Showing papers in "Plant Cell Tissue and Organ Culture in 2005"

Marker assisted selection in crop plants

Abstract: Genetic mapping of major genes and quantitative traits loci (QTLs) for many important agricultural traits is increasing the integration of biotechnology with the conventional breeding process. Exploitation of the information derived from the map position of traits with agronomical importance and of the linked molecular markers, can be achieved through marker assisted selection (MAS) of the traits during the breeding process. However, empirical applications of this procedure have shown that the success of MAS depends upon several factors, including the genetic base of the trait, the degree of the association between the molecular marker and the target gene, the number of individuals that can be analyzed and the genetic background in which the target gene has to be transferred. MAS for simply inherited traits is gaining increasing importance in breeding programs, allowing an acceleration of the breeding process. Traits related to disease resistance to pathogens and to the quality of some crop products are offering some important examples of a possible routinary application of MAS. For more complex traits, like yield and abiotic stress tolerance, a number of constraints have determined severe limitations on an efficient utilization of MAS in plant breeding, even if there are a few successful applications in improving quantitative traits. Recent advances in genotyping technologies together with comparative and functional genomic approaches are providing useful tools for the selection of genotypes with superior agronomical performancies.

Application of bioreactor systems for large scale production of horticultural and medicinal plants

Abstract: Automation of micropropagation via organogenesis or somatic embryogenesis in a bioreactor has been advanced as a possible way of reducing costs. Micropropagation by conventional techniques is typically a labour-intensive means of clonal propagation. The paper describes lower cost and less labour-intensive clonal propagation through the use of modified air-lift, bubble column, bioreactors (a balloon-type bubble bioreactor), together with temporary immersion systems for the propagation of shoots, bud-clusters and somatic embryos. Propagation of Anoectochilus, apple, Chrysanthemum, garlic, ginseng, grape, Lilium, Phalaenopsis and potato is described. In this chapter, features of bioreactors and bioreactor process design specifically for automated mass propagation of several plant crops are described, and recent research aimed at maximizing automation of the bioreactor production process is highlighted.

Genetic Engineering in Floriculture

Abstract: The global flower industry thrives on novelty. Genetic engineering is providing a valuable means of expanding the floriculture gene pool so promoting the generation of new commercial varieties. Commercialisation of genetically engineered flowers is currently confined to novel coloured carnations. However, further products are expected given the level of activity in the field. In general terms engineered traits are valuable to either the consumer or the producer. At present only consumer traits appear able to provide a return capable of supporting what is still a relatively expensive molecular breeding tool. The biosynthesis of floral pigments, particularly anthocyanins, has been elucidated in great detail in model flowers such as petunia. This knowledge is now being applied to an understanding of a wide range of other flowers and providing a means of targeting colour modification in these species. The engineering of novel traits in a given variety also rests on capabilities in plant transformation that are continuing to expand at a rapid rate. The expression of genes transferred across genera is not always predictable and so requires considerable trial and error to arrive at stable phenotype of commercial interest. Manipulation of metabolic pathways, often requiring introduction of multiple genes can also be problematic. This is a reflection of the complexity of interactions within and between cells at a gene and gene product level. An understanding of gene function is only an essential first step in engineering novel traits. The production of novel flower colour has been the first success story in floriculture genetic engineering. Other traits that have received attention include floral scent, floral and plant morphology, senescence of flowers both on the plant and post-harvest and disease resistance.

Shoot multiplication in Rauvolfia tetraphylla L. using thidiazuron

Abstract: The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10μM) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5μM TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5μM indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.

Intergeneric somatic hybridization and its application to crop genetic improvement

Abstract: Related or distant species of cultivated cs are a large pool of many desirable genes. Gene transfer from these species through conventional breeding is difficult owing to post- and pre-zygotic sexual incompatibilities. Somatic hybridization via protoplast fusion is a possible alternative for gene transfer from these species to cultivated crops. Since the early days of somatic hybridization many intergeneric somatic hybrids have been developed through symmetric fusion, asymmetric fusion and microfusion. Somatic hybrids are mainly selected by using markers such as specific media or fusion parents with special features, biochemical mutants, antibiotic resistance and complementation strategy. The hybridity of the regenerants is determined based on morphological, cytological and molecular analysis. The inheritance patterns of nuclear and cytoplasmic genomes in the somatic hybrids are diverse. Nuclear DNA from both fusion parents co-exists congruously in some hybrids with translocation and rearrangement of chromosomes, but spontaneous elimination of chromosomes from either or both fusion parents has been observed very often. In asymmetric fusion, chromosome elimination is an important issue that is a complicated process influenced by many factors, such as irradiation dose, phylogenetic relatedness, ploidy level of fusion parent and regenerants. As for chloroplast genome, uniparental segregation is mainly detected, though co-existence is also reported in some cases. The mitochondrial genome, in contrast to chloroplast, undergoes recombination and very frequent rearrangements. Somatic cell fusion has potential applications for crop genetic improvement by overcoming sexual incompatibility or reproductive barriers, and by realizing novel combinations of nuclear and/or cytoplasmic genomes.

Simple bioreactors for mass propagation of plants

Abstract: Bioreactors provide a rapid and efficient plant propagation system for many agricultural and forestry species, utilizing liquid media to avoid intensive manual handling. Large-scale liquid cultures have been used for micropropagation through organogenesis or somatic embryogenesis pathways. Various types of bioreactors with gas-sparged mixing are suitable for the production of clusters of buds, meristems or protocorms. A simple glass bubble-column bioreactor for the proliferation of ornamental and vegetable crop species resulted in biomass increase of 3 to 6-fold in 3–4 weeks. An internal loop bioreactor was used for asparagus, celery and cucumber embryogenic cultures. However, as the biomass increased, the mixing and circulation were not optimal and growth was reduced. A disposable pre-sterilized plastic bioreactor (2–5-l volume) was used for the proliferation of meristematic clusters of several ornamental, vegetable and woody plant species. The plastic bioreactor induced minimal shearing and foaming, resulting in an increase in biomass as compared to the glass bubble-column bioreactor. A major issue related to the use of liquid media in bioreactors is hyperhydricity, that is, morphogenic malformation. Liquid cultures impose stress signals that are expressed in developmental aberrations. Submerged tissues exhibit oxidative stress, with elevated concentrations of reactive oxygen species associated with a change in antioxidant enzyme activity. These changes affect the anatomy and physiology of the plants and their survival. Malformation was controlled by adding growth retardants to decrease rapid proliferation. Growth retardants ancymidol or paclobutrazol reduced water uptake during cell proliferation, decreased vacuolation and intercellular spaces, shortened the stems and inhibited leaf expansion, inducing the formation of clusters. Using a two-stage bioreactor process, the medium was changed in the second stage to a medium lacking growth retardants to induce development of the meristematic clusters into buds or somatic embryos. Cluster biomass increased 10–15-fold during a period of 25–30 days depending on the species. Potato bud clusters cultured in 1.5 1 of medium in a 2-l capacity bioreactor, increased during 10–30 days. Poplar in vitro roots regenerated buds in the presence of thidiazuron (TDZ); the biomass increased 12-fold in 30 days. Bioreactor-regenerated clusters were separated with a manual cutter, producing small propagule units that formed shoots and initiated roots. Clusters of buds or meristematic nodules with reduced shoots, as well as arrested leaf growth, had less distortion and were optimal for automated cutting and dispensing. In tuber-, bulb- and corm-producing plants, growth retardants and elevated sucrose concentrations in the media were found to enhance storage organ formation, providing a better propagule for transplanting or storage. Bioreactor-cultures have several advantages compared with agar-based cultures, with a better control of the contact of the plant tissue with the culture medium, and optimal nutrient and growth regulator supply, as well as aeration and medium circulation, the filtration of the medium and the scaling-up of the cultures. Micropropagation in bioreactors for optimal plant production will depend on a better understanding of plant responses to signals from the microenvironment and on specific culture manipulation to control the morphogenesis of plants in liquid cultures.

Major mutation-assisted plant breeding programs supported by FAO/IAEA

Abstract: Under the Joint FAO/IAEA programme, radiation-induced mutations are used for genetic improvement of both seed and vegetatively propagated plants. The FAO/IAEA programme maintains a database of officially released mutant varieties worldwide (http://www-mvd.iaea.org/). Currently, over 2300 mutant varieties are registered in our database. Coordinated Research Projects (CRPs) and Technical Co-operation Projects (TCP) are two major activities at IAEA that serve Member States at the national, regional and interregional levels. This article highlights CRPs on banana, underutilized and neglected crops, and tropical and subtropical fruits. CRPs on banana and underutilized and neglected crops have already been concluded. TCPs in South East Asia (Thailand, and Malaysia), Africa (Algeria, Morocco, Tunisia, Ghana), and the Middle East (Yemen) are discussed. The main projects in South East Asia are on genetic improvement of ornamental plants, fruits and cereals. In Africa, projects are on cassava, date palm, salinity and drought. In the Middle East, funded projects are related to salinity, and drought. In this article, major achievements are highlighted through CRPs and TCPs on low cost tissue culture, banana, underutilised and neglected crops, tropical and subtropical fruits.

Optimization of plantain (Musa AAB) micropropagation by temporary immersion system

Abstract: The positive and reliable effect of temporary immersion systems on in vitroshoot proliferation was already proved for different plant genera and it is now presented as an alternative for plantain micropropagation. Some culture parameters affecting the efficiency of the twin flasks system or temporary immersion bioreactor (Escalona et al., 1999) were investigated. Three different cytokinins (benzyladenine, thidiazuron and meta-topolin) were added to the culture medium and meta-topolin at a concentration of 4.4 μM was proved to be the most efficient. Successive subcultures (28 days per subculture) were performed on medium supplemented with meta-topolin, revealing a decrease in multiplication after the 6th subculture. Multiplication rate was not changed within the ranges of immersion times (4, 12 or 22 min) and frequencies (every 3, 5 or 7 h) tested. The size of the bioreactor (250, 1,000, 5,000 or 10,000 ml) and the volume of medium per inoculum (10, 20 or 30 ml) were also evaluated and appeared to have an influence on the multiplication. A proportion of 25–100 ml of headspace per inoculum and 30 ml of medium per inoculum resulted in a multiplication rate > 13 in 28 days.

The effect of salt stress and abscisic acid on proline production, chlorophyll content and growth of in vitro propagated shoots of Eucalyptus camaldulensis

Abstract: Three clones, selected for their variation in salt tolerance, were examined regarding their growth and physiological responses on exposure to salt (NaCl) and abscisic acid (ABA) in vitro. The shoot proline levels significantly increased in two salt tolerant clones when exposed to 100 mM NaCl in the shoot multiplication medium. In contrast, proline in a salt sensitive clone did not change in comparison to the control treatment. When 10 μM ABA was included in the medium all clones had an increase in proline regardless of whether they were salt tolerant or salt sensitive, linking proline production to the stress hormone ABA. Callus production was so variable that it was not possible to produce callus of consistent texture, colour and growth for all three clones. For the two clones where consistent growth was achievable, both the salt tolerant and salt sensitive clones increased proline production when exposed to salt. This response, however, was greater in the salt tolerant clone. Other parameters examined were growth (dry weight) and shoot chlorophyll content. These characteristics did not correlate with the salt tolerance of the clones, with similar weights being produced on non salt and salt media and similar chlorophyll in both salt sensitive and salt tolerant clones regardless of the medium in which they were grown. The production of proline is considered with regard to selection for differences in salt tolerance in vitro.

Factors affecting transformation efficiency of embryogenic callus of upland cotton (gossypium hirsutum) with Agrobacterium tumefaciens

Abstract: A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pΔgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 108 cells ml−1 (OD600=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l−1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.

Use of the temporary immersion bioreactor system (RITA®) for production of commercial Eucalyptus clones in Mondi Forests (SA)

Abstract: In order to optimise tissue culture systems and to meet production targets, Mondi Forests’ biotechnology programme has in the last 2 years concentrated efforts on the use of the RITA® temporary immersion bioreactor system. Protocols have been established for six Eucalyptus clones. Results indicate a four- to six-fold increase in yield, in half the time, with the RITA® system when compared with axillary bud proliferation on semi-solid media. Furthermore, plants produced from the RITA® system are hardier and acclimatize better, giving higher yields of hardened-off plants. The establishment of aseptic axillary shoots into the RITA® system is from shoots in the semi-solid system. Highest multiplication was achieved using 30-second flushes of medium every 10 min, starting with 50 shoots per vessel. The multiplication cycles in RITA® are between 14 and 18 days, compared with 25–28 days in a semi-solid system. There is minimal callus evident on the leaves and bases of the stems of plants in the RITA® system and, in addition, cold-tolerant plants have a greater rooting competence when compared with plants coming from the semi-solid system. Ex vitro rooting of RITA® – derived plantlets is substantially better than the plants from the semi-solid media.

