Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Summary Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.     

Response to NaCl of alfalfa plants regenerated from non-saline callus cultures

Salinity restricts crop productivity in many arid environments. Inadvertent selection for tolerance to osmotic stress may occur under cell or tissue culture conditions and could affect the performance of regenerated plants. The effect of NaCl on forage produced by alfalfa (Medicago sativa L.) plants regenerated from non-saline callus cultures was examined in this study. Plants of Regen-S, which was selected for improved callus growth and regeneration in non-saline cultures, had higher forage weight when grown on SHII medium at NaCl levels up to 100 mM compared to its parental cultivars, Saranac and DuPuits. Five additional original-regenerant plant pairs, each derived from non-saline callus cultures of different alfalfa plants, were evaluated in a solid (soil-like) substrate under saline and non-saline conditions. Weight of forage produced by rooted stem cuttings of regenerated plants was 33% higher at 50 mM NaCl compared to cuttings of explant donor plants. Self progenies from four of five regenerants had higher relative forage weight at 100 mM NaCl (percent of 0 NaCl treatment) than the original plants indicating increased NaCl tolerance.     

Cytogenetic analysis of plants regenerated from colchicine-treated callus cultures of an interspecificHordeum hybrid

Summary Hybrids of Hordeum vulgare (HV) x H. jubatum (HJ) were synthesized for purposes of introgressive breeding, but were sterile and the recovery of pure diploid tillers by colchicine applications in vivo was difficult. Plant regeneration from colchicine-treated callus cultures of the hybrid (HV x HJ) was investigated as a means to produce high numbers of pure diploid, fertile intermediates. 10 of 50 plants regenerated in this manner exhibited variable chromosome numbers with means of approximately 37 (expected diploid number = 42). Cytological examinations of microsporogenesis in all such plants revealed a high incidence of bivalent formation at metaphase I (as compared to nearly complete asynapsis in the F₁), but spindle and chromosome abnormalities in later meiotic stages led to complete sterility. Approximately 40% of HJ plants regenerated from colchicine-treated calli appeared to be pure tetraploids of high fertility. These techniques are hence useful for high frequency production of diploid or polyploid plants.     

Mitochondrial DMA variation in plants regenerated from embryogenic callus cultures of CMS triticale

The mitochondrial DNA (mtDNA) organization of primary hexaploid cytoplasmic male-sterile (CMS) triticale regenerants containing Triticum timopheevi cytoplasm was analysed by hybridization experiments and compared with the mitochondrial genome organization of the corresponding regenerants with maintainer cytoplasm. Callus cultures had been derived from immature embryos, and 623 triticale plants were regenerated via somatic embryogenesis after three to four subcultures. The chondriome of 159 regenerants was investigated with regard to somaclonal variation. Six different mitochondrial gene probes and four different restriction enzymes were used for Southern blot analyses by the non-radioactive digoxigenin labeling technique. Alloplasmic regenerants showed a gain or loss of hybridization signals up to a high percentage, while euplasmic ones revealed only minor variability with respect to band stoichiometries. In 24 cases rearrangements in the mtDNA were proved. We suppose that recombination processes and selective amplification events are responsible for these findings.     

Genetic and chromosomal variation inPetunia hybrida plants regenerated from protoplast and callus cultures

Plants regenerated from callus cultures derived from leaf discs and mesophyll protoplasts ofPetunia hybrida cv. Rose of Heaven exhibit a high frequency of genetic and chromosomal variation. Of twelve leaf disc-derived plants examined, only three had the normal diploid chromosome number (2n=14) while seven were tetraploid and two were aneuploid (16 and 27 chromosomes). Of seventeen plants derived from two protoplasts, none had the diploid chromosome number. Most had 28 chromosomes, one 29, two 27, one 26 and one had variable numbers (14–28) in different root tip cells. In all cases aneuploidy was associated with developmental abnormality. In addition, heritable differences in growth, morphology and flower pigmentation were observed in callus-derived tetraploids and diploids, including one diploid which differed from parent plants in at least four characters. These results are discussed in terms of the importance ofPetunia in genetics research and for studies of somaclonal variation.     

Monoterpene synthesis in shoots regenerated from callus cultures

-Shoots regenerated from two-year old callus cultures of Lavandula angustifolia and Rosmarinus officinalis accumulated monoterpenes characteristic of the parent tissue. No such compounds could be detected in undifferentiated callus maintained under a variety of conditions.     

Correlation between the nicotine content of tobacco plants and callus cultures

Callus cultures of two low-alkaloid lines of Nicotiana tabacum L. had considerably lower nicotine contents than cultures from the respective highalkaloid cultivars which were isogenic except for the two loci for alkaloid accumulation. Thus, there was a strong correlation between the nicotine content of callus cultures and the plants from which they were derived.     

