Callus Initiation Medium Research Articles

Analysis of potential sources of variation in tissue culture derived celery plants

SummarySomaclonal variation derived from tissue culture is a potential source of variation that can be used in crop improvement programmes. The characteristics of this variation are first shown in the regenerant generation and their heritability is then confirmed by examination of the progeny. There would be savings of time, space and labour if this variation could be detected in vitro using easily assessed visual cues. The aim of this study was to relate variation in the source of explant and the morphology of the newly initiated callus to the characteristics of the regenerant plant, of which the most important was resistance to leaf spot disease caused by Septoria apiicola. Associations were investigated by isolating four stem explants from each of 564 surface sterile seedlings, var. Celebrity, on a callus initiation medium (MS medium, 30 g litre‘1sucrose, 0.5 mg litre’12,4‐D, 0.6 mg litre‘1kinetin) and assessing the morphology and colour of the callus. After this initial culture (8 wk), each callus was transferred to a regeneration medium (MS medium, 30 g litre“1sucrose). Plantlets were regenerated from many of the callus cultures and these were transferred to the glasshouse. When all of the surviving regenerant plants (276) were mature, leaf shape, amount and composition of the essential oils and resistance to late blight were assessed. Statistical analysis revealed that the character of the newly initiated callus (width, height, colour, organogenesis) showed poor correlation with all aspects of the regenerated plant measured. However, it was shown that increased variation resulted from different seedlings more than from plants derived from within seedlings or within callus.

Regeneration of plants from cultured guard cell protoplasts of Nicotiana glauca (Graham)

Experiments were performed to optimize conditions for culturing guard cell protoplasts of Nicotiana glauca and to determine whether cultured guard cell protoplasts were totipotent. Guard cell protoplasts were isolated from adaxial epidermal tissue of leaves of Nicotiana glauca grown under fluorescent light (800–900 μmol m −2 s −1 of photons of photosynthetically active radiation). To increase the probability that guard cells were of uniform osmotic potential at the time leaves were harvested, leaves were collected in darkness, just before the beginning of each light period. Protoplasts were cultured in liquid media similar to those used for culturing mesophyll cell protoplasts of Nicotiana tabacum but of modified pH and KCl, CaCl 2, sucrose and glycine concentrations. Concentrations of growth regulators were 0.3 mg 1 −1 α-napthaleneacetic acid (NAA) and 0.075 mg 1 −1 of 6-benzylaminopurine (BAP). Protoplasts were incubated in darkness at 25°C in 8-well microchamber slides at a density of 1.25 × 10 5 cells ml −1. Cell divisions began within 72–96 h of initiation of cultures. After 96 h, cell survival averaged 57% of the initial number of cells ( n, 20; range, 36–84%). After 8–10 weeks of culture, cell colonies were transferred to a callus initiation medium containing agar and incubated under continuous white fluorescent light (25 μmol m −2 s −1 of photons of photosynthetically active radiation) for another 8–10 weeks. Green callus tissue was then transferred to a commercial callus growth medium and incubated under similar conditions. After 8–10 weeks of further growth, callus was transferred to a commercial shoot differentiation medium and incubated similarly. Multiple shoots appeared within 2 weeks. Shoots were transferred to a root differentiation medium and cultured under the conditions described above. When roots were sufficiently developed (6–8 weeks), plants were transplanted to soil and grown in a growth chamber. We conclude that guard cell protoplasts can be made to survive in culture at high percentages and that cultured guard cell protoplasts of Nicotiana glauca are totipotent.

