NaCl-Dependent growth, ion content and regeneration of calluses initiated from the mangrove plant,Bruguiera sexangula

Calluses initiated from leaves and seedlings of the mangrove,Bruguiera sexangula, were isolated from the original tissues and subcultured. Effects of NaCl on growth and ion content of each callus were measured. The growth rate of calluses derived from leaves (leaf callus) gradually decreased as the NaCl concentration in the medium increased, while that of calluses derived from seedlings (seedling callus) was highest in the medium containing 100 mM NaCl. Concentrations of Na and Cl in both calluses increased with increasing the NaCl concentration in the culture medium. The concentration of K of leaf calluses greatly decreased at 300 mM NaCl, while the K concentration of seedling calluses decreased only slightly and remained relatively high even in the presence of 300 mM NaCl. Transient treatment of leaf calluses with media containing high concentrations of NaCl frequently induced regeneration of adventitious tissues.     

卫星搭载红豆草后代中耐盐细胞系的筛选及鉴定

将红豆草种子搭载于940703返地卫星,经田间繁殖得后代种子,先将种子在1.5%NaCl上筛选、并在该盐浓度下诱导愈伤组织和筛选,在无盐培养基上恢复生长后再在1.2%NaCl上筛选得到耐盐变异系.变异系具有正常的分化能力并表现出对PEG胁迫的交叉抗性.变异系在无胁迫条件下脯氨酸含量较低但在有盐胁迫时具有高效积累脯氨酸的能力.后者可能对红豆草耐盐系更为重要.变异系中脯氨酸的这种合成机理可能是由于一些基因在调控中对水的敏感性改变引起.梯度聚丙烯酰胺凝胶电泳表明耐盐系的SOD和酯酶分别出现175kD和75kD的新形式.说明空间诱变和组织培养相结合可以筛选耐盐变异系.     

Efficient callus initiation from leaf of mangrove plant,Bruguiera sexangula in amino acid medium: Effect of NaCl on callus initiation

Experimental conditions for efficient callus initiation from mangrove plants were investigated. As a source explant, leaf ofBruguiera sexangula was used. Mangrove plant is one of the most famous woody plants which can grow at the salty area. The initiated callus can be a suitable material for the investigation of salt tolerant mechanisms of mangrove plants. Leaf pieces cultured in an Amino Acid medium supplemented with 2 μM 2,4-dichlorophenoxyacetic acid and 2 μMN-(2-chloro-4-pyridyl)-N′-phenylurea at 30 C developed calluses. Microscopic observation suggested that the callus was initiated from the tissue in the vascular bundles in the leaf. We also examined the effect of NaCl on callus initiation and short-term culture of the calluses on the leaves. Callus initiation rate decreased with increasing NaCl concentration higher than 100 mM in the culture media. The medium containing 100 mM NaCl produced the largest callus on the leaf, compared with higher or lower concentrations of NaCl.     

Short-term in vitro storage of t Miscanthus x ogiformis Honda 'Giganteus' as affected by medium composition,temperature, and photon flux density

Miscanthus x ogiformis Honda 'Giganteus' shoot cultures were stored in vitro on proliferation or rooting medium for up to 27 weeks at temperatures of 8, 12, 16, or 20 °C and photosynthetic photon flux densities of 5, 10, or 20 μmol m⁻² s⁻¹. Plants survived storage much better on rooting medium than on proliferation medium. Plants stored on rooting medium for 1 week survived well when survival was assessed immediately after storage or after 14 days of acclimatization, but had the lowest survival 28 days after transplantation. With increasing storage period on rooting medium increasing survival was found 28 days after transplantation. This was probably a result of the development of rhizomes and/or roots during storage. Best survival was observed at 20 μmol m⁻² s⁻¹ and a temperature of 8-16 °C. Increasing the temperature to 25 °C during the last week of storage improved survival considerably. Root formation was slow at 8 °C, but after 27 weeks of storage the rooting percentage was the same at all storage temperatures. An increasing number of shoots per plant 28 days after transplantation was found with increasing PPFD during storage.Miscanthus shoot cultures can be stored in vitro for at least 27 weeks with limited losses when stored on rooting medium at 20 μmol m⁻² s⁻¹, a temperature of 16 °C, and given a 1-week end-of-storage treatment of 25 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphogenic studies in tissue cultures of the parasiteSantalum album L.

Whole seeds, excised embryos, and excised endosperm ofSantalum album were aseptically cultured with a view to studying seed germination in isolation from the host species, and to establishing callus cultures from both embryo and endosperm for comparative studies et their morphogenesis. Seed germination and seedling formation occurred normally only on modified White's medium supplemented with casein hydrolysate or coconut milk, or with both substances. Neither the excised embryo nor the endosperm grew on any of the culture media tested. However in about 17 per cent seed cultures on White's medium supplemented with 2,4-D, kinetin, and yeast extract, the endosperm degenerated, whereas the embryo callused and subsequently differentiated into innumerable embryoids; eventually the embryoids developed into normal plantlets. Callusing of the endosperm occurred also in seed cultures on four media supplemented variously with 2,4-D, kinetin, and yeast extract. Although the endosperm tissue grew through several passages no organ fornation was observed.     

