Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

GFP-FABD2和GFP-MBD标记蛋白对拟南芥悬浮细胞培养及应激响应能力的影响

以转GFP-FABD2和GFP-MBD基因的拟南芥为材料,研究了GFP-FABD2和GFP-MBD这两种细胞骨架标记蛋白对拟南芥愈伤组织诱导、悬浮细胞培养及应激响应能力的影响.结果表明:(1)GFP-MBD标记蛋白延长愈伤的出愈时间,改变愈伤形态,使转基因拟南芥种子的出愈量减少为野生型的59%、悬浮细胞的长短轴比缩小为1.20±0.21、第7天细胞活力下降为0.66±0.09,影响细胞的生长曲线.(2)GFP-FABD标记蛋白虽对愈伤生长影响不大,但却使悬浮细胞的长短轴比显著增加为2.49±1.18、第7天细胞的活力下降为0.87±0.06,造成悬浮细胞生长曲线的改变.(3)通过调整培养条件的激素水平,以上两种细胞骨架标记蛋白对悬浮细胞生长的影响可以得到修复.(4)检测优化条件下培养的GFP-FABD2或GFP-MBD悬浮细胞对温度、渗透压、机械应力等环境改变的应激响应能力,结果未发现与野生型有明显区别.     

A genetic analysis of cell culture traits in tomato

Summary Tomato genotypes superior in regenerating plants from protoplast and callus cultures were obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding. The genetics of regeneration and callus growth have been studied in selfed and backcross progenies of a selected plant (MsK93) which has 25% L. peruvianum in its ancestry. Segregation data showed that the favourable cell culture traits of L. peruvianum are dominant. Regeneration capacity from established callus cultures was controlled by two dominant genes. Callus growth on primary expiants, callus growth of established cultures and shoot regeneration from explants had high heritabilities (0.47, 0.78, 0.87, respectively). Callus growth and regeneration capacity were not correlated within the populations studied.     

The biology ofAzospirillum-sugarcane association II. Ultrastructure

Summary Tissue cultures of sugarcane support abundant growth ofAzospirillum brasilense (SP 7). Visible after 1–2 weeks as a white or pink slime, this growth reaches 2×10⁸ bacteria/mm² on the surface of callus. Growth of the bacterium is strictly extracellular in viable callus, and instances of intracellular growth result from rupture of the cell wall during senescence of callus tissue. A significant proportion of the bacterial population on callus is pleomorphic. Varying the nitrogen source in the nutrient medium caused no obvious effect on callus cell structure. The presence of the bacterium caused structural alterations in callus cells which did not inhibit overall growth of the bacterium. Growth of callus as tight groups of cells lacking intercellular spaces may be important for the establishment of a long-term association withAzospirillum. The interface of bacteria and live callus tissue is at the surface of tight cell groups. Browning of the surface cell layers of these groups in the presence ofAzospirillum is not of the rapid nature known for hypersensitivity reactions. Rather, this production of phenolics appears to be due to the accumulation of extracellular bacterial metabolites. The ultrastructure of this and other callus reactions is described. As evidenced by organogenesis, the associated cultures have remained viable for at least 18–20 months.Florida Agricultural Experiment Station Journal Series No. 1695.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of antioxidants from <Emphasis Type="Italic">Hemidesmus indicus</Emphasis> R. Br. cultures

Summary Shoot cultures and callus cultures from roots and leaves of Hemidesmus indicus R. Br (Asclepiadaceae) were established on Murashige and Skoog medium with various hormonal combinations. The production of antioxidants (lupeol, vanillin, and rutin) in shoot cultures, callus cultures derived from leaf cells and root cells, was compared with root and aerial portions of the parent plant. Shoot cultures and leaf callus cultures produced more antioxidants than root callus cultures. In vitro culture of this species might ofter an alternative method for production of these important pharmaccuticals, which would reduce the collection pressure on this rare plant.     

Heritability of regeneration in tissue cultures of sweet potato (Ipomoea batatas L.)

