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.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

Somatic embryogenesis and plant regeneration from leaf-derived cell suspension of a mature tree — Thevetia peruviana L.

Cell suspension cultures, which retained embryogenic potential for almost 2 years, were established from young, expanding, juvenile leaves of a mature Thevetia peruviana L. tree. Calli were obtained by culturing young leaf discs on MS medium supplemented with 2 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.1 mg/L kinetin. Suspension cultures were initiated by transfer of calli to liquid medium containing 1 mg/L 2,4-D + 0.1 mg/L kinetin, and the cultures were maintained by subculturing to fresh medium at 2 week intervals. Embryogenic frequency of cell aggregates was more than 80% when plated on semi-solid medium containing 0.1 mg/L 2, 4-D and 2 mg/L 2-isopentenyladenine (2-iP). Cell aggregates with developing embryos were transferred to fresh medium lacking growth regulators for embryo maturation. Early embryo development was synchronous and a large number of somatic embryos were produced. These somatic embryos developed into plantlets upon subsequent transfer to modified half-strength MS medium. More than 200 green and rooted plants, at an average of 80 plants per 100 mg of embryogenic callus, were obtained with 60% survival under glass house conditions.Abbreviations 2, 4-D 2 4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine - IAA Indole — 3 — acetic acid - KN Kinetin - MS Murashige and Skoog (1962) basal medium - NAA 1 -Napthalene acetic acid     

Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)

Embryogenic cultures and somatic embryos were obtained from immature zygotic embryos of oak (Quercus robur L.) cultured on a modified MS medium and WPM containing BAP (1 mg·l^–1) and GA₃ (1 mg·l^–1) or BAP and IBA. Germination and conversion of oak somatic embryos into plantlets was achieved on WPM containing a reduced concentration of cytokinin. Linden (Tilia cordata Mill.) somatic embryos developed in embryogenic tissues initiated from immature zygotic embryos cultured on a modified MS medium supplemented with 2,4-D (0.3-2.0 mg·l^–1). Germination of linden somatic embryos and plantlet formation occurred on MS medium containing a low concentration of IBA. Oak and linden plantlets produced from somatic embryos were successfully established in soil. Somatic embryos and plantlets were also regenerated from embryogenic cultures of Quercus petraea and Tilia platyphyllos.Abbreviations BAP 6-benzyIaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - WPM woody plant medium     

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.     

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     

Plant regeneration from embryogenic suspension cultures ofCamellia Japonica

Summary Two methods (I and II) for somatic embryo production from embryogenic suspension cultures ofCamellia japonica are presented. Method I, embryogenic suspension cultures, was established from suspension cultures initiated from leaf-derived callus. These cultures were maintained by reducing agitation and increasing subculture interval. Induction of somatic embryogenesis was achieved in MS28 medium, 6, 12, 24, and 36 mo. after culture establishment. Embryo production decreased after 1 yr of culture. Method II, suspensions of single embryogenic cells and proembryos, was obtained from leaves cultured in liquid MS13 medium 6 wk after culture initiation. Embryo production was 23 embryos/ml. Germination of cell suspension-derived embryos on MS56 medium was 16.7 % (±4.2%) for method I, and 35.4% (±5.1%) for method II. The embryos germinated into plantlets with 0 to 7 axillary shoots.     

Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7%?±?1.3% for anthers and 4.8%?±?2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9%?±?4.8% to 83.9%?±?8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7%?±?9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.     

The somatic embryogenesis and establishment of transformation experiment system in Larix principis-Rupprechtii]

Larix principis-Rupprechtii is one of the superior afforestation forest trees growing in north China. Embryogenic cultures were initiated from immature zygotic embryos of Larix principis-Rupprechtii on S culture medium containing 2, 4-D 0-2.2 mg/L, KT and BA each at 0-0. 8 mg/L. Embryogenic calli were subcultured and multiplicated on S + B culture medium containing dropping off each hormone concentration. We set up 33 steady-going embryogenic cell lines; We studied on the growth stage and genotype differences of every embryogenic cell lines; and Finded more than 10 high-frequency somatic embryogenesis cell lines such as 2K, 2T, 2I, 2J, 3C etc.. The number of 2T somatic embryos reaches 314/per gram of embryogenic tissue and the number of 3C somatic embryos is 185/per gram of embryogenic tissue. The re-induction method of Larix principis-Rupprechtii from somatic embryos was used to produce renewable embryogenic cultures and steady-going embryogenic cell lines effectively. Mature somatic embryos can germinate and develop further into plantlets when they are isolated and cultured on a hormone-free WPM culture medium. The regeneration plantlets were obtained. Furthermore, the transformation with a truncated gene of Bacillus thuringensis (B. t) were carried out, the PCR showed positive results, because of this, embryogenic cell line of Larix principis-Rupprechtii can be used for transformation experiments to support further breeding in forestry.     

Somatic embryogenesis in wild cherry (Prunus avium)

Indirect somatic embryogenesis was obtained inPrunus avium L. from either somatic or zygotic embryos. An embryogenic line was established by reinduction of embryogenic calluses from somatic embryos. The line was maintained for more than 3 years through 6 generations of embryogenic cultures. In the last 2 generations, more than 50% of the explants were embryogenic. Embryos at different stages of development were produced. Among cotyledonary-stage embryos, 50% had two cotyledons and a distinct hypocotyl, 43% had one or more than 2 cotyledons and 7% had fused cotyledons. Most of the embryos were translucent and conversion into plantlets was very rare. Secondary embryos could be observed to occur with low frequency from cultured somatic embryos and from embryos emerging from calluses. Indirect somatic embryogenesis was also induced from immature zygotic embryos. From one donor tree, 51% of the explants were embryogenic when cultured on a medium containing 0.9 μM kinetin, 0.9 μM BA and 0.5 μM NAA. This revised version was published online in June 2006 with corrections to the Cover Date.     

华北落叶松体细胞胚胎发生及遗传转化实验系统的建立(简报)

华北落叶松(Larix principis-Rupprechtii)是我国北方中高山地区重要的针叶速生用材树种,进行其体细胞胚胎发生和植株再生的研究,在针叶树无性快速繁殖及基因工程育种上有其特殊的用途,既可为针叶树无性系林业提供产业化途径,也可作为目的基因遗传转化实验系统。针叶树的基因转化相对较难,再生更属不易,Lelu等报道过杂种落叶松与欧洲落叶松体细胞胚胎发生方面的研究;而我国尚未见有落叶松体细胞胚胎发生的研究报道。我们     

High frequency somatic embryogenesis and plant regeneration in zygotic embryo cultures of Japanese honeysuckle

Japanese honeysuckle plant (Lonicera japonica Thunb.) is rich in iridoid secologanin and is a potentially useful model for the study of secologanin biosynthesis. Culture conditions for high frequency plant regeneration via somatic embryogenesis from zygotic embryo cultures and zygotic embryo-derived embryogenic cell suspension cultures of this species are described. Mature zygotic embryos formed embryogenic calluses at a frequency of 46.7% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 M 2,4-dichloro-phenoxyacetic acid (2,4-D). Cell suspension cultures were established with embryogenic calluses using liquid MS medium with 4.52 M 2,4-D. Upon plating onto MS basal medium, embryogenic cell suspension cultures produced numerous somatic embryos, which subsequently developed into plantlets at a frequency of 68%. Regenerated plantlets were transplanted to potting soil and grown to maturity in a greenhouse.     

Somatic embryogenesis from leaf derived callus of Tylophora indica (Burm. f.) Merrill

Mature leaf explant derived callus of Tylophora indica (Burm. f.) Merrill yielded somatic embryos on MS medium supplied with BA(1-2 mg/L) or kinetin(1-5 mg/L) or kinetin/BA (1-2 mg/L) used along with IAA(0.1-1 mg/L). Maximum somatic embryos (30) could be recovered from 100 mg of embryogenic callus within 60 days at an optimum concentration of 2 mg/L of BA which was also best suited for providing the maximum conversion rate (90%) of embryoids to plantlets. Kinetin (1-5 mg/L), used as the sole growth hormone, induced the development of embryoids showing either shoot or root primordia in 30% of the cultures. However, embryoids with shoot primordia developed roots upon transfer to medium containing IAA(0.1 mg/L) and kinetin(2 mg/L). Embryoids from all cultures germinated in the initiation medium and were transplanted to sterile vermiculite for hardening. After two weeks of hardening, the plantlets were transferred to the green house where they grew and established well showing a high rate of survival (90%).     

Somatic embryogenesis and plant regeneration in zygotic embryo cultures of balloon flower

Mature zygotic embryos of balloon flower (Platycodon grandiflorum) formed embryogenic calluses at a frequency of 43% when cultured on Murashige and Skoog medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Cell suspension cultures were established from embryogenic calluses using MS liquid medium with 4.52 μM 2,4-D. Following transfer to solid MS basal medium, cell suspension cultures gave rise to somatic embryos, which then developed into plantlets. Plantlets were transplanted to potting soil and grown to maturity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from mesocotyl and leaf-base segments of <Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L., a minor millet

Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N₆) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N₆ medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N₆ regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N₆ medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.     

In Vitro Regeneration of Onion through Repetitive Somatic Embryogenesis

A reliable protocol for the regeneration of onion through repetitive somatic embryogenesis was established. Embryogenic callus was derived from mature seeds on Murashige and Skoog (MS) medium supplemented with 2 mg dm-3 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos aroused on the surface of calli cultures and formed plantlets after the removal of 2,4-D or its substitution with 1 mg dm-3 kinetin (Kin). Reculturing the somatic embryos on 2,4-D containing medium led to secondary embryos formation. The embryogenic cultures which were preserved for five months on maintenance medium containing 2 mg dm-3 2,4-D + 0.5 mg dm-3 Kin have retained their ability for regeneration, while those kept on 2,4-D only, failed to form plantlets. Electrophoretic analysis of total soluble proteins revealed that the competence for successful conversion of somatic embryos into plantlets is associated with the expression of new set of proteins (112, 58 and 30 kD). The regenerated plants were successfully transferred to the soil.     

Somatic embryogenesis from immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.     

Recovery of somatic embryos and plantlets from protoplast cultures of Larix × eurolepis

Summary Somatic embryos and plantlets were regenerated from protoplasts of hybrid larch (Larix × eurolepis) isolated from two embryogenic callus and cell suspension culture lines (L1 and L2). L2, which was highly embryogenic, consistently yielded protoplasts that gave rise to somatic embryos. Centrifugation on a discontinuous medium/Percoll density gradient resulted in accumulation of embryogenic protoplasts in one of the Percoll interfaces. First division frequencies were in the range of 28–39% in line 1 and 18–20% in line 2 in both liquid and agarose-solidified culture media. The critical factor in maintaining high viability of cultures was lowering of osmotic pressure by dilution of the initial medium. The first somatic embryos were detected in 23- to 28-day-old cultures. Some of these developed into plants that were transferred to soil.     

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.     

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.