Genetic and physiological variability among cultured cells and regenerated plants of Nicotiana tabacum

Summary Tobacco cell lines selected for resistance to picloram (4-amino-3,5,6-trichloropicolinic acid) and plants regenerated from these cell lines manifest several traits not shown by the parental strains. Genetics analyses of the regenerated plants have permitted the sources of this variability to be identified.Tricotyledenous seedlings appeared at a much higher frequency among the progeny of a heterozygous mutant plant (PmR1/+) regenerated from culture than they did among progeny of normal regenerated plants. In crosses with the regenerated heterozygous mutant plant and with homozygous progeny of this plant (PmR1/PmR1) the frequency of tricotyly was influenced more by the generation than by the genotype of the parent plant. Therefore, it is concluded that tricotyly is a physiological response to passage through cell culture.More than half of the picloram-resistant cell lines isolated were also resistant to hydroxyurea. Segregation of these two resistances was analyzed in progeny of crosses with regenerated plants. In all cases hydroxyurea-resistance was genetically stable and inherited as a single dominant nuclear mutation (designated HuR). In crosses with plants PmR1/+ and PmR7/+ the HuR and PmR mutations assorted independently. In contrast, the HuR mutation recovered from plant PmR6/+ was linked to the PmR6 mutation.     

Chromosomal variability in tissue cultures and regenerated plants of Hordeum

Summary Spontaneous polyploidy, aneuploidy, and chromosomal rearrangements were observed in callus and suspension cultures of Hordeum vulgare, H. jubatum, and their interspecific hybrid. The extent to which each class of chromosomal variability was present in a culture depended upon differentiated state, age, and history. Cytological and isozymic analysis of subdivided callus cultures revealed spatial segregation of chromosomal variability. Cytogenetic analyses were performed to determine the expression of this in vitro chromosomal variability in corresponding regenerated plant tissues. A complete loss of polyploidy and a decrease in aneuploidy and chromosomal rearrangements were observed. Analyses of specific isozyme activities in regenerates suggested that a quantitative segregation of H. vulgare and H. jubatum genomes had occurred in tissue cultures of their interspecific hybrid. Possible uses of in vitro chromosomal variability for plant breeding and genetical studies are discussed.Work supported by a grant to Project 3195 from the Michigan Agricultural Experiment Station and by Grant E4-76-S-02-7528. A004 to P.S. Carlson from the US Department of Energy     

Cytogenetic variation of Ungernia victoris cell lines during cultivation on culture media of different composition

Chromosome numbers of U. victoris cell lines obtained from the same bulb and cultured for a long time on different agar-solidified and liquid nutrient media differed significantly. The components of the nutrient media including phytohormones did not influence the ratio of cells with different ploidy levels in various lines while transfer of the calluses to the liquid media resulted in the increase of diploid metaphase frequencies.     

The influence of the extract of Ungernia victoris cultured cells and of some metal cations on Escherichia coli transformation with plasmid DNA

In the system of cation-induced E. coli transformation by the plasmid pBR322 the effects of the extract derived from the biomass of cultured cells of U. victoris on the correlation between yield of transformants, viability of CaCl2 - treated cells and plasmid DNA conformation alterations has been investigated. The data obtained have been compared with effects of some salts of one- and divalent metals on the same parameters. The presence of different mechanisms of the variations of cell population viability and yield of transformants depending on utilization of salts or plant extracts has been shown.     

Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation

Shoot-tips from in vitro cultured Hypericum perforatum L. genotypes were subjected to assessments of developmental competence, genetic stability, and biosynthetic ability to identify critical points during cryopreservation. Survival rate, chromosome number stability, alteration in VNTR sequences and hypericin content were evaluated, in plants after pre-culture, and two subsequent cryogenic steps (cryoprotection and cooling) and those recovered from cryopreserved meristems. Pre-culture and cryoprotection treatments, did not reveal any significant differences, in these studied characteristics. Genetic stability was assessed by chromosome counts and analysis of variability in the VNTR sequences. No changes in chromosome number were detected in comparison with the untreated control but minor alterations were revealed in non-coding sequences. The content of hypericin after the recovery of cryopreserved meristems remained comparable with the unfrozen control. The controlled rate freezing technique used for cryopreservation was relevant for restoration of genetic and biochemical stability in Hypericum perforatum L. shoot-tips.     

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.     

