Showing papers in "Plant Cell Tissue and Organ Culture in 2001"
TL;DR: Positive achievement recorded in other species seem to support the hypothesis that in vitro mutation induction has high potential also for fruit improvement, and the possible contribution of a well-pondered and coordinated use of the numerous mutation induction, mutant selection, and field validation procedures available to advances in fruit breeding is discussed.
Abstract: This review describes in vitro mutation induction methods in fruits and the in vitro selection procedures available for early screening. Results obtained through in vitro mutation techniques, including somaclonal variation, are reviewed and compared with the current achievements and future prospects of transgenic breeding. Plant improvement based on mutations, which change one or a few specific traits of a cultivar, can contribute to fruit improvement without altering the requirements of fruit industry. Induced mutations have well defined limitations in fruit breeding applications, but their possibilities may be expanded by the use of in vitro techniques. Tissue culture increases the efficiency of mutagenic treatments for variation induction, handling of large populations, use of ready selection methods, and rapid cloning of selected variants. Molecular techniques can provide a better understanding of the potential and limitations of mutation breeding e.g. molecular marker-assisted selection, which can lead to the early identification of useful variants. The relatively high number of research reports compared with the low number of cultivars released suggests that mutagenesis in combination with tissue culture is either ineffective or has yet to be exploited in fruits. Positive achievement recorded in other species seem to support the hypothesis that in vitro mutation induction has high potential also for fruit improvement. The possible contribution of a well-pondered and coordinated use of the numerous mutation induction, mutant selection, and field validation procedures available to advances in fruit breeding is discussed.
267 citations
TL;DR: It is hypothesised much of the variability expressed in microplants may be the consequence of, or related to, oxidative stress damage caused to the plant tissues during explant preparation, and in culture, due to media and environmental factors.
Abstract: A number of well defined problems in physiological, epigenetic and genetic quality are associated with the culture of plant cell, tissue and organs in vitro, namely, absence or loss of organogenic potential (recalcitrance), hyperhydricity (`vitrification') and somaclonal variation These broad terms are used to describe complex phenomena that are known to be genotype and environment dependent These phenomena affect the practical application of plant tissue culture in plant propagation and in plant genetic manipulation Here it is hypothesised much of the variability expressed in microplants may be the consequence of, or related to, oxidative stress damage caused to the plant tissues during explant preparation, and in culture, due to media and environmental factors The characteristics of these phenomena are described and causes discussed in terms of the known effects of oxidative stress on eukaryote genomes Parameters to characterise the phenomena are described and methods to remediate the causes proposed
264 citations
TL;DR: In the approximately 70 year-old history of induced mutations, there are many examples on the development of new and valuable alteration in plant characters significantly contributing to increased yield potential of specific crops as discussed by the authors.
Abstract: In the approximately 70 year-old history of induced mutations, there are many examples on the development of new and valuable alteration in plant characters significantly contributing to increased yield potential of specific crops. However, knowledge on the success of induced mutations in crop improvement among geneticists and breeders is usually limited to species of their interest. The present paper contains a comprehensive list of officially released mutant varieties, based on information from plant breeders. The number of mutant varieties officially released and recorded in the FAO/IAEA Mutant Varieties Database before the end of 2000 is 2,252. Almost half of these varieties have been released during the last 15 years. Considering a significant delay in the dissemination of information on newly released varieties and difficulties in the collection of such data, there has been a renaissance in the use of mutation techniques in crop improvement. At the demand of geneticists, plant breeders, and more recently molecular geneticists, for information on released mutant varieties of specific crops, the MVD was transferred to the web site of the FAO/IAEA Joint Division. The MVD will be available on our web pages early in 2001. (author)
204 citations
TL;DR: Efforts to improve disease or insect resistance of these cereal plants by genetic engineering using genes for PR-proteins also are discussed, and chromosomal location of the PR-protein genes indicates that members of the same family of PR- protein genes or sometimes even several families are clustered in the cereal genome, suggesting coordinate regulation.
Abstract: Pathogenesis-related proteins (PR-proteins) are induced in plants in response to attack by microbial or insect pests. They have been classified into several groups (PR-1 through PR-14 at present) based on their amino acid sequences and biochemical functions. Many of these proteins that have been purified from infected plants or seed extracts possess antifungal or insecticidal activity. Genes and cDNA clones for all classes of PR-proteins have been isolated from a variety of cereals. Some of these genes/cDNAs have been used to transform cereals. This review presents a summary of the PR-proteins and their genes characterized from rice, wheat, barley, sorghum and maize. Efforts to improve disease or insect resistance of these cereal plants by genetic engineering using genes for PR-proteins also are discussed. In many cases, the expression of the PR-proteins either singly or in combination appears to improve resistance to fungi or insects. In addition, chromosomal location of the PR-protein genes indicates that members of the same family of PR-protein genes or sometimes even several families of PR-protein genes often are clustered in the cereal genome, suggesting coordinate regulation. Some of these PR-protein genes map closely to quantitative traits loci. Some concerns regarding the use of genes encoding PR-proteins for genetic modification of cereals also are addressed.
