Isolation and culture of protoplasts from cell suspension cultures of Duboisia myoporoides with subsequent plant regeneration

Protoplasts were isolated from suspension cultures of various cell lines of Duboisia myoporoides R. Br. There were differences among cell lines with respect to optimal conditions for protoplast isolation including the amount and kind of enzymes and the osmoticum concentration. Protoplasts isolated from one cell line were successfully cultured and induced to form cell colonies in liquid modified B5 medium. Addition of conditioned medium, coconut milk and glucose as an osmoticum to protoplast culture medium as well as maintenance of high protoplast density in culture (> 10⁵/ml) were essential to obtain protocolony formation. Reduction of osmoticum concentration and deletion of coconut milk and conditioned medium from the culture medium were necessary to allow further colony development leading to cellus formation. Intact plants regenerated from calli derived from protoplasts were successfully transferred to pots.     

The effects of interactions of culture environment with genotype on wheat (Triticum aestivum) anther culture response

Summary The interactions of genotype and several variables related to culture environment, including temperature pretreatment, conditioned medium and agar concentrations were examined in a series of experiments for their effects on percent anthers producing callus and number of embryoids produced per 100 anthers scored. Significant genotypic interaction was observed for both traits with all environmental variables except methods of medium conditioning. Such interactions involve both changes of response magnitude and changes of rank order of genotypes. The highest response frequencies observed were in excess of 30% of anthers callusing. Most lines examined responded relatively well to a culture regime utilizing a 4°C treatment for 7–14 d prior to anther excision, followed by float culture, without transfer and without preconditioning of the culture medium. The results indicate, however, that particular genotypes may have specific requirements with respect to various environmental conditions so that culture conditions may need to be adjusted, especially for the least responsive genotypes.     

Experimental Studies of Young Ginkgo Embryos—the Effect of Coconut Milk on the Embryos Cultured in Vitro

The present investigation deals with the effect of coconut milk upon the growth of in vitro culture of young Ginkgo embryos. The basic medium consists of White mineral salts in which Fe-citrate was used instead of Fe2(SO4)3 and vitamins (B1, 0.5 ppm, B6, 0.5 ppm, Ca-pantothenate, 0.5 ppm, niacin, 1 ppm and glycine, 2.5 ppm). 8% sucrose was used for younger embryos as the carbon source and 5% for the larger ones. The pH value of the medium was adjusted to about 6. 0.7% agar was used. The cultures were kept in an incubator (about 20–23 ℃) and no artificial light was used. The coconut milk, both filtered and autoclaved, was tested for its effect on the growth and structure of the young embryos. The important results obtained are summarized as follows: 1. The coconut milk, used both filtered and autoclaved, greatly promoted the growth and differentiation of the Ginkgo embryos cultured in vitro. 2. The optimum concentration of the coconut milk is 10%–20% for the younger embryos (900–1,600μ), while the higher concentrations (30% and over) may induce callus formation. 3. For the larger embryos the coconut milk was less effective, no callus formation occurred for the embryos over 2,800μ at isolation and for them 40% coconut milk was found more effective than 10% coconut milk. 4. There was no significant difference of the effect between the filtered and the autoclaved coconut milk. 5. From the experimental data obtained the authors conclude that the coconut milk at adequate concentration greatly promotes the rate of cell division and may initiate the meristematic regions around the shoot apex and over the whole surface of the cotyledons of the treated embryos.     

大花蕙兰组培快繁技术研究

选用大花蕙兰杂交新品种1053为外植体,采用1/2MS为基本培养基进行大花蕙兰组培快繁技术研究,结果表明：类原球茎诱导的最佳外植体为大花蕙兰鳞茎,培养基最佳激素浓度配比为0.5 mg/L 6-BA＋0.2 mg/L NAA;类原球茎增殖与分化芽最佳激素浓度配比为1.0 mg/L 6-BA＋0.3 mg/L NAA;幼芽增殖的最佳激素浓度配比为2.0 mg/L 6-BA＋0.5 mg/L NAA,最佳有机添加物为150 ml/L椰汁,幼芽增殖正交试验得出影响因素主次顺序：6-BA〉椰汁添加物〉NAA,优组合为2 mg/L 6-BA、0.5 mg/L NAA、添加物椰汁150 ml/L;诱导生根的最佳激素浓度配比为0.3 mg/L IBA＋0.2 mg/L NAA,添加香蕉泥100 g/L对幼苗根生长有显著的促进作用。优化后的大花蕙兰组培快繁技术参数,可为大规模工厂化生产提供参考。     