Karyotypic variability in plants of Solanum melongena regenerated from callus grown in presence of culture filtrate of Verticillium dahliae

Summary Plantlets were regenerated from calli derived from leaf expiants of three genotypes of Solanum melongena (two parental genotypes and their hybrid). The cytological analysis showed that a) plants regenerated were all mixoploid, b) toxic medium (basal medium added with filtrate culture of Verticillium dahliae) was able to evidence karyotypic differences between genotypes not displayed by plants regenerated from callus grown on control medium, c) chromosomal mosaicism persists up to plant maturity and also in the selfed progeny. The results are discussed in terms of a selective process involving genes controlling chromosome number and/or a direct effect of toxic medium on the activity of the same genes.This research is supported by a grant from ERSO (Ente per la Ricerca e Sperimentazione in Ortoflorifrutticoltura e Sementi) — Regione Emilia Romagna     

Putative homozygous mutations in regenerated plants of rice

Summary Both normal and putative homozygous mutant (dwarf mutant) rice plants were regenerated from diploid seed callus, cultured in the presence of 1% NaCl. This trait was transmitted at least through the eighth genration (D₈) of regenerated plants (D₁) by self-pollination, as a homozygous mutation. However, the trait disappeared in the F₁, F₂, F₃ and F₄ obtained by reciprocal crosses of mutant plants with either control plants or with progeny of normal regenerated plants. Chimeric reversion of the homozygous mutant trait was observed and the revertant phenotype was transmitted stably to at least three successive generations. Similar dwarf types of homzygous mutation were observed independently in the two varieties, Norin 8 and Nipponbare, in an experimental series of ca. 3000 D₁ plants. The frequency of mutations among regenerated plants was calculated to be 1.8×10^-2. The mechanism responsible for these phenomena may be heritable gene inactivation induced by in vitro culture.     

Karyotypic changes in potato plants regenerated from protoplasts

Over two hundred plants were regenerated from shoot-culture derived proto-plasts of potato (Solanum tuberosum L. cv. Majestic). Some had grossly aberrant phenotypes but the majority were similar to, or indistinguishable from normal control Majestic. Cytological examination showed that on average, 57% of the regenerants had the normal chromosome number (2n=4x=48). The remainder were aneuploids and fell into two classes in approximately equal numbers. The first class was limited at about the euploid level (ie, 2n=44–49). The second class contained plants with higher chromosome numbers ranging from 2n=73 to the octaploid level (2n=8x=96). The overall results represent an improvement over our earlier studies on chromosome variation in protoplast-derived potato plants. In addition, three cases of structural chromosome variation were observed.     

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.     

Diploid somatic-hybrid plants regenerated from rice cultivars

Summary Somatic hybrid plants were obtained between rice cultivars Yamahoushi and Murasakidaikoku. Since Murasakidaikoku is a double mutant having both dominant (purple coloration) and recessive (dwarf) markers, the somatic hybrids can be easily distinguished from their parents. Protoplasts were isolated from anther-derived calli, and electrofused protoplasts were cultured without selection of hybrid cells. Out of 27 regenerated plants, 9 proved to be hybrids based on their purple coloration and normal plant type, traits which were identical to those of the sexual F₁ hybrid between the same parental cultivars. The somatic hybrids included three diploid and six triploid plants. Segregation of parental markers was observed in the selfed progenies. These results demonstrated that diploid hybrids of rice could be obtained through somatic hybridization between haploid anther-derived cells instead of by sexual hybridization.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

Somaclonal variation in soybean plants regenerated from tissue culture

Callus cultures of soybean (Glycine max (L.) Merr.) genotypes PI 88788, PI 438489B, and cultivar Bedford were initiated in vitro from seedling explants consisting of the cotyledonary node plus epicotyl from germinated mature seed. Plants were regenerated from these callus cultures and subsequently evaluated for qualitative variation in three to four subsequent generations. Variant phenotypes observed that have not been previously reported from tissue culture include lanceolate leaves, leaf variegation (chimeral variegated plants), pod variegation on otherwise normal plants, and change in growth habit from indeterminate to determinate. The lanceolate leaf, chimeral variegated plant, and change from indeterminate to determinate growth habit characters were inherited through at least three generations (R₀-R₂), and segregation occurred in each generation. Pod variegation was inherited through the two generations tested thus far and segregation occurred in each generation. No variation was observed in control plants derived from normal seed. Variants appeared more frequently in regenerants from PI 88788 and PI 438489B than from Bedford. These results confirm and extend the finding that certain tissue culture techniques may be used to induce novel plant formation from somatic tissue of soybean.Missouri Agricultural Experiment Station, University of Missouri, Columbia, Missouri, USAMention of tradenames does not constitute a guarantee or warranty of the product by University of Missouri or USDA-ARS and does not imply their approval to the exclusion of other products.     

