Plant regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp. pekinensis)

Isolated microspores of Chinese cabbage (Brassica campestris ssp. pekinensis) were incubated in modified NN medium containing 10% sucrose in darkness at 33°C for one day followed by culture at 25°C. After 14 days of culture, microspores developed into embryos ranging from globular to cotyledonary stage. Plants were regenerated after transfer of embryos to medium containing 3% sucrose and no plant growth regulators.Abbreviations NN Nitsch and Nitsch - MS Murashige and Skoog - NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

Meristematic nodule culture: a new pathway for in vitro propagation of<Emphasis Type="Italic"> Eucalyptus globulus</Emphasis>

Morphogenesis was induced in Eucalyptus globulus seeds, cotyledons, hypocotyls and leaves from in vitro clonal plantlets. Globular structures were observed after 2 weeks induction on B5 culture medium supplemented with 10% coconut water, 0.05–0.5 mg l^?1 6-benzylaminopurine (BAP) and 0.5 mg l^?1 indole-3-butyric acid (IBA). These continued to proliferate under dark conditions until the 2nd to 3rd subculture. Following transfer to a photoperiod of 16 h light, shoots evolved from these globular structures and developed further to plantlets. The influence of several factors, including culture medium composition, sucrose concentration, the type, concentration and combination of growth regulators and the presence of coconut water was studied. The percentage of explants showing globular structure formation and the number of globular structures per explant were evaluated. Macroscopic, histological and scanning electron microscopic studies revealed that the morphogenic process involved mainly organogenic nodules with fewer globular somatic embryos. The nodules gave rise to shoots and subsequently complete plants following incubation on B5 Gamborg medium containing 0.5 mg l^?1 IBA and 30 g l^?1 sucrose, which promoted root formation.     

Plant regeneration through somatic embryogenesis in protoplast cultures of sandalwood (Santalum album L.)

Summary Protoplasts were isolated from embryogenic cell suspension cultures derived from proliferating shoot segments of a 20-year-old sandalwood tree (Santalum album Linn.). Under appropriate conditions, isolated protoplasts divided in liquid culture medium and produced embryogenic cell aggregates and globular embryos. Plating of cell aggregates on a fresh medium facilitated the differentiation of somatic emryos which further developed into plantlets.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA indoleacetic acid - IBA indolebutrytic acid - MES 2-(N-morpholino)ethane sulfonic acid - MS Murashige and Skoog's medium as modified in the text     

In vitro Culture of Immature Embryos of Cytisus laburnum

Immature embryos of Cytisus laburnum L. were cultivated in vitro and four culture media, different techniques of substrate preparation, sucrose concentration and the effect of suspensor removal were tested. The best results were obtained with N6 medium supplemented with 2 mg dm⁻³ glycine and set up using a double-layer culture system, in which the top layer had a higher osmotic potential than the bottom one. These conditions allowed normal embryogenic development in up to 45 % of early globular embryos, that were able to develop until a complete maturity. Osmotic potential and mineral nutrients of the medium demonstrated to be crucial for the successful culture and their effects were dependent on embryo age at the time of excision. The presence of an intact suspensor showed to be beneficial only for early globular embryos while older developmental stage embryos were not significantly affected. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

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     

Spontaneous somatic embryogenesis and plant regeneration from root cultures of Peucedanum palustre

The regeneration of Peucedanum palustre (L.) Moench (milk parsley) was established for the first time via somatic embryogenesis from primary root cultures. Callus formation occurred on the root cultures and showed spontaneous embryogenic capability on B5 basal medium supplemented with a low concentration of indoleacetic acid (5.5 × 10^–7 M). 2,4-Dichlorophenoxyacetic acid was not needed for the initiation of embryogenesis. The somatic embryos germinated and formed plantlets on hormone-free B5 medium. These plantlets were easily transferable to pots, and are presently passing their second growing season in the greenhouse.Development of the somatic embryos progressed through the globular, heart-shaped, torpedo-shaped, and cotyledonary stages, typical of zygotic embryos. Synchronization performed by sieving the embryos did not affect the development time. The culture has retained its embryogenic capacity for 25 months.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA 3-indolebutyric acid - BAP 6-benzylaminopurine     

In vitro regeneration of Camellia sinensis (L.) O. Kuntze by somatic embryogenesis

Within 3 weeks of culture, excised cotyledon expiants of Camellia sinensis (L.) O. Kuntze produced somatic embryos without intermediate callus when cultured in Murashige and Skoog's basal medium with 30 g^–1 sucrose. In medium without plant growth regulators, up to 60% of the cultures developed somatic embryos. Embryogenic competence was reduced by increasing concentrations of plant growth regulators tested (i.e. kinetin, 6-benzylaminopurine, and indole butyric acid). The somatic embryos developed, grew to maturity without being subcultured within 6–8 weeks. Secondary embryogenesis was not observed. Germination of isolated mature somatic embryos was low in medium without plant growth regulators. Up to 53% and 60% germination occurred when medium impregnated with kinetin at 1.8 mgl^–1 or 1.0 mgl^–1 6-benzylaminopurine were used respectively. Callus was also routinely produced when cotyledons were cultured in MS basal medium with auxins (2,4-dichlorophenoxyacetic acid and indole acetic acid). Callus induction was however, also achieved in plant growth regulator free medium. Indirect somatic embryogenesis was not induced in the present study.Abbreviations K kinetin - BAP 6-benzylaminopurine - IBA indole butyric acid - IAA indole acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - Fe-EDTA Ethylenediaminetetra-acetic acid (Ferric monosodium salt)     

Anther culture in Helianthus annuus L., influence of genotype and culture conditions on embryo induction and plant regeneration

Summary Production of microspore-derived embryos from cultured anthers is now a well established technique for the isolation of homozygous lines in many crop plants. We describe here a culture method for embryo induction and plant regeneration from anthers of four sunflower genotypes. For preliminary experiments, anthers of uninucleate microspores were cultured on four types of basal media viz., Murashige and Skoog's MS, Gamborg's B5, Nitsch and Nitsch, and White's W, supplemented with 1.0 mg/l 2,4 dichlorophenoxy acetic acid and 0.5 mg/l 6-benzylaminopurine and 40 g/l sucrose. MS basal medium, being more responsive for embryo induction, was used for further experimentation. To optimise the culture requirement MS basal medium was supplemented with 0.2–2.0 mg/l 2,4 dichlorophenoxy acetic acid and 0.5 and 1.0 mg/l 6-benzylaminopurine. The effect of cold pretreatment, hormone regime and sucrose concentration were tested for embryogenic efficiency. Genotype had a significant effect on the capacity of embryo induction. Addition of silver nitrate (2.5 mg/l), an ethylene inhibitor, stimulated embryo germination. Plantlets were obtained (10–15%) from embryos of only one genotype.Abbreviations 2,4-D 2,4 dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - IAA indole-3-aceticacid - BAP 6-benzylaminopurine - KN Kinetin - ABA abscisic acid - GA₃ gibberellic acid     

Efficient plant regeneration from protoplasts of Arachis paraguariensis Chod. et Hassl. using a nurse culture method

Factors influencing in vitro growth rates of soybean embryos were investigated using embryos isolated in the cotyledon stage. The influence of these factors on final plant recovery from the embryos cultured was tested. Sucrose and glucose could serve as carbon sources with final plant yields being higher with sucrose than with glucose. A culture medium containing only KNO₃ (25 mM) as the nitrogen source supported embryo growth. Adding glutamine (10 mM) to the medium containing KNO₃ increased final plant recovery to 25%. Of several vitamin supplements tested a combination of pyridoxine-HCl, nicotinic acid and pantothenic acid (0.5; 1.0; 0.5 mg l^-1) provided the best growth and plant yield. Of the plant growth regulators tested IAA, BAP and GA₃ stimulated embryo growth and plant development when added to the medium at a low concentration (0.1 M). The optimal temperature for in vitro growth of cotyledon stage embryos was 27°C. Temperatures above 30°C caused growth retardation and reduced plant yield. A protocol for culturing soybean cotyledon stage embryos under conditions ensuring high plant recovery is proposed.Abbreviations IBA indole-3-butyric acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid     

From callus to embryo: a proteomic view on the development and maturation of somatic embryos in <Emphasis Type="Italic">Cyclamen persicum</Emphasis>