Summary A population of open-pollinated progeny from 12 parents, and the 12 parents, was surveyed for in vitro growth and regeneration characteristics. Four different tissue culture procedures involving different media and the use of different explants to initiate the cultures were used. Petiole explants from young leaves were used as explants for initiation of callus cultures. These were evaluated for callus growth rate, friability, and callus color and texture, before transferring to each of three different regeneration media for evaluation of morphogenetic potential. Small shoot tips also were used to initiate callus cultures, which were evaluated for the same growth characteristics and transferred to growth-regulator free regeneration media. Regeneration occurred through root or shoot regeneration or through embryogenesis. Tissue culture treatment effects, as well as genotypic effects, were highly significant in determining: the types of callus produced, callus growth rates, color and texture on the two types of media used for the second and third subcultures. The family x treatment interaction was generally not statistically significant, affecting only callus color. Estimates of narrow sense heritability for callus growth rate in both the second and third subcultures were high enough (0.35 and 0.63, respectively) for the evaluation of parental lines for selection procedures. These characteristics were also the only early culture callus traits that were consistently correlated with later morphogenesis of the cultures. They were negatively correlated with root or shoot regeneration. The occurence of somatic embryogenesis was not correlated with early callus growth characteristics. Genetic and treatment effects were highly significant in the evaluation of morphogenetic potential, through root or shoot regeneration, or through embryogenesis. Regeneration of all types was of low frequency for all procedures, expressed in 11% of the cultures of the total population.Paper No. 9906 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA. From a thesis submitted by the senior in partial fulfillment of the requirements for the Ph.D. degree     

Callus cultures and bark from Norway spruce clones show similar cellular features and relative resistance to fungal pathogens

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.     

Role of origin and endophyte infection in browning of bud-derived tissue cultures of Scots pine (Pinus sylvestris L.)

Callus tissues originating from buds of mature Scots pine (Pinus sylvestris L.) trees exhibit the typical problem of browning, which leads to degeneration and death of the tissues. The effects of medium, origin (tree and location) and endophyte infection were studied on the browning and growth of bud-derived tissue cultures. The calli growing on medium with higher kinetin content and source of organic nitrogen, and originating from the southern location grew better and exhibited less browning. Endophytic microbial cells were detected in the brown callus tissues by transmission electron microscopy. The natural endophyte infection frequency of Scots pine buds was studied and found dependent on the tree, but not on the location. A well-growing, green callus line was artificially infected by an endophytic strain of Methylobacterium extorquens, and browning was not observed on solid media compared to the uninfected control clones of the same callus. However, suspension cultures started from the infected callus died faster than cultures started from the uninfected callus. The endophyte species composition and plant genotype together with tissue culture conditions are the key factors for gaining plant tissue cultures with high regeneration capacity.     

Production of pyrroloquinazoline alkaloid from leaf and petiole-derived callus cultures of <Emphasis Type="Italic">Adhatoda zeylanica</Emphasis>

Summary Callus cultures of Adhatoda zeylanica Medicus were established from leaf and petiole explants. Accumulation of a bioactive pyrroloquinazoline alkaloid, vasicine, in callus cultures was detected and confirmed by thin layer chromatography, electron-ionization mass spectra, ¹³C NMR and high-pressure liquid chromatography analysis. The mass of vasicine obtained from leaf-derived callus cultures was found as 188 and this is comparable to that of the authentic sample. The retention time for leaf-derived extract was 10.065 and for the petiole-derived extract was 9.78 (authentic sample had 9.6 retention time) on high-performance liquid chromatography. The mass and NMR spectra were compared with the spectra obtained from the authentic sample of vasicine. Different growth regulators greatly influenced the growth of callus cultures. The accumulation of vasicine was more in leaf-derived callus grown on Murashige and Skoog (MS) medium with 2.3 μM kinetin, and 4.5 μM 2,4-dichlorophenoxyacetic acid. This is the first report on in vitro production of a pharmacologically important compound vasicine and its characterization by mass spectrometry and ¹³C NMR studies from callus cultures of Adhatoda zeylanica.     

