<Emphasis Type="Italic">In vitro</Emphasis> induction and identification of autotetraploids of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

Dioscorea zingiberensis is an important medicinal plant and a source of diosgenin in China. We report research on the induction, characteristics, and chemical assays of polyploid plants of D. zingiberensis. Immersing calli in 0.3% colchicine solution for 16 h prior to culture induced a high number of autotetraploid plants. The induction rate reached as high as 36.7% of treated calli. More than 50 lines of autotetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Six elite lines have been selected for further selection and breeding into new varieties for commercial production.     

In vitro regeneration ability of diploid and autotetraploid plants of Cichorium intybus L.

Polyploidy has played a significant role in the evolutionary history of plants and is a valuable tool for obtaining useful characteristics. Because of the novelty of polyploids, comparison of their in vitro culture responses with diploids would be notable. In this study, leaf explants from diploid, autotetraploid and mixoploid plants of Cichorium intybus L. were cultured in vitro on the similar media and under same conditions. The ploidy level of the obtained calluses and regenerants were determined by flow cytometry analysis. The callogenic response of leaf explants cultured on the callus induction medium did not depend on the ploidy level of their parental plants. According to the flow cytometry analysis, the increased ploidy levels (4x) and (8x) were observed in the callus cultures with diploid and tetraploid origin, respectively. A considerable difference was observed between the ploidy level of mixoploid plants and their calluses, indicating the dominance of diploid cells in the callus tissue. The results showed that polyploidy led to the loss of organogenic potential as the tetraploid origin calluses failed to regenerate, while the diploid origin calluses successfully regenerated to whole plants.     

Efficient and stable regeneration from protoplasts of <Emphasis Type="Italic">Cyclamen coum</Emphasis> Miller via somatic embryogenesis

Embryogenic cultures of Cyclamen coum were established on solid media and in suspension, and their growth characteristics in response to different concentrations of plant growth regulators (PGRs) were evaluated. Embryogenic cultures exhibited a high regeneration capacity of 876 somatic embryos per gram fresh mass. Up to 4.24 × 10⁵ protoplasts per gram of fresh mass were isolated from somatic embryos and embryogenic suspension cultures. Protoplasts derived from both embryos and suspension cultures were successfully cultured in vitro and regenerated into plants via somatic embryogenesis. Phenotypic analyses and flow cytometric measurements revealed that some regenerated plants were tetraploid. About 20% of the protoplast-derived calluses used for regeneration were tetraploid, while tetraploidy was found in 0.9% of the plants regenerated from the embryogenic cultures.     

In vitro induction and identification of tetraploid plants of <Emphasis Type="Italic">Paulownia tomentosa</Emphasis>

Polyploidization is a major trend in plant evolution that has many advantages over diploid. In particular, the enlargement and lower fertility of polyploids are very attractive traits in forest tree breeding programs. We report here a system for the in vitro induction and identification of tetraploid plants of Paulownia tomentosa induced by colchicine treatment. Embryonic calluses derived from placentas were transferred to liquid Murashige and Skoog (MS) medium containing different concentrations of colchicine (0.01, 0.05, or 0.1%) and incubated for 24, 48, or 72 h on an orbital shaker at 110 rpm. The best result in terms of the production of tetraploid plantlets was obtained in the 48 h + 0.05% colchicine treatment, with more than 100 tetraploid plantlets being produced. The ploidy level of plantlets was verified by chromosome counts, flow cytometry, and morphology. The chromosome number of tetraploids was 2n = 4x = 80 and that of diploid plantlets was 2n = 2x = 40. The relative fluorescence intensity of tetraploids was twofold higher than that of diploids. The tetraploid and diploid plantlets differed significantly in leaf shape, with those of the former being round and those of the latter pentagonal. The mean length of the stomata was longer in tetraploid plants than diploid plants, and stomatal frequency was reduced with the increased ploidy level. The tetraploids had large floral organs that were easily distinguishable from those of diploid plants.     