Micropropagation ofPenstemon serrulatus and iridoid formation in regenerated plants

A method for the micropropagation ofPenstemon serrulatus Menz. from shoot tips or nodal segments was developed. Multiple microshoot cultures (up to 20 shoots from a single explant) were obtained by maintenance of shoot tip explants on Schenk & Hildebrandt medium (SH) supplemented with 4.4 µM benzyladenine (BA) or 8.9 µM BA and 0.57 µM indole-3-acetic acid (IAA). Microshoots developed into numerous, normal shoots when explants were transferred to SH medium containing 2.9 µM IAA or 2.5 µM indole-3-butyric acid (IBA). Shoot cultures were also established from nodal segments (max. 6.8 shoots per segment) when they were placed on SH medium with 0.49 µM IBA and 2.2 µM BA. Rooting of shoots was better on SH medium containing auxin (IBA, NAA or IAA) than on SH medium without growth regulators. The plantlets were then transferred to pots and grown in the greenhouse. Four-month-old regenerated plants demonstrated similar iridoid content (leaves contained 3.83% dry wt. penstemide and 1.8% dry wt. serrulatoloside) as the original plants.     

Genetic and epigenetic integrity assessment of acclimatised papaya plants regenerated directly from shoot-tips following short- and long-term cryopreservation

A vitrification-based cryopreservation protocol was applied to in vitro sourced shoot-tips of four genotypes of Carica papaya; two female (70 and Z6) and two male (B2 and B4). Regeneration of ~58?% (70) and ~59?% (Z6) was recorded for the female genotypes confirming previously published results. Regeneration was at ~77 and ~53?% for the two male genotypes B2 and B4 respectively. Cryo-tube storage and regeneration was tested after 2?C18?months storage in one male (B2) and one female (70) genotype. Regeneration post cryo-storage was similar to 1?h exposure to liquid nitrogen. Individual shoot-tips from the two female and two male genotypes were grown into complete in vitro plants, potted and acclimatised without micropropagation to provide material for randomly amplified DNA fingerprinting (RAF) and amplified DNA methylation polymorphism (AMP) analysis of multiple individuals from in vitro control, plant vitrification solution 2 (PVS2) cryoprotectant control and short (1?h) and long-term cryopreservation treatment plants. No variations were detected for genotype Z6 control and treatment individuals and no RAF variations were detected in any individuals of genotype B2. Small numbers of RAF and AMP variations were detected in some individuals from genotypes B2 (AMP variation only), B4 and 70, but these were also found in controls. Genotype 70 showed the greatest level of variation; genomic DNA variation (RAF) was detected in control and cryopreservation treatment individuals, and the PVS2 control group was the only treatment group without variations for the respective AMP analysis. The variations observed could not be correlated with any phenotypic characteristics 2?months after acclimatisation.     

Occurrence of 5-methoxypodophyllotoxin in plants,cell cultures and regenerated plants of Linum flavum

The 5-methoxypodophyllotoxin contents of plants, cell cultures and regenerated plants of Linum flavum are compared. It is demonstrated that cell cultures are able to produce amounts of 5-methoxypodophyllotoxin that are comparable to the concentration in fully differentiated plants. The production of 5-methoxy-podophyllotoxin depends on the hormonal balance of the growth medium. The use of 2,4-dichlorophenoxyacetic acid as the growth regulator is favourable for 5-methoxypodophyllotoxin production when compared to naphthylacetic acid. The 5-methoxypodophyllotoxin accumulation appears to be positively related to the internal cell volume.     

Chromosome variation in wheat plants regenerated from cultured immature embryos

Summary A cytological study has been made of plants regenerated from cultured immature embryos of four wheat cultivars (Triticum aestivum, 2n = 6x = 42). In total, 29% of the 192 plants examined were aneuploid with a range in chromosome numbers of 38–45. Evidence of chromosome structural changes was also found. This variation occurred in regenerants of all four cultivars, but there were large differences in the proportions of aneuploids arising from individual cultures which meant that no significant differences could be demonstrated between cultivars. Chromosome abnormalities were present in plants regenerated both from embryogenic cultures and from cultures in which the origin of shoots could not be distinctly defined.     

T-DNA analysis of plants regenerated from hairy root tumors

Summary Plants regenerated from hairy root tumors induced on Nicotiana glauca and Nicotiana tabacum by Agrobacterium rhizogenes strain A4 were examined for the presence of T-DNA. Regenerated N. tabacum plants contained intact copies of both TL-DNA and TR-DNA. However, plants regenerated from N. glauca tumors did not contain the TR-DNA region corresponding to the tms (auxin synthesis) genes. Some of the regenerants exhibited an abnormal phenotype which is characterized by severe leaf wrinkling. This phenotype is correlated with the presence of TL-DNA, but not TR-DNA.