Isolation,Culture and Callus Formation of lpomoea batatas Protoplasts

Numerous viable protoplasts from stem callus cells of Ipomoea batatas tissue culture have been isolated by enzyme treatment involving cellulase EA3 867 (2.0%), CaC12·2H2O (20 mM) and mineral constituent of medium A at pH5.4 in 0.8 M mannitol in 5 hours at 25±1℃. The protoplasts were cultured at a density of 1-2 × 105/ml in solid agar medium E supplemented with 2, 4-D (0.1mg/l) and kinetin (0.1 mg/l), or NAA (0.3 mg/l) and kinetin (0.1 mg/l) in petri dishes, and placed in a controlled growth cabinet maintained at 27 ℃, and illuminated with floureseent light. They regenerated new cell wails after 7 days of culture. The first cell division was observed after 10 days. Ceil division continued thereafter, and after 40 days of culture small white calli (size about 0.2–0.3 mm) were visible in the petri dishes small calli were inoculated in the same nutrients as the protoplasts culture media, but without mannitol. They developed into large calli.     

Sugarcane tissue and protoplast culture

Experiments are described which improve the protocols for initiating in vitro cultures of sugarcane and allowing efficient regeneration of plants even after 30 months of callus proliferation. Procedures adopted included use of leaf base explants, CS medium with 3 mg/l 2, 4-D and 0.25 mg/l kinetin for callus initiation and growth, MS medium with 0.5 mg/l IAA and 1 mg/l BAP for shoots, MS medium with 5 mg/l NAA and 7% (wt/vol) sucrose for rooting of shoots. Casein hydrolysate (N-Z amine) significantly shortened the lag period in the growth of sugarcane suspension cultures, but did not increase the rate of growth following the lag phase. Protoplasts isolated from two types of cultures could be grown to re-establish cell cultures but no plants have yet been regenerated derived from isolated protoplasts.     

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     

Plant regeneration from protoplasts of peppermint (Mentha piperita L.)

Summary Enzymatically isolated leaf-derived protoplasts of peppermint (Mentha piperita L.) were cultured in modified B5 medium containing 1 mg/l NAA, 0.4 mg/l BA, 0.5% sucrose, 0.5 M mannitol and 0.1% Gelrite (first medium). After 30 d culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. Gelrite medium blocks were transferred into liquid medium to promote further growth. Colonies of 0.5 mm transferred to 0.2% Gelrite solidified medium (same components as first medium) formed green calli (1–2 mm) under incubation in the light. Green calli transferred to differentiation medium (B5, 0.1 mg/l NAA, 5 mg/l BA, 2% sucrose, 0.2 M mannitol, 0.2% Gelrite) developed shoot buds after 3–4 weeks. Whole plants were recovered following rooting of shoots in B5 medium without hormones.Abbreviations BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - KIN kinetin - ZEA zeatin - CPW cell and protoplast wash solution - B5 Gamborg et al. (1968) mineral elements - MS Murashige and Skoog (1962) mineral elements     

Growth and ultrastructural characteristics of Citrus cells grown in medium containing NaCl

Changes in growth and structural properties of Citrus cell line Carvalhal acclimated to 100 mM NaCl in the medium were compared to unacclimated control cells and cells exposed to 100 mM NaCl. Transmission electron microscopy (TEM) showed presence of ring-shaped mitochondria, increase in the number of amyloplasts and lipid bodies, higher cell wall thickness and partitioned vacuoles in acclimated cells.     

Efficient callus formation and plant regeneration of goosegrass [<Emphasis Type="Italic">Eleusine indica </Emphasis>(L.) Gaertn.]

Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [ Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn.     

Growth characteristics and chemical analysis of Psychotria carthagenensis cell suspension cultures

Callus and cell suspension cultures of Psychotria carthagenensis have been established in Gamborg's B5 medium supplemented, respectively, with 3% sucrose, 0.2 mg/l kinetin, and 1.0 mg/l 2,4-D and 2% sucrose, 2.0 mg/l 2,4-D, 0.2 mg/l kinetin, and 50 mg/l cysteine. Suspension culture presented a typical growth curve with the complete cycle of ca. 18 days and the maximum specific growth rate (μ) was 0.0099 day. The presence of different secondary metabolite pathways was determined by measuring the enzyme activity of phenylalanine ammonia lyase (PAL), tryptophan decarboxylase (TDC), strictosidine synthase (STR), strictosidine-beta-glucosidase (SG), and geraniol-10-hydroxylase (G10H). Activity could only be measured for SG (14.55 pkatal/mg protein) and G10H (0.3 pkatal/mg protein). Analysis of extracts from leaves, callus and cell suspension cultures demonstrated the presence of two major triterpenes: beta-sitosterol and ursolic acid.(2)     