Regeneration and characterization of plants from potato root lines transformed by Agrobacterium rhizogenes

Summary Transformed root lines were obtained after infection of leaf segments and tuber discs of tetraploid potato cvs Bintje and Desirée with Agrobacterium rhizogenes. In response to shoot induction, about 10% of the root lines produced shoots through callus formation. The tests for opine suggest that all 26 shoot lines of cv Bintje (Ri-Bintje) and 13 of Desirée (Ri-Desirée) were transformed. All shoot lines were tetraploid except for one octoploid subshoot line of cv Desirée; no aneuploids were observed. With the exception of two shoot lines derived from the same root line, all other Ri-Bintje plants showed a pattern of phenotypic variation, generally observed among transformed plants. In contrast, the phenotype of Ri-Desirée plants was uniform and normal; variation was observed in tuber form and size. Phenotypic variation observed among Ri-plants appeared to be mainly root line-dependent, particularly for height of plants and tuber size and form. Variation was also observed within root and shoot lines and was more pronounced among the Ri-Bintje plants. Segregation of phenotypic characteristics was observed among transformed plants, resulting in the occurrence of phenotypes resembling the control. Chromosomal stability and the frequent reversion to normal phenotype of Ri-plants make A. rhizogenes particularly suitable as a virulence vector in the binary transformation system for the transfer of desirable genes.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Studies on the Culture Conditions of Higher Plant Cells in Suspension Culture

To study the influence of cultural conditions on higher plant cells in suspension culture, the effects of nutritional conditions on the growth of suspended cells were investigated. Calluses were induced from 39 species of Nicotiana plants and 6 species of Populus plants on agar slant media, then these were transferred to suspension cultures. Concentrations of 2,4-D and kinetin suitable for incubation of callus from each plant were investigated and species having high growth rates in the appropriate medium were selected.

The effects of concentrations of auxins and kinetin, a variety of carbon and nitrogen sources, thiamin and myo-inositol on growth of the selected calluses were also studied. Of these calluses studied, N. glutinosa, N. tabacum var. Xanthi ova and P. hybrids were selected as calluses having high growth rates. Myo-inositol had no effect on any callus growth, and thiamin gave a distinct effect on Populus callus only. Nitrate as a nitrogen and sucrose as a carbon sources, and 2,4-D as an auxin were most effective in all calluses studied. Kinetin was essential for N. glutinosa among the calluses studied. Although high sugar concentrations tended to lengthen the lag period in the growth curve, there was no difference in the growth rates of the logarithmic phase among the concentrations.     

Increased induction and chromosome doubling of androgenetic haploid rye

Summary Further progress of studies aimed at increasing production of androgenetic Secale cereale plants via the culture of anthers is described. Two culture media initially developed for rice and wheat anther culture have been shown to have pronounced influence on rye. It has been possible to increase the average percentages of responsive anthers (i.e. those producing embryoids or calluses) from 0.26% to 10% with a maximum in certain experiments of over 40 %. Of nearly 400 plants produced in 1976, 1/4 are green and can be grown further by transfer to potting compost; 3/4 are albino. Stable green haploid lines were present amongst the plants, and after vegetative propagation of the lines representative samples have been treated with colchicine resulting in diploid, triploid and tetraploid plants. The influence of the genetic background of the donor plants on the success rate of anther culture and on the percentage of albino formation is discussed.     

Plant regeneration from leaf petioles in <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Summary Camptothecin, produced by Camptotheca acuminata, is a pharmaceutically important compound. Transgenic technology has potential uses for the enhancement of camptothecin production; however, an efficient plant regeneration protocol for C. acuminata is not currently available. Factors that affected successful seedling germination were evaluated. The regeneration potential of various parts of seedlings was tested. Camptothecin production in regenerated plants was compared to its production in calluses and the original seedlings. Dark incubation and seed coat removal led to a higher germination rate and a higher survival rate after germination. The best shoot induction medium was found to be Gamborg's B5 medium+8.9 μM benzyladenine. Among the calluses induced from various parts of seedlings, leaf petiole calluses, leaf dise calluses, and cotyledon calluses regenerated shoots, but internode calluses did not. Furthermore, leaf petiole calluses and leaf dise calluses regenerated normal shoots, while cotyledon calluses regenerated hyperhydric shoots. Moreover, leaf petiole calluses had a higher shoot regeneration rate, 50% versus 9%, and a higher shoot number, 6.2±0.5 versus 2.0±0.3, than did leaf dise calluses on the best shoot induction medium. It took 4–6 wk to regenerate shoots after transfer into shoot induction media. Camptothecin concentration in the regenerated plants was significantly higher than that in the calluses and similar to that in the original seedlings. In conclusion, leaf petioles provide efficient plant regeneration of C. acuminata.     

