Plant regeneration from somatic embryogenic suspension cultures of cotton (Gossypium hirsutum L.)

Maintainable, highly embryogenic suspension cultures of cotton (Gossypium hirsutum L. cv. Coker 310) have been obtained. Callus cultures were initiated from cotyledonary tissues from aseptically-germinated seedlings. To establish the suspension cultures, callus tissue was placed in a liquid medium containing either 0.5 mg/l picloram or 0.1 mg/l 2,4-dichlorophenoxyacetic acid. For proliferation of the embryogenic suspension, 5 mg/l of 2,4-dichlorophenoxyacetic acid was used. Embryo development took place when the embryogenic tissue was transferred to an auxin-free liquid medium containing 15 mM glutamine. Early embryo development was fairly synchronous and large numbers of somatic embryos were produced. Regenerated plants were fertile and smaller than seed-derived plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid     

Development and characterization of embryogenic suspension cultures of pecan

A method for the establishment and proliferation of developmentally stable, embryogenic suspension cultures in pecan is described, and the growth and development of cultures characterized. Suspension cultures were generated from somatic embryos derived from zygotic embryo cotyledon explants induced on a solidified medium with naphthaleneacetic acid. Cultures were repetitively embryogenic and proliferated in growth-regulator-free medium. The suspensions consisted of a mixture of globular stage embryo-aggregates, freely suspended globular embryos and pre-globular stage embryo masses. Culture growth and proembryo production were evaluated with respect to several liquid media and pH conditions. Significant differences in growth and productivity were observed between cultures. Pre-globular stage embryo masses collected on filter paper and overlaid on solidified medium continued ontological development and converted into plants. Thus a method has been developed for pecan suspension culture, which presents a major improvement in embryogenic tissue culture within the Juglandaceae. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment of embryogenic suspension cultures of a wild relative of cotton (Gossypium klotzschianum Anderss.)

Summary Maintainable, highly embryogenic suspension cultures of a wild relative of cotton (Gossypium klotzschianum Anderss.) have been obtained. Callus with no apparent organization was used to establish the liquid culture. Callus growth conditions as well as suspension medium composition were optimized. A visual selection scheme was beneficial for the maintenance of the embryogenic suspension. These liquid cultures have been maintained for over 10 mo. with no loss in embryogenic capacity. The somatic embryos developed after transfer of the embryogenic tissues to a hormone-free liquid medium. Salaries and research support were provided by State and Federal funds appropriated to OSU-OARDC. This is journal article No. 71-87.     

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.     

Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.)

Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0°C/min). A cooling rate of 0.5°C/min provided a satisfactory recovery rate (30%) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of 1°C/min. Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soil.Abbreviations DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) - MMS modified MS - NAA -naphthaleneacetic acid     

Plant regeneration from embryogenic suspension cultures of dune reed

Embryogenic callus, derived from mature seeds of dune reed (Phragmites communisTrinius) was used to establish suspension culture. Green shoot-forming type and albino shoot-forming type embryogenic callus of dune reed were selected carefully by the difference of shape and color of callus growing under light and mechanically dispersed before suspending in liquid MS medium supplemented with 1.0 mg l^–12,4-D. They were subcultured every 5 days to remove mucilaginous material in the early culture stage. Both fine albino and green shoot-forming cell suspension lines of dune reed were composed of rapidly growing small cell aggregates that were densely cytoplasmic and potentially embryogenic. Globular somatic embryos were continuously produced in each liquid medium containing 1.0 mg l^–1 2,4-D. The cell aggregates in fine albino cell suspension line (size below 300 m) were smaller than that of green shoot-forming cell suspension line (size between 300 and 800 m). Following transfer to a differentiation medium, both suspension cultures formed regenerating plants with normal roots and albinotic or green shoots, respectively.     

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.     

Growth characteristics and transformability of soybean embryogenic cultures

Embryogenic cultures of soybean [Glycine max (L.) Merr. cv. Jack and Asgrow A2872] were established in liquid Finer and Nagasawa medium, maintained by transfer to fresh medium at biweekly intervals, and subjected to microprojectile bombardment over time. Cultures were not amenable to transformation until they were at least 6 months old. Over time, different cell lines of the same genotype acquired very different culture phenotypes. Histological analysis of cell lines differing in transformation ability showed that the most transformable cultures had cytoplasmic-rich cells in the outermost layers of the tissue. In contrast, the outer layers of less transformable cultures contained cells with prominent vacuoles. Although fresh weight accumulation of the cultures was curvilinear during the 2-week subculture period, a burst of mitotic activity was evident shortly after transfer to fresh medium. This activity usually lasted from the 2nd to the 6th day following subculture, and peaked on the 4th day. Tissues at or near this stage always produced more transient expression of a reporter gene than did bombardments at other times. In addition, the cell lines most amenable to transformation also exhibited the highest mitotic index. Thus any treatment to increase the mitotic index, especially when the cell lines are less than 6 months old, may facilitate the transformation of cell lines from which efficient recovery of transgenic plants is still possible. Received: 16 September 1997 / Revision received: 29 January 1998 / Accepted: 21 February 1998     

Polyphenoloxidase-activity and -activation in embryogenic and non-embryogenic suspension cultures of Euphorbia pulcherrima

The activity and activation potential of polyphenoloxidase (PPO, E.C. 1.10.3.1.) of tissue from shoot tips, adult leaves and embryogenic and non-embryogenic cell suspension cultures of Euphorbia pulcherrima was investigated using an oxygen probe technique. PPO derived from differentiated in vivo plant tissue (shoot tips, leaves) cannot be activated either by storage at 0–4°, freezing and thawing, incubation with CaCl₂, sodium dodecyl sulfate or by incubation with trypsin. Embryogenic cells are characterized by high initial PPO activity and strong activation potential of membrane bound enzyme. Non-embryogenic material reveals low phenolase activity and low activation potential. An activation quotient (based on the ratio between PPO-activity determined after sodium dodecyl sulfate incubation to PPO-activity determined after CaCl₂-incubation) was calculated. This is independent of absolute enzyme activity and can be used for characterization of the embryogenic status of cells.Abbreviations fw fresh weight - MS Murashige and Skoog - PPO polyphenoloxidase - SDS sodium dodecyl sulfate     

High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures

Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment, was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with abnormal morphology and a high degree of sterility. Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999     

Cryopreservation of embryogenic suspension cultures of Cyclamen persicum Mill.

We have developed a simple protocol for the cryopreservation of embryogenic suspension cultures of Cyclamen persicum. Embryogenic suspension cultures in the linear growth phase 7–10 days after subculture were used for cryopreservation. Of the different cryoprotectants tested during a 2-day pre-culture, 0.6 M sucrose resulted in the highest re-growth rates of 75%. An additional pre-treatment with 0.6 M sucrose and 10% DMSO (dimethylsulfoxide) for 1 h also positively affected re-growth. Microscopic studies on viability revealed that only few small embryogenic cells survived cryopreservation, while vacuolated single cells died. Experiments in which the duration of the pre-culture period—i.e. the length of time the embryogenic suspension cells were exposed to 0.6 M sucrose—was varied showed that 2–4 days was the most optimal exposure time to 0.6 M sucrose. Callus re-grown after cryopreservation showed growth rates similar to that of unfrozen callus and regenerated even higher numbers of somatic embryos than unfrozen callus.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - DM Dry mass - DMSO Dimethylsulfoxide - FDA Fluorescein diacetate - FM Fresh mass - 2IP 6-(,-Dimethylallylamino)purine - LN Liquid nitrogen - rpm Rounds per minute - SCV Sedimented cell volume     

Establishment and characterization of long-term embryogenic maize callus and cell suspension cultures

Friable callus (type 2) was selected from three genotypes (A188, hybrid A188 × B73, and hybrid B73 × A188) of Zea mays L. The three genotypes of type 2 callus doubled in fresh weight after 1 week, and growth was better on N6 than on Murashige-Skoog (MS) medium. Type 2 callus of hybrid B73 × A188 was maintained in culture longer than A188 type 2 callus, and it regenerated higher numbers of plants than the other two genotypes. Type 2 callus of the hybrid B73 × A188 was used to establish cell suspensions. Suspension cells initially grew better on N6 than on MS medium, but after several months of subculture, cells in either N6 or MS medium grew at similar rates. Suspension cells were in mid-log phase by 5–7 days and in stationary phase by about 10 days depending on inoculum density. Growth rate was optimal when cells were transferred at mid-low phase and dry weight of the suspension cells increased at least 10-fold during a 10-day period. Suspension cells from 9-month-old cultures plated on solid medium regenerated plants at an efficiency similar to that of the friable type 2 callus but with more phenotypic abnormalities. Thus, cell suspensions derived from type 2 B73 × A188 callus, in culture for over 1 year, were capable of regenerating plants when 9-months old.     

Differences in compounds released by embryogenic and non-embryogenic suspension cultures of Euphorbia pulcherrima

Media from embryogenic and non-embryogenic cell suspension cultures were analysed for protein content, electrophoretic protein patterns, glycoproteins and activity of peroxidases and β-glucosidases in order to characterize the physiological status of the cultures. On a dry mass basis the amount of extracellular proteins per cell was greater in embryogenic suspensions than in non-embryogenic suspensions. Non-embryogenic suspensions contained unidentified slimy compounds which were not present inembryogenic cultures. The extracellular Concanavalin A-specific glycoproteins gave different isoelectric focussing patterns and thus enabled embryogenic and non-embryogenic cultures to be differentiated. The extracellular peroxidase activity per cell dry mass was far greater in embryogenic than in non-embryogenic cultures. The isoenzymes differed in number and composition of the anionic bands. β-glucosidases were found in the same range of activity in both culture types, but the time course of enzyme activity during cultivation was significantly different. In the embryogenic culture the activity was correlated with dry mass increase, whereas in the non-embryogenic suspension the activity reached maximum during the linear growth phase. Polyphenoloxidase which was recently recognized as an intracellular marker for embryogenic stages was not released into culture media. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gibberellic acid-induced cell elongation in cotton suspension cultures

Gibberellic acid (GA₃) causes cell elongation in cotton suspension cultures derived from cotton ovule callus tissue of both auxin-dependent and-independent lines. Cell elongation was more pronounced in auxin-dependent cultures. Cells were cultured for a period of 14 days but differences in cell lengths could be detected after 6 days in culture. Cell elongation took place in cultures in which GA₃ was present throughout the culture period or only for the first 3 days. Auxins and cytokinin alone or in the presence of GA₃ did not promote cotton cell elongation above the value for the treatment with GA₃ alone.     

Gibberellic acid-induced cell elongation in cotton suspension cultures

Gibberellic acid (GA₃) causes cell elongation in cotton suspension cultures derived from cotton ovule callus tissue of both auxin-dependent and-independent lines. Cell elongation was more pronounced in auxin-dependent cultures. Cells were cultured for a period of 14 days but differences in cell lengths could be detected after 6 days in culture. Cell elongation took place in cultures in which GA₃ was present throughout the culture period or only for the first 3 days. Auxins and cytokinin alone or in the presence of GA₃ did not promote cotton cell elongation above the value for the treatment with GA₃ alone.Names of companies of commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned.     

Somatic embryogenesis and plant regeneration from embryogenic suspension cultures of perennial ryegrass

Summary Embryogenic callus induced from mature caryopses of perennial ryegrass (Lolium perenne L.) were placed in liquid half-strength Murashige and Skoog (MS) basal medium and supplemented with 6.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 3 g/l (w/v) casein hydrolysate (CH), and B5 vitamins, to initiate fast-growing highly embryogenic cell suspension cultures. Newly initiated suspension cultures contained a high level of large non-embryogenic cells (NE) with relatively few embryogenic (E) cells. Cell types were separated by discontinuous Percolls gradients or by filtering the newly initiated cultures through 31-μm nylon mesh. The growth conditions of the E cell were optimized by testing various media components including 2,4-D and sucrose, and subculture diluton ratio. Optimal shoot formation occurred after pretreatment of the embryogenic cells on solidified callus maintenance medium supplemented with 60 mg/l cefotaxime for 4 weeks prior to transfer to regeneration medium Regeneration media consisted of half-strength MS basal medium supplemented with B5 vitamins, 0.5 mg/l fluridone, and 0.5 mg/l BA. Most plants regenerated were albino with only a few green plants. Journal Paper number MAES 2959 of the Massachusetts Agricultural Experiment Station.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

Extracellular proteins in embryogenic suspension cultures of Norway spruce (Picea abies)

Embryogenic cell lines of Norway spruce ( Picea abies ) varying in growth habit and morphology were compared as regards profiles of extracellular proteins. Similar proteins were detected in the culture medium by SDS PAGE and in vivo labeling experiments, indicating that the proteins were secreted. Approximately 20 protein bands could be detected in the medium of each cell line. Three of the bands represented glycosylated proteins, as revealed by Concanavalin A staining. Some of the secreted proteins were similar for all tested embryogenic lines of Norway spruce, others were either specific for a group of cell lines or for individual cell lines. A correlation was observed between the morphology of the somatic embryos in a cell line and the presence of secreted proteins. The embryogenic cell lines of Norway spruce can be divided into two main groups. A and B, where A is characterized by somatic embryos with dense embryoheads and B by somatic embryos with loosely aggregated cells in their embryoheads. When proteins secreted from a cell line belonging to group A were added to cell lines belonging to group B, the somatic embryos of the B type developed further and became more similar in morphology to A-type embryos. These observations indicate that cell lines belonging to group A secrete certain proteins to the culture medium that are essential for the development of somatic embryos of Norway spruce.     

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.     

Peroxidase activities and contents of phenolic acids in embryogenic and nonembryogenic alfalfa cell suspension cultures

Contents of phenolic acids, peroxidase activities and growth curves showed significant differences in embryogenic (EC) and nonembryogenic (NEC) suspension cultures ofMedicago sativa L. NEC gave a typical growth curve while in EC the distinct phases were absent. The total content of phenolic acids was higher in NEC (related to EC), changed during the growth cycle and most of the acids occurred in ester-bound methanol soluble form. The level of phenolic acids in EC was significantly lower and did not change during 12-d cultivation. The major fraction was formed by phenolic acids ester-bound to the cell wall. The cytoplasmic peroxidase activity in NEC increased continuously during the growth and reached the maximum value at the end of exponential phase. In EC the extremely low cytoplasmic peroxidase activity did not change during cultivation. Ionically bound peroxidases in NEC represented 14 to 30% of the total extracted activity in dependence on the growth phase while in EC formed about 50% of the total activity and did not change during studied period. A possible participation of ionic peroxidase in the incorporation of phenolics into the cell wall is discussed.