Carotenoids synthesized in citrus callus of different genotypes

Eight carotenoids, such as phytoene, α-carotene, violaxanthin, etc., synthesized in citrus callus of 31 genotypes were identified and determined. Though varied with genotypes, the carotenoids composition of callus derived from a certain genotype was stable, while carotenoids contents altered between sub-cultures. Some specific carotenoids were produced in calluses of limited genotypes: β-citraurin was only synthesized in calluses of Nianju tangerine (Citrus reticulata Blanco) and Page tangelo (C. reticulata × C. paradisi); while 9-Z-violaxanthin was only detected in Nianju tangerine and Skaggs Bonanza navel orange (C. sinensis L. Osbeck). Notably, the only carotenoid detected in calluses of Natsudaidai (C. aurantium L.) and other two sweet oranges (C. sinensis L. Osbeck) was phytoene. It implied that citrus calluses could be employed to produce specific carotenoids in the future. To further elucidate the characters of callus carotenoids profile, comparisons of carotenoids profiles was made among calluses, fruit tissues and leaves of four selected citrus genotypes. Results showed that lycopene was not detected in leaves and calluses; nevertheless, both citrus fruits and calluses accumulated phytoene, whereas leaves did not except those of Cara Cara navel orange. It is postulated that citrus callus featured its carotenoids profile different from fruit tissues and leaves. In conclusion, the advantages of using citrus callus as an alternative model research system in understanding the regulation of carotenogenesis have been discussed.     

In vitro evaluation of phenolic and osmolite compounds,ionic content,and antioxidant activity in safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.) under salinity stress

Carthamus tinctorius L., rich in antioxidant compounds, is a herbal medicine. Biochemical mechanisms of adaptation to salinity stress in safflower are still poorly understood at the cellular level. For this purpose, callus cultures of four different genotypes of safflower were used in this study to evaluate changes in their biochemical (ionic content, proline, and glycine betaine), total phenolics content (TPC), total flavonoids content (TFD), antioxidant responses (2,2-diphenyl-1-picrylhydrazyl: DPPH assay and carotenoid content), and lipid peroxidation (malon dialdehyde content: MDA) under salinity stress. The calluses derived from hypocotyls were exposed to in vitro salt stress at different concentrations of sodium chloride (0, 100, 200, and 300 mM). A reducing trend was observed in K⁺ and carotenoid reserves of the calluses with increasing NaCl concentration while an increasing trend was observed in Na⁺ content, proline, MDA, TPC, TFD, and DPPH activity under the same conditions. Callus glycine betaine content was found to decrease in the medium containing 100 mM NaCl but increased beyond this concentration up to 300 mM NaCl. Positive and significant correlations were recognized between DPPH and total phenolics as well as DPPH and total flavonoid contents, demonstrating that phenolics are the main contributors to the potential antioxidant activity of safflower at the cellular level. Overall, the salt-tolerant genotypes of Mex.2-137 and Mex.2-138 were found capable of being processed for the production of secondary metabolites via NaCl elicitation.     

Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.     

Genetic analysis of callus formation in a diversity panel of 96 rose genotypes

Plant Cell, Tissue and Organ Culture (PCTOC) - In a diversity panel of 96 rose genotypes, variation in the capacity to form calluses on leaf explants in vitro was investigated, and a genome-wide...     

Somatic embryogenesis and organogenesis in apomictic and sexual Brachiaria brizantha

Brachiaria brizantha (syn. Urochloa brizantha) is an important tropical forage grass widely cultivated in Brazil. In order to optimize tissue culture conditions for B. brizantha, in vitro culture of mature seeds, basal segments and leaf segments from in vitro plants of an apomictic and a sexual genotype of B. brizantha was performed. When cultured on different media, leaf segments yielded non-embryogenic calluses which formed several roots. Friable calluses from mature seeds and basal segments explants incubated on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine yielded 80% compact and nodular embryogenic structures. Calluses with such compact embryogenic structures were highly regenerable upon transfer to medium supplemented with kinetin and naphthalene acetic acid. They produced isolated somatic embryos, multiple fused scutelli or isolated scutellum with polyembryos that germinated into isolated or multiple shoots. Green and morphologically normal plants were obtained for the two genotypes. Changing the media from pH 5.8 to pH 4.0 increased the number of explants that formed calluses as well as the number of shoots per explant. When embryogenic calluses from mature seeds were successively sub-cultured for 4 months, aiming at repetitive somatic embryogenesis, all the regenerated plants were albinos. The embryogenic nature of the compact structure was confirmed by scanning electron microscopy.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Indica rice genotypes: an assessment of factors affecting the transformation efficiency