Production of androgenic dihaploid lines of the disomic tetraploid potato species Solanum acaule ssp. acaule

Over 250 dihaploid lines derived from a disomic tetraploid genotype of Solanum acaule ssp. acaule Bitt. (acc. PI 472655) were produced via androgenesis. The anther donor plant had previously shown immunity to bacterial ring rot caused by Clavibacter michiganensis ssp. sepedonicus (Spieck. and Kotth.) Davis et al., and has now been shown to have high embryogenic capacity in anther culture. In total, 370 shoots were regenerated from 4,011 anthers cultured. The ploidy level of the 287 regenerants was determined from greenhouse-grown plants using flow cytometry. Of these plants, 274 (95%) were dihaploids with an average DNA content of 1.68 pg, approximately half that of the tetraploid anther donor (2.95 pg). The remainder of the anther-derived regenerants (5%) were tetraploid, hexaploid or mixoploid. Chromosome counts confirmed the results obtained by flow cytometry. In the greenhouse, none of the 33 dihaploid lines analysed produced berries but showed low (2%) male fertility. This contrasted with five greenhouse-grown tetraploid anther-derived plants which produced berries and seeds. Comparison of the general leaf morphology and floral characteristics of the tetraploid anther donor, S. acaule, and the dihaploids indicated that little variation exists in this species. Received: 28 August 1997 / Revision received: 22 December 1997 / Accepted: 27 July 1998     

In vitro induction and identification of autotetraploid of <Emphasis Type="Italic">Bletilla striata (Thunb.) Reichb.f</Emphasis>. by colchicine treatment

Polyploidy breeding has proved to be a valuable approach for acquiring the high yield superior varieties in medicinal plants. An effective protocol for obtaining Bletilla striata autotetraploid is in vitro induction of protocorms with colchicine. The protocorms of B.striata were soaked in different concentrations of colchicine solution [0.05, 0.1 and 0.2% (w/v)] for 12, 24, 36, 48 and 60 h, and the ploidy of the seedlings was identified by chromosome counting and flow cytometry analysis. The results showed that the optimal condition for induction of autotetraploid of B. striata protocorms was treated with 0.2% colchicine for 36 h with the induction rate reached as high as 26.7%. In addition, the morphological and anatomical characteristics were observed and compared between the diploid and tetraploid plants. And we found that the features of tetraploid plants were significantly different from diploid plants, such as tetraploid plants possessed thicker and deeper green leaves, larger stomata and more chloroplast number, which could be used as simple and efficient parameters for screening tetraploid. This study laid a foundation for breeding superior varieties of B. striata.     

Induction of tetraploidy in Buddleia globosa

The aim was to produce a tetraploid form of Buddleia globosa to facilitate introgression of yellow flower colour into B. davidii, which is naturally tetraploid. Protocols were established for the micropropagation of B. globosa and tetraploid plants were obtained by application in vitro of colchicine to pre-cultured excised nodal sections. Three concentrations of colchicine were applied (0.01%, 0.05% and 0.1% w/v) for 1, 2 or 3 days. At 0.01% tetraploids were produced only after 2 days of application. All other treatments produced at least one tetraploid. The colchicine technique was extremely effective: of 29 lines tested, 19 were tetraploid and 5 were mixoploid. The vegetative characteristics of these tetraploids are described and the flowering characteristics of the three that flowered. This revised version was published online in August 2006 with corrections to the Cover Date.     