Rapid multiplication of Sapium sebiferum Roxb. by tissue culture

Multiple shoots formation and elongation was induced from stem explants of Sapium seedlings on media containing cytokinins. Leaf explants produced callus on a medium containing cytokinins, auxin, casein hydrolysate and coconut milk, which could be induced to form multiple shoots on transfer to a medium lacking casein hydrolysate, coconut milk and auxin. Rooting of isolated shoots by treatment with an auxin mixture (indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid) and transfer of the plantlets to field have also been successful.     

Reduction of hyperhydricity in sunflower tissue culture

In order to reduce the occurrence of hyperhydrated shoots, the response of three sunflower inbred lines was examined on regeneration media containing various concentrations of kinetin, silver nitrate, and casein hydrolysate, calcium nitrate and cobalt nitrate. There were differences among the inbred lines for all the parameters taken into account to outline the in vitro efficiency. Percentage of hyperhydrated primordia, average number of shoots and of primordia per total explants, and percentage of hyperhydrated shoot traits differed among all media. The genotype × culture media interaction was significant for average number of shoots and primordia per total explants, regeneration percentage and percentage of hyperhydrated primordia. Among all media tested, those containing silver nitrate significantly reduced hyperhydricity in a dose-dependent way. The addition of silver nitrate showed to be useful in improving the quality of sunflower micropropagated plants by reducing this undesirable phenomenon.     

The effect of genotype and explant age on somatic embryogenesis of coffee

We demonstrate that embryogenic capacity of coffee (Coffea arabica) depends on the genotype, and that it is a character fixed at the early generations F3 or F4. Therefore, the embryogenic capacity F5 lines can be predicted in a breeding program if it is possible to evaluate the F3 of F4 ancestors, or lines with the same parents on those generations.Regeneration via somatic embryogenesis of genotypes from a C. arabica cv. Caturra by Timor Hybrid cross were evaluated, to determine the effect of the pedigree, month of leaf collection, as well as its interaction with the genotype. Large variations in embryogenic capacity among genotypes were detected with rates ranging from 4.8 to 72.7. There was association between embryogenic response of the progenies and the progenitor from which they proceeded. Significant differences were also found in embryogenic responses depending on the month of leaf explant collection, as well as a significant interaction of genotype by planting month.The results are relevant for breeding and tissue culture purposes of coffee because they will allow speed up of the breeding program and save effort by being able to predict embryogenic capacity of a line, based on the response of either its progenitors or the F3 or F4 lines of the same origin. In addition, high embryogenic capacity lines can be considered directly for genetic transformation or for massive multiplication (bioreactors).     

Callus Formation, Plant Regeneration and Clonal Propagation in Vitro of Gynura aurantiaca (Blume) DC.

Methods for culture in vitro of Gynura aurantiaca (Blume) DC.are described. Callus formation from gynura explants was initiallydifficult in spite of the multiple combinations of plant growthregulators assayed. In order to induce high growth rate, incorporationof several organic addenda to a basal medium was necessary,the best of these being coconut milk. Unorganized cell linescould be obtained by subculturing on C medium supplemented with10% coconut milk, and transfers had to be done at short intervals. A morphogenetic cell line without apparent loss in its organogenicpotential through more than 8 months of subculturing was establishedfollowing a sequence of culture media. Methods for rapid and efficient clonal propagation in vitroof this plant species are also reported. The possible utilization of these tissue culture techniquesdeveloped for gynura, a suitable indicator host for Citrus ExocortisViroid, in further studies concerning pathogenesis and replicationof this viroid is discussed. (Received April 22, 1985; Accepted October 11, 1985)     

Cytoplasmic-nuclear interaction and medium effect for protoplast culture in sunflower

Protoplasts of 6 alloplasmic and 2 euplasmic sunflower inbred lines were isolated from dark grown seedling hypocotyls with a density of 2×10⁴ protoplasts/ml. The protoplast suspension was mixed with a solution of 0.5% agarose (sigma – type 1), then pipetted in droplets of about 1000 protoplasts. Droplets were surrounded by two different liquid media. After 30 days droplets from both media were transferred to solid differentiation medium. Protoplast division, microcolony frequency and the number of calluses produced were strongly dependent on medium composition and genotype. The number of calluses per 1000 protoplasts plated range from 0.3 to 5.0 according to the genotype and the method used. The alloplasmic line RHA274-PEF1, was the best responding genotype for calluses produced in both media used. In all cases, the percentage of calluses for alloplasmic lines were significantly higher when compared with the nucleus donor genotype. H. petiolaris fallax cytoplasm increased both the number of calluses produced and the percentage of microcolonies. The complex interaction among genotypes tested indicates that protoplast culture responses are affected independently by nuclear-cytoplasm interactions. Some nucleus-cytoplasm combinations can improve the protoplast culture responses in sunflower. This revised version was published online in June 2006 with corrections to the Cover Date.     

Wheat anther culture: effect of genotype and environmental conditions

Twenty-two cultivars and lines of winter and spring wheat (Triticum aestivum L.) were studied, most for the first time, for their anther culture response. The response was genotype dependent. Plants grown in the field gave higher callus induction frequency than those grown in the greenhouse and the controlled environment chamber. Donor plants grown in a season of low drought stress as compared to a season of severe drought stress resulted in a higher frequency of callus induction. Spherical microcalli were observed in two wheat genotypes in some of only those anthers that were placed with only one loculus in contact with the medium. Wheat lines that were more responsive to anther culture were identified.     

Evaluation of Brassica rapa L. genotypes for microspore culture response and identification of a highly embryogenic line

Isolated microspore culture techniques are being widely used in Brassica breeding programs to generate haploid and doubled haploid plants. A number of factors influence regeneration response in vitro including genotype. In order to assess the effect of genotype on microspore embryogenesis in B. rapa L. var. oleifera, 17 cultivars and breeding lines were evaluated. Embryos developed from all but one genotype when using NLN medium with 17% sucrose, followed by a reduction in sucrose concentration to 10%, 48 h later. The number of embryos /100 buds differed between genotypes, ranging from 0 to 70. Further studies indicated that sucrose concentration and incubation time influenced embryogenesis. Selection studies carried out with an Agriculture and Agri-Food Canada breeding line have resulted in the identification of a highly embryogenic B. rapa line. This line produced thousands of microspore-derived embryos /100 buds and will be useful in mutant selection and gene transfer as well as biochemical and developmental studies.     

Effect of culture density on the polyteny degree of giant chromosomes in inbred lines and hybrids of Drosophila melanogaster

The influence of culture density and genotype on the polyteny degree of giant chromosomes (PDC) in Drosophila melanogaster was investigated. The reliable depression of the polytene chromosomes endoreduplication function under increased culture density was revealed. The essential dependence of the PDC on the genotype was shown. Correlation between the PDC and the number of adaptive features was established. The certain parallelism between the increased heterosis effect on the number of quantitative characters and the superiority of the hybrids above the inbred lines on the PDC in conditions of high culture density was found out. The sex-dependent distinctions and maternal effect at the inheritance of the PDC in drosophila were shown.     

One hundred years of zygotic embryo culture investigations

Summary Isolation of zygotic embryos from seeds and their culture in a defined medium, initiated by Hanning in 1904, has proved to be a promising method to study the factors that control growth and differentiation of embryos. Using this technique, several investigations have focused on the carbohydrate and nitrogen nutrition during germination of cultured seed embryos and on the effects of plant hormones on their morphogenesis. Culture of immature embryos leads to their germination into weak seeldings, skipping the later stages of embryogenesis, by a process known as precocious germination. Progressively smaller embryos have been cultured by supplementation of the medium with coconut milk or hormonal additives or by osmotic adjustment of the medium by high concentrations of sucrose or mannitol. Although methods have not been developed for large-scale isolation and culture of zygotes, zygotes of maize isolated from embryo sacs and those obtained by in vitro fertilization have been grown in culture into full-term embryos. Embryo culture techniques are widely used to rescue embryos from seed of wide crosses which usually abort and to overcome dormancy of recalcitrant seeds.     

GROWTH OF EXCISED PINE EMBRYOS AND THE ROLE OF THE COTYLEDONS DURING GERMINATION IN VITRO

A study was made of the role of the cotyledons, embryo orientation, surgical treatment, darkness, light, and autoclaved coconut milk on the growth of Pinus lambertiana Dougl. embryos in vitro. The embryos did not require an haustorial function of the cotyledons in vitro. Removal of the shoot meristem drastically altered the developmental physiology of the embryo and the function of the root meristem was severly inhibited. Positional effects on embryo growth were reversed by darkness. In the light vertical-inverted tube cultures displayed better growth than horizontal-inverted tube cultures, whereas in the dark the horizontal-inverted tube cultures displayed better growth than the ver ical-inverted tube cultures. Autoclaved coconut milk had no statistically demonstrable effect on embryo growth as measun d by the analysis of variance and Student-Newman-Keul's range test; however, the graphical analysis suggests that, in conjunction with the presence of the shoot meristem, there may be a slight beneficial response to autoclaved coconut milk.     

Anther culture of Solanum genotypes

Anther culture response was examined in five Solanum genotypes. Large genotypic differences exist in response to culture and liquid culture media were found to be superior to agar solidified media systems. Pre-treatment effects on embryoid induction were also investigated and two of the genotypes displayed differential response to temperature stress pretreatment. In general the beneficial effect of charcoal in the media was confirmed. Successful embryoid production and plantlet regeneration was obtained from the wild potato species, Solanum papita. The influence of sucrose concentration on embryoid production was also investigated. Large genotype by sucrose interactions were detected and this was mainly due to the differential response of the tuberosum clones to increasing sucrose concentration when compared with S. papita. Embryoids were produced from this species in media containing 15% sucrose although the optimum concentration of sucrose for embryoid production was 9%. The possible role of anther culture techniques in gene introgression and potato improvement is discussed.     

Auxin and gibberellin-like substances in coconut milk and malt extract

Auxin-like and gibberellin-like activity was detected in both coconut milk and malt extract. Indole-3-acetic acid was tentatively identified as being present in malt extract. The results obtained correlate well with the responses reported by researchers using coconut milk and malt extract as additives to basal nutrient media in tissue culture experiments.     

GROWTH INDUCTION IN CULTURES OF HAPLOPAPPUS GRACILIS. I. THE BEHAVIOR OF THE CULTURED CELLS

Blakely , L. M., and F. C. Steward . (Cornell U., Ithaca, New York.) Growth induction in cultures of Haplopappus gracilis. I. The behavior of the cultured cells. Amer. Jour. Bot. 48(5): 351–358. Illus. 1961.—Because of the unusual cytology of Haplopappus gracilis (2n = 4), a study has been made of the growth of its stem tissue in culture. Although growth may occur on a basal medium supplemented in various ways, it was stimulated for present purposes by the use of a basal medium containing casein hydrolysate, coconut milk (2–10% by volume) and naphthaleneacetic acid (0.5 p.p.m.). A definite synergism between coconut milk and naphthaleneacetic acid was demonstrated. The general form of the colonies so obtained responds to the composition of the medium, and certain effects of pigmentation indicate that the biochemistry of the cultured tissue is also a function of the conditions. The Haplopappus cultures were maintained in liquid culture either in the form of free cells or of the small cell clusters to which they readily gave rise. The form of typical cells and cell clusters is described, and stress is laid upon the range of growth forms that are encountered. Variations in suspensions of cells, or small cell clusters, may be investigated by the application of simple microbiological techniques. Haplopappus gracilis is thus a useful material for the further study of growth and morphogenesis by tissue culture techniques.     