In this study, the proteome structures following the pathway in somatic embryogenesis of Cyclamen persicum were analysed via high-resolution 2D-SDS-PAGE with two objectives: (1) to identify the significant physiological processes during somatic embryogenesis in Cyclamen and (2) to improve the maturation of somatic embryos. Therefore, the effects of maturation-promoting plant growth regulator abscisic acid (ABA) and high sucrose levels on torpedo-shaped embryos were investigated. In total, 108 proteins of differential abundance were identified using a combination of tandem mass spectrometry and a digital proteome reference map. In callus, enzymes related to energy supply were especially distinct, most likely due to energy demand caused by fast growth and cell division. The switch from callus to globular embryo as well as from globular to torpedo-shaped embryo was associated with controlled proteolysis via the ubiquitin-26S proteasome pathway. Storage compound accumulation was first detected 21 days after transfer to plant growth regulator (PGR)-free medium in early torpedo-shaped embryos. Increase in abundance of auxin-amidohydrolase during embryogenesis suggests a possible increase in auxin release in the late embryo stages of Cyclamen. A development-specific isoelectric point switch of catalases has been reported for the first time for somatic embryogenesis. Several proteins were identified to represent markers for the different developmental stages analysed. High sucrose levels and ABA treatment promoted the accumulation of storage compounds in torpedo-shaped embryos. Additionally, proteins of the primary metabolic pathways were decreased in the proteomes of ABA-treated embryos. Thus, ABA and high sucrose concentration in the culture medium improved maturation and consequently the quality of somatic embryos in C. persicum.     

Growth characteristics of Glycyrrhiza glabra cell suspension cultures

Sweet potato (Ipomoea batatas (L.) Lam.) breeding has been hampered by self-and cross-incompatibilities that are frequently encountered among the plants in the section Batatas. Ovule culture techniques were developed to assist in overcoming some of these incompatibilities. Ovules that contain embryos at the late globular to heart shaped stage of development were cultured on MS medium containing full strength or one-half strength salts with 3%, 8% or 12% sucrose. Ovules were cultured either intact or after slicing. Ovules of I. triloba and I. trifida were successfully cultured as early as 3 and 4 days after pollination while sweet potato ovules were successfully cultured 5 and 6 days after pollination. The percentage of ovules with developing embryos on the media tested ranged from 27.8% to 50.2%. The highest percentage of embryos developed when the ovules were sliced and cultured on medium containing one-half MS salts and 8% sucrose. Three plants were recovered from cultured ovules of incompatible interspecific crosses.Abbreviations DAP days after pollination - MS medium Murashige and Skoog (1962) medium     

In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [Cajanus cajan (L.) Millsp.] cv. JKR105

In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L⁻¹ 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L⁻¹ 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds.     

Interactions of ancymidol with sucrose and α-naphthaleneacetic acid in promoting asparagus (Asparagus officinalis L.) somatic embryogenesis

Interactions of varying ancymidol concentrations with those of α-naphthaleneacetic acid (NAA) or sucrose in embryo induction medium were related to the production and development of asparagus (Asparagus officinalis L.) somatic embryos, and to the ability of these embryos to germinate. A significant sucrose×ancymidol interaction was observed only for the production of bipolar embryos; 4% sucrose with 0.75 mg l^–1 ancymidol gave the best result, 78 g^–1 callus. The frequency of globular embryos decreased as sucrose or ancymidol concentrations increased. Sucrose concentration affected embryo germination; 3% and 4% sucrose were optimal with approximately 60% and 40% of bipolar and globular embryos germinating, respectively. Significant ancymidol×NAA interactions were observed for the production of bipolar and globular embryos and their germination. Varying ancymidol concentrations affected embryo production and germination in combination with 0.1 mg l^–1 NAA, but not with 1.0 mg l^–1 NAA. The treatment combination of 0.1 mg l^–1 NAA with 0.75 mg l^–1 ancymidol produced the most bipolar embryos, 64 g^–1 callus, and the greatest percentages of bipolar and globular embryos germinated, 63% and 42%, respectively. Received: 19 August 1996 / Revision received: 24 April 1997 / Accepted: 22 May 1997     

Somatic embryogenesis from leaf explants of Australian fan flower,Scaevola aemula R. Br.

Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2–0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium—from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and -naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.Abbreviations ABA Abscisic acid - BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -Naphthaleneacetic acid - PIPES Piperazine-N, N-(2-ethanesulfonic acid) Communicated by R.J. Rose     

Somatic embryogenesis inGnetum ula Brongn. (Gnetum edule) (Willd) Blume

Summary Somatic embryos ofGnetum ula (Gnetum edule) an endangered gymnosperm closely related to the angiosperms have been induced in vitro. Megagametophyte tissue with immature embryos was cultured on Murashige and Skoog medium. A mucilaginous, translucent embryogenic callus was obtained with 5 mg/l BA. Callus induced with 2,4-D was non-embryogenic. The embryogenic callus in liquid half strength Murashige and Skoog medium without inorganic nitrates supplemented with 2.5 g/l casein hydrolysate and 0.5 g/l L-glutamine gave rise to immature embryos. The embryos matured when treated with 60 g/l sucrose and 10 mg/l abscisic acid.Abbreviations MS Murashige and Skoog - BA 6-benzylaminopurine - 2,4-D 2,4 - dichlorophenoxyacetic acid - ABA abscisic acid     

Plant regeneration from protoplasts ofVicia narbonensis via somatic embryogenesis and shoot organogenesis

Protoplasts ofVicia narbonensis isolated from epicotyls and shoot tips of etiolated seedlings were embedded in 1.4% sodium-alginate at a final density of 2.5×10⁵ protoplasts/ml and cultivated in Kao and Michayluk-medium containing 0.5 mg/I of each of 2,4- dichlorophenoxyacetic acid, naphthylacetic acid and 6 -benzylaminopurine. A division frequency of 36% and a plating efficiency of 0.40–0.5% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred onto gelrite-solidified Murashige and Skoog-media containing various growth regulators. Regeneration of plants was achieved via two morphologically distinguishable pathways. A two step protocol (initially on medium with a high auxin concentration followed by a culture phase with lowered auxin amount) was used to regenerate somatic embryos, whereas cultivation on medium containing thidiazuron and naphthylacetic acid resulted in shoot morphogenesis. Mature plants were recovered from both somatic embryos as well as from thidiazuron-induced shoots.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6 -benzylaminopurine - CH casein hydrolysate - FDA fluorescein diacetate - IBA indole-3-butyric acid - KM Kao and Michayluk's medium (1975) - MS Murashige and Skoog's medium (1962) - NAA naphthylacetic acid - TDZ thidiazuron     

Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis (Orchidaceae)

Summary Embryogenic calluses were induced from 73% of Phalaenopsis shoot-tip explants excised from flower stalk buds by culturing for 7 mo. on New Dogashima Medium (NDM) containing 0.5 μM α-naphthaleneacetic acid (NAA), 4.4 μM 6-benzylaminopurine and 29.2 mM sucrose. The sucrose concentration was increased to 58.4 mM 4 mo. after initiation of the callus culture. These calluses were successfully subcultured as cell suspension cultures in liquid NDM supplemented with 5.4μM NAA and 58.4 mM sucrose. By simply reducing the sucrose concentration to 29.2 mM, the cells grew into plantlets through a developmental process similar to that of Phalaenopsis seedlings. The occurrence of somaclonal variants was less than 10% in six out of eight genotypes examined. These results suggest that the embryogenic callus and cell suspension culture could be utilized as the materials for micropropagation and breeding of Phalaenopsis orchids.     

Somatic embryogenesis and plantlet regeneration in Cornus florida

Somatic embryos were initiated from 12 to 15 weeks postanthesis (WPA) zygotic embryos of Cornus florida L. (flowering dogwood) cultured on Murashige-Skoog (MS) or Schenk and Hildebrandt (SH) medium amended with either 3 mg/L 2,4-D or 5 mg/L 2,4-D and 1 mg/L kinetin. White, opaque globular and early cotyledonary stage embryos were formed directly on detached cotyledons from 2 of the 5 trees sampled after 7 weeks of culture. Morphologically mature embryos developed after an additional 4 weeks incubation on medium without growth regulators; however, many of the embryos were fused in pairs along the entire length of the hypocotyl-radicle axis. Indirect embryogenesis was observed from callus cultures initiated from 9 to 15 WPA zygotic embryos. These cultures have continued to produce embryos for 16 months. Many of the embryos formed roots on germination medium, but only 12% formed plantlets and none developed past the first true leaf stage.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - FPA Formalin-propionic acid-ethanol (50%) - WPA weeks post-anthesis     

Regeneration of diploid annual medics via direct somatic embryogenesis promoted by thidiazuron and benzylaminopurine

 The development of a simple and rapid procedure for direct somatic embryogenesis from wild Medicago spp. (M. truncatula, M. littoralis, M. murex, M. polymorpha) has exploited various explants including meristematic zones. Phytogel-solidified medium supplemented with thidiazuron or 6-benzylaminopurine at different concentrations effectively promoted this process. The first somatic structures emerged within 20 days of culture initiation. Histological analyses confirmed the nature of the directly formed embryos. Secondary embryogenesis was also observed. Cuttings of clusters of primary and secondary embryos were used for cyclic production of new embryo generations. Regenerated plants with well-developed root systems on medium with reduced levels of macroelements and sucrose were easily adapted to a greenhouse. Received: 23 March 1998 / Revision received: 10 August 1998 / Accepted: 7 December 1998