Root hair protoplasts ofLotus corniculatus L. (birdsfoot trefoil) express their totipotency

Treatment of the roots of 24–48 h old seedlings of the forage legumeLotus corniculatus with 1.0% Cellulase YC, and 0.1% Pectolyase Y-23 in 4.2% mannitol solution released protoplasts from the tips of root hairs within 30–40 sec of enzyme incubation. Roots from approximately 1000 seedlings yielded 1.7×10⁵ protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10⁵ ml^–1 in droplets of KM8P medium with 0.6% Sea Plaque agarose. Colonies formed callus on UM agar medium; protoplast-derived tissues produced shoots on B5 medium containing 0.05 mg 1^–1 of BAP. Regenerated plants were phenotypically and cytologically normal (2n=2x=24±2), and produced nitrogen fixing root nodules following inoculation withRhizobium. These results confirm the totipotency of protoplasts isolated from specialised epidermal cells of seedling roots ofLotus corniculatus.     

Plant regeneration from protoplasts of Panax ginseng (C.A. Meyer) through somatic embryogenesis

Summary Protoplasts of Panax ginseng were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22–25 × 10⁶ protoplast / g tissue were obtained following 5–6 h digestion with 2% Cellulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of 1×10⁵ protoplasts /ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus, which regenerated into plantlets.Abbreviations NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberellic acid - MS Murashige and Skoog medium     

Isolation and cell regeneration of protoplasts from sugar pine (Pinus lambertiana)

Protoplasts were isolated at high yields from actively growing callus and cell suspensions of cotyledons and needles of mature trees. The best protoplast growth response was obtained from cell suspensions of cotyledon and needle callus. Lower protoplast yields were obtained directly from young needles of flushing buds on explants from mature shoots (30-year-old trees) growing in vitro. In all cases, the first divisions, promoted by dimethyl sulfoxide, were observed in 10–45% of the protoplasts by 7–10 days. After 25–30 days, colonies of 8–10 cells were established. Browning of protoplast-derived cell cultures was observed within 40–45 days (cotyledons) and 20–25 days (mature tree sources).Abbreviations BA N⁶-Benzyladenine - DCR Douglas-fir cotyledon revised medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - FDA Fluorescein diacetate - Mes 2-(N-morpholino) ethanesulfonic acid - NAA -naphthaleneacetic acid     

In vitro culture of Cucumis sativus L. XVIII. Plants from protoplasts through direct somatic embryogenesis

A procedure is described for the isolation and culture of protoplasts from embryogenic callus (gel-like callus — GLC) and embryogenic suspension cultures (ESC) of Cucumis sativus c.v. Borszczagowski. Maximal protoplast yields from GLC and ESC were 5×10⁶ and 1×10⁷ protoplasts/g tissue respectively. They were obtained following 14–16 h digestion with 1.2% Cellulase Onozuka R-10, 1.2% Macerozyme R-10 and 0.3% Driselase. At a plating density of 2×10⁵ / ml, first divisions occurred in 4–5 days and 7–8 days in ESC-and GLC-derived protoplasts respectively. The highest percentage of direct embryogenesis (over 80%) was observed with ESC. It was possible to obtain approximately 5000 embryo structures / g tissue. Some embryos converted into plants after 6 weeks, but most of them after 2 months of culture. ESC-derived plants, when transferred into the glasshouse, bloomed normally, and set seeds.Abbreviations CMS Murashige & Skoog (1962) medium for cucumber - GLC gel-like callus - ESC established embryogenic suspension culture - 2,4-d 2,4-dichlorophenoxyacetic acid     

Development of laticifer cells in callus cultures of Calotropis procera (Ait.) R. Br.