Callus growth and somatic embryogenesis in thalamus tissue ofRanunculus asiaticus L. CultivatedIn vitro: Cytokinin effect and phenol metabolism

Summary The effect of cytokinin on growth and plant regeneration of thalamus-derived calluses ofRanunculus asiaticus L. has been investigated with various concentrations of 6-benzyladenine and 6-furfurylaminopurine (kinetin), in a medium containing 2,4-dichlorophenoxyacetic acid levels, which was decreased to 0 over three subcultures. Cytokinins, although not essential, for initiating callus production, improved subsequent callus growth and plant regeneration. No somatic embryogenesis was observed on calluses grown on media lacking cytokinins or containing only kinetin. Calluses manifested embryogenesis on media containing 6-benzyladenie plus kinetin or only 6-benzyladenine. Nondifferentiating callus was characterized by a high content of phenolic polymers and an elevated peroxidase and polyphenol oxidase activity in comparison with differentiating callus. Differences in simple phenol concentrations were observed in the two kinds of callus.     

Selection and Characterization of High pH Resistant or Salt Resistant Variants from Haploid Triticale callus (n=28)

Haploid Triticale callus did not grow on N6 medium with pH adjusted to 10 by 1% NaOH or with 1% NaC1, and was killed totally after culture for 40–50 days. However, living spots could be found in few dead inoculated calluses. When these living spots were transferred to the same medium, some of them will continue to grow and others dying off. These media are called screening media and the calluses growing more or less normally on them, variants. It is supposed that each variant came from a single callus cell. One gram of fresh callus contains about 1.65×106 cells, and the variation rate for resistance to 1% NaCl, as calculated from total quantity of inoculum, is 0.86 per million cells, and for resistance to pH 10, 0.81. R4 is a variant resistant to pH 10. The variation rate of IL for resistance to pH 11 is 1.01 per million cells. If selection is made directly from the normal callus, the variation rate for resistance to pH 11 could be estimated as (0.81×10–6)× (1.01×10–6), or 0,82 per 10" cells. For such a low rate of variation, it would be impossible to isolate them under the usual condition of laboratory. However, it could be achieved easily if a stepwise selection schedule is adopted. The variation rate can be increased one to two tithes by pretreatment of callues cells with 0.4% EMS aqueous solution for two hours. When the variants were cultured in normal medium far 3 to 5 passages (28 days between two subcultures), 23–33% of variants could not maintain their resistance, However, the two-third or more variants are stable and maintain their resistance through passages in normal media. Besides, plantlets induced from the callus of pH 10 resistant line TR4, could grow on the pH 10 medium. And callus induced from these plantlets explant maintains the resistance. These facts indicate that the stable resistant variants selected from the callus cells would be valuable for breeding purpose.     

Evaluation of salt tolerance in cultivated and wild tomato species through in vitro shoot apex culture

The possibility of using in vitro shoot apex culture to evaluate salt tolerance of cultivated (Lycopersicon esculentum Mill.) and wild (Lycopersicon pennellii (Correll) D'Arcy) tomato species was determined and related to the response obtained by callus culture. Both apices and calluses were grown on media supplemented with 0, 35, 70, 105, 140, 175 and 210 mM NaCl, and growth and physiological traits were determined. Most apices of L. esculentum did not develop roots from low NaCl levels, whereas the apices of L. pennellii were able to develop roots at the different salt levels. This different degree of salt tolerance between L. esculentum and L. pennellii was not, however, clearly shown on the basis of the shoot growth of the plantlets. The callus response was similar to that shown by the rooting parameters, as callus growth in response to increased salinity was much greater in L. pennellii than in the tomato cultivar. K⁺decreased more and proline accumulated less with salinity in shoots of L. esculentum compared to L. pennellii, whereas the opposite response was obtained in calluses. The results obtained in this study suggest that rooting parameters are the most useful traits for rapid evaluation and screening of tomato species and segregating populations through in vitro shoot apex culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Latent infections of in vitro anthurium caused by Xanthomonas campestris pv. dieffenbachiae

Latent infections of tissue-cultured Anthurium andraeanum Lind. caused by the blight pathogen, Xanthomonas campestris pv. dieffenbachiae (McCulloch & Pirone) Dye, were examined. The pathogen survived in or on callus for over 4 months without producing symptoms in callus or turbidity in the medium. The pathogen survived for more than 1 year on or within stage II shoots without producing symptoms and was successively transferred three times as latently infected shoots were multiplied. The pathogen did not grow or survive for more than 2 weeks in Murashige and Skoog medium lacking plant material. The addition of coconut water enhanced bacterial growth and produced turbidity in culture media. Latently infected in vitro anthuriums may be inoculum sources for subsequent outbreaks of the disease.     

Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and Gracilaria perplexa (Gracilariales, Rhodophyta)

The effects of auxins and cytokinin on callus formation, growth and regeneration of Gracilaria tenuistipitata Chang et Xia and G. perplexa Byrne et Zuccarello (Gracilariales, Rhodophyta) are reported. Plant growth regulators (PGR) in concentrations ranging from 0.1 to 100.0 μmol of indole‐3‐acetic acid, 2,4‐dichlorophenoxyacetic acid (2,4‐D), and kinetin (K) were added to the ASP 12‐NTA solid medium (0.7% agar), and apical and intercalary segments (5 mm long) were inoculated as initial explants. K stimulated growth rates of intercalary segments of G. tenuistipitata in a linear relation, and 2,4‐D (1.0 μmol) and K (10.0 μmol) stimulated growth rates of apical and intercalary segments of G. perplexa, respectively. The simultaneous formation of apical, basal, and intermediate calluses is reported for the first time in axenic tissue cultures of red algae. With intercalary segments of G. tenuistipitata, basal callus induction rates were higher than those of apical and intermediate calluses in the majority of treatments, and auxins had stimulatory effects on the formation of all callus types. In apical segments of G. perplexa, intermediate callus formation was stimulated only by treatment with 1.0 μmol of K, while apical callus formation was stimulated by indole‐3‐acetic acid (1.0–10.0 μmol), 2,4‐D (10.0–100.0 μmol), or K (0.1 μmol). Intercalary segments of G. perplexa developed only intermediate calluses, and the majority of treatments with PGR stimulated higher rates than those presented by apical segments. Potential for regeneration (development of adventitious plantlets originated from callus cells) was higher in apical calluses than in basal and intermediate calluses developed in intercalary segments of G. tenuistipitata. Moreover, auxins and cytokinin were essential to the induction of regeneration in intermediate calluses, while specific concentrations stimulated regeneration from basal and apical calluses. Plant regeneration in G. perplexa was observed only after transferring calluses from solid to liquid medium, and the majority of treatments with PGR had stimulatory effects. Regenerating plants of G. perplexa developed tetrasporangia, and released tetraspores giving rise to adult gametophytes. Our results indicate that auxins and cytokinin have a regulatory role in the growth and morphogenesis in G. tenuistipitata and G. perplexa, and diversity of responses presented by both species is related to specific developmental systems.     

Salt tolerance in rice in vitro: Implication of accumulation of Na+, K+ and proline

The implication of accumulation of both inorganic (Na⁺, K⁺) and organic (proline) solutes were evaluated in unadapted and NaCl-adapted callus of a salt-sensitive (Basmati 370) and a salt-tolerant (SR-26B) cultivar of rice (Oryza sativa L.) after a NaCl shock. Accumulation of Na⁺,K⁺ and/or proline in callus was co relatable and the relative presence of these components in tissues after shock treatment was found to be important factors to support differential regrowth capacities of the shock treated calluses. Presence or retention of K⁺ in rice callus was a key factor for salt tolerance as it was observed to be positively correlated with growth in both the varieties. The results indicated that K⁺ was the first candidate to counteract the negative water potential of outside milieu, while proline was probably the last metabolic device that rice calluses opted for when exposed to salt stress.     

Effect of salicylic acid on protein composition of Tatar buckwheat Fagopyrum tataricum calluses with different ability for morphogenesis

The effect of salicylic acid on the content of soluble proteins and individual polypeptides in Tatar buckwheat Fagopyrum tataricum calluses differing in ability for morphogenesis was studied. Changes in the protein composition of the calluses cultivated in the dark and in the light indicated the higher sensitivity of the non-morphogenic callus. Different response of callus cultures to salicylic acid and conditions of cultivation (light, darkness) is suggested to be associated with the antioxidant defense system, which is, in particular, characterized by the hydrogen peroxide content in the calluses. Salicylic acid increased the H₂O₂ content in non-morphogenic calluses more strongly than in morphogenic calluses, and the difference was more significant for the calluses cultivated in the light.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 390–396.Original Russian Text Copyright © 2005 by Maksyutova, Galeeva, Rumyantseva, Viktorova.     

Studies on the Culture Conditions of Higher Plant Cells in Suspension Culture

To study the influence of cultural conditions on higher plant cells in suspension culture, the effects of nutritional conditions on the growth of suspended cells were investigated. Calluses were induced from 39 species of Nicotiana plants and 6 species of Populus plants on agar slant media, then these were transferred to suspension cultures. Concentrations of 2,4-D and kinetin suitable for incubation of callus from each plant were investigated and species having high growth rates in the appropriate medium were selected.

The effects of concentrations of auxins and kinetin, a variety of carbon and nitrogen sources, thiamin and myo-inositol on growth of the selected calluses were also studied. Of these calluses studied, N. glutinosa, N. tabacum var. Xanthi ova and P. hybrids were selected as calluses having high growth rates. Myo-inositol had no effect on any callus growth, and thiamin gave a distinct effect on Populus callus only. Nitrate as a nitrogen and sucrose as a carbon sources, and 2,4-D as an auxin were most effective in all calluses studied. Kinetin was essential for N. glutinosa among the calluses studied. Although high sugar concentrations tended to lengthen the lag period in the growth curve, there was no difference in the growth rates of the logarithmic phase among the concentrations.     

Normalization of the DNA content of telophase cells from wheat calli by nutrient modifications

Summary Cytophotometric analyses were conducted to determine whether the DNA content of wheat callus varied by tissue culture medium or age of callus. Wheat,Triticum aestivum L. line PCYT-20, was cultured on three variations of the Murashige and Skoog (1962) growth medium. At the end of 2, 4, 6 and 8 weeks, samples were collected and prepared for Feulgen cytophotometry. Standards for the DNA measurements were readings from 100 telophase nuclei in wheat meristematic root tips. Amounts of DNA per nucleus present in telophase cells from callus grown on single-strength MS indicated that ploidy level increased 52%, 74% and 39%, respectively, over time from 2, 4, and 6 weeks as compared to the double-strength MS medium, and 29%, 60% and 32%, respectively, when coconut water was added to the single-strength MS culture medium. The shape of the mitotically-active cells in callus was more variable than in root tips cells. Callus grown on double-strength MS medium produced more shoots than callus grown on single-strength MS. Double-strength MS medium and, to a lesser extent, additional sucrose and organic nitrogen overcame the effects of 2,4-D on DNA amplification. Improved media may reduce the somaclonal variation induced by tissue culture.     

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.     

Tissue culture inHaworthia

Effects of three auxins and kinetin on growth of the calluses of two species ofHaworthia, H. aristala andH. setata, were investigated. Stock calluses derived from the flower buds of these species were maintained for two years on RM-1964 agar medium containing 5 mg/l NAA. Small pieces of the stock calluses were transferred to the basal medium containing either auxin, IAA, NAA or 2,4-D in six different concentrations (0.1–50 mg/l) combined with three concentrations (0–2 mg/l) of kinetin; in total, 54 kinds of media were used. Fresh weight of the calluses was measured 0 to 30 days from transfer and transformed to the natural logarithm. The linearity of their growth curves against the culture period was tested. The growth curves of theH. aristata calluses grown in dark and under continuous light and that of theH. setata callus grown in dark gave similar regression coefficients of 0.07 to 0.11, indicating that the doubling time of the callus mass was about 6.3 to 10.1 days. After 42 to 50 days from inoculation, the fresh weight of each individual callus was recorded, and the data were statistically analyzed. All auxins at the concentration of 50 mg/l significantly inhibited callus growth. Kinetin did not affect growth of theH. aristata callus in dark, while its effect on theH. setata callus was detected under light. Interaction of kinetin was found with IAA and 2,4-D inH. aristata and with IAA and NAA inH. setata. REsponses of theH. aristata callus to auxins and kinetin, when grown in dark, were different in several points from those of theH. setata callus grown under light. The best callus growth was observed in the following media; 0.2 mg/l kinetin supplemented with 1 mg/l IAA, or 0.5 mg/l 2,4-D, and 2 mg/l kinetin with 0.5 mg/l NAA inH. aristata, and 0.2 mg/l kinetin supplemented with 1 mg/l IAA, 5 mg/l NAA or 0.1 mg/l 2,4-D inH. setata. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 413.     

Organogenesis and somatic embryogenesis from callus cultures in Muscari armeniacum Leichtl. ex Bak.

Summary Establishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Effects of salt and proline on Medicago sativa callus

In this study, two cultivars of Medicago sativa (cv. Yazdi and cv. Hamedani) were used for callus production. Calluses were transferred to MS medium containing 0, 30, 60, 90, and 120 mM NaCl and 0, 5, 10 mM proline. After 4–5 weeks dry weight and intracellular free proline of the calluses were measured. The growth of callus in both cultivars decreased with increasing salt concentration. Addition of exogenous proline to the culture medium increased the dry weight and free proline content of callus. The difference between control and treated calluses with 10 mM exogenous proline in the medium was significant. The data obtained from experiments indicated that the responses of two Medicago cultivars was genotype dependent.