Variability in APOE genotype status in human-derived cell lines: a cause for concern in cell culture studies?

Although cell culture studies have provided landmark discoveries in the basic and applied life sciences, it is often under-appreciated that cells grown in culture are prone to generating artifacts. Here, we introduce the genotype status (exemplified by apolipoprotein E) of human-derived cells as a further important parameter that requires attention in cell culture experiments. Epidemiological and clinical studies indicate that variations from the main apolipoprotein E3/E3 genotype might alter the risk of developing chronic diseases, especially neurodegeneration, cardiovascular disease, and cancer. Whereas the apolipoprotein E allele distribution in human populations is well characterized, the apolipoprotein E genotype of human-derived cell lines is only rarely considered in interpreting cell culture data. However, we find that primary and immortalized human cell lines show substantial variation in their apolipoprotein E genotype status. We argue that the apolipoprotein E genotype status and corresponding gene expression level of human-derived cell lines should be considered to better avoid (or at least account for) inconsistencies in cell culture studies when different cell lines of the same tissue or organ are used and before extrapolating cell culture data to human physiology in health and disease.     

First stages of microspore reprogramming to embryogenesis through anther culture in Prunus armeniaca L.

Prunus armeniaca L. is a worldwide known species, very important particularly in the Mediterranean basin. Microspore embryogenesis through in vitro anther culture is a widely used method to obtain haploid and doubled haploid (DHs) plants which are being routinely used in breeding programmes for new superior cultivar development in many crops. Haploid-diploidization through gametic embryogenesis allows single-step development of complete homozygous lines from heterozygous parents. In the case of fruit crops, with long reproductive cycle, a high degree of heterozygosity, large size, and, often, self-incompatibility, there is no way to obtain haploidization through conventional methods. Induction of microspore embryogenesis in vitro is switched by a stress treatment. In many species, heat or cold stress has been reported to trigger pollen embryogenesis, the response being genotype dependent. In the present work we analyzed whether microspore reprogramming could be induced in apricot cultivars by cold stress through anther culture. We report the development of an in vitro anther culture protocol in P. armeniaca L. and analyse the response of several cultivars to stress treatments and culture media for inducing pollen embryogenesis. Results showed the formation of multicellular pollen and proembryos. The effect of two culture media in the embryogenic response was also analyzed, being the responses genotype-dependent. Monitoring of the cellular changes on the microspores was performed by structural and confocal microscopy analyses. Results indicated that the reprogramming of the microspore and the first steps of the embryogenic pathway have been achieved in different varieties of P. armeniaca, which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and DH plants, for future potential applications in breeding programmes of this economically important fruit tree.     

Genetic and non-genetic factors affecting anther culture of Brussels sprouts (Brassica oleracea var. gemmifera)

Summary Eight inbred lines of Brussels sprouts and ten F₁ hybrids derived from them were tested for their response to anther culture. From 5–19 plants per genotype were tested, and each plant was tested on 3–6 separate occasions. Results from the inbred lines were broadly similar to those from the F₁ hybrids, despite the inbreds producing fewer buds and having a higher frequency of anther deformities. The maximum embryo yield from an inbred line was 215 embryos per 100 anthers, and from a hybrid was 275. From estimation of the variance components it was calculated that, for both inbreds and hybrids, about half the total variation was genetic whereas variation due to plants within genotypes and to occasions within plants were each about 13% of the total. The narrow sense heritability of responsiveness to anther culture (estimated by the proportion of variation between inbred lines which was genetic) was 0.48, and there was partial dominance for this character. In three cases the hybrid outyielded the better inbred, and this heterosis may well be due to dispersed dominant genes.