Tissue cultures were established from stem explants of Calotropis procera, a hydrocarbon yielding desert shrub on Murashige and Skoog's medium supplemented with 1.5 mg. 1^–01 2,4-D + 0.5 mg.1^–1 kinetin and polyvinylpyrrolidone. Laticifer cells were not present in young callus but were observed after 4 weeks of callus growth when examined histochemically. These young laticifers were detected in the 5th week of culture and were distinguished from surrounding cells by the presence of characteristic cytoplasm and thin walls. A group of cells with extensive branching was developed after 8 weeks of growth of the callus cultures. These cells were thick walled and contained latex particles in coagulated masses. Positive Liebermann-Burchard test proved the presence of terpenoids in these laticifers.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN Kinetin - PVP Polyvinylpyrrolidone - HHS Heidenhain's Haematoxylin and safranin     

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     

Production of reserpine and its optimization in cultured Rauwolfia serpentina Benth. cells

Cultured R. serpentina cells have been maintained on modified Linsmaier-Skoog medium for over 13 years. These cultured cells produced much more ajmaline (0.005–0.012% dW) than reserpine (0–0.003% dW). Selection of callus which survived the stress induced by alteration of the medium composition including hormones, was repeated over several generations. Surviving callus was then transferred back to the original liquid growth medium and subculture continued, during which time the cells exhibited a return to their pre- stress rate of growth, enhanced reserpine production, and a decrease in ajmaline production. R. serpentina cell suspension cultures selected as described and serially subcultured in fresh growth medium every 3 weeks consistently produce reserpine at a yield of approximately 0.03–0.06% dW.Abbreviations LS Linsmaier Skoog(1965)medium - ML Modified Linsmaier Skoog medium - B5 Gamborg(1968)medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - IAA Indole-3-acetic acid - BA Benzyl adenine     

Plant regeneration from hypocotyl-derived protoplasts of Brassica juncea (L.) Czern and Coss

Protoplasts isolated from etiolated hypocotyls of 6-day-old seedlings of Brassica juncea cv RLM 198 were cultured in a modified V₄₇ medium containing 7% mannitol, 2% sucrose, 1.0 mg/l 2,4-D, 0.1 mg/l NAA and 0.4 mg/l BAP, at a density of 5×10⁴ protoplasts per ml of medium. Cultures were incubated in the dark at 25+1°C. After 7 d of culture, cell colonies were diluted with 8_p medium containing 5% mannitol and a similar hormone combination as described earlier. After 14 d, cell colonies were embedded in 8_p medium containing agarose and 3.5% mannitol. Immediately upon gelling, liquid 8_p medium was added to each Petri dish as an overlayer, and cultures were incubated in the light. After a total of 3 to 4 weeks in culture, microcalli were obtained. A modified MS medium with 2% sucrose, 1.0 mg/l 2,4-D and 0.1 mg/l kinetin solidified with 0.5% agarose was used for growing microcalli into callus lines. On MS medium containing 2% sucrose, 0.1 mg/l IAA, 2.0 mg/l zeatin riboside and 2.0 mg/l BAP, solidified with 0.5% agarose, about 35% of the calli regenerated multiple shoots. The time required from culture of protoplasts to multiple shoot regeneration was about 10 weeks. Regenerated shoots were rooted and plants were re-established in a growth chamber at high frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - IBA Indole-3-butyric acid     

Isolation,culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense)

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2–3 cells in G. hirsutum and 5–8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - p CPA p-chlorophenoxyacetic acid - MES 2[N-morpholino]ethanesulfonic acid     

Plant regeneration from callus-derived protoplasts of Pelargonium x domesticum

A protocol for regenerating plants from callus-derived protoplasts of Pelargonium x domesticum (rega l geranium cv. Melissa) has been developed. Protoplasts were isolated from leaf-derived callus tissue on MS medium supplemented with 3.0 mg/l naphthalene acetic acid, 2.0 mg/l 6- benzylaminopurine, and 3.0% sucrose. This callus yielded 2.7×10⁵ protoplasts/gram of tissue after a 6 hr incubation in an enzyme solution consisting of 2.0% cellulysin, 0.5% macerase, and 0.5 M sucrose. Protoplasts were plated at 1×10⁵ protoplasts/ml in a mixture (11 v/v) of KMP8/KP liquid medium layered on the same medium solidified with 0.6% agarose. Protoplast division was initiated within 2 days, and colonies of 15 to 50 cells developed 8 wk after plating. P-calli 1–2 mm³ developed 15 wk after plating, and plants regenerated from the p-calli have been transferred to the greenhouse.Abbreviations NAA naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - CW Calcofluor White - FDA fluorescein diacetate     