Isozyme patterns in callus cultures and in plants regenerated from calli ofCereus peruvianus (Cactaceae)

Electrophoretic patterns for isocitrate dehydrogenase (IDH; EC 1.1.1.42), acid phosphatase (ACP; EC 3.1.3.2), peroxidase (PER; EC 1.11.1.7), and esterase (EST; EC 3.1.1.1) isozymes were determined inCereus peruvianus tissues and used as markers of genetic uniformity of calli and of the plants regenerated from callus cultures. One IDH, six ACP, six PER, and six EST isozymes were induced in cultured callus tissues in medium containing three 2,4-dichlorophenoxyacetic acid and kinetin combinations. Four ACP, two PER, and three EST isozymes were still present in all regenerated plantsin vitro and therefore can be used as markers of theC. peruvianus plants regenerated from callus tissues. The differential patterns of ACP and IDH isozymes and the similar zymograms for PER and EST isozymes presented by callus tissues were used in a comparison of callus tissues cultured for 2 years. The comparative analysis of zymograms within each enzyme system indicated a mean heterogeneity coefficient of 0.33 forC. peruvianus calli cultured for 2 years. Because of the isozyme variations, which developed in culture medium and were transferred to the regenerated plants, the IDH, ACP, PER, and EST enzyme systems can be considered to be good markers for investigating possible genetic variations in plant populations ofC. peruvianus obtainedin vitro from callus culture.This research was supported by the CNPq     

Methylation of ribosomal RNA genes inPetunia hybrida plants,callus cultures and regenerated shoots

The extent of methylation of the 18S–25S ribosomal RNA genes ofPetunia hybrida has been investigated using methylation-sensitive restriction endonucleases and Southern hybridisation. The genes in leaves, ovaries, petals, stigmas, anthers, and seed are methylated to a similar extent, but those in roots are hypomethylated. Genes in adventitious roots formed from stem cuttings are also hypomethylated. Genes in callus cultures initiated from leaf discs vary widely in the extent of methylation, but the pattern obtained is stable during culture. Callus cells containing hypomethylated genes can give rise to shoots in which the extent of methylation has reverted to that of the original leaf explant. These results demonstrate the striking plasticity of ribosomal RNA gene methylation inPetunia.     

The effect of barban on shoot formation in tobacco callus cultures

Shoot formation in tobacco callus was completely inhibited bythe presence of barban in the media during the first 2 daysof culture. Callus transferred to media containing barban from4th to the 12th day showed progressively less inhibition. Similarresults were obtained with GA₃. ¹Present address: Biology Department, Chung Chi College, TheChinese Univ. of Hong Kong, Shatin, N.T., Hong Kong ²Present address: Plant Hormone & Regulator Pioneering ResearchLab., U.S. Dept. Agric, Crops Res. Div., Beltsville, Md., U.S.A. (Received April 21, 1970; )     

The production of strawberry plants from callus cultures

Shoots were regenerated from callus of the commercially important strawberry varieties Bogota, Brighton, Cambridge Favourite, Hapil, Ostara, Rapella, Red Gauntlet and JILA33 which is a promising selection from a current breeding programme.The callus was initiated from explants of petiole or lamina of leaves of micropropagated shoots in vitro or of lamina or peduncle from greenhouse plants. There was more shoot regeneration with callus from lamina than from petiole although with the variety Hapil, regeneration occurred only with callus from peduncle.With seven of the varieties, shoot regeneration occurred on culture media with BAP and 2,4-D whilst with the remaining variety, Cambridge Favourite, it occurred only with medium which contained 1AA- alanine conjugate in place of 2,4-D.Regenerated shoots rooted readily and the plants produced are being studied for somaclonal variation.     

Morphactin-kinetin interaction on growth and shoot formation in tobacco callus cultures

Pith-derived calluses of Nicotiana tabacum L. cv. Wisconsinno. 38 were inoculated on an RM-1964 medium containing variousconcentrations of a morphactin, chlorflurenol (CFl) and kinetin(KIN). An addition of KIN (0.1–2 mg/liter) alone was effectivefor shoot formation from the calluses, but a high dose (10 mg/liter)resulted in the inhibition of growth and in no differentiation.The inhibitory effect of a high dose of KIN was counteractedwith CFl. Three combinations of KIN and CFl; CFl (0.1 mg/liter)$KIN(2.0 mg/liter); CFl (0.1 mg/liter)$KIN (10.0 mg/liter) and CFl(1.0 mg/liter) $KIN (2.0 mg/liter), were successful for 100%shoot redifferentiation in inoculated calluses. An appropriatebalance between CFl and KIN seems to be involved in shoot formation.The present results can best be interpreted by assuming thatCFl acts as an auxin in cultured tissues. (Received January 16, 1975; )     

Somaclonal variation in plants regenerated from cultures of soybean

Plants were regenerated from embryogenic and organogenic cultures derived from immature embryos of nine soybean (Glycine max L. Merr.) genotypes and extensive qualitative variation was noted in different regenerated families. Three lethal sectoral albinos were seen in the regenerated plants (R₀). Variants observed in later selfed generations included twin seeds, multiple shoots, dwarfs, abnormal leaf morphology, abnormal leaflet number, wrinkled leaves, chlorophyll deficiency, partial sterility and complete sterility. The frequency of possible mutations ranged from 0 to 4% in R plants as determined by studies of corresponding R₁, R₂, R₃ and R₄ families. No significant differences were seen in the frequencies of possible mutations for embryogenic as compared to organogenic culture derived plants. Chlorophyll deficiency, sterility and wrinkled leaf traits were followed in two or more generations and showed that these traits were inherited stably. The known traits of this nature are controlled by single recessive nuclear genes. Other traits occurred more randomly and not in all generations. The genetic basis of the random variation is not known at the present time. This study indicates that heritable somaclonal variation does occur in tissue culture derived plants of soybean.Abbreviations R₀ Original regenerated plant - R₁ Selfed seeds of R₀ plants - R₂ Selfed seeds of R₁ plants - R₃ Selfed seeds of R₂ plants This research was supported by funds from the Illinois Agricultural Experiment Station and Agrigenetics Inc.     