Long-term culture of embryogenic sugarcane callus

Embryogenic calluses of sugarcane capable of regenerating green plants after long-term culture were sought. The largest quantities of embryogenic calluses were produced on Murashige & Skoog medium, but cultures maintained on Chu N6 medium remained embryogenic and totipotent longer. Both media contained 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-d). The effect of supplements on somatic embryogenesis was examined. Kinetin (0.5 μM) and 10% (v/v) coconut water in callus initiation medium were inhibitory to subsequent embryogenesis. Embryogenic calluses on N6 medium increased in fresh weight with proline concentration up to 90 mM. Maximum fresh weight was achieved with 5% sucrose. Although genotypic differences were observed, embryogenesis occurred in all 17 sugarcane clones tested. Embryogenic calluses of one cultivar regenerated green plants after 16 months, but suspensions were totipotent for only 8 months. Total number of regenerated plants decreased with time in culture, while the number of pale green plants increased starting after 5 months in culture.

Plant regeneration and chromosome instability in tissue culture of Elymus canadensis × E. trachycaulus F1 hybrid

Callus was induced from pieces of immature inflorescence of a sterile F1 hybrid (2n = 4x = 28) between Elymus canadensis and E. trachycaulus on MS medium supplemented with 2 mg/L of 2,4-D. Calli were of two types: compact or soft. Regeneration occurred predominantly from the compact callus. Although some plantlets were obtained on the callus initiation medium, the frequency of regeneration increased considerably when the callus was transferred to an auxin-free MS medium. Deliberate aging of callus induced cytological instability and variation in chromosome number of the regenerants. Five of the 43 regenerated plants had deviant chromosome numbers, including an octoploid (2n = 8x = 56). Chromosome pairing in the octoploid plant suggests that it originated through chromosome doubling.Key words: callus culture, variation, chromosome number.

The effects of kinetin on callus characters in alfalfa (Medicago sativa L.)

Three callus initiation media, B2-k, B2, and 7951, were used to study the effects of kinetin on callus initiation, morphology, histology, and regenerability in alfalfa (Medicago sativa L.). The presence of kinetin in callus initiation media retarded callus initiation, but enhanced division and differentiation of callus cells. Calluses induced on kinetin-containing media (B2 and 7951) had many compact cell aggregations, which were considered meristematic regions that might differentiate to plantlets on a regeneration medium. Visually, these calluses were compact and had many nodular structures. In contrast, most calluses induced on a kinetin-free medium were composed of large, individual cells and had friable structures without nodules. After transfer to a hormone-free medium, calluses induced on kinetin-containing media regenerated more frequently than those induced on a kinetin-free medium, but cytokinin (kinetin) autotrophism also occurred. Autotrophism was sexually transmissable and especially affected by the female parent.

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.

GROWTH REGULATOR EFFECTS ON BUD INITIATION IN CALLUS CULTURES OF MEDICAGO SATIVA

Influence of growth regulators on bud initiation in callus of alfalfa (Medicago sativa L.) was studied by varying levels and combinations in the first medium of a two‐medium sequence used to obtain whole plants. Callus of tetraploid clone S‐4 (cv. ‘Saranac‘) was initiated from immature ovaries on a modified Blaydes' basal medium containing all combinations of six concentrations (0–36 μM) of kinetin (K), six concentrations (0–44 μM) of naphthaleneacetic acid (NAA), and seven concentrations (0–36 μM) of 2,4‐dichlorophenoxyacetic acid (2,4‐D). After 28 days the callus was challenged to form buds by transfer to the modified Blaydes' medium containing 2.0 g/liter yeast extract and 0.57 mM inositol. No buds were produced in the absence of 2,4‐D in the first medium, and the frequency of bud formation on the second medium was directly proportional to the 2,4‐D concentration in the range 2.3–54 μM in the preceding medium. Buds were produced in the absence of kinetin in the first medium, but its presence in the range 2.3–36 μM markedly increased bud formation. NAA was not required for bud formation, and the budding frequency increased only slightly with increasing NAA concentration in the first medium. Budding of callus of two other alfalfa clones was also influenced by the 2,4‐D concentration in the initial medium. There were several indications that many of the buds were initiated on the first medium and completed development on the second medium. These included the differential effect on budding of combinations of 2,4‐D, NAA and kinetin in the callus initiation medium, the specific media sequence required, and the presence of embryoids on the callus which after transfer to the yeast extract‐inositol medium produced buds.