Sugarcane protoplasts: factors affecting division and plant regeneration

Summary Sugarcane cell suspensions were initiated from leaf callus and sub-cultured every 7 to 10 days by alternate transfer to MS based medium with 3.0 and 1.0 mg 1^–12,4-D. Suspensions older than 3 months gave the most reproducible yields of protoplasts. Isolated protoplasts required 50 mM Ca²⁺ in the washing solution and 100 mM Ca²⁺ in the culture medium to prevent lysis. At plating densities of 2.0–3.0×10⁵ ml^–1, 18% or more of the isolated protoplasts produced cell colonies when cultured in droplets or sectors of Kao and Michayluk (1975) based medium with 1.2% w/v Sea Plaque agarose. Cell colonies were of two morphological types. Those consisting of small, tightly packed cells developed into morphogenic callus. The latter produced an abundance of green meristems from which shoots and whole plants were regenerated.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - MS Murashige and Skoog (1962)     

Isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato

Optimum conditions for the isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato (Ipomoea batatas) were investigated. Growth phase of callus and the ratio of callus-enzyme solution affected the yield of protoplasts. Composition of the medium and protoplast density were examined for protoplast culture.Small colonies were regenerated from the protoplasts. Upon transfer to light a high amount of anthocyanin was accumulated in these colonies.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

Cauliflower inflorescence protoplast culture and plant regeneration

A yield of 2–4×10⁶ protoplasts/g F.W. could be obtained when fresh cauliflower inflorescence segments were digested with 2% cellulase Onozuka R-10, 1% cellulase RS and 0.4% Macerozyme R-10 in CPW18S for 7 to 10 h. Purified protoplasts were cultured in K8p liquid and agarose medium. Although protoplasts in liquid medium divided earlier than in agarose, protoplast-derived cells in liquid culture could not avoid browning. With agarose culture, sustained division and callus formation could be achieved. After 20 days, calli were transferred onto B5 agar medium with ZT 1.5 mg l^-1, BA 0.5 mg l^-1 and IAA 0.1 mg l^-1 for shoot formation. The frequency of bud formation varied from 56.7% for calli of 1mm in size to 5.6% for 5mm calli. The shoots formed were rooted in B5 medium containing 0.5 mg l^-1 IBA, and the regenerated plants were transplanted to pots and grew normally. It took about two months from protoplasts to the regenerated plants.Abbreviations Ade adenine - BA 6-benzyl aminopurine - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4,-dichlorophenoxyacetic acid - GA₃ gibberellic acid - Gln glutamine - NAA -naphthylacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - ZT zeatin     

Plant regeneration from mesophyll protoplasts of Williams' Bon Chretien (syn. Bartlett) pear (Pyrus communis L.)

Leaf protoplasts of axenic shoot cultures of Pyrus communis L. cv. Williams' Bon Chretien (syn. Bartlett) underwent cell wall regeneration and division to give multicellular colonies in a modified Murashige and Skoog medium which lacked ammonium ions, but supplemented with 1-naphthaleneacetic acid (NAA), 4-indole-3yl-acetic acid, 6-benzylaminopurine (BAP) and casein hydrolysate. Protoplast-derived colonies gave callus on Murashige and Skoog salts medium with NAA and BAP and exhibited shoot regeneration on half-strength Murashige and Skoog medium supplemented with 0.2 mg 1^–1 4-indole-3yl-butyric acid, 2.0 mg 1^–1 BAP, 0.2 mg 1^–1 gibberellic acid, 50 mg 1^–1 casein hydrolysate and 10 mg 1^–1 Ca-pantothenate. Following rooting, protoplast-derived plants of pear were transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - GA₃ gibberellic acid - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-but yric acid - IPE initial plating efficiency - NAA 1-naphthaleneacetic acid - f.wt. fresh weight - MES 2-N-morpholinoethane sulfonic acid - MS Murashige and Skoog (1962) - %PE % plating efficiency - PVP-10 polyvinylpyrrolidone (Av. MW 10,000) - FDA fluorescein diacetate     

Isolation and culture of protoplasts from embryogenic suspension cultures of red fescue (Festuca rubva L.)

