using a simple bioassay system and identified to be phytosulfokines (PSKs), sulfated oligopeptide growth factors originally isolated from a CM derived from asparagus (Asparagus officinalis L.) mesophyll culture. Quantification studies using a competition ELISA system employing an anti-PSK-alpha polyclonal antibody showed that PSK production might be related to growth of cells, rather than development of somatic embryos. Thus the stimulatory effect of PSK on somatic embryo formation might be due to promotion of cell proliferation.     

Primary and repetitive secondary somatic embryogenesis of <Emphasis Type="Italic">Lepidosperma drummondii</Emphasis> (Cyperaceae) and <Emphasis Type="Italic">Baloskion tetraphyllum</Emphasis> (Restionaceae) for land restoration and horticulture

Somatic embryogenesis was developed as a method of mass propagation for Lepidosperma drummondii (Cyperaceae), a difficult to propagate but important species for post-mining restoration in a region of high plant biodiversity, in the southwest of Western Australia. Cultures were initiated from excised zygotic embryos, shoot cultures to rhizomes. Only zygotic embryos of L. drummondii developed somatic embryos, with half strength Murashige and Skoog basal medium (BM) and 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) being the most effective combination. The first culture cycle yielded a mean of 30 somatic embryos per excised zygotic embryo forming an embryo cluster. After a further 6 wk in culture (on fresh BM with 1 μM 2,4-D), approximately 350 somatic embryos per starting embryo cluster were recorded. Following regular sub-culturing of primary somatic embryo clusters onto fresh media (every 4 wk), more than 74,000 secondary somatic embryos were estimated to have been produced after eight subculture periods. This translates to between 1,000 and 2,000 somatic embryos produced from an estimated 45 mg of starting tissue per culture plate or potentially 22,0000–44,000 somatic embryos per gram of tissue. This is a significant improvement over all previous methods used to propagate L. drummondii, in which typical in vitro shoot multiplication rates are as low as 1.43 per 8 wk. This also compared favourably with published data and concurrent experiments undertaken in this study (as an extra control measure) on somatic embryo production for a related species Baloskion tetraphyllum (using the same BM with 1 μM 2,4-D and coleoptile segments as explants). Various media combinations were investigated for efficacy in converting somatic embryos into plants with best results ranging from 86% to 100% conversion for B. tetraphyllum on BM without plant growth regulators. Development of L. drummondii somatic embryos into plants was not observed on BM without plant growth regulators. However, a best result of 39% conversion to plants was observed on BM with 1 μM thidiazuron. This is the first report of successful development of somatic embryogenesis and conversion of somatic embryos into plants using thidiazuron for the Australian cyperale L. drummondii.