Photoperiodically independent flowering of Pharbitis nil plants regenerated from flower buds

Summary Flower buds and anthers of the short-day plant Pharbitis nil were treated either with thermic shock (7 or 35°C) or osmotic/trophic shock (12% sucrose) for 24 h. Explants were transferred either to Murashige and Skoog medium (MS) with addition of 6-benzylaminopurine (BA; 4.4μM) and 6% sucrose or to the same growth medium containing 22 μM BA and 3% sucrose. Both media were supplemented with α-naphthaleneacetic acid (NAA; 0.55 μM). Osmotic/trophic shock stimulated the occurrence of shoots on flower buds grown on medium containing 22 μM BA. Thermic shock (7 and 35°C) inhibited this process on both types of explants. Regenerated plantlets were transferred to MS medium supplemented with 6% sucrose, gibberellic acid (GA₃; 1.44μM), NAA (0.55 μM) and Ca²⁺ (0.66 mgl⁻¹). After 3–4 wk they were able to produce flowers without photoperiodic induction.     

Karyotype analysis of regenerated plants from callus cultures of interspecific hybrids of cultivated barley (Hordeum vulgare L.)

Summary The karyotype of 82 regenerated plants from callus cultures of interspecific hybrids between cultivated barley (Hordeum vulgare L.) and seven polyploid wild barley species was examined by C-banding or Feulgen staining. The karyotypic changes observed in 46 plants included aneuploidy, double haploidy, amphidiploidy, deletions, inversions, extra C-bands, and extra euchromatic segments. Apparently, chromosome 5, 6, and 7 of H. vulgare were more frequently exposed to elimination or structural change than the other chromosomes of this species. Irradiation of calli seemed to enhance the occurrence of karyotypic variants.     

Shoot histogenesis in tobacco callus cultures

Summary The earliest histological event observed in light-grown shoot-forming tobacco (Nicotiana tabacum L. cv. Wisconsin 38) callus was the deposition of safranin-stainable substances (probably suberin) on cut, exposed cell surfaces. This was followed by the initiation of cell files and the appearance of starch granules. Nodules with lignified tracheary elements also were observed in the upper part of the callus. Pronounced starch accumulation occurred in the lower part of the callus in which protrusions of tissue into the medium occurred. Meristemoids were found in these protrusions as well as elsewhere. In between meristemoids, parenchyma cells with starch granules of varying sizes were observed. Cell strands that connected with the meristemoids also were observed. These strands often terminated at the surface of the protrusion at which point shoot apices originated. The earliest shoots were formed in these protrusions. With time, additiional shoots were formed in other parts of the bottom of the callus and finally in the top part of the callus on prolonged culture. The determination of the loci at which shoot primordia were formed sequentially, was interpreted in relation to the physiological gradient concept. This work was carried out while E. M. was a Visiting Scientist at the University of Calgary under the Scientific Exchange Program between the National Research Council of Canada and the Japan Society for the Promotion of Science. Support for this study was provided by N.R.C. (Canada) Grant A-6467 to T. A. T.     

The frequency of marker changes in sugarcane plants regenerated from callus culture

This study investigates the frequency of apparent and permanent expression of marker change following two types of tissue culture, conventional callus and direct regeneration cultures, and for two markers it relates this frequency to that following breeding. Each clone was used for only one marker. After conventional callus culture, plants of the sugarcane clone Arundoid B, a clone having a growth habit with shortened internodes and leaves, were freed of this marker at a rate of 1 in 172 plants. Marker remission in a second clone with a leaf blotch was enhanced in the presence of a mutagen. Callus culture alone gave a remission rate of 1/280 plants, while treatment of callus with ethyl methanesulfonate gave a remission rate of 1/42 plants. Of two markers subjected to vegetative and sexual transmission, the first, a leaf marker, was stable in callus culture with no remissions; crossing with non-marker parents produced progeny with 54% lacking the marker. The second, a stalk marker (multibud), showed epigenetic effects during two generations of vegetative propagation; plants lacking the multibud marker produced vegetative progeny in which the marker reappeared. Nine crosses to nonmarker parents produced progeny of which an average of 29% had the marker. The use of stalk chimeras as markers demonstrated that passage through conventional callus or direct regeneration culture resulted in the loss of the donor phenotype in all plants regenerated. Phenotypic variation in plants derived from callus culture appears to arise from several sources; chimeral segregants, epigenetic transients, and mutational variants.