Assessment of genetic stability of the germplasm lines of medicinal plant <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi (Huang-qin) in long-term,in vitro maintained cultures

An approach of combining flow cytometry (FCM) analysis with morphological and chemical profiling was used to assess the genetic stability and bioactive compound diversity in a Scutellaria baicalensis Georgi (Huang-qin) germplasm collection that was clonally maintained in in vitro for a period of over 6 years. Based on the FCM analysis of nuclei samples from young shoots, the nuclear DNA content of S. baicalensis was calculated as 0.84 pg/2C. FCM analysis showed no significant variation in the nuclear DNA contents and ploidy levels in the long-term in vitro maintained germplasm lines. Germplasm lines, acclimatized to ex vitro conditions, exhibited distinctive plant growth and bioactive compound production capacities. The high level of genetic stability observed in in vitro maintained S. baicalensis lines opens up a variety of opportunities such as allowing long-term aseptic preservation and easy distribution of well-characterized germplasm lines of this medicinal plant species. This study represents a novel approach for continuous maintenance, monitoring, and production of medicinal plant tissues with specific chemistry.     

An improved system to establish highly embryogenic haploid cell and protoplast cultures from pollen calluses of maize (Zea mays L.)

Regenerable, embryogenic haploid cell suspensions were initiated and established from type II pollen calluses of two selected Chinese maize genotypes (No 592 Y and 592.A2 LY). The induction frequency of friable, embryogenic callus (type II) was highly dependent on three factors: genotype, medium, cold pretreatment, and on their interactions. Repeated callus and cell selection during the culture procedure led to stable haploid suspensions consisting of fine clusters each containing 20–50 cells. The selected cell lines were able to maintain their morphogenic ability during long-term subculture (2 years). Protoplasts were successfully isolated from subcultured, friable, embryogenic pollen calluses and cultured on N₆BM and N₆K media using a feeder layer, obtained from 2-day-old suspension culture. Healthy plants were regenerated from protoplast-derived calluses.     

High efficient transgenic plant regeneration from embryogenic calluses of <Emphasis Type="Italic">Citrus sinensis</Emphasis>

Transformation and high efficient regeneration of transgenic plants from embryogenic calluses of Bingtang sweet orange [Citrus sinensis (L.) Osbeck] was reported. Embryogenic calluses were inoculated with Agrobacterium tumefaciens strain EHA105, harboring the binary Ti plasmid pROK II and carrying a neomycin phosphotransferase II (NPTII) gene, an intron β-glucuronidase (GUS) gene and the Arabidopsis APETALA1 (AP1) gene. Transformation treatment was with inoculation time of 30 min, co-culture of 3 d at 23 °C and supplementation of the co-culture medium with 2 mg dm⁻³ acetosyringone (AS). Kanamycin (50 mg dm⁻³) was effective to inhibit the growth of non-transformed calluses while it did not affect the transformed ones. The total number of transformed callus lines was 7 with 100 % embryo induction. High efficient regeneration of the transgenic embryos (88 % with 4–5 shoots per embryoid) was realized within 3 months. Integration of the transgene into the citrus genome was confirmed by histochemical GUS staining, polymerase chain reaction (PCR) analysis with AP1-specific primer and Southern blot hybridization with a 712 bp PCR fragment of AP1 as the probe.     

A cytogenetic and phenotypic characterization of somatic hybrid plants obtained after fusion of two different dihaploid clones of potato (Solatium tuberosum L.)

Summary Somatic hybrid plants of various ploidy levels obtained after chemical fusion between two dihaploid clones of potato Solanum tuberosum L. have been analysed by cytological, morphological and molecular methods. The hybrid nature of tetraploid and hexaploid plants and the genome dosage in hexaploid hybrids were confirmed by Giemsa C-banding. Tetraploid and hexaploid hybrids showed numerical as well as structural chromosome mutations. The latter occurred mainly in the nuclear organizing chromosome. The tetraploid hybrids were more vigorous than the dihaploid parents as demonstrated by an increase in height, enlargement of leaves, increase in the number of internodes, restored potential for flowering and increased tuber yield. The grouping of tetraploid somatic hybrids into various classes on the basis of leaf morphology revealed that plants with a full chromosome complement were more uniform than aneuploids. Many hexaploid somatic hybrids were also more vigorous than the dihaploid parents and could be grouped into two different classes on the basis of floral colour and tuber characteristics, the differences being due to their different dosage of parental genomes. Most of the tetraploid somatic hybrids showed pollen development halted at the tetrad stage as one of the parental clones contained a S. Stoloniferum cytoplasm. However, one tetraploid plant produced pollen grains with high viability. The chloroplast genome in the hybrid plants was determined by RFLP analysis. All of the hybrids had a cpDNA pattern identical to one parent, which contained either S. Tuberosum or S. Stoloniferum cpDNA. A slight preference for S. Tuberosum plastids were observed in hybrid plants. No correlation between pollen development and plastid type could be detected.     

