Factors promoting sustained divisions of mesophyll protoplasts isolated from dihaploid clones of potato (Solanum tuberosum L.) and a cytological analysis of regenerated plants

A culture protocol has been developed for mesophyll protoplasts isolated from various dihaploid clones of potato. A special effort was made to promote the growth of initially dividing cells to form cell colonies and calli. An increase in plating efficiency in 3 different dihaploid clones and one doubled dihaploid clone was obtained after serial dilution of cultures with a suitable amount and type of medium at different stages of cell colony development. Plating on a refined semi-solid medium after 14 days of culture further improved both the yield and the quality of calli obtained. The refined plating medium also enhanced shoot regeneration ability from 67 to 90% in one of the dihaploid clones (67:9). The refined culture protocol could also be used without causing a decrease in plating efficiency at a low population density adjusted after 3 days of culture. The ploidy level of plants regenerated from dihaploid protoplasts were determined by chromosome counting and DNA analysis by flow cytometry. Most of the plants were aneuploid or tetraploid although, some dihaploid plants were obtained after protoplast culture of 2 dihaploid clones derived from the same cultivar (cv. Stina).Abbreviations BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid     

Ploidy doubling by callus culture of potato dihaploid leaf explants and the variation in regenerated plants

Somatic chromosome doubling of potato dihaploids was achieved by culturing callus from leaf pieces derived from glasshouse and in vitro grown plants. The glasshouse-grown leaves produced better callus on average but there was no significant difference between the average number of plantlets per callus regenerated from the two types of material. Mixtures of 2x and 4x plants were obtained from callus culture and the proportions of each ploidy type varied with the dihaploid genotype. Leaflet length/breadth ratios were chiefly determined but ploidy but there was variation within ploidy groups. There were also differences in blight and cyst nematode resistance between tetraploids derived from the same dihaploid.     

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.     

Karyotype variation in aminoethylcysteine resistant cell and callus cultures and regenerated plants of a dihaploid potato (Solanum tuberosum)

Karyotypic studies on cell suspensions and calli of an S-(2-aminoethyl)cysteine resistant cell line of the interdihaploid potato H²578 (2n=24) revealed a high degree of variation in the number of chromosomes (33–217) and dicentric chromosomes (0–8). The suspensions also exhibited megachromosomes and fused chromosomes. Differential staining showed that in suspensions dicentrics survived mitotic cycles mainly by parallel separation of the chromatids during anaphase and hardly by the breakage-fusion-bridge cycle. Two phenotypes of plantlets regenerated, each with 34 or 35 chromosomes with gross structural mutations and with the triploid amount of DNA. Chromosomal variation was related to the degree of tissue organization.     

An efficient genotype-independent method for regeneration of potato plants from leaf tissue

The regeneration of plants from leaf explants of a number of potato cultivars using a number of published one-, two- and three-step methods was assessed. A method using a pretreatment with high levels of auxin and cytokinin coupled with silver thiosulphate in the regeneration medium proved the most rapid and efficient for the eight cultivars examined.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid - STS silver thiosulphate     

Effect of temperature on carbohydrate content,ADP glucose pyrophosphorylase,and ATP- and PPi-dependent phosphofructokinase activity of potato tuber callus tissue

Callus derived from Lemhi Russet and Russet Burbank tuber tissue was incubated at 20°C and 30°C on a high sucrose medium for starch-formation and subjected to simulated storage and reconditioning treatments at 5°C and 25°C after transfer of the callus to a medium without a carbon source. Percent dry weight of callus from both cultivars averaged about 21% after starch formation and 5% after storage and reconditioning treatments. Total sugars were higher in callus incubated on the starch forming treatment. Lemhi Russet callus contained predominantly reducing sugars, while Russet Burbank callus contained mostly non-reducing sugars. Total sugar content was generally lower for both cultivars after the storage and reconditioning treatment in callus after starch formation at 20°C. Starch content was generally higher in Lemhi Russet tissue. After starch formation at 20°C Lemhi Russet had higher starch after the storage and reconditioning treatment than tissue from 30°C, while the opposite trend was found in Russet Burbank tissue. Total protein declined in Russet Burbank tissue during the storage and reconditioning treatment regardless of prior incubation conditions, while this decline only occurred in Lemhi Russet tissue initially incubated at 30°C during the starch formation treatment. In tissue of both cultivars, ATP- and PPi-dependent phosphofructokinase activities were inversely proportional to total sugar concentrations, while in RB callus ADP glucose pyrophosphorylase activities were proportional to starch content.Research Paper 91B1 of the Idaho Agricultural Experiment Station.     

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     

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.     

Regeneration of plants from callus tissue of Desmodium affine and Desmodium uncinatum

Plants were in vitro regenerated from leaf callus of Desmodium affine and D. uncinatum. Leaf explants were induced to form callus when aseptically cultured on Murashige and Skoog medium (MS) supplemented with 6 mg dm-3 6-benzylaminopurine (BAP) in combination with 1 mg dm-3 naphthaleneacetic acid (NAA). Regeneration of shoots was induced when callus was cultured on MS medium supplemented with 6 mg dm-3 BAP and 0.01 mg dm-3 NAA. Roots regenerated in high frequency when differentiated shoots were subcultured on MS medium supplemented only with 0.01 mg dm-3 NAA. The regenerated plantlets were successfully grown in pots. Calli from D. incanum failed to regenerate shoots. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sugarcane mosaic virus in plantlets regenerated from diseased leaf tissue

Plantlets produced from sugarcane leaf tissue were examined to determine the effect of propagation on the frequency of occurrence of sugarcane mosaic virus (SCMV).Explants from immature leaf tissues of the sugarcane variety CP 72-356 (Saccharum interspecific hybrid), healthy or SCMV-infected, were cultured on Murashige-Skoog medium to which a combination of cytokinin and auxin had been added. Plantlets developed on healthy and infected leaf tissue within 6 weeks. The juice from plantlets was assayed for SCMV on Rio sorghum (Sorghum bicolor (L.) Moench, var. Rio) seedlings and on sugarcane varieties CP 31-294 and CO 31-588 for SCMV-strain identification. Results indicated that SCMV strain H was transmitted from the donor tissue to the regenerated plantlets. Observation on plantlets reared in the greenhouse showed that 23% had symptoms of SCMV. In a second replicated experiment, the leaf tissue from plants of POJ 234 free of mosaic or infected with SCMV strain A, B, D, H, or I was cultured. Each of the five strains was transmitted from donor to plantlet as indicated by assays on sorghum and sugarcane varieties. From 11 to 88% of the plantlets had mosaic symptoms, depending on the strain infecting the donor plant. In this experiment, SCMV-strain M was transmitted from an unidentified donor variety to 23% of the regenerated plantlets.Portions of this paper have been presented to the American Society of Sugar Cane Technologists, at the meeting in Clearwater, Florida in June, 1984.     

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.     

Colorado potato beetle resistant somatic hybrid potato plants produced via protoplast electrofusion

Leptines are natural glycoalkaloids found only in certain selections of the wild potato speciesSolanum chacoense. These rare glycoalkaloids have been identified to be phytochemical defensive agents against insect herbivores such as the Colorado potato beetle (CPB). In an attempt to introduce this CPB resistance into the cultivated potatoS. tuberosum, interspecific somatic hybrid plants were developed between a dihaploid ofS. tuberosum and a high leptine-producing germplasm selection ofS. chacoense. The somatic hybrid was fused using protoplast electrofusion and regeneration techniques. Selection of interspecies fusion cell lines was based on hybrid vigor in protoplast-callus (p-callus) growth, on shoot regeneration from p-calli, and on characteristic appearance of anthocyanin pigment. This selection system was highly efficient and 12 of 13 fully regenerated plants were identified as somatic interspecies hybrids, as determined by the analyses of morphologic biochemical, and isozyme markers. In vitro insect bioassays demonstrated that the hybrids averaged a threefold reduction in leaf consumption by the CPB when compared to cultivated potatoes.     

Resistance to potato leaf roll virus and potato virus Y in somatic hybrids between dihaploid Solanum tuberosum and S. brevidens

Summary Many somatic fusion hybrids have been produced between a dihaploid potato Solanum tuberosum and the sexually-incompatible wild species S. brevidens using both chemical and electrical fusion techniques. S. brevidens was resistant to both potato leaf roll virus (PLRV) and potato virus Y (PVY), the viruses being either at low (PLRV) or undetectable (PVY) concentrations as determined by enzyme-linked immunosorbent assay (ELISA). The S. tuberosum parent was susceptible to both viruses. A wide range of resistance, expressed as a decrease in virus concentration to both viruses was found amongst fusion hybrids, four of which were especially resistant. The practicality of introducing virus resistance from S. brevidens into cultivated potatoes by somatic hybridisation is discussed.     

Wheat regenerated from scutellum callus as a source of material for transformation

Reports of wheat transformation efficiencies vary from less than 1% to more than 5% for individual experiments. Rarely are negative experiments reported though we estimate that between one in two and one in three of all experiments fail to produce transformed plants. Consequently if transformation efficiencies were calculated from the total number of scutellum bombarded rather than from only those experiments which produced transformed plants there would be a significant fall in reported efficiencies. The use of scutellum-derived material from plants regenerated from scutellum callus and grown in a controlled environment room significantly reduced the number of experiments failing to produce plants. Though there is a small but significant increase in transformation efficiencies for individual experiments, the recovery of plants, as a direct consequence of the reduction in the number of failed experiments, increases nearly 350%, from 4.8 plants/1000 scutella from seed gown plants bombarded to 17 plants/1000 embryos bombarded from tissue culture regenerated plants. There appears to be no additional gains to be made from plants which are cycled through tissue culture more than one time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chromosome doubling in monohaploid and dihaploid potatoes by regeneration from cultured leaf explants

Plants were regenerated from cultured excised leaf segments of monohaploid (2n=x=12) and diphaloid (2n=2x=24) potato (Solanum tuberosum L.) and a sample has been studied cytologically. In the case of monohaploids, a single leaf regeneration cycle resulted in almost total recovery of doubled monohaploid plants (2n=2x=24), whilst 50% of the plants regenerated from doubled monohaploid leaves had doubled again to the doubled double monohaploid (or homozygous tetraploid, 2n=4x=48) level. Regeneration from dihaploid leaf pieces also gave a good proportion (60%) of doubled genotypes. Very few mixoploids and very few aneuploids were found. These results, together with the general applicability of the method to a large number of potato cultivars, suggest that it can be used as a simple and reliable method of obtaining homozygous tetraploid potatoes.     