An efficient Agrobacterium-mediated method for transformation of popular Bangladeshi Indica rice genotypes has been developed. Mature embryo-derived calluses as well as immature embryos were used as the target material. Transgenic plant production frequency was higher using the immature embryos than mature embryo-derived calluses. However, 3-week-old mature embryo-derived calluses served as an excellent starting material. The super-binary vector (pTOK233) was generally more effective than the binary vector (pC1301-Xa21mSS) particularly with recalcitrant Bangladeshi genotypes such as BR22. However, transformation of the Japonica cultivar Taipei-309 was equally effective with either plasmid. Inclusion of acetosyringone (200M) in co-cultivation media proved essential for successful transformation and the optimum co-cultivation period found was to be 3days. A large number of morphologically normal, fertile transgenic plants were obtained which expressed gus as determined by histochemical staining. Integration of the hpt gene into the genome of transgenic plants was confirmed by molecular analysis. Mendelian inheritance of transgenes (hpt and gus gene) was observed in T1 progeny.     

Anther culture with different genotypes of opium poppy (Papaver somniferum L.): effect of cold treatment

This study concerns anther culture and the production of microspore-derived calluses and plants of the opium poppy (Papaver somniferum L.). It was confirmed that growth regulators were necessary for microspore callus production. Cold treatment (7 d at 7°C) of the buds prior to culture lead to a twofold increase in the frequency of responsive anthers and in the number of calluses per 100 anthers plated. Callus was produced from cultured anthers of several genotypes, covering a wide genetic background. Step by step removal of growth regulators from the culture medium promoted organogenesis and plant regeneration. Most regenerated plants were diploid. The overall process of microspore embryogenesis closely resembled that described in previous reports on somatic callus production and plant regeneration from poppy hypocotyls in vitro.     

Genetic transformation of commercial breeding lines of alfalfa (Medicago sativa)

Bio-engineering technologies are now routinely used for the genetic improvement of many agricultural crops. However, breeding lines of Medicago sativa are not easily amenable to genetic transformation and therefore cannot benefit from the molecular tools that have been developed for genetic manipulations. This paper describes a strategy that has been developed to transfer DNA into commercially important breeding lines of winter-hardy alfalfa via Agrobacterium infection. Three highly regenerative genotypes have been selected from ca 1000 genotypes within 11 breeding lines. They have been used as basic material for an extensive genetic transformation trial. Combinations of genotypes (11.9, 8.8, 1.5) expression vectors (pGA482, pGA643, pBibKan) and bacterial strains (C58, A281, LBA4404) were tested for their ability to produce stable transgenic material. Putative transgenic plantlets were further screened by nptII-specific PCR amplification, Southern hybridization and recallusing assays. One genotype (1.5) gave only one transformant out of 432 individual trials. With the two other genotypes, efficiency of transformation (kanamycin-resistant calluses obtained/explant tested) ranged from 0 to 0.92 depending on the strain/vector combination used. Statistical interactions underline the possibility of obtaining good genotype-strain-vector combinations for alfalfa transformation. Predicted transformation probability indicates that with strain LBA4404 containing the vector pGA482 and genotype 11.9, transformation efficiency is above 60% and 10% or more of the calluses retain embryogenic potential. PCR amplification and Southern hybridization of randomly chosen regenerated plantlets demonstrated that all embryos developing on 50 g ml^-1 kanamycin had a stable genomic insertion of nptII. Sexual crosses with untransformed genotypes showed that segregation of the transgenic trait followed Mendelian heredity.     