Cycle characteristics in a temporary immersion bioreactor affect regeneration, morphology, water and mineral status of coffee (Coffea arabica) somatic embryos

Abstract: Mass regeneration of Coffea arabica L. somatic embryos using a temporary immersion bioreactor was improved by optimizing the immersion cycles, i.e. both the duration and the frequency of immersions. It was demonstrated that increasing the frequency of short immersions (1 min immersions every 24, 12 and 4 h) stimulated embryo production (480, 2,094 and 3,081 embryos/1-l bioreactor, respectively) and improved quality (60, 79 and 85 of torpedo shaped embryos, respectively). On the other hand, an increase in the immersion duration (1, 5 and 15 min) inhibited embryo regeneration (from 2,094 to 428 embryos per 1-l bioreactor) and negatively affected their morphological quality (from 79 to 49 torpedo-shaped embryos) and the conversion of embryos into plants (from 70 to 33). A 15 min immersion duration applied every 4 h produced hyperhydric symptoms in 90 of the embryos. Hyperhydric embryos were characterized by higher fresh weight and water content, more negative values for water potential and higher K+ content when compared to normal torpedo-shaped embryos. Micrographs showed structural problems in the globular stage, such as the existence of an irregular epidermis and an absence of reserves. Whatever the immersion cycle used, the somatic embryos exhibited water and mineral characteristics very different from those of their zygotic counterparts. The use of 1 min immersions every 4 h led to the production of the largest quantities of torpedo-shaped embryos without hyperhydricity that succeeded in regenerating plants (75 conversion).

Agrobacterium -mediated transformation of cotton (Gossypium hirsutum) using a heterologous bean chitinase gene

Abstract: Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l−1 kanamycin and 200 mg l−1 cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T1 plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T0 transformants and progeny plants (T1) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.

Effect of cytokinins on shoot regeneration from cotyledon and leaf segment of stem mustard (Brassica juncea var. tsatsai)

Abstract: Cotyledon and leaf segments of stem mustard (Brassica juncea var. tsatsai) were cultured on Murashige and Skoog medium supplemented with various concentrations of different cytokinins [6-benzyladenine (BA), N-(2-chloro-4-pyridyl)-n-phenylurea (CPPU), 6-furfurylaminopurine (KT) and thidiazuron (TDZ)] in combinations with different levels of α-naphthalene acetic acid (NAA). The shoot regeneration frequency of cotyledon and leaf segment was dependent on the kinds and concentrations of cytokinins used in the medium, while in most cases cotyledon gave high regeneration frequency than leaf segment. TDZ proved to be the best cytokinin to induce shoot from both cotyledon and leaf segments compared to BA, KT and CPPU. The highest frequency of shoot regeneration was 61.3–67.9 % in cotyledon and 40.7–52.4% in leaf segment respectively when 2.27 or 4.54 μM TDZ was combined with 5.37 μM NAA. Next to TDZ, CPPU was also very suitable to induce shoot formation both in cotyledon and leaf segment. When 1.61 μM CPPU was combined with 2.69 μM NAA, shoot regeneration frequency was 45.0% in cotyledon and 36.4% in leaf segment, respectively. It was also shown that KT and BA affected shoot regeneration from cotyledon and leaf segment, the shoot regeneration was greatly increased when NAA was added together with cytokinins. The efficient and reliable shoot regeneration system was developed in both cotyledon and leaf segments. This regeneration protocol may be applicable to the improvement of this crop by genetic engineering in the future.

Micropropagation of zedoary (Curcuma zedoaria Roscoe)--a valuable medicinal plant

Abstract: Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l−1 sucrose and 5 g l−1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l−1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l−1 BA, and 0.5 mg l−1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l−1 sucrose and 8 g l−1 agar, supplemented with 20 (v/v) CW and 2 mg l−1 NAA. More than 95 of the rooted plants were established in pots after hardening.