Heterozygosity in tetraploid potatoes revealed by rDNA polymorphism analysis of their dihaploid progenies: a contribution to chromosome assignment

Restriction map and organization of rDNA was inferred from analysis of dihaploid progenies of two tetraploid genotypes of cultivated potato. Each tetraploid genotype was characterized by a specific distribution of different types of rDNA repetition units on their four homologous chromosomesII. The genotypes were heterozygous and differed by the kind of units carried by each chromosomeII. Models for the generation of the observed organization are discussed and supported by first cloning studies.     

Mitochondrial DNA variation in maize plants regenerated during tissue culture selection

Summary Plants resistant to Helminthosporium maydis race T were obtained following selection for H. maydis pathotoxin resistance in tissue cultures of susceptible, Texas male-sterile (T) cytoplasm maize. The selected lines transmitted H. maydis resistance to their sexual progeny as an extranuclear trait. Of 167 resistant, regenerated plants, 97 were male fertile and 70 were classified male sterile for reasons that included abnormal plant, tassel, anther or pollen development. No progeny were obtained from these male-sterile, resistant plants. Male fertility and resistance to the Phyllosticta maydis pathotoxin that specifically affects T cytoplasm maize were co-transmitted with H. maydis resistance to progeny of male-fertile, resistant plants. These three traits previously were associated only with the normal (N) male-fertile cytoplasm condition in maize. Three generations of progeny testing provided no indication that the cytoplasmic association of male sterility and toxin susceptibility had been broken by this selection and regeneration procedure. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) revealed that three selected, resistant lines had distinct mtDNA organization that distinguished them from each other, from T and from N cytoplasm maize. Restriction patterns of the selected resistant lines were similar to those from T cytoplasm mtDNA; these patterns had not been observed in any previous analyses of various sources of T cytoplasm. The mtDNA analyses indicated that the male-fertile, toxin-resistant lines did not originate from selection of N mitochondrial genomes coexisting previously with T genomes in the T cytoplasm line used for selection.Scientific Journal Series Article no. 11,185 of the Minnesota Agricultural Experiment Station and no. 2295 of the Florida Agricultural Experiment Station. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee of warrantly of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 M picloram as the only PGR. Ten M picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 M picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige-Skoog - NAA naphthaleneacetic acid - PGR plant growth regulator Research paper #9053 of the Idaho Agricultural Experiment Station.     

The use of antibiotics to eliminate latent bacterial contamination in potato tissue cultures

A simple leaf bioassay was developed to screen for slow-growing latent bacteria found in potato nodal cuttings. The potato cultures were established to provide a source of sterile leaf tissue for protoplast isolation and the bacteria were only detected after numerous generations in culture. In an attempt to solve this problem cultures were screened before and after various treatments. Two antibiotic mixtures were tested for their efficacy in eliminating bacteria: ABM1 contained penicillin, streptomycin, amphotericin and NaCl while ABM2 contained erythromycin, streptomycin and carbenicillin at various concentrations. Both cocktails, when added to the micropropagation medium, reduced plant growth, induced chlorosis at higher levels and did not eliminate contamination. In terms of protoplast isolation, the only effective treatment was when ABM1 was incorporated into the plasmolysis and enzyme media. It removed contamination at all levels tested and had a differential effect on cell division such that, at low levels of ABM1, protoplasts regenerated successfully.     

Root and shoot initiation by leaf,stem, and storage root explants of sweet potato

Explants from stem, leaf, and storage root tissue of sweet potato (Ipomoea batatas L.) cv. Jewel, were placed on media conaining 0.1, 1.0, and 10 mg/1NAA with 0.1, 1.0, or 10 mg/1BA in a factorial experiment. Some callus formed in every treatment, but the best callus growth was on media containing 1.0 mg/1NAA and 10 mg/1BA. Roots formed over a range of treatments but were most prolific on the medium containing 1.0 mg/1NAA and 0.1 mg/1BA. Some de novo formed roots subsequently produced shoot buds in culture. Shoot formation increased the longer the original explants remained in culture without subculture. Roots could be subcultured indefinitely on agar solidified medium, but shoot regeneration did not occur after two subcultures. Shoot formation was greatest when the roots were subcultured on medium containing 1.0 mg/NAA and 0.1 mg/1BA. The cultivar Caromex followed the same regeneration pathway, but the number of shoots formed was considerably less. Regeneration in both Jewel and Caromex explants was enhanced by light.Paper No. 8292 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.This work was done as a partial requirement for the M.S. degree at North Carolina State University.