Influence of Mechanical Stress on Auxin-Stimulated Growth of Excised Pea Stem Sections

The auxin to cytokinin ratios are described for promoting growth in the in vitro cultures of soybean (Glycine max (L.) Merr. cv. Bragg) and perennial clover (Trifolium repens L. cv. Regal Ladinc). Callus growth was induced on somatic tissue with 50:1 auxin to cytokinin (w/w) ratio. A 5:1 ratio served for initiation of cell suspensions from callus and for subsequent growth of callus from cells in suspension. A 1:2 ratio served for regeneration of buds and plantlets from the callus grown from cells. Although (2,4-dichlorophenoxy) acetic acid was the auxin for suspension and regenerative cultures, (2,4,5-trichlorophenoxy)acetic acid was the more effective auxin for initiation of callus on somatic tissue. All cultures were grown with 6-furfurylaminopurine as the cytokinin. The phytohormones strongly influenced the rates of culture growth, but determination of culture type was augmented by dl-alpha tocopherol acetate and iron. Tocopherol and a relatively high complement of iron promoted growth of juvenile cultures, whereas low level of iron and absence of tocopherol favored growth to comparatively more differentiated cultures. Without tocopherol, no callus formed on somatic tissue during the allotted period of incubation. Tocopherol plus a complement of low iron enabled growth of callus on rapidly growing somatic tissue. A high level of iron enabled comparatively more callus growth but suppressed growth of somatic tissue. In suspension cultures tocopherol and a high iron level enhanced dispersion of cells. A low iron complement in the absence of tocopherol induced growth of callus from cells and subsequent regeneration of buds and plantlets from the callus.     

Programmed cell death in Datura innoxia Mill. callus cultures cultivated in light and in dark

It has been established that dimedone in solid culture medium influencedthe growth of Datura innoxia Mill. callus tissues and theapoptotic processes of cells. This formaldehyde (HCHO) capture reagent appearsto modify the metabolism of plant cells, resulting in quantitative changes inthe apoptotic index (A_i). Apoptotic cells were detected insix-week-old callus tissues by the TUNEL reaction. The amount of TUNEL positivenuclei showed a characteristic spatial distribution. Enhanced DNA fragmentationwas observed in the cell layers close to the surface of the cultures. ElevatedA_i was determined in cultures grown in the dark compared to thetissues grown in the light. High doses of dimedone considerably decreasedapoptosis in tissue cultures under both light and dark conditions.     

Development of callus and suspension cultures of potato resistant to NaCl and mannitol and their response to stress

Callus and suspension cultures adapted to various concentrations of NaCl or mannitol were developed from the cultivated potato Solanum tuberosum cv. Desire. Growth of the calli was less inhibited by mannitol than by iso-osmotic concentrations of NaCl. Reduction of growth by both NaCl and mannitol was considerably lower in osmotically adapted calli than in non-adapted ones. Salt-adapted suspension cultures that grew in the medium to which they had been originally adapted had a shorter lag in growth as well as a shorter time required to achieve the maximum growth, as compared with non-adapted cells. Suspension cultures adapted to NaCl concentrations higher than 150 mM were obtained only after preadaptation to osmotic stress. Adaptation of these cells was found to be stable. Accumulation of Na⁺ was lower and level of K⁺ was more stable in osmotically adapted than in non-adapted calli, when both were exposed to salt. Potassium level in NaCl-adapted calli exposed to saline medium was lower than that in non-adapted calli in standard medium. The maximum of Cl^– and Na⁺ accumulation was reached at higher external salt concentration in salt-adapted than in non-adapted suspension cultures. In both callus and suspension cultures, Cl^– accumulated more than Na⁺. Potassium level decreased more in non-adapted than in NaCl-adapted suspension cultures. The decrease of osmotic potential in osmotically adapted calli exposed to mannitol and in salt-adapted calli and suspension cultures exposed to salt was correlated to the increase of the external concentration. Such a correlation was not found in osmotically adapted calli exposed to salt. Non-electrolytes were found to be the main contributors to the decrease is osmotic potential in both callus and suspension cultures.     