Effect of physical desiccation on plant regeneration efficiency in rice (Oryza sativa L.) variety super basmati

This experiment assessed the effect of partial physical desiccation on plant regeneration efficiency in scutellum-derived embryogenic calluses of rice (Oryza sativa L.) variety Super basmati. A number of callusing cultures were developed, and efficient callus induction was observed on MS (Murashige and Skoog) basal medium supplemented with 2.0 mg/L 2,4-dichlorophenoxy acetic acid. The calluses were proliferated on the same medium for 3 weeks and then shifted to dehydration desiccation treatment for 72 h. The desiccated calluses were cultured on different media for somatic embryogenesis and plant regeneration. A medium with 2.0 mg/L α-napthaleneacetic acid, 10.0 mg/L abscisic acid , 2.0 mg/L kinetin was best for somatic embryogenesis only, but not for further plant development. After 10 d, differentiated calluses were sub-cultured on medium with various concentrations and types of carbohydrates (carbon source) in ¹MS_2j medium. A large number of plantlets (14.51±2.81 and 8.56±2.90 plants/callus) were regenerated via chemical desiccation, on MS with 3% maltose+3% sorbitol and 6% sucrose, respectively. Under dehydration on only simple MS (3% sucrose), 11.23±3.22 plants/callus were developed. Under conditions of dehydration and chemical desiccation, plant regeneration rates were higher than the calluses cultured on simple MS medium in the presence of plant growth regulator. After somatic embryogenesis, >25% plants were sterile. The protocol used here may allow maximum regeneration of normal and fertile plantlets of super basmati rice within 3 months.     

Stable transformation of barley callus using biolistic® particle bombardment and the phosphinothricin acetyltransferase (bar) gene

We have previously studied chromosomal and morphological variation in protoplast cultures of diploid petunia (Petunia hybrida) plants. We found that 85% of the regenerants were tetraploid (2n=4x=28). These plants flowered and set seeds.In the present study, the cytological stability of plants regenerated from leaf mesophyll protoplast cultures derived from the progeny of the self-fertile tetraploid plants was assessed on the basis of mitotic analysis, morphological characters, and protein patterns. When we analyzed the root tip chromosomes of 117 regenerants derived from 39 protoclone calluses, all of the regenerants tested retained the parental chromosome number of 2n=4x=28. One hundred regenerants were further analyzed and displayed normal vegetative morphology and retained the floral characteristics of the seed-derived plants from which they were derived. No significant variations in any character were observed among regenerants. When leaf protein patterns from four regenerated tetraploid protoclones were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those of seed-derived plants, the protein patterns exhibited great similarity.The data suggest that tetraploidization of petunia plant increases cytological stability during further in vitro cultures and may play an important role in the genetic stability of regenerant populations.Abbreviations BA benzylaminopurine - IAA indole-3-acetic acid - IEF isoelectric-focusing - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs

Summary The techniques of microspore and protoplast regeneration starting from dihaploid Solanum tuberosum plants has been improved to such an extent that the production of more than 2000 microspore derived A₁ plant lines and of several hundred protoplast derived plantlets has become possible. Further, from the dihaploid Solanum species S. phureja the regeneration of microspores to plants, and from the species S. infundibuliforme, S. sparsipilum and S. tarijense the regeneration of protoplasts to calluses, has been achieved. The plants descending from the two single cell culture systems are compared with reference to phenotypic markers and economic qualities. Some principles characteristic for either microspore or protoplast derived plants are examined and their significance is discussed. The results are compiled into an extended analytical synthetic breeding scheme based on a stepwise reduction of the autotetraploid to the monohaploid level and a subsequent controlled combination to a new synthetic completely heterozygous tetraploid potato.