Thidiazuron stimulates shoot regeneration of sugarcane embryogenic callus

Summary Efficient shoot regeneration of sugarcane (Saccharum spp. hybrid cv. CP84-1198) from embryogenic callus cultures has been obtained using thidiazuron (TDZ). Callus was placed on modified Murashige and Skoog (MS) medium containing 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D), or 9.3 μM kinetin and 22.3 μM naphthaleneacetic acid (NAA) and compared with the same MS medium supplemented with 0.5, 1.0, 2.5, 5.0 or 10.0 μMTDZ, A11 TDZ treatments resulted in faster shoot regeneration than the kinetin/NAA treatment, and more shoot production than either the 2,4-D or kinetin/NAA treatments. Maximum response, as determined by total number of shoots (26 per explant) and number of shoots greater than 1 cm (4 per explant) 4 wk after initiation, was obtained with 1.0 μM TDZ. The shoots rooted efficiently on MS medium supplemented with 19.7 μM indole-3-butyric acid (IBA). These results indicate that TDZ effectively stimulates sugarcane plant regeneration from embryogenic callus, and may be suitable to use in genetic transformation studies to enhance regeneration of transgenic plants.     

In vitro culture of switchgrass: Influence of 2,4-d and picloram in combination with benzyladenine on callus initiation and regeneration

The objective of this study was to determine the types and concentrations of auxins and cytokinins that would result in effective culture initiation and plant regeneration of switchgrass (Panicum virgatum L.). Whole caryopses and young seedling explants were cultured on MS medium containing different concentrations of picloram or 2,4-dichlorophenoxyacetic acid (2,4-d) in combination with benzyladenine. Best results from mature caryopses were obtained with 11.3–45.0 M 2,4-d in combination with 15.0 or 45.0 M benzyladenine (BA). More than 300 plants were obtained per embryogenic callus from some treatments after the second transfer to regeneration medium (90 days after initiation of the cultures). Regeneration was obtained from young seedling explants with both auxins. However, picloram was more effective over a wider range of BA concentrations than 2,4-d. Protocols developed during this study were used to regenerate hundreds of plants which could easily be established in the field.     

Long-term culture of embryogenic sugarcane callus

Embryogenic calluses of sugarcane capable of regenerating green plants after long-term culture were sought. The largest quantities of embryogenic calluses were produced on Murashige & Skoog medium, but cultures maintained on Chu N6 medium remained embryogenic and totipotent longer. Both media contained 4.5 M 2,4-dichlorophenoxyacetic acid (2,4-d). The effect of supplements on somatic embryogenesis was examined. Kinetin (0.5 M) and 10% (v/v) coconut water in callus initiation medium were inhibitory to subsequent embryogenesis. Embryogenic calluses on N6 medium increased in fresh weight with proline concentration up to 90 mM. Maximum fresh weight was achieved with 5% sucrose. Although genotypic differences were observed, embryogenesis occurred in all 17 sugarcane clones tested. Embryogenic calluses of one cultivar regenerated green plants after 16 months, but suspensions were totipotent for only 8 months. Total number of regenerated plants decreased with time in culture, while the number of pale green plants increased starting after 5 months in culture.Published as Paper No. 785 in the journal series of the Experiment Station, HSPA     

Effect of BA,NAA, and 2,4-D on red maple callus growth

Established red maple (Acer rubrum L.) callus was cultured on media varying in auxin (NAA or 2,4-D) and cytokinin (BA) concentrations. Callus growth was positively affected by the presence of both an auxin and cytokinin in the medium. Optimal growth depended on the ratio of cytokinin/auxin as well as the total amount of plant growth regulators in the medium.Abbreviations (NAA) naphthaleneacetic acid - (2,4-D) 2,4-dichlorophenoxyacetic acid - (BA) and 6-benzylaminopurine     

Effects of 2,4-D removal on the synthesis of specific proteins by Catharanthus roseus cell cultures

Total and neosynthesized proteins of periwinkle cell suspensions (Catharanthus roseus) were first investigated in cells grown in a 2,4-D-containing medium. Analysis of total (silver-stained) proteins by two-dimensional gel electrophoresis revealed that the levels of seventeen polypeptides were altered during the growth cycle of the cells. Analysis of in vivo [³⁵S]-methionine labeled polypeptides revealed differences in the synthesis of at least 35 polypeptides. Three polypeptides with molecular masses of 30, 35 and 39 kDa appeared to be specific markers of the early stationary phase. In a second sequence of experiments, cells were grown in a 2,4-D-free medium. Alterations in protein synthesis were observed: several polypeptides were expressed earlier in the 2,4-D-starved cells than in control cells; the synthesis of at least two specific polypeptides was increased in cells grown in 2,4-D-free medium, whereas the synthesis of three other polypeptides (molecular masses 33, 34 and 52.5 kDa) was switched on in these cells. As previous studies showed that 2,4-D depletion increased the alkaloid production in C. roseus cells, the present results may suggest that these polypeptides are implicated in the regulation of the alkaloid pathway.     

Shoot regeneration in long-term callus cultures derived from mature flowering plants of Cyclamen persicum Mill.

Summary In long-term callus cultures of Cyclamen persicum Mill. two types of tissue could be distinguished. One type featured a brown suberised outer layer and was poorly organogenic. The other type was yellowish in appearance and gave rise to many shoot buds. Both types co-existed on the same callus, the former prevailing. Selection for organogenic tissue resulted in cultures yielding approximately three times more petioles than random subcultures. Callus-derived shoots could be rooted and established in the greenhouse. The method allowed for the production of thousands of plants but the regenerants often showed deviant phenotypes and genotypes.Abbreviations BA 6-benzylaminopurine - BMP basal medium propagation - BMR basal medium rooting - DAPI 4,6-diamino-2-phenylindole - KIBA potassium salt of indole-3-butyric acid - kinetin 6-furfurylaminopurine - MS Murashige and Skoog - NAA 1-naphthaleneacetic acid     

Effect of various growth regulators on shoot regeneration of sugarcane

Summary Sugarcane (Saccharum spp. hybrid cv. CP 84-1198) embryogenic calluses were induced from young leaves cultured on modified Murashige and Skoog basal medium supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid. Five concentrations, 0.5, 1.0, 2.5, 5.0, and 10.0 μM, of five different growth regulators, 6-benzylaminopurine, kinetin, 6-γ,γ-(dimethylallylamino)purine, zeatin, and thidiazuron, were tested with or without 22.5 μM α-naphthaleneacetic acid to compare their ability to induce regeneration from embryogenic callus. After 4 wk on medium, the percentage of shoot meristem induction was evaluated, and after 10 wk the total number of shoots produced, as well as the percentage of shoots greater than 1 cm in length, was obtained. Although it had the lowest percentage of elongated shoots, medium containing thidiazuron alone performed better than all other growth regulators tested, with the highest percentage of shoot induction and the largest number of shoots, particularly at a concentration of 2.5 μM.     

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 M picloram as the only PGR. Ten M picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 M picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige-Skoog - NAA naphthaleneacetic acid - PGR plant growth regulator Research paper #9053 of the Idaho Agricultural Experiment Station.     

The frequency of marker changes in sugarcane plants regenerated from callus culture

Leaf tissue from five sugarcane clones with distinctive markers was cultured on a medium favoring callus growth. Transferred to a differentiation medium, calli produced over 5000 plants. Plants differentiated from two clones with stem markers exhibited a high rate of remission of the marker, but the marker reappeared in the vegetative progeny of these plants, and remission was, therefore, transient. Plants differentiated from callus from two clones with leaf markers showed a low rate of remission (2 or 3 per thousand) of the marker and the vegetative progeny was stable. A clone with variegated leaves produced plants with the majority having green leaves, some were albino, and some variegated, suggesting that plant differentiation may start with more than one cell. Permanent phenotypic change may result from tissue culture, but the results suggest that such changes are not frequent and may be confounded by temporary alterations or by chimeras formed in the process of differentiation.     

Gradual Depletion of 2,4-D in the Culture Medium for Indirect Shoot Regeneration from Leaf Explants of Camellia sinensis (L.) O. Kuntze

Adventitious shoot regeneration via callus phase from in vitro leaf explants is reported for the first time in tea. Callus was obtained on Murashige and Skoog medium supplemented with varied concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5, 5.0, 7.5 and 10.0 mg/l). Rhizogenesis was observed at all concentrations of 2,4-D. Adventitious shoot buds developed indirectly on leaf explants after prolonged culture for 16 weeks on medium supplemented with 10.0 mg/l 2,4-D. GC analysis of the medium and the tissues at different stages of development showed that specific levels of 2,4-D in the tissue were responsible for morphogenesis. Shoot buds developed on rhizogenic calli, only when 2,4-D declined to undetectable or negligible concentrations in the tissue probably due to detoxification and metabolism. Alternatively, shoot buds could also be evoked when rhizogenic calli were transferred to medium supplemented with low concentration of 2,4-D (1.5 mg/l). The adventitious nature of the shoots was confirmed through histological studies.     

In vitro assay for 2,4-D resistance in transgenic cotton

Summary 2,4-Dichlorophenoxyacetic acid (2,4-D) resistant plants of transgenic cotton (Gossypium hirsutum L.) were produced using Agrobacterium tumefaciens containing a plasmid carrying the neomycin phosphotransferase II (npt II) and 2,4-D monooxygenase (tfd A) genes. An in vitro assay was performed to determine the sensitivity of seed germination, and the growth of seedlings of transgenic and non-transgenic cotton to various concentrations of kanamycin and 2,4-D. The results indicated that kanamycin caused the cotyledons of non-transgenic plants to turn white, but transgenic plants grew normally. Seed germination and seedling growth of non-transgenic plants were strongly inhibited by 2,4-D, but only slightly for transgenic plants. Transgenic plants and non-transgenic plants can be clearly distinguished by the use of 2 mg l⁻¹ 2,4-D in seed germination medium. There was a high correlation between the response of seed germination and the growth of seedlings to kanamycin or 2,4-D, based on the germination ration, albino ratio, dry weight or fresh weight. On this basis, we development a rapid method for identifying transgenic plants that has been verified in the field. These findings will allow identification of cotton transformants at an early stage of plant development, saving time and improving cultivars containing the 2,4-D resistance trait.     

The frequency of marker changes in sugarcane plants regenerated from callus culture

This study investigates the frequency of apparent and permanent expression of marker change following two types of tissue culture, conventional callus and direct regeneration cultures, and for two markers it relates this frequency to that following breeding. Each clone was used for only one marker. After conventional callus culture, plants of the sugarcane clone Arundoid B, a clone having a growth habit with shortened internodes and leaves, were freed of this marker at a rate of 1 in 172 plants. Marker remission in a second clone with a leaf blotch was enhanced in the presence of a mutagen. Callus culture alone gave a remission rate of 1/280 plants, while treatment of callus with ethyl methanesulfonate gave a remission rate of 1/42 plants. Of two markers subjected to vegetative and sexual transmission, the first, a leaf marker, was stable in callus culture with no remissions; crossing with non-marker parents produced progeny with 54% lacking the marker. The second, a stalk marker (multibud), showed epigenetic effects during two generations of vegetative propagation; plants lacking the multibud marker produced vegetative progeny in which the marker reappeared. Nine crosses to nonmarker parents produced progeny of which an average of 29% had the marker. The use of stalk chimeras as markers demonstrated that passage through conventional callus or direct regeneration culture resulted in the loss of the donor phenotype in all plants regenerated. Phenotypic variation in plants derived from callus culture appears to arise from several sources; chimeral segregants, epigenetic transients, and mutational variants.     

Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts

Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. Dissa) and anthers (cv. Igri) was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0.1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds.     

Retention of phytoalexin regulation in legume callus cultures

Legume callus cultures were examined to assess whether regulation of phytoalexin biosynthetic pathways is retained in cultured tissues. Callus tissue cultures ofCanavalia ensiformis (jackbean),Medicago sativa (alfalfa), and nine species ofTrifolium (clover) were established (six clover species for the first time) and maintained on modified Gamborg's B5 medium. Phytoalexins educed in cultures incubated for 48 h with an abiotic elicitor (3.15 mM HgCl₂) were detected by their antifungal activity and were purified by column chromatography and high-performance liquid chromatography. Following crystallization, phytoalexins were identified by ultraviolet and proton nuclear magnetic resonance spectroscopy. None of the treated cultures yielded the same complement of phytoalexins reported for fungal-inoculated leaves of the corresponding plants. Callus from all species exceptT. pratense yielded medicarpin, the only phytoalexin reported in treated leaves of all the corresponding plants. A second phytoalexin, maackiain, was found in treatedT. pratense andT. medium calli; maackiain has been reported in fungal-inoculated leaves of those plant species as well asT. hybridum. The phytoalexins sativan and vestitol were not found in treated callus tissues even though they were reported to be present in fungal-inoculated leaves of the same species. These results suggest that (a) the pathway for medicarpin biosynthesis is of central importance for this group of legumes, (b) some phytoalexin anabolic pathways contain metabolic blocks in cells of cultured tissue, and (c) the mechanism for regulating phytoalexin accumulation in tissues is not lost in culture. Contribution no 8113 of the US Regional Pasture Research Laboratory, USDA-ARS, University Park, PA, USA     

Plant regeneration from tissue cultures of Pokkali rice is promoted by optimizing callus to medium volume ratio and by a medium-conditioning factor produced by embryogenic callus

The frequency of plant regeneration from seed-derived Pokkali rice callus has been substantially increased. Four conclusions were drawn from the study: (1) Non-embryogenic callus consisting of elongated, highly-vacuolated cells did not produce regenerated plants. Embryogenic callus consisting of small, non-vacuolated cells produced somatic embryos and regenerated plants. (2) The numbers of plants could be markedly increased by optimizing a medium for embryogenic callus production and a second medium for plant regeneration from embryogenic callus. (3) The optimization of callus to medium volume ratio of 6.5 mg embryogenic callus per 1.0 ml of medium significantly increase plant production on regeneration medium. (4) A further significant increase was obtained by using regeneration medium previously conditioned for one or two weeks by optimal amounts of embryogenic callus. At present, the callus derived from a single seed in six months could theoretically be used in the seventh month to produce 127500 plants.This research was supported by the Agency for International Development under Contract No. AID/DSAN-C-0273     

Induction of embryogenic mango cultures as affected by genotype,explanting, 2,4-D and embryogenic nurse culture

The nucellus was removed from immature seeds of 4 mango genotypes, andcultured under different induction conditions. The mango genotypes includedpolyembryonic ‘Hindi’ and ‘Nam Doc Mai’ and monoembryonic ‘Lippens’ and’Tommy Atkins‘. Nucellar explants were cultured on modified B5 basal mediumunder the following inductive conditions: 1) 4.52 μM 2,4-D; 2) nogrowth regulator (control); 3) 4.52 μM 2,4-D + embryogenic ‘Parris‘nurse culture; 4) no growth regulator + embryogenic ‘Parris’ nurse culture.Induction of embryogenic competence was mediated by 4 factors: genotype,explanting, 2,4-D and the presence of a highly embryogenic nurse culture,although there was considerable difference in genotype response. ‘Hindi’ hadthe greatest embryogenic potential, followed by ‘Lippens’, ‘Tommy Atkins‘and ‘Nam Doc Mai’, respectively. Induction of embryogenic cultures of allgenotypes at low frequency occurred as a result of explanting excisednucellus onto control medium. The most effective treatment for inducingembryogenic cultures was 2,4-D + embryogenic ‘Parris’ nurse culture with’Hindi’, ‘Lippens’ and ‘Nam doc Mai’, with the exception of ‘Tommy Atkins’,in which the treatment with 2,4-D alone was most effective. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selection of callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to salt stress

Stable callus cultures tolerant to NaCl (68 mM) were developed from salt-sensitive sugarcane cultivar CP65-357 by in vitro selection process. The accumulation of both inorganic (Na⁺, Cl⁻ and K⁺) and organic (proline and soluble sugars) solutes was determined in selected and non-selected calli after a NaCl shock in order to evaluate their implication in in vitro salt tolerance of the selected lines. Both salt-tolerant and non-selected calli showed similar relative fresh weight growth in the absence of NaCl. No growth reduction was observed in salt-tolerant calli while a significant reduction about 32% was observed in nonselected ones when both were cultivated on 68 mM NaCl. Accumulation of Na⁺ was similar in both salt-tolerant and non-selected calli in the presence of NaCl. Accumulation of Cl⁻ was lower in NaCl-tolerant than in non-selected calli while proline and soluble sugars were more accumulated in salt-tolerant than in non-selected calli when both were exposed to salt. K⁺ level decreased more severely in non-selected calli than in NaCl-tolerant ones after NaCl shock. The results indicated that K⁺ and Cl⁻ may play a key role in in vitro salt-tolerance in sugarcance cell lines obtained by in vitro selection and that organic solutes could contribute mainly to counteract the negative water potential of the outside medium.     

Sucrose accumulation and enzyme activities in callus culture of sugarcane

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) were measured in callus cultures of four Mexican sugarcane cultivars (Saccharum spp.) with a different capacity to accumulate sucrose in stem parenchyma cells. The results indicated that sucrose accumulation in callus was positively correlated to the activity of SPS and SUSY and negatively to the activity of SAI and NI while SPS explained most of the variation found for sucrose accumulation and NI least.The research was funded by the department of Biotechnology and Bioengineering CINVESTAV Mexico City, and F. G.-M. received grant-aided support from CONACyT, Mexico.     

Shoot regeneration of hybrid seed geranium (Pelargonium x hortorum) and regal geranium (Pelargonium x domesticum) from primary callus cultures

Procedures have been developed that increase the rate of shoot regeneration of hybrid seed geranium from month-old primary callus cultures. Hybrid geranium callus tissue covered with green nodular structures was initiated by placing shoot tip explants on solidified Murashige & Skoog medium (MS) supplemented with 2.0 mgl^-1 zeatin and 1.9 mgl^-1 indoleacetic acid. Hybrids Red Orbit, White Orbit and Scarlet Orbit were shown to produce 5–50 shoot primordia per explant when callus was initiated on this medium. Regal geranium callus was initiated by placing leaf explants on MS medium supplemented with 2.0 mgl^-1 6-benzylaminopurine and 2.0 mgl^-1 naphthaleneacetic acid. Regal geranium cultivars Tiny Tot and Lavender Grand Slam were shown to produce between 2–50 shoot primordia per explant when initiated on the same medium.     

Effects of light conditions and 2,4-D concentration in soybean anther culture

The morphogenic response of anther walls and connective tissue is the greatest obstacle to androgenesis in soybean anther culture. Whereas induction to microspore embryogenesis occurs in the dark in almost all plant species, soybean anthers have been cultured under light. In an attempt to establish culture conditions that simultaneously stimulate microspore embryogenesis and inhibit epidermal and connective cell proliferation, the effect of light and two 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations (2 and 10 mg l^–1) on the induction process was investigated. Higher 2,4-D concentration speeded up microspore plasmolysis and did not improve androgenesis. Callogenesis and embryogenesis induction from sporophytic cells were significantly lower in the dark, and some microspores showed major alterations in the sporoderm. Auxin 2,4-D and induction under light contributed to the morphogenic response of the anther walls and connective tissue under the conditions previously recommended to trigger microspore embryogenesis.