Protoplasts were isolated from fast-growing embryogenic suspension cultures of red fescue cv. Dawson (Festuca rubra L.) without agitation. The enzyme isolation solution was highly efficient at releasing protoplasts of greater than 95% viability (5×10⁶–10⁷ protoplasts per ml of packed cell volume). A three step procedure was followed for washing and transferring protoplasts from a solution high in inorganic salts to a medium containing glucose and sucrose. The addition of 30 mM sodium thiosulfate to the wash and culture media was found to be helpful in reducing the number of lysed protoplasts. Isolated protoplasts began to divide within 48–72 h when protoplasts were plated in agarose squares and surrounded by nurse cells (mixed nurse plating technique). Maximum colony formation (plating efficiency) was approximately 1%. Many of the colonies continued to grow and produced embryos when transferred to a medium consisting of half-strength MS salts, 4 mg/l 2,4-D, 3 g/l casein hydrolysate and 30 g/l sucrose. Upon transfer to hormone-free medium and exposure to light 16 h/day, many of the embryos germinated to produce green leaves and roots.Abbreviations BA Benzylaminopurine - 2,4-D 2,4-dicholorophenoxyacetic acid - DMSO dimethyl sulfoxide - MES 2-(N-morpholino)-ethanesulfonicn acid - MS Murashige and Skoog medium (1962) - UGC Ultraclone Growth Chamber - KM Kao and Michayluk medium (1975) - NAA Naphthalene acetic acid     

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium     

Plant regeneration from mesophyll protoplasts ofDianthus superbus

Leaf mesophyll protoplasts ofDianthus superbus were cultured at a density of 5 × 10⁴ protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 E m^–2 sec^–1) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N₆-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.Abbreviations MS Murashige and Skoog - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - N₆ Chu basal salt mixture - MES 2-N-morpholinoethanesulfonic acid     

Direct somatic embryogenesis from protoplasts of Citrus mitis Blanco

Protoplasts isolated from embryogenic suspension cultures of Citrus mitis were cultured in a medium without any plant growth substances. Somatic embryos developed directly from protoplasts without an obvious intervening callus phase. As many as 1,800 somatic embryos developed from 4 ml of protoplast suspension (density 2×10⁶/ml) cultured for 35 days. Upon transferring the embryoids to medium with 1 mgl^–1 GA₃, they developed into plant-lets. Rooted plantlets were obtained in 3 months after protoplast isolation.Abbreviations BAP Benzylaminopurine - GA₃ Gibberellic acid - MT Murashige and Tucker medium (1969) - FDA Fluorescein diacetate     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

Marker proteins for embryogenic differentiation patterns in pea callus

Polypeptide pattern alterations during somatic embryogenesis were investigated using callus cultures of two Pisum sativum genotypes. Both genotypes show the formation of two different callus lines from the same explant after six to eight weeks in culture: a nodular yellowish callus line, which forms somatic embryoids in suspension cultures (e⁺) and a white compact callus line with no regenerative capacity (e^–). The cytosol proteins of the two different callus lines were separated in a semi-preparative two-dimensional system and the polypeptide patterns were compared. Two protein bands were found (P1: M_r=45000 D, pI=7.0–7.1; P2: M_r=7000 D, pI=<4.5), which were characteristic of the putatively embryogenic (e⁺) callus line in all tissues investigated (two genotypes × two explant sources). These proteins found in nodular (e⁺) pea cultures are very similar to two proteins found in Daucus carota suspension cultures preceding the formation of somatic embryos.Abbreviations BA 6-benzyl-aminopurine - Bistris 2-(bis(2-hydroxyethyl)imino)-2-(hydroxymethyl)-1.3-propanediol - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - TCA trichloroacetic acid - Tris tris(hydroxymethyl)-aminomethane