155 citations
TL;DR: In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 4.9 μM IBA and almost 100% transplantation success in the field.
Abstract: A mass in vitro propagation system for Bacopa monniera (L) Wettst (Scrophulariaceae), a medicinally important plant, has been developed A range of cytokinins have been investigated for multiple shoot induction with node, internode and leaf explants Of the four cytokinins (6-benzyladenine, thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (68 μM) and 6-benzyladenine (89 μM) proved superior to other treatments Optimum adventitious shoot buds induction occurred at 68 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation However, subculture of leaf explants on medium containing 22 μM benzyladenine yielded a higher number (1291) of adventitious shoot buds by the end of third subculture The percentage shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles, facilitating their simultaneous harvest for rooting In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 49 μM IBA After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field
151 citations
TL;DR: The result indicates that variation at DNA level has occurred during in vitro culture and green shoot primordia were seen to differentiate from the surface of the callus.
Abstract: Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoog's basal medium (MS) supplemented with dicamba, picloram (2 mg l−1) or 1-naphthaleneacetic acid (NAA) (5 mg l−1) in combination with benzyladenine (BA) (0.5 mg l−1). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l−1) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l−1), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture.
147 citations
TL;DR: Only resistance against insects, and parthenocarpic fruit development have successfully been developed in eggplant using Agrobacterium tumefasciens transformation, however, some work on genetic engineering of eggplant for other biotic and abiotic stresses has recently been initiated.
Abstract: Eggplant (Solanum melongena L.), an economically important vegetable crop in many countries in Asia and Africa, often has insufficient levels of resistance to biotic and abiotic stresses. Genetic resources of eggplant have been assessed for resistance against its most serious diseases and pests (bacterial and fungal wilts, nematodes and shoot and fruit borer). Attempts at crossing eggplant with its wild relatives resulted in limited success due to sexual incompatibilities. However, the ability of eggplant to respond well in tissue culture, notably plant regeneration, has allowed the application of biotechnology, particularly the exploitation of somaclonal variation, haploidisation, somatic hybridisation and genetic transformation for gene transfer. Somaclonal variation has been used to obtain lines with increased resistance to salt and little leaf disease. Traits of resistance against bacterial and fungal wilts have successfully been introduced into the cultivated eggplant through somatic hybridisation. However, most somatic hybrids were sterile when the parental lines were distantly related. In contrast, the use of close relatives as fusion partners or highly asymmetric fusion resulted in the production of fertile hybrids with resistance traits and a morphology close to the cultivated eggplant, thus avoiding the series of backcrosses necessary for introgression of desired traits into eggplant. As far as molecular markers and genetic engineering are concerned, the information available for eggplant is very scanty. Two genetic linkage maps have been established by using RAPD and RFLP markers. In order to analyse the genetic relationships between eggplant and its relatives, some studies based on AFLP and ctDNA analyses have also been conducted. So far only resistance against insects, and parthenocarpic fruit development have successfully been developed in eggplant using Agrobacterium tumefasciens transformation. However, some work on genetic engineering of eggplant for other biotic and abiotic stresses has recently been initiated.
145 citations
TL;DR: A simple, rapid and effective system to regenerate Arabidopsis plants via direct somatic embryogenesis has been established and the regenerates showed normal morphological characteristics and were fertile.
Abstract: A simple, rapid and effective system to regenerate Arabidopsis plants via direct somatic embryogenesis has been established. Somatic embryogenesis was induced directly during culture of immature zygotic embryos. The frequency of somatic embryogenesis was strongly influenced by the stage of development of the explants. Explants in different developmental stages were cultured on B5 agar medium containing 5 μM 2,4-dichlorophenoxyacetic acid and the highest frequency (up to 90%) of somatic embryogenesis was observed in zygotic embryos with fully-developed cotyledons. The first somatic embryos developing directly from explant tissue were noticed after 8 days of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all six genotypes tested (Columbia, C-24, RLD, Wassilewskaja, Landsberg erecta and Wilna). Subculture of somatic embryos onto auxin-free medium resulted in their conversion into plants with an average frequency of 79.5%. The regenerates showed normal morphological characteristics and were fertile. All 56 analysed plants displayed a diploid number of chromosomes and two out of 96 (2.1%) tested plants carried a chlorophyll or embryo-lethal mutation.