In vitro regeneration of Bambusa balcooa Roxb.: Factors affecting changes of morphogenetic competence in the axillary buds

Abstract: Axillary buds from field-grown culms of Bambusa balcooa were used as explants to induce multiple shoots in liquid Murashige and Skoog’s (MS) medium supplemented with 11.25 μM of 6-benzylaminopurine (BAP) and 4.5 μM kinetin (Kn). A clump of at least 3 shoots was used for root induction in half strength MS medium with 1.0 μM of 3-indolebutyric acid (IBA). Morphogenetic competence of the axillary buds varied widely in different months of two consecutive calendar years. The highest morphogenetic potentials were observed in October. The major problem encountered was presence of systemic fungal contaminants. Perhaps, rainfall positively contributed to induce morphogenetic competence. A moderately high phenolic content of the nodal explant was also detrimental for in vitro morphogenesis. The morphogenetic competence of B. balcooa correlated with the season in which the explants were excised from the natural stands. To the best of our knowledge this is the first report on in vitro regeneration of B. balcooa from mature field-grown axillary buds.

Rosmarinic acid production by Coleus forskohlii hairy root cultures

Abstract: Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.

Storability and germination of sodium alginate encapsulated somatic embryos derived from the vegetative shoot apices of mature Pinus patula trees

Abstract: Storability and germination of sodium alginate encapsulated somatic embryos derived from vegetative shoot apices of mature Pinus patula trees were tested on half strength DCR basal medium without growth regulators. The germination percentage of encapsulated somatic embryos was affected significantly by the concentration of sodium alginate and the duration of exposure to calcium chloride. Somatic embryos encapsulated with 2.5 sodium alginate dissolved in DCR basal salts gave significantly higher germination (89) than other treatments. Short (5 min) incubation of the alginate encapsulated embryos in calcium chloride solution proved to be the best encapsulation procedure and the embryos subsequently gave the highest germination (89). Synthetic seeds could be stored at 2 °C for 120 days without a reduction in germination as opposed to non-encapsulated somatic embryos which showed only 9 germination after 20 days at 2 °C. Germinated synthetic seeds produced normal plantlets. This study reports for the first time the storability of encapsulated somatic embryos generated from vegetative shoot apices of mature Pinus patula trees. This has potential for application in forestry.

Plant cell and biotechnology studies in Linum usitatissimum - A review

Abstract: The species Linum usitatissimum (flax/linseed) has been the focus of a great deal of both basic and applied research effort in plant cell and biotechnology studies in recent years. In this review we consider applications of the techniques of plant biotechnology in this species under several distinct headings. Plant cell and tissue regeneration strategies and applications are discussed, and the applications of the techniques of somatic embryogenesis, protoplast isolation, culture and fusion and cell suspension cultures in this species are described. A major area of study is the use of anther and microspore culture where clear advantages to breeding programmes could be applied. In addition, embryo and ovary culture studies have resulted in significant findings. The more recent technologies of gene transfer and expression by genetic transformation are reviewed, and a section on strategies for improvements in technological quality is also included. Finally we propose conclusions and future prospects for this ancient, but still highly relevant crop.

Successful Agrobacterium-Mediated Genetic Transformation of Maize Elite Inbred lines

Abstract: An efficient transformation system was developed for maize (Zea mays L.) elite inbred lines using Agrobacterium-mediated gene transfer by identifying important factors that affected transformation efficiency. The hypervirulent Agrobacterium tumefaciens strain EHA105 proved to be better than octopine LBA4404 and nopaline GV3101. Improved transformation efficiencies were obtained when immature embryos were inocubated with Agrobacterium suspension cells (A600 = 0.8) for 20 min in the presence of 0.1% (v/v) of a surfactant (Tween20) in the infection medium. Optimized cocultivation was performed in the acidic medium (pH5.4) at 22 °C in the dark for 3 days. Using the optimized system, we obtained 42 morphologically normal, independent transgenic plants in four maize elite inbred lines representing different genetic backgrounds. Most of them (about 85%) are fertile. The transformation frequency (the number of independent, PCR-positive transgenic plants per 100 embryos infected) ranged from 2.35 to 5.26%. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to three copies of the transgene were integrated into the maize nuclear genome. About 70% of the transgenic plants received a single insertion of the transgenes based on Southern analysis of 10 transformed events. T1 plants were analyzed and transmission of transgenes to the T1 generation in a Mendelian fashion was verified. This system should facilitate the introduction of agronomically important genes into commercial genotypes.

In vitro regeneration of the medicinal woody plant Phellodendron amurense Rupr. through excised leaves

Abstract: Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm2 sections of about 10-day-old leaves on MS medium enriched with 4.4 μM BAP and 1.0 μM NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 μM TDZ and 4.0 μM 2,4-D or 4.0 μM NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 μM BAP and 1.0 μM NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 μM IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.

In vitro induction of polyploidy in annatto ( Bixa orellana )

Abstract: The present work aims to establish a protocol for in vitro polyploidization using hypocotyl segments or cotyledonary nodes from in vitro grown annatto seedlings. The culture medium used to induce polyploidization was supplemented with MS salts, B5 vitamin complex, 100 mg l− myo-inositol, 3% (w/v) sucrose, 2.28 μ M ZEA and 0.30 μ M IAA (hypocotyl segments) or 4.56 μ M ZEA (cotyledonary nodes), 0.8% (w/v) agar, and different concentrations of microtubule depolymerising agents, namely colchicine (0, 25, 250 and 1250 μ M) and oryzalin (0, 5, 15 and 30 μ M). To determine the optimum duration of either colchicine or oryzalin treatment for the induction of tetraploids, explants were treated for 15 or 30 days on regeneration medium. High frequencies of polyploidy in regenerated shoots from cotyledonary nodes were achieved in culture medium supplemented with 15 μ M oryzalin, for 15 days. Ploidy determination was based on chromosome counting in metaphasic cells from apical buds, and in the number of pairs of heterochromatic markers on the biggest chromosome, as visualized in interphasic nuclei, detection being easier in the latter. Among the characteristics evaluated, the measurements based on stomata length, width, area and frequency enabled greater discrimination between diploid and polyploid regenerated shoots.

Mass propagation of conifer trees in liquid cultures — progress towards commercialization

Abstract: At Weyerhaeuser Company, somatic embryo production in liquid medium and manufactured seed delivery has been developed to reduce labor costs and increase efficiency of mass clonal propagation We have scaled-up embryonal suspensor masses (ESM) of Douglas-fir in 1-l liquid medium flasks Over 10,000 somatic embryos have been produced from a single flask We have cryostored ESM of over 700 genotypes in liquid nitrogen Somatic embryos of Douglas-fir have also been produced from liquid medium in a bioreactor Different types of bioreactors are required for embryo multiplication and for cotyledonary embryo development Over 250,000 Douglas-fir somatic seedlings from a large number of genotypes have been produced for clonal field tests

Micropropagation of Hagenia abyssinica: a multipurpose tree

Abstract: An in vitro propagation protocol has been developed for Hagenia abyssinica using original material from both juvenile and mature trees. Juvenile explants were obtained from seedlings, as well as shoots and meristems from 5 to 7-month-old greenhouse grown plants. Shoots collected from stem bases of five genotypes were used to establish cultures from mature trees. Explants of seedling origin were used to optimize the multiplication medium and growth regulators concentration. The best result was obtained from shoots subcultured on either MS or WPM medium supplemented with 4.4 μM BAP and 0.49 μM IBA. The initiated shoots from all the different explants were multiplied on these media. Rooting of shoots was achieved using MS medium containing macronutrients at one-third strength supplemented with 4.9 μM IBA. The shoots were kept in the dark for 4 days and transferred to medium of the same composition but containing 0.3% activated charcoal without growth regulators. Up to 100% rooting was achieved depending on genotype. Shoots multiplied on MS medium rooted better than those multiplied on WPM. Plantlets were transferred to pots containing a mixture of soil and perlite in a 2:1 ratio, respectively, and were maintained in the greenhouse. Increased irradiance reduced stem and leaf lengths and increased branch number of micropropagated plants.

The effect of magnetic field on Paulownia tissue cultures

Abstract: In this study, in vitro tissue cultures of Paulownia tomentosa and Paulownia fortunei were prepared and then exposed to a magnetic flow density of 2.9–4.8 mT and 1 m s−1 flow rate for a period of 0, 2.2, 6.6 and 19.8 s. The magnetic field (MF) increased the regeneration capability of Paulownia cultures and shortened the regeneration time. On the 28th day of culture, the positive effect of magnetic field on plant fresh weight, length, number of leaves and chlorophyll content in node explants of P. tomentosa and P. fortunei was observed. It was found that this effect varied with exposure time. When the cultures were exposed to a magnetic field with strength of 2.9–4.8 mT for 19.8 s, the regenerated P. tomentosa and P. fortunei plants dominated the control plants.

Optimisation of growing conditions for the apple rootstock M26 grown in RITA containers using temporary immersion principle

Abstract: The use of bioreactors may provide an efficient and economic tool for mass clonal propagation of plants if technical problems can be solved. In this paper, we report the results of experiments aimed at optimising conditions for apple rootstock M26 grown in RITA containers using the temporary immersion principle. We tested different types and sizes of explants, different concentrations of plant growth regulators (BAP, kinetin and IBA) in the multiplication and elongation phases, and medium exchange during the shoot elongation period. The results show that the higher concentrations of cytokinins were required during the shoot multiplication phase, while the lower concentrations were better during the shoot elongation phase. Hyperhydricity was increased with increasing concentration in of cytokinins during both shoot multiplication and shoot elongation phases. The best shoot production in terms of shoot number and shoot quality was obtained using 4.4 μmol BAP and 0.5 μmol IBA during the shoot multiplication phase and 1.1 μmol BAP and 0.25 μmol IBA during the shoot elongation phase. Medium exchange twice during the shoot elongation phase resulted in higher shoot production compared with no exchange of the medium. However, it also resulted in increased hyperhydricity. Immersion frequency of 16 times per day gave a higher multiplication rate and longer shoots than 8 times per day. The explant size of 0.5 cm or 1 cm resulted in a significantly higher shoot production rate compared with that of 1.5 cm, but shoot length and hyperhydricity were not affected by the explant size. Shoot cultures from the liquid media rooted normally in the RITA containers with more than 90% rooting and the rooted plantlets acclimatised well in the greenhouse.

Tissue culture propagation of Mongolian cherry (Prunus fruticosa) and Nanking cherry (Prunus tomentosa)

Abstract: In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 μM indole-3-butyric acid (IBA) and either 4.44 or 8.88 μM 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 μM BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 μM thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 μM) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.

Isolated microspore culture of Canadian 6× triticale cultivars

Abstract: Isolated microspore culture was conducted on nine Canadian triticale cultivars (X Triticosecale Wittmack) using two induction media developed for wheat, with or without 100 g l−1 Ficoll. Significant interactions were observed for the number of embryos and calluses induced, green and albino plantlets regenerated and fertility of green plants. Ficoll was beneficial in both media to increase numbers of embryos and green plants for all cultivars. Overall, medium NPB99 supplemented with ficoll provided the most suitable condition for most cultivars. AC Alta performed slightly better on CHB3 supplemented with Ficoll. Only cv. Wapiti was not amenable to androgenesis. The cultivars AC Certa, AC Copia, AC Alta, Sandro, Ultima, Frank, Pronghorn and Banjo produced respectively, 10, 9, 6, 5, 4, 3, 3 and 1 green plants per Petri dish (35,000 microspores), on their optimum treatment. Twenty-two percent of total lines produced were fertile, and considered doubled haploids. The application of isolated microspore culture to triticale, opens new possibilities in breeding triticale, for the utilization of in vitro selection and genetic engineering.

Rapid micropropagation of Curcuma longa using bud explants pre-cultured in thidiazuron-supplemented liquid medium

Abstract: Multiple shoots of Curcuma longa were induced by culture of bud explants for 1 week in Murashige and Skoog (MS) liquid medium supplemented with 72.64 μM thidiazuron (TDZ) prior to culture on MS gelled medium without growth regulator for 8 weeks. The regeneration rate was up to 11.4 ± 1.7 shoots/explant. Rooting was spontaneous and the regenerated plants were successfully transferred to soil. This protocol can be an alternative for rapid micropropagation of C. longa used for phytomedicine raw material production.

Putrescine enhances somatic embryogenesis and plant regeneration in upland cotton

Abstract: Improvement in somatic embryogenesis has been achieved in several cotton lines (Gossypium hirsutumL.) from the Georgia and Pee Dee germplasm with culture media containing various Putrescine concentrations. The best results were obtained with the α-naphthalene acetic acid (NAA)-based treatments, S15 g.05 NAA and EMMS2, as compared to the 2,4-dichlorophenoxyacetic acid (2,4-D)-based culture medium, EMMS4. Inclusion of 0.5 mg l−1 Putrescine improved somatic embryo (SE) induction for most treatments and lines tested. An 8-and 2-fold improvement was achieved in SE production on the EMMS2-0.5 Putrescine treatment as compared to EMMS2 alone for cotton lines PD 97019 and GA 98033, respectively. A significant increase in SE number (53-fold) was obtained with the addition of 0.5 mg l−1Putrescine to EMMS2 for PD 97021, which was essentially recalcitrant without Putrescine treatment. Conversion of SEs into plants was both genotype- and culture medium-dependent.