Endogenous hormone concentrations and embryogenic callus development in wheat

Immature zygotic embryos of two wheat (Triticum aestivum L.) genotypes, known for their different ability to generate embryogenic callus, were used as initial explants to establish callus cultures. Embryogenic and non-embryogenic calluses were obtained from the competent genotype (`Combi'), while only non-embryogenic callus was produced by the incompetent one (`Devon'). The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. The endogenous hormone concentrations (free indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins ₁, ₃ and ₂₀ [GAs], zeatin/zeatin riboside [Z/ZR] and N ⁶[²-isopentenyl] adenine/ N ⁶[²-isopentenyl] adenosine; [iP/iPA]) of the initial explants were determined by means of radio-immunoassay and showed that the only difference was the higher concentration of ABA found in the embryos of the most competent genotype; whose embryos showed a reduced rate of precocious germination. When analysing the endogenous hormone concentrations in the various callus types generated in each genotype, it was found that only differences in the free IAA concentrations were associated with variations in the morphogenic properties of the calluses. Higher concentrations of endogenous free IAA were typical of embryogenic callus cultures. It was also observed that a loss in the embryogenic competence of the calluses, due to a prolonged time of culture, occurred concomitantly with a reduction in free IAA concentrations, practically to the concentrations found in the non-embryogenic calluses.     

不同状态银杏愈伤组织蛋白质电泳分析及电镜观察

通过对同一来源3种银杏愈伤组织中蛋白质、黄酮含量测定,蛋白质SDS-PAGE电泳分析,细胞超微结构观察等研究银杏不同状态愈伤组织的生长情况。结果表明,银杏的这3种愈伤组织代表愈伤组织细胞生长的3个阶段。绿色愈伤组织处于分生组织状态,细胞生长旺盛,蛋白质较多,蛋白质条带宽,细胞内物质丰富,但黄酮的含量并不是最高;微黄色愈伤组织细胞处于初生代谢向次生代谢转化的阶段,蛋白质含量开始减少,45.7、38.4、33.4kD的蛋白质开始减少,核膜不完整,淀粉含量较高,其黄酮含量高于绿色愈伤,属次生代谢的阶段;褐色愈伤组织蛋白质含量很低,条带数减少,有5条蛋白质条带消失,且黄酮的含量最低,细胞结构发生质壁分离现象,内含物解体,细胞核解体,是衰亡的主要特征。微黄色愈伤组织是获取高黄酮含量的最好材料。     

Statistical Parameters Reflecting Morphogenetic Capacity of Soft Spring Wheat Calluses

Callus cultures of soft spring wheat were subcultured without separation into explants to follow the line one excised embryo–one callus. This approach revealed the following statistical correlations. Within every cultivar of Triticum aestivum L. and within a row of cultivars arranged in ascending order according to the frequency of embryogenic callus formation, positive correlations (at P = 95) were found between the proliferative activity of callus cells and the frequency of embryogenic callus formation. A reliable intraspecies correlation (significant at P = 95) between multiple regenerations of plants from calluses and the tillering trait (bushiness) of donor plants was also found. We assessed the importance of various statistical parameters of callus cultures for preliminary estimation of morphogenesis efficiency at early stages of culturing. Frequencies of callusogenesis and the growth curves for randomly selected calluses turned out to be noninformative characteristics, unless the morphogenetic activity of calluses was taken into account. The following statistical parameters were found to correlate with the morphogenetic capacity of wheat calluses: gradually increasing coefficients of variation in fresh weight of primary calluses, a larger callus size, and higher fresh weight gain in potentially morphogenetic calluses.     

High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ thidiazuron (TDZ) and 1 mg l⁻¹ α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation. J. Seo and S.W. Kim contributed equally to this work.     

In vitro culture of tobacco callus on medium containing peptone and phytate leads to growth improvement and higher genetic stability

Growth and genetic stability of Nicotiana tabacum L. callus were strongly improved by replacing the inorganic nitrogen and phosphorus of the Murashige and Skoog's medium by a soybean peptone and phytate, respectively. Cell proliferation after subcultivation on the modified medium was highly stimulated as evidenced by a strong biomass increase; this improvement was mainly due to the organic N source. In addition, while calluses grown under standard conditions displayed various cell sizes and DNA contents, subcultivation on the modified medium led to homogeneous cell size distribution and stable 4C–8C DNA contents through several subcultures. This improved genetic stability was due to replacement of inorganic P by phytate, provided the presence of peptone. Such new media composition could be useful for slow-growing cell suspensions or calluses.     