145 citations
TL;DR: Findings demonstrate that the 2-C-methyl-D-erythritol 4-phosphate pathway, not the mevalonic acid pathway, is involved in the formation of isopentenyl diphosphate, which constitutes ring C of anthraquinones in the Rubiaceae.
Abstract: Plants and their derived cell and tissue cultures in the family Rubiaceae accumulate a number of anthraquinones. There are two main biosynthetic pathways leading to anthraquinones in higher plants: the polyketide pathway and the chorismate/o-succinylbenzoic acid pathway. The latter occurs in the Rubiaceae for the biosynthesis of Rubia type anthraquinones. In this pathway, ring A and B of the Rubia type anthraquinones are derived from shikimic acid, α-ketoglutarate via o-succinylbenzoate, whereas ring C is derived from isopentenyl diphosphate, a universal building block for all isoprenoids. At present, it is known that isopentenyl diphosphate is formed via the mevalonic acid pathway or the 2-C-methyl-D-erythritol 4-phosphate pathway. Recent findings demonstrate that the 2-C-methyl-D-erythritol 4-phosphate pathway, not the mevalonic acid pathway, is involved in the formation of isopentenyl diphosphate, which constitutes ring C of anthraquinones in the Rubiaceae. This review summarizes the latest results of studies on the biosynthetic pathways, the enzymology and regulation of anthraquinone biosynthesis, as well as aspects of the metabolic engineering. Furthermore, biochemical and molecular approaches in functional genomics, which facilitate elucidation of anthraquinone biosynthetic pathways, are briefly described.
132 citations
TL;DR: Axillary buds of field plants of Cunila galioides Benth were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growing, and repeated subcultures of shoot tips and single nodes enabled mass multiplication of shoots without any evidence of decline.
Abstract: Axillary buds of field plants of Cunila galioides Benth. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growing. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with 8.8 μM of benzyladenine. Repeated subcultures of shoot tips and single nodes at 4-week intervals for eight months on the above medium enabled mass multiplication of shoots without any evidence of decline. The best conditions for rooting were MS medium plus 0.5 to 2.5 μM of indolebutyric acid. The rooted plants were successfully transferred to soil, exhibiting a normal development.
127 citations
TL;DR: The current challenges for genetic engineering of plants will be to understand and control factors causing transgene silencing, instability and rearrangement, which are often seen in transgenic plants and highly undesirable in lines to be used for crop development.
Abstract: Genetic improvement of crops has traditionally been achieved through sexual hybridization between related species, which has resulted in numerous cultivars with high yields and superior agronomic performance. Conventional plant breeding, sometimes combined with classical cytogenetic techniques, continues to be the main method of cereal crop improvement. More recently, through the introduction of new tools of biotechnology, crossing barriers have been overcome, and genes from unrelated sources have become available to be introduced asexually into plants. Cereal crops were initially difficult to genetically engineer, mainly due to their recalcitrance to in vitro regeneration and their resistance to Agrobacterium infection. Systematic screening of cultivars and explant tissues for regeneration potential, development of various DNA delivery methods and optimization of gene expression cassettes have produced transformation protocols for the major cereals, although some elite cultivars still remain recalcitrant to transformation. Most of the transgenic cereals developed for commercial purpose exhibit herbicide and/or insect resistance; traits that are often controlled by a single gene. In recent years, more complex traits, such as dough functionality in wheat and nutritional quality of rice have been improved by the use of biotechnology. The current challenges for genetic engineering of plants will be to understand and control factors causing transgene silencing, instability and rearrangement, which are often seen in transgenic plants and highly undesirable in lines to be used for crop development. Further improvement of current cereal cultivars is expected to benefit greatly from information emerging from the areas of genomics, proteomics and bioinformatics.
TL;DR: Microrhizomes produced were of a wide range in size and readily regenerated when isolated and cultured in vitro on culture initiation medium or shoot multiplication medium and, under in vivo conditions, Plantlets developed from big microrhZomes grew faster.
Abstract: In vitro microrhizome production was obtained in turmeric (Curcuma longa Linn.). Freshly sprouted buds with small rhizome portions excised from stored mature rhizomes were cultured on semi-solid culture initiation medium — MS basal medium + 0.88M BAP (6-benzylaminopurine) + 0.92M kinetin + 5% coconut water + 2% sucrose + 0.5% agar — resulting in bud elongation. Multiple shoots were produced from these elongated buds by culturing in liquid shoot multiplication medium — MS basal medium + 2.2 MB AP +0 .92M kinetin + 5% coconut water + 2% sucrose — at 251C and 16-h light (at 11.7mol m 2 s 1 )/8-h dark cycles. Clumps of four to five multiple shoots/single shoots were used in various experiments. Cultures were incubated in the dark at 251C. Half strength MS basal medium supplemented with 80 g l 1 sucrose was found to be optimal for microrhizome production. Cytokinin BAP had an inhibitory effect on microrhizome production. At the highest concentration of BAP tried (35.2M) microrhizome production was totally inhibited. Microrhizome production depended on the size of the multiple shoots used. Microrhizomes produced were of a wide range in size (0.1‐2.0 g) and, readily regenerated when isolated and cultured in vitro on culture initiation medium or shoot multiplication medium. Under in vivo conditions, small (0.1‐0.4 g), medium (0.41‐0.8 g) and big (>0.81 g) microrhizomes regenerated. Plantlets developed from big microrhizomes grew faster.
TL;DR: A wheat regeneration system was developed using mature embryos and the highest embryogenic calli induction rate was obtained when 2,4-dichlorophenoxyacetic acid was suppressed after a 3–4 week induction period.
Abstract: A wheat regeneration system was developed using mature embryos. Embryos were removed from surface-sterilised mature caryopses (winter wheat Odeon cultivar and spring wheat Minaret cultivar) and ground to pieces through a sterile nylon mesh. The fragments were characterised by means of the image analysis technique. They were 500 μM mean diameter and most of them were elongated. They were used as explants to initiate embryogenic calli on solid medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid. The morphogenic pathway of the initiated calli was followed for a 40-day culture period. Active cellular division occurred within 24 hours of cultivation. Several hundred calli were produced from 100 fragmented embryos within 3 days. A 90% callus induction rate was achieved and proembryos appeared by the 8th day of culture. The highest embryogenic calli induction rate of 47% was obtained when 2,4-dichlorophenoxyacetic acid was suppressed after a 3–4 week induction period. Two regeneration methods were finally compared. A total of 513 plantlets were produced. The optimal protocol produced 25–30 plants per 100 embryos. This regeneration method may be suitable for transformation applications.
TL;DR: A 90% resource cost reduction in tissue culture of banana was achieved by replacing tissue culture grade sucrose and Gelrite in the medium with locally available commercial sugar and a starch/Gelrite mixture and by using sun light instead of artificial light.
Abstract: A 90% resource cost reduction in tissue culture of banana was achieved by replacing tissue culture grade sucrose and Gelrite in the medium with locally available commercial sugar and a starch/Gelrite mixture and by using sun light instead of artificial light. The micropropagation of Musa `Grande Naine' by shoot tip culture was used as model. Thirteen commercial sugars from different countries were tested. Best results were achieved using white and light brown sugars with low electrical conductivity. Sugars of cane or sugar beet origin were suitable. Starches of corn or potato could partially substitute for Gelrite and agar. In all experiments, micropropagation rates under natural light conditions were equal to or higher than under the controlled conditions of a growth room with PPFD of 65 μmol m−2 s−1 and a 16-h photoperiod. Plants were exposed to average PPFD levels of 58–96 μmol m−2 s−1 and photoperiods ranged from 8–16 hours.
TL;DR: To improve the regeneration efficiency of cassava in vitro, the effect of silver nitrate (AgNO3) on shoot organogenesis from somatic cotyledons was assessed and adding AgNO3 to the regeneration medium improved the regeneration frequency and reduced callus formation in all tested cultivars.
Abstract: To improve the regeneration efficiency of cassava (Manihot esculenta Crantz) in vitro, the effect of silver nitrate (AgNO3) on shoot organogenesis from somatic cotyledons was assessed Adding AgNO3 to the regeneration medium improved the regeneration frequency and reduced callus formation in all tested cultivars Both the extent of the response to and the optimum concentration of AgNO3 were cultivar dependent In the model cultivar MCol22, the use of AgNO3 at concentrations between 4 and 12 mg l−1 increased shoot organogenesis frequency and the number of shoot primordia per explant, the maximum effect being observed on a medium containing 12 mg l−1 AgNO3 At this concentration, the frequency of shoot organogenesis was enhanced from 60 to 90%, while callus formation decreased from 100 to 5% The highest shