Somatic embryogenesis and plant regeneration from leaf callus of Ocimum basilicum L

A effective protocol for complete plant regeneration via somatic embryogenesis has been developed for Ocimum basilicum L. Callus was initiated from leaf explant of young plant on supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) 1.0 mg l(-1), 3% sucrose and 0.9% agar. The calli showed differentiation of globular structure embryos when transferred to MS medium containing 2,4-D 0.5 mg l(-1) and BAP 1.0 mg l(-1). The maximum globular structure embryos were further enlarged and produced somatic embryos in MS basal medium supplemented with BAP 1.0 mg l(-1)+NAA 1.0 mg l(-1) + KN 0.5 mg l(-1). Continued formation of globular embryo and germination of embryos occurred in this medium. Complete plantlets were transferred onto specially made plastic cup containing soilrite followed by their transfer to the garden soil. Survival rate of the plantlets under ex vitro condition was 80%.     

In vitro induction of pollen embryos and plantlets in Aesculus carnea Hayne through anther culture

Uninuclear microspores in red horse chestnut anther cultures formed pollen embryos and plantlents in MS agar medium supplemented with varying 2,4-D concentrations (1.0, 1.5 or 2.0 mg l^-1) and 1.0 mg l^-1 Kin. The highest number of embryogenic anthers (38%) was obtained in MS medium containing 1.0 mg l^-1 of each 2,4-D and Kin. The ability of pollen embryos to germinate was closely correlated with normal embryo morphology and was influenced by hormone content in the medium (MS+;1.0 mg l^-1 IAA+1.0 mg l^-1 GA₃+0.1 mg l^-1 Kin+400 mg l^-1 glutamine). Pollen embryos and plantlets had the haploid chromosome number (x=n=40). Cytological examinations demonstrated pollen dimorphism of this Aesculus species.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin 6-furfurylaminopurine - GA₃ gibberellic acid - MS Murashige and Skoog     

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     

Plant regeneration from zygotic embryo-derived embryogenic cell suspension cultures of Ranunculus kazusensis

Culture conditions for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Ranunculus kazusensis are described. Zygotic embryos formed white nodular structures and pale-yellow calluses at a frequency of 84.9% when cultured on half-strength Schenk and Hildebrandt (SH) medium supplemented with 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). However, the frequency of white nodular structure and off-white callus formation decreased with an increasing concentration of 2,4-D up to 10 mg l⁻¹, when the frequency reached 25%. Cell suspension cultures were established from zygotic embryo-derived pale-yellow calluses using half-strength SH medium supplemented with 0.1 mg l⁻¹ of 2,4-D. Upon plating onto half-strength SH basal medium, over 90% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted to potting soil and grown to maturity at a survival rate of over 90% in a growth chamber. The plant regeneration system established in this study can be applied to mass propagation and conservation of this species.     

High frequency plant regeneration from immature ovule-derived embryogenic cell suspension cultures of Chelidonium majus var. asiaticum

Culture conditions for high frequency plant regeneration via somatic embryogenesis in cell suspension cultures of Chelidonium majus var. asiaticum are described. Immature ovules formed embryogenic calluses at a frequency of 40% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimum ovule size for embryogenic callus formation ranged from 1 to 1.5 mm in length. Cell suspension cultures were established from embryogenic calluses using MS liquid medium containing 4.52 μM 2,4-D. Upon plating onto MS basal medium, cell aggregates from cell suspension cultures produced somatic embryos which then developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to maturity in a growth chamber. This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency somatic embryogenesis and plant regeneration in tissue cultures of Codonopsis lanceolata

Culture conditions for high frequency somatic embryogenesis and plant regeneration from cotyledonary explants of Codonopsis lanceolata are described. The maximum induction frequency of somatic embryos from cotyledonary explants was 80% on Murashige and Skoog (MS) medium containing 6% sucrose with 1 mg/l 2,4-dichlorophenoxyacetic acid and 10% coconut water. Upon transfer onto MS basal medium containing 3% sucrose, most somatic embryos developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin a₃ - MS Murashige and Skoog     

Somatic embryogenesis and plant regeneration in Anthurium andraeanum hybrids

Summary A method for the production of somatic embryos and subsequent plant regeneration for Anthurium andraeanum Linden ex André (Monocotyledonae) hybrids is described. Whole leaf blade explants, derived from plantlets grown in vitro, formed translucent embryogénic calli at their basal ends within one month of culture in the dark. Secondary somatic embryos formed frequently and without an intervening callus on surfaces of primary embryos. Embryogenesis was induced with three genotypes using a modified half-strength Murashige and Skoog (MS) medium supplemented with 1.0 to 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.33 to 1.0 mg l^–1 kinetin. A combination of 2% sucrose with 1% glucose in the medium favored embryogenesis over 3% sucrose alone. Whole leaf blades on medium solidified with 0.18% Gelrite produced more somatic embryos than leaves on medium with 0.7% Bacto-agar. Within two to three months after culture initiation, embryos were transferred to modified MS medium containing 0.2 mg l^–1 6-benzyladenine (BA) and 2% sucrose and placed in the light for conversion into plantlets. Rooted plantlets were recovered and transferred into pots with tree fern fiber medium and grown in the greenhouse.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4 dichlorophenoxyacetic acid - BA 6-benzyladenine     

Plant regeneration from embryogenic cell suspension cultures of wild sorghum (Sorghum dimidiatum Stapf.)

A simple and efficient protocol is described for regeneration of wild sorghum (Sorghum dimidiatum) from cell suspension cultures. Fast-growing cell suspensions were established from shoot-meristem-derived callus. Plating of the suspension on Murashige and Skoog agar medium supplemented with 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in the formation of embryogenic calli. High-frequency (80%) somatic embryogenesis from small cell clusters (300–400 μm) was observed when the cultures were initially maintained in liquid medium with reduced levels of 2,4-D (0.25 mg l^–1), followed by transfer to regeneration medium. Direct plating of these small clusters on regeneration medium or transfer to liquid regeneration medium containing kinetin and 6-benzylaminopurine resulted in the development of mature somatic embryos and plantlets. The regenerants developed to maturity and were all phenotypically and cytologically normal. Received: 20 May 1998 / Revision received: 1 September 1998 / Accepted: 23 September 1998     

Somatic embryogenesis,encapsulation, and plant regeneration of <Emphasis Type="Italic">Rotula aquatica</Emphasis> lour., a rare rhoeophytic woody medicinal plant

Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.     

High frequency plant regeneration from zygotic-embryo-derived embryogenic cell suspension cultures of watershield (Brasenia schreberi)

An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when cultured on half-strength MS medium supplemented with 0.3 mg l⁻¹ 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly with increasing concentrations of 2,4-D up to 3 mg l⁻¹, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryo-derived white friable callus were established using half-strength MS medium supplemented with 0.3 mg l⁻¹ 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield, which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could be useful for the mass propagation and transformation of selected elite lines.     

新疆天山雪莲体胚诱导与分化研究

以新疆天山雪莲的叶片为外植体,分别用不同配方培养基诱导愈伤组织,后进行体胚诱导和分化培养形成再生雪莲植株.结果表明,诱导愈伤组织的最适培养基为MS 2,4-D 0.5 mg/L BA 1.5 mg/L,诱导率可达到100%;愈伤组织转移至MS 2,4-D 0.5 mg/L BA 1.5 mg/L培养基进行继代培养,增殖后的愈伤组织转移到MS 2,4-D 0.2 mg/L的液体培养基后成功诱导出雪莲体胚,出胚率达40%;将体胚接至MS ABA 0.5 mg/L培养基后,结果分化生长出大量的再生雪莲幼苗.     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.)

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B₅ vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B₅ medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B₅ medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-Benzyl-aminopurine - IAA IndoIe-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - Kinetin 6-furfuryl aminopurine - Zeatin 6-(4-hydroxy-3-methylbut-2-enylamino)-purine     

Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and petiole expiants has been developed in sweetpotato [Ipomoea batatas L. (Lam.)]. The optimal somatic embryogenic response was obtained in the genotype PI 318846-3 with a two-step protocol: (1) stage I-incubation of expiants in the dark for 2 weeks on Murashige Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l) and 6-benzylaminopurine (0.25 mg/l) and, (2) stage II-culture in the light on MS medium with abscisic acid (ABA) (2.5 mg/l). The addition of ABA was critical for enhanced production of somatic embryos. Secondary somatic embryos were produced from the primary embryos cultured on MS medium with 2,4-D at 0.2 mg/l. The somatic embryos were converted into normal plantlets when cultured on basal MS medium. Upon transfer to soil, plants grew well and appeared normal with no mortality. The system of somatic embryogenesis described here will facilitate tissue culture, germplasm conservation and gene transfer research of sweetpotato due to its rapidity (6 to 10 weeks), prolific plant production by direct embryogenesis, ease of secondary somatic embryo production and reproducibility.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine, 2,4-D-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - KIN kinetin - MS medium of Murashige and Skoog (1962) - NAA 1-naph-thaleneacetic acid - PIC picolinic acid - TDZ thidiazuron     

Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.     

In vitro propagation of Podophyllum peltatum L. by the cultures of embrya and divided embrya

Excised embrya and subsequently divided embrya of Podophyllum peltatum were cultured on the Murashige and Skoog medium supplemented with different growth regulators, because traditional methods of breaking seed dormancy failed. The growth of excised embrya was stimulated by 1 or 0.1 mg dm-3gibberellic acid (GA3), 0.1 mg dm-3 GA3 + 0.2 mg dm-3 kinetin (kin), or 0.2mg dm-3 kin. GA3 (1 mg dm-3) showed the best effect; after 5 weeks the plantlets had 1.5 - 2 cm long cotyledons, 5 - 6 cm long roots, 88 % of embrya germinated and developed further. The addition of 0.5 mg dm-3 zeatin + 0.2 mg dm-3 naphtaleneacetic acid (NAA), 0.2 mg dm-3 NAA, and 1 mg dm-3 kinetin inhibited the growth of embrya. 1 mg dm-3 kinetin + 0.1 mg dm-3 NAA, 0.1 mg dm-3 zeatin and 0.2 mg dm-3 6-benzylaminopurine resulted in a compact appearance of plantlets and a lower germination rate. Divided embryo cultures produced plantlets via somatic embryogenesis which occurred only on the 2,4-dichlorophenoxyacetic acid containing media. The maturation of somatic embrya was observed on media without any auxin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l^?1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.     

Secondary somatic embryogenesis and plant regeneration in myrtle (Myrtus communis L.)

Immature seeds, as well as hypocotyls and cotyledons excised from seedlings of Myrtus communis L., were cultured on media containing half-strength Murashige and Skoog macronutrients (MS/2) with combinations of auxins and cytokinins, in order to study their morphogenetic competence. Somatic embryogenesis was obtained from cotyledons, hypocotyls and 2-month-old immature seeds with 0.1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The percentage of explants showing this primary somatic embryogenesis ranged from 4% for hypocotyls to 12% for 2-month-old immature seeds. In the latter, somatic embryogenesis was also obtained in media containing 2,4-D plus a cytokinin, and with only a cytokinin. Somatic embryos obtained from hypocotyls, cotyledons or immature seeds were able to develop on MS/2 medium without plant growth regulators. Subculture of primary somatic embryos obtained from immature seeds on MS/2 medium without plant growth regulators gave rise to clusters with secondary somatic embryos and embryogenic calli. These clusters were subcultured every 8 weeks, and they were the source of highly embryogenic cultures. An average of 10% of the secondary somatic embryos developed into plantlets in each subculture. Therefore, the same culture on MS/2 medium without growth regulators yielded both plantlets and de novo secondary embryos. Received: 6 April 1998 / Revision received: 10 July 1998 / Accepted: 21 July 1998     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium