Plant regeneration from protoplasts of Triticum aestivum L. cv. Nakasoushu

A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l^-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l^-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Regeneration of shoots from cell suspension-derived protoplasts ofAllium cepa

Callus from pre-selected regenerating lines ofAllium cepa andA. fistulosum were used to initiate cell suspensions. Small clusters of callus selected for greater friability were placed into BDS liquid medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), and were subcultured biweekly. Rapidly growing, finely dispersed lines were used for protoplast isolation. The highest yields came from 3–4 month old cell suspension lines. Protoplasts were cultured in modified K8P liquid medium. Microcalli recovery depended on the number of weeks the cell suspension had been in culture with highest recovery from 4–5 month old cell suspensions. Microcalli were moved to semisolid media when they were approximately 2 mm in diameter. After 4–6 weeks, embryogenic calli thus recovered were moved to variations of standard onion regeneration media containing picloram and BA. Elongating shoots were obtained from up to 88 % of the microcalli of one line, and 40–50 % of the shoots were further multiplied in culture.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - BA 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - BDS modified B5 medium of Dunstan and Short (1977a) - K8P medium of Kao and Michayluk (1975) - MES 2-(Nmorpholino)ethanesulfonic acid     

Initiation of morphogenic cell-suspension and protoplast cultures of barley (Hordeum vulgare L.)

Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p culture media; see Material and methods - cv chltivar - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

High frequency plant regeneration from protoplasts in cotton <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM₈P medium with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 μM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 μM indole-3-butyric acid (IBA) and 0.93 μM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.     

Efficient plant regeneration from rice protoplasts in general medium

Summary We established an efficient and reproducible procedure for protoplast propagation and fertile plant regeneration of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Selection of scutellum-derived secondary calli, the use of General medium and nurse culture were all found to be critical in the procedure. When 5 basal media (Murashige and Skoog, RY-2, modified R2, Amino Acid and General media) were compared, suspension callus growth rate, protoplast yield and plating efficiency were all about 30% higher in General medium than in the second-best R2 medium. Only one month was required to develop suspension cultures for protoplast isolation using General medium. A plating efficiency as high as 17% and a plant regeneration frequency of 67% were achieved by the improved procedure. Agronomic traits of protoplast- and seed-derived plants were found to be similar.Abbreviations AA Amino Acid medium (Muller and Grafe 1978) - MS Murashige and Skoog (1962) medium     

Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids)

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 10⁶ protoplasts per gram fresh weight of cells [gfwt^-1]) compared to heterogeneous cell suspension cultures (0.1 × 10⁶ protoplasts gfwt^-1). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 10⁶ protoplasts gfwt^-1. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 10⁶ protoplasts gfwt^-1. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Q110 and Q138.     

Differentiation of somatic embryos from protoplasts isolated from embryogenic suspension cultures of Abies alba L.

Embryogenic suspension cultures of Abies alba were established using an embryogenic suspensor mass culture originating from the zygotic embryo in immature seed explants (Schuller et al. 1989). Protoplasts were isolated from the suspension material. The protoplasts were immobilized in alginate layers in order to follow the development of single protoplasts. During the first days of protoplast culture a modified Kao and Michayluk (1975) medium proved to be necessary for subsequent divisions. The formation of proembryos succeeded within 2–3 weeks when subcultured with a modified Schenk and Hildebrandt (1972) liquid medium. Light, enhanced sugar concentration, and the addition of abscisic acid led to the formation of slightly green torpedo-shaped somatic embryos after 6–8 weeks from protoplast isolation.Abbreviations ABA abscisic acid - BAP N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ESM Embryonal suspensor mass (Gupta and Durzan 1986) - KM Kao and Michayluk (1975) - LP (von Arnold and Eriksson 1977) - MES 2-(N-morpholino)ethane-sulfonic acid - NAA 1-naphthalene-acetic acid (sodium salt) - PVP polyvinylpyrrolidone - SH Schenk and Hildebrandt (1972) - Tween 80 polyoxyethylene-sorbitan-monooleate     

Growth and regenerability of long-term suspension cultures of the U.S. rice cultivar Mercury

Suspension cultures of the U.S. rice cultivar Mercury have been maintained in modified General Medium for more than 3 years. These suspensions have continued to have high and relatively stable regeneration rates. Two different explants, immature panicles and seeds, were compared during the development of these embryogenic suspensions. Initial formation of secondary embryogenic callus from immature panicles on induction medium was greater than that from seeds. Suspensions of these two cell lines, however, did not differ morphologically and maintained similar regeneration rates. After 5 months in culture the rates of regeneration began to decline. The suspensions were plated onto regeneration medium without growth regulators for 2 weeks and then embryogenic cells were manually selected and used to develop secondary suspensions. Through this simple rejuvenation procedure, the suspensions retained high and stable regeneration rates. Variability in suspension growth, however, was observed during the culture period. Slower growth occurred at weeks 13, 15, 27, and 29 and was associated with a decrease in regeneration rates. Reproductive fertility of regenerated plants remained high for 3.5 years but then declined.Abbreviations CH casein (acid hydrolysate) - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige & Skoog basal medium - SE standard error     

High plating efficiency and plant regeneration frequency in low density protoplast cultures derived from an embryogenic Brassica napus cell suspension

Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv. Jet Neuf. Protoplast yield varied with the cell suspension growth medium. Optimization of protoplast plating density, manipulation of culture medium, carbon source and medium matrix, and inclusion of Ficoll resulted in protoplast plating efficiencies close to 30%. Placement of the protoplasts close to the gas interface contributed greatly to the elevated plating efficiency. Low density cultures could be induced to regenerate calli at optimum plating efficiencies if grown in the presence of nurse culture. This is of great advantage for manipulation of individual protoplasts or for microinjection. Plants were regenerated directly from the cell suspension or from the protoplast cultures.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid     

Effect of nurse cultures on the production of macro-calli and fertile plants from maize embryogenic suspension culture protoplasts

Fertile plants have been obtained from maize (Zea mays L.) embryogenic suspension culture protoplasts. Friable, embryogenic callus initiated from an immature embryo from a cross involving the genotypes A188, B73, and Black Mexican sweetcorn was used to establish a rapidly growing embryogenic suspension culture. After nine months in culture, high yields of viable protoplasts (30×10⁶/ gram fresh weight) were obtained following a 1.5 hour enzymatic digestion. Protoplasts cultured with feeder cells divided and formed embryogenic callus, from which male and female fertile plants were regenerated. Protoplast-derived R₁ plants were self-pollinated and immature R₂ embryos isolated for callus initiation. Female fertile plants have also been produced from protoplasts isolated from an R₂-derived suspension culture. Significant interactions between protoplast and feeder-cell lines were observed.Abbreviations BC backcross - BMS Black Mexican Sweetcorn - 2,4-D 2,4-dichlorophenoxyacetic acid - PWS protoplast wash solution (0.2 M mannitol, 80 mM CaCl₂) - FDA fluorescein diacetate - ABA abscisic acid     

High efficiency shoot regeneration from callus of quaking aspen (Populus tremuloides Michx.)

Callus was induced from leaf segments of aspen (Populus tremuloides Michx.) on modified B5 (mB5) medium with 0.1 mg/1 benzyladenine (BA) and 0.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting callus was either subcultured to solidified Woody Plant Medium (WPM) with 0.5 mg/1 BA directly for shoot regeneration or sieved into liquid mB5 medium for suspension culture. After 3 weeks of suspension culture, when the callus clumps grew to 3–4 mm in diameter, they were transferred onto solidified WPM with 0.5 mg/1 BA for shoot regeneration. Almost 100% of the clumps formed shoots on WPM when subcultured directly from mB5 with an average number of 6 shoots per callus. When transferred from suspension culture in mB5 to WPM, an average of 6 shoots per callus were produced from 51% of calli. These shoots could be easily rooted on either mB5 or WPM with 0.2 mg/1 indole-3-butyric acid (IBA) and transferred to pots. Transplanted plants were kept under intermittent mist for 2–4 weeks before normal growth in the green house.Abbreviations BA 6-Benzyl-adenine - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - mB5 medium modified B5 medium - WPM Woody Plant medium     

Plant regeneration from protoplasts isolated from embryogenic suspension cultures of creeping bentgrass (Agrostis palustris Huds.)

A rapidly growing embryogenic suspension culture cell line of creeping bentgrass cv Penncross (Agrostis palustris Huds.) was established from callus derived from the culture of mature seeds. High concentrations of 2,4-D were required for the induction of callus (3 mg/1) as well as for the maintenance of the cell Une (2 mg/1) on modified B5 medium of Gamborg. Protoplasts isolated from the suspension cultures were successfully cultured in Murashige and Skoog's basal medium supplemented with only 0.1 mg/1 2,4-D. Although protoplast plating efficiency was rather low (0.36%), 30% of the protocalli formed normal green plants that were successfully established in soil.Abbreviations BA 6, benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - MES 2, (N-morpholino)-ethane sulfonic acid - MS Murashige and Skoog medium (1962) - B5 Gamborg medium (1968)     

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3–5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N₆ regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.Abbreviations 2,4-D 2,4-Dichorophenoxyacetic acid - FDA Fluorescein diacetate - FW Fresh weight - GA₃ Gibberellic acid - Kinetin 6-Furfurylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid     

Plant regeneration from indica rice (Oryza sativa L.) protoplasts

Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1·10⁶ to 15·10⁶ viable protoplasts/1 g fresh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0%; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N⁶-benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N₆ or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about three-fourths the height of seed-grown plants.Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-week-old primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pcy packed cell volume - BAP N⁶-benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - IAA indole-3-acetic acid Media AA Muller and Grafe (1978) - CPW Frearson et al. (1973) - Kao^* Kao (1977) - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - N₆ Chu et al. (1975) - PCM Ludwig et al. (1985)     

Somatic embryogenesis and plant regeneration from isolated protoplasts of Lavatera thuringiaca

Embryogenic cell suspensions of Lavatera thuringiaca L. were established from leaf petiole and shoot regeneration was achieved when cells were plated on medium without growth regulators. We tested three methods for protoplast culture, isolated from a one-year old embryogenic cell suspension, to determine the best conditions for L. thuringiaca protoplast culture and shoot regeneration. The highest protoplast plating efficiency was obtained with the agaroseembedded method, reaching 30%, while the nursing culture method gave 5% when the protoplasts were plated over Whatman paper No. 2. However, the same nursing culture failed to produce protoplast-derived microcalluses when the protoplasts were plated on a nitrocellulose filter. The liquid thin layer method gave the lowest plating efficiency with only 0.5%. Shoot regeneration from protoplast-derived microcalluses was achieved in two steps; first, globular embryo development was favored in medium low in auxin (2,4-d and BA at 0.01 and 0.05 mg 1^-1, respectively), second, the globular embryos further differentiate into shoots in medium without growth regulators or in medium containing GA₃ (0.5 to 1.0 mg 1^-1).Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - IBA indole-3-butyric acid     

Efficient plant regeneration from Indica (group 1) rice protoplasts of one advanced breeding line and three varieties

Regenerable embryogenic suspensions were established from one Indica (group 1) rice advanced breeding line and 9 Indica (group 1) rice varieties in 6–8 weeks. Four were chosen for protoplast culture and plant regeneration. About 4–7×10⁷ protoplasts were isolated from one gram of 8-week-old cell suspension. High plating efficiency (30.5%) and colony formation (13.7%) were obtained using nurse culture methods. A high plant regeneration frequency (67.5%) was observed for line IR57311-95-2-3. In total, 322 plants were regenerated. All the regenerated plants were fertile.Abbreviation 2,4-D 2,4-dichlorophenoxy acetic acid - NAA 1-naphtalene acetic acid     

Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium

Three unselected cell lines of C. roseus maintained on a growth-associated alkaloid production medium were studied over a period of 2 to 5.5 years for the stability of alkaloid production (serpentine and ajmalicine). Large fluctuations in the total alkaloid content of 20-day-old cells were found for all three cell lines at each subculture over a two-year period. Growth rates increased during prolonged subculture and one cell line became unproductive after five years culture. By selection of small autofluorescent aggregates, high alkaloid production was restored in this cell line, while the parent line was found to be unresponsive to alkaloid induction treatments. The instability in both alkaloid production and spectrum and the loss of alkaloid productivity are discussed in relation to the selection pressures present during long-term maintenance of cell suspension cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - nHS n-heptane sulphonate