In vitro induction of tetraploid garlic with trifluralin

Garlic (Allium sativum) is propagated asexually. Since sexual cross breeding is almost impossible, means for effective breeding are not currently available and the available production cultivars are seriously aged and degenerated. A possible alternative for breeding is chemical induction. Trifluralin, a type of herbicide, has been reported to provoke chromosome doubling. However, this chemical had not been tested on garlic. We tested various trifluralin concentrations and treatment durations for efficiency in the induction of tetraploid garlic. A clove base of garlic with a stem cv. Gailiang was used as the ex-plant to induce calluses on Murashige and Skoog (MS) medium; the calluses were then inoculated onto MS medium containing different levels of trifluralin and cultured to induce chromosome number variation in vitro. Garlic calluses were effectively induced via the ex-plant and both shoots and roots differentiated well on MS medium containing 6-benzylaminopurine at 3.0 mg/L and indole-3-acetic acid at 0.1 mg/L. However, increases in trifluralin concentration and treatment duration reduced the survival rate and differentiation rate of calluses. Garlic callus cultured for 15 days on medium containing 100 μM trifluralin gave the highest rate of chromosome doubling. Through observation of chromosome number in the root apical cells and the morphology of guard cells on the leaf epidermis of the regenerated plantlets, it was clear that chromosome number variation was induced and tetraploids were produced in vitro by trifluralin treatment.     

Nitrogen compounds in embryogenic and non-embryogenic calluses of <Emphasis Type="Italic">Medicago arborea</Emphasis> L.

Nitrogen (N) metabolism during embryogenesis may be fundamental in the embryogenic response. We used different explants of Medicago arborea L. subsp. arborea seedlings: cotyledons, petioles and leaves, which form calluses with different embryogenic responses. The endogenous contents of total nitrogen, nitrate, nitrite and ammonia and nitrate reductase activity were determined in embryogenic and non-embryogenic calluses induced from the different explants. The endogenous total N content decreased in the calluses as the culture time progressed, this decrease being more pronounced in the more embryogenic calluses obtained from petioles with the H8 and F0 media. Inorganic N decreased during embryogenesis, coinciding with an increase in organic N. Thus, N metabolism somehow seems to be essential in embryogenesis. The N detected in calluses, at the start of culture, was mainly metabolised to nitrite. This metabolism was very pronounced; especially in embryogenic calluses obtained from cotyledons and petioles. That is, the metabolism of N seemed to be more marked in the calluses in which embryogenesis was greater. The nitrite content decreased in all the calluses, the contents being lower, especially in the last months of culture, in the more embryogenic calluses obtained from petioles. In many calluses, ammonia levels did not follow any general pattern. Neither was it possible to detect changes in ammonia levels between the embryogenic and non-embryogenic calluses. Regarding nitrate reductase activity, no clear differences between embryogenic and non-embryogenic calluses were found.     

Temperature and genotype affect asparagus somatic embryogenesis

Summary Calluses from five asparagus genotypes G14, G32, G171, G203, and G447 and hybrid Jersey Giant (JG) were incubated at three temperature regimes (24, 27, and 30°C) on embryo induction medium to assess somatic embryo development and conversion to plantlets. The calluses from three genotypes (G14, G32, and G171) were not responsive, failing to produce somatic embryos at any temperature regime. For three responsive genotypes (G203, G447, and JG), both incubation temperature and genotype significantly affected the numbers of somatic embryos produced. The calluses produced the most and the least numbers of total, bipolar, and globular embryos when incubated at 27°C and 24°C, respectively. When incubated at 27°C, G203 produced the highest numbers of total and globular embryos, 178 g⁻¹ callus and 142 g⁻¹ callus, respectively while G447 produced the highest number of bipolar embryos, 77 g⁻¹ callus. Incubation temperature but not genotype significantly affected the conversion of somatic embryos to plantlets. The somatic embryos recovered from the three responsive genotypes incubated at 27°C also converted to plantlets at the highest frequencies, 60–63% of the bipolar embryos and 42–43% of the globular embryos converted to plantlets, while the somatic embryos recovered from the calluses incubated at 24°C converted to plantlets at the lowest frequencies.     