Spectral composition of irradiation regulates the cell growth and flavonoids biosynthesis in callus cultures of Saussurea medusa Maxim

Cell growth, flavonoids biosynthesis and L-phenylalanine ammonia-lyase (PAL) activity were studied in callus cultures of Saussurea medusa Maxim. under different types of spectral radiance. After 21 days, red light significantly improved the callus growth, but inhibited the biosynthesis of flavonoids in callus cultures. However, blue light was found to enhance the biosynthesis of flavonoids, although callus growth under this spectrum was comparable with that under white and other coloured spectra, such as green and yellow. The accumulation of flavonoids in callus cultures was related to the PAL activity, which was found to be stimulated by the spectral composition of irradiation.     

Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast

The tissue culture of phycocolloid yielding seaweeds included preparation of axenic explants, callus induction, subculture of excised callus and regeneration of plantlets from pigmented callus in the laboratory. Treatment of algal material with 0.1–0.5% detergent for 10 min and 1–2% betadine for 1–5 min and 3–5% antibiotic treatment for 48–72 h successively enabled viable axenic explants to be obtained as high as 60% for Gracilaria corticata, Sargassum tenerrimum and Turbinaria conoides and 10% for Hypnea musciformis. Callus induction was more conspicuous in T. conoides than in the other three species investigated. Of the irradiances investigated, 30 μmol photons m⁻² s⁻¹ produced calluses in as many as 40% explants in G. corticata and T. conoides and 10% in H. musciformis and S. tenerrimum. The explants cultured at 5 and 70 μmol photons m⁻² s⁻¹ did not produce any callus in all the species studied except for H. musciformis in which 10% explants developed callus at 5 μmol photons m⁻² s⁻¹. Most of the species investigated showed uniseriate filamentous Type of growths and buds from cut ends and from all over the surface of explants. Nevertheless, T. conoides had three Types of callus developments, namely (1) uniseriate filamentous Type of outgrowths from the centre of the cut end of explant, (2) bubbly Type of callus and (3) club-shaped callus clumps. The subculture of T. conoides callus embedded in 0.4% agar produced two Types of filamentous growth, namely filiform (with elongated cells) and moniliform filaments (with round cells) in the 2 months period after inoculation. Further, friable callus with loose cells was also found associated with excised callus. The moniliform filaments showed prolific growth of micro-colonies resembling to somatic embryo-like growth which, in liquid cultures, differentiated and developed into propagules with deformed shoots and distinct rhizoids. The shoots of these propagules remained stunted with abnormal leaf stalks without forming triangular shaped leaves as the parental plant and rhizoids had prolific growth in the laboratory cultures. The excised callus of G. corticata continued to grow when transferred to liquid cultures and showed differentiation of new shoots within 10 days. The shoots grew to a maximum length of 5–6 cm in the 2 months period in aerated cultures in the laboratory. Dedicated to the memory of Late Dr. Rangarajan.     

<Emphasis Type="Italic">Cupressus</Emphasis> callus and cell suspension cultures: Effect of seiridins on their growth and sensitivity

Summary Cupressus macrocarpa and C. arizonica were examined for callus and cell culture production in vitro. Both species produced callus on agar-solidified MSCY medium supplemented with vitamins, antioxidants, 0.14 μM kinetin (KIN), and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures of both species were established in liquid MSCY medium. Seiridin (SE) and iso-seiridin (ISE), two phytotoxic butenolides produced by Seiridium cardinale, S. cupressi, and S. unicorne, the causal agents of many canker diseases of cypress, were tested on callus or cell suspension cultures. In the medium without other plant growth regulators (PGR), SE promoted cell proliferation of cypress better than ISE, for callus initiation, callus maintenance, and cell suspension cultures. The growth rates of cypress callus tissues and suspension cultures of both cypress species on media containing 50–150 μM SE or ISE were measured. At concentrations of 50 μM and higher, growth rates increased exponentially with the SE concentration. A comparison with KIN and 2,4-D indicated that 50 μM SE promoted growth of callus tissues and cell suspension cultures more than 100 μM ISE. SE can also interact with, or counteract, KIN and 2,4-D. It was demonstrated that SE could replace KIN in the medium for C. arizonica. SE could be involved in cell enlargement and proliferation processes. The less susceptible cypress species (C. arizonica) had a high content of terpenoids than that of the more susceptible species (C. macrocarpa). SE could be a useful tool as a phytohormonal-like regulator to manipulate physiological changes at the cellular level and as an elicitor of sensitivity or tolerance of cypress germplasm to the phytotoxin.     