Studies on the Growth in Culture of Plant Cells: VII. EFFECTS OF KINETIN ON THE CARBOHYDRATE AND NITROGEN METABOLISM OF ACER PSEUDOPLATANUS, L CELLS GROWN IN SUSPENSION CULTURE

Aspects of the carbohydrate and nitrogen metabolism of Acerpseudcplatanus, L cell suspension cultures grown on a syntheticmedium containing 2 per cent glucose and 1.0 mg/l 2,4-dichlorophenoxyaceticacid and kinetin either at 0.25 mg/l (low kinetin) or at 2.5mg/l (high kinetin) are described. High kinetin inhibits growthas measured by increase in cell number, packed-cell volume,and cell dry weight. Although not inhibitory to glucose utdization,high kinetin inhibits the O₂ uptake of the cells. Such cellscontain only a trace amount of fructose and their rate of O₂uptake can be raised to that of the low kinetin cells by a periodof fructose feeding. The O₂ uptake of both kinds of cell issensitive to malonate but the stimulation of O₂ uptake inducedby bis(hexafiuoroacetonyl)-acetone (‘1799’) at 0.2mM is much less with the high-kinetin than the low-kinetin cells.The enzymes phosphoglucoseiseomerase and glucose-6-phosphatedehydrogenase are much less active in the high-kinetin cells.Mitochondria isolated from both kinds of cells show good respiratorycontrol although slightly lower values for Q_O2(N), ADP/O ratioand control ratio are recorded with mitochondria from the highkinetin cells. Kinetin at 2.5 mg/l slightly reduces the ADP/Oratio of isolated mitochondria but at 4.0 mg/l their responseto ADP is completely suppressed. Extracellular hemicelluloseformed in presence of high kinetin has a reduced content ofgalactose and xylose and an increased content of glucose. Theseobservations indicate that the inhibition of respiration byhigh kinetin is mainly due to suppression of glucose conversionto other sugars rather than to inhibition of glycolysis or terminalrespiration. High kinetin decreases the rate of protein but not of amino-acidsynthesis. Suppression of the synthesis of particular proteinsmay be an important factor responsible for the reduced cellyield of the cultures in presence of high kinetin. The significanceof these observations to our understanding of the critical metaboliceffects of cytokinina is discussed. Acer pseudoplatanus cells release amino acids into their culturemedium early in the period of batch culture and largely reabsorbthem as incubation proceeds.     

Behaviour in culture of isolated protoplasts from “Paul's scarlet” rose suspension culture cells

Summary Protoplasts were enzymatically isolated from Paul's scarlet rose suspension culture cells. They were cultured in medium similar to that used to culture the cells from which they were isolated with the addition of sucrose as an osmotic stabiliser. They were studied by light and electron microscopy and their changes in size and number per culture were recorded. Expansion was greater when the protoplasts were cultured in medium plus 12% sucrose than with 24% sucrose. Budding was observed. In medium plus 12% sucrose about 45% of the protoplasts divided but in medium plus 24% sucrose far fewer divided. Cytokinesis was abnormal: the phragmoplast disappeared soon after cytokinesis began and the cell plate became a groove and then a fibril-lined or filled tongue which progressed across the vacuole, unconnected by strands to other parts of the protoplast. The wall regenerated after several days culture in medium plus 12% sucrose fluoresced with calcofluor. The wall regenerated in medium with 24% sucrose fluoresced usually only after several weeks culture. Cytokinesis hastened formation of a wall fluorescing with calcofluor. In the electron microscope the wall was seen to contain fibrils and non-fibrillar material. The latter was the minor component in medium plus 12% sucrose but was usually the major component in medium plus 24% sucrose. The growth in plasmolysing and nonplasmolysing medium of the cells from which protoplasts are isolated was also studied.It appears that loss of the wall alters the potential of protoplasts to expand and possibly also to regenerate a wall and to divide. Wall regeneration is initially linked with expansion and cytokinesis. Osmotic pressure of the external medium is also an important factor.This work formed part of a thesis by one of us (R.S.P.) approved for the degree of Ph. D. in the University of Nottingham. The work was supported by the Agricultural Research Council.     

Plantlets from mesophyll protoplasts of Solanum xanthocarpum

Young leaves of Solanum xanthocarpum from axenic shoot cultures released viable protoplasts when treated with appropriate enzymes. The protoplasts on culture in modified Murashige and Skoog (1962) medium supplemented with 2,4-dichlorophenoxy-acetic acid (0.5 mg/l), naphtha leneacetic acid (1 mg/l), kinetin (1 mg/l) and organic nutrients of KM (Kao and Michayluk 1975) regenerated to form callus tissue as a result of repeated divisions. Protoplast-derived calli differentiated into shoots on MS medium enriched with kinetin (0.5 mg/l) and rooting could be initiated by transferring the shoot-buds to basal medium.     