Production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (Hordeum vulgare)

The in vitro plant regeneration frequencies for immature scutella, leaf-bases/apical meristems (LB/AM) and mature embryos of four commercially important barley genotypes were compared. Production of shoots from mature embryos or calluses of LB/AM incubated on media containing 1.0 or 2.0 mg l^–1 6-benzylaminopurine (BA) were comparable to regeneration frequencies obtained for scutella-derived calluses of the same genotypes. Incubation of excised mature embryos and LB/AM on media containing the plant growth regulator, thidiazuron (TDZ), resulted in an increased shoot production. However, TDZ treatment did not stimulate plant regeneration from calluses derived from scutella or LB/AM. Shoots formed from TDZ-treated mature embryos and LB/AM were induced without a callus interphase and the in vitro culture system gave a three- to eight-fold higher regeneration frequency than recorded for scutella-derived calluses on BA medium. The simplicity and rapid development of shoots using the mature embryo system could potentially be used for the regeneration and genetic transformation of barley over alternative regeneration systems.     

Sugar beet (Beta vulgaris L.) morphogenesis in vitro: Effects of phytohormone type and concentration in the culture medium, type of explants, and plant genotype on shoot regeneration frequency

In vitro regeneration techniques have been optimized for seven strains and cultivars of sugar beet (Beta vulgaris L.) bred in Russia. The frequency of shoot regeneration from somatic cells and tissues of sugar beet varies from 10 to 97% depending on the explant type, culture-medium composition, and genotype. The in vitro regeneration potential has been estimated in plants with different genotypes. The effect of medium composition (phytohormones and carbohydrates) on the frequency of the formation of a morphogenic callus competent for plant regeneration has been determined. The effect of the types and concentrations of various cytokines (zeatin, kinetin, and 6-benzylaminopurine) on direct shoot regeneration from cotyledon nodes has been estimated. The culture-medium composition has been optimized for direct shoot regeneration from petioles. The effects of different concentrations of abscisic acid on the frequency of shoot regeneration from a morphogenic callus has been studied. Micropropagation has been used to obtain petiole explants and reproduce the shoots obtained by direct regeneration from cotyledonnodes, petioles, and calluses. Improved shoot-regeneration methods can be used for both agrobacterial and bioballistic genetic transformation of the sugar beet genotypes studied.     

Sugar beet (Beta vulgaris L.) morphogenesis in vitro: effects of phytohormone type and concentration in the culture medium, type of explants, and plant genotype on shoot regeneration frequency

In vitro regeneration techniques have been optimized for seven strains and cultivars of sugar beet (Beta vulgaris L.) bred in Russia. The frequency of shoot regeneration from somatic cells and tissues of sugar beet varies from 10 to 97% depending on the explant type, culture-medium composition, and genotype. The in vitro regeneration potential has been estimated in plants with different genotypes. The effect of medium composition (phytohormones and carbohydrates) on the frequency of the formation of a morphogenic callus competent for plant regeneration has been determined. The effect of the types and concentrations of various cytokines (zeatin, kinetin, and 6-benzylaminopurine) on direct shoot regeneration from cotyledon nodes has been estimated. The culture-medium composition has been optimized for direct shoot regeneration from petioles. The effects of different concentrations of abscisic acid on the frequency of shoot regeneration from a morphogenic callus has been studied. Micropropagation has been used to obtain petiole explants and reproduce the shoots obtained by direct regeneration from cotyledon nodes, petioles, and calluses. Improved shoot-regeneration methods can be used for both agrobacterial and bioballistic genetic transformation of the sugar beet genotypes studied.     

小麦成熟胚愈伤组织诱导及分化研究

以2个小麦品种成熟胚为外植体进行离体培养,研究了不同预处理、不同2,4-D浓度及与KT组合、不同蔗糖浓度等因素对愈伤组织诱导及分化的影响。结果表明:4℃低温预处理可提高愈伤组织的出愈率及再生苗率,2个材料的出愈率及再生苗率均达到90%和30%以上;在不同预处理条件下,2,4-D浓度对出愈率及再生苗率的影响与基因型有关,2,4-D浓度为1~2 mg/L更有利于愈伤组织诱导及分化;附加KT能缓解高浓度2,4-D对再生苗率的抑制作用,而对于在1、2 mg/L 2,4-D的培养基中附加KT则不表现这种作用;蔗糖浓度则在30 g/L条件下更有利于愈伤组织诱导。因此通过4℃低温预处理,在MS基本培养基中附加1~2mg/L 2,4-D及30 g/L蔗糖亦可促进小麦成熟胚愈伤组织的诱导和分化。