Growth of callus and suspension culture cells from cotton varieties (Gossypium hirsutum L.) resistant and susceptible toxanthomonas malvacearum (E. F. Sm.) dows

Summary In vitro conditions are defined for starting and maintaining callus and suspension, cells from two cotton (Gossypium hirsitum L.) varieties, Im 216 and Acala 44, which are resistant and susceptible, respectively, to the bacterial pathogenXanthomonas malvacearum (E. F. Sm.) Dows. A light, friable callus was easily obtained and has been maintained for over 4 years. Whether stems or leaves, the explant source for callus initiation made no difference for growth of callus tissue. Acala 44 callus had a fresh-weight doubling time of 4 to 5 days, and Im 216 callus had a fresh-weight doubling time of 4 to 9 days; however, in suspension culture the fresh-weight doubling times for Im 216 and Acala 44 were 6 days. The pH of the suspension medium dropped to 4.7 during the exponential growth phase and rose to 5.4 at the stationary phase. Attempts to induce root and shoot initiation from these callus cells were unsuccessful; however, greening of the callus tissue did occur. Schenk and Hildebrandt medium was used for both callus and suspension cultures. Inoculation of Im 216 and Acala 44 callus tissues with two races ofX. malvacearum resulted in a resistant and susceptible response, respectively. This research was supported in part by C. S. R. S. Grant 315-16-96 and the Agricultural Experiment Station of Oklahoma State University.     

Effects of ionizing radiation (60Co gamma rays) on growth and morphogenesis ofHaworthia mirabilis,haw. Callus tissue

Summary The effects of γ-radiation on growth and morphogenesis ofHaworthia callus in vitro were determined. The doses ranged from 100 to 5000 rads. Survival, growth pattern, growth rate, and differentiation of vegetative buds and roots in both irradiated and nonirradiated callus were compared. Growth data up to 24 weeks for irradiated and control cultures were analyzed. The dose range between 800 to 2500 rads produced compact callus as compared to the controls which were friable. After 12 weeks all control cultures differentiated vegetative buds with roots, whereas callus exposed to 800 to 2500 rads continued to grow with little or no organogenesis. However, it was observed that the wet and dry weights of callus receiving 1000 to 1500 rads ultimately exceeded those of nonirradiated controls.     

Plant regeneration from somatic embryos in black pepper

Embryogenic callus derived from zygotic embryos of black pepper (Piper nigrum Linn.) were induced to form somatic embryos on solid and liquid Schenk and Hildebrandt basal medium. Callus proliferation, somatic embryo-genesis and germination of embryos were achieved in about 8 months in static cultures while it took only 8 weeks in liquid suspension cultures. The highest number of embryos and plantlets was produced from cells grown as suspension cultures raised in half-strength medium without growth regulators and sucrose level reduced from 3% to 1.5%. Regenerated plants were established in soil.     

In vitro propagation of two species of commelinaceae from bud cultures and repeated subcultures on a growth regulator-free medium

Summary In our wide-ranging research on in vitro propagation of some monocotyledonous plants, two Commelinaceae species were studied: Tradescantia fluminensis var. foliis variegatis and Tradescantia pallida. Initial cultures were established successfully using nodal and apical meristems that produced single shoots, many roots, and no callus, by utilizing growth regulator-free MS medium. Addition of growth regulators did not increase the activity of explants that produced single or multiple shoots, atypical roots, and no callus. Consecutive cultures were possible using the apical and nodal meristems of the previous generation. The behavior of the different generations in culture did not change and was similar to the initial cultures. Their growth capacity was maintained over many months, also on a growth regulator-free medium. In both species, the chromosome number in the root tips of the mother plant and all morphologically stable in vitro plantlets confirmed a constant ploidy level, in T. fluminensis 2n=72, and in T. pallida 2n=24.