The Growth of Acer pseudoplatanus Cells in a Synthetic Liquid Medium: Response to the Carbohydrate, Nitrogenous and Growth Hormone Constituents

A synthetic culture medium which supports a high level of growth of a scrially propagated cell suspension culture of Acer pseudoplatanus is described. The sucrose of this medium can be effectively replaced by glucose or fructose or a mixture of glucose and fructose or galactose or maltose or soluble starch. When the carbohydrate is glucose or fructose no other sugars appear in the culture medium in significant amounts. Glucose is absorbed in greater quantity than fructose from an equimolar mixture of these sugars. When sucrose is supplied both glucose and fructose appear in the medium. Glucose appears in maltose medium, and maltose and glucose in soluble starch medium. Under the standard conditions of culture, media containing 2 % sucrose or 2 % glucose become depleted of sugar before the 25th day of incubation. Enhanced yield of the cultures can be obtained by raising the initial sucrose concentration to 6 %. – A supply of nitrate is essential for maximum yield and healthy growth. Growth, in the presence of nitrate, is significantly enhanced by a supply of urea. Addition of casein hydrolysate or of a mixture of amino acids enhances growth in the presence of nitrate and urea and particularly when nitrate is omitted. – When kinetin is omitted or incorporated at the standard level (0.25 mg/I), 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0 mg/l is essential for continuation of growth at a high level. It cannot be replaced by indol-3yl-acetic acid (IAA). 1-naphthaleneacetic acid (NAA) at 10 mg/l permits of a low level of growth with abnormal aggregation. When the level of kinetin is raised to 10 mg/l a high level of growth occurs in the absence of added auxin but the cultures become brown and tend to show increasing aggregation on subculture.     

Rooting and the Metabolism of Nicotine in Tobacco Callus Cultures

The usefulness of exogenous nicotine as a factor in the induction of morphogenesis in a tobacco tissue culture medium has been demonstrated. Nicotiana rustica callus cell cultures were grown on a modified Murashige and Skoog medium with 2 mg/l indoleacetic acid (IAA) and 0.2 mg/l kinetin (MMS). Root morphogenesis was induced in roller tube callus cell cultures and solid callus cell cultures grown on MMS without kinetin supplemented with 10–100 mg/l nicotine. Optimal nicotine concentration for root induction was 50 mg/l. Other tests using varying combinations of IAA, kinetin and nicotine produced no obvious morphogenesis, although some changes in the amount of callus growth and endogenous protein concentration did correlate with nicotine concentration relative to the presence of IAA and/or kinetin. In liquid MMS medium, ¹⁴C-nicotine was primarily incorporated into the protein fraction of cultured cells while primarily incorporated into the cell wall and/or cell membrane fraction of cells cultured on MMS without kinetin in the medium. In MMS without IAA and MMS without both IAA and kinetin, there was incorporation, but to a lesser extent in both the protein and the cell wall and/or cell membrane fractions.     

Plant regeneration from mesophyll protoplast culture of cabbage (Brassica oleracea var ‘capitata’)

Summary Protoplasts were enzymatically isolated from the first leaves of cabbage (Brassica oleracea var capitata, F1 hybrid Baochun). Sustained cell division and somatic embryogenesis were obtained after culturing the protoplasts in modified liquid DPD medium supplemented with CaCl₂ · 2H₂O 800 mg/l, 2,4-D 0.5 mg/l, kinetin 1 mg/l, 0.3 M mannitol and sucrose 20 g/l. Upon transferring cell colonies onto a modified Murashige and Skoog (MS) agar medium, small calli were gradually formed. Callus proliferated on MS medium supplemented with hormone combinations of 2,4-D 0.1–0.5 mg/l and kinetin 3–4 mg/l. Multiple shoots were induced on differentiation medium supplemented with 3 mg/l of kinetin and 0.1 mg/l of gibberellic acid GA₃. After transferring differentiated shoots onto MS medium supplemented with indoleacetic acid (IAA), kinetin, GA₃ at 0.1 mg/l each and 500 mg/l of N.Z. amine, intact plants were eventually produced.     

Expression of α-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient

A rice suspension cell culture system has been established to study how sugar depletion regulates α-amylase expression, carbohydrate metabolism, and other physiological and cellular changes. It is shown here that a group of 44 kDa α-amylases are constitutively expressed whether or not the cells are starved of sucrose. However, expression of a new group of α-amylases of 46 kDa is dramatically induced when cells are starved of sucrose. Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells. Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplasts. Immunocytochemical studies revealed that α-amylases are localized in starch granules within amyloplasts, in cell walls, and in some of the vacuoles. The presence of putative signal sequences in the N-termini of nine rice α-amylases suggests hitherto unidentified pathways for import of α-amylases into amyloplasts. The studies show that differential α-amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium. As the starved suspension cells exhibit some sugar-regulated characteristics of α-amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of α-amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants.     

In Vitro Culture and Regeneration of Solanum khasianum and Extraction of Solasodine

Leaf explants of Solanum khasianum regenerated on MS medium containing 2, 4-D (3 mg/l) and kinetin (1 mg/l). Shoots could be induced from these calli on medium containing BAP (3 mg/l) alone. Rhizogenesis of these shoots occurred when transferred to medium containing 2 mg/l NAA. The yield of solasodine — a pharmaceutically important compound, from 4-month-old callus tissue was remarkable at 2 per cent of dry weight.     

The isolation, culture and division of protoplasts from citrus cotyledons

High yields (2.3 × 10⁵ to 1.3 × 10⁶ protoplasts/g.f.wt.) of isolated protoplasts were obtained from cotyledons of Cirus sinensis (L.) Osb. 'Valencia'. Osmotic potential of the medium and enzyme concentrations were important in obtaining high viability of preparations as indicated by FDA fluorescence. Adding malt extract to a Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the rate or duration of cell division. However, modifying the NAA and kinetin concentration optimized plating efficiencies (up to 20%) of protoplasts and also the rate or duration of cell division. The highest plating efficiency and number of cells per colony were obtained on a defined medium containing NAA (15 μ M ). and kinetin (4.6 μ M ). Coincidence of percentage protoplast viability after 13 days (assessed by FDA fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is an accurate indicator of citrus protoplast viability.     

Morphogenesis of the root cap in adventitious roots of Salix viminalis

The preformed root primordia in stems of Salk viminalis L. consist of undifferentiated cells. Forty-eight hours after activation of the primordia in cuttings a root cap meristem was initiated four to five cell tiers from the surface of the primordia. The cells distal to the meristem divided only in an anticlinal plane, while in the meristem they divided mostly periclinally but sometimes anticlinally. After 72 hours a columella was established and the amyloplasts began to sediment in response to gravity. Shortly after this stage the roots began to bend slightly downward, probably as a geo-tropic response. Six days after activation the root cap consisted of up to 15 tiers of cells. The ultrastucture of the cap cells just prior to emergence was studied in more detail. The plastids in the cells adjoining the root proper were typical proplastids. Distal to this cell tier starch accumulated in the plastids. In the fifth tier the amyloplasts were fully sedimented to the lowermost cell walls. The amount of ER increased with the distance from the initial cells and most of it was located at the distal periclinal cell wall. The nucleus and the vacuoles in the geo-sensitive cells occurred in the space above the sedimented amyloplasts. The cytoplasm was less electron opaque than in the initial cells and the mitochondria had more cristae. In the distal cells of the columella and the lateral root cap secretion of mucilage seemed to have started. Numerous large dictyosomes were associated with large vesicles containing a fibrillar or granular material. The plasmalemma lining the distal periclinal cell wall had separated from the wall. A fibrillar material was present between the plasmalemma and the wall and also in intercellular spaces outside the root cap.     

Fertile indica and japonica rice plants regenerated from protoplasts isolated from embryogenic haploid suspension cultures

Summary A system to regenerate fertile rice (Oryza sativa L.) plants (both indica and japonica varieties) from protoplasts isolated from anther-derived embryogenic haploid suspension cultures has been established. Green plants were regenerated from protoplast-derived cell clusters five months after suspension culture initiation. Protoplast yields and subsequent growth of the protoplast-derived microcalli were enhanced by transferring suspension cells into AA medium (Muller et al. 1978) three to four days prior to protoplast isolation. Protoplasts were cultured initially in Kao medium (Kao et al. 1977) and in association with nurse cells for four weeks. Protoplast-derived microcalli were transferred onto N6 (Chu et al. 1975) or MS (Murashige and Skoog 1962) media for callus proliferation. Callus growth was more rapid and the calli were more enbryogenic when grown on N6 medium. The 2,4-D concentration used to develop the suspension culture was important. Cell cultures grown in medium containing 0.5 mg/l 2,4-D released protoplasts whose plating efficiency was higher than for protoplasts obtained from suspension cultures grown in 2.0 mg/l 2,4-D. However, suspension cells grown in 2.0 mg/l 2,4-D were superior with regard to the ability of protoplast-derived calli to regenerate green plants. Amongst several hormone treatments evaluated, a combination of 0.5 mg/l NAA + 5.0 mg/l BAP resulted in the largest number of green plants regenerated. There were no significant differences between BAP or kinetin regarding total number of plants regenerated. More than 200 green plants have been produced form six independently initiated suspension cell lines. The number of regenerated plants per 10⁶ protoplats plated anged from 0.4 to 20.0, and the average seed fertility of single panicles of these R_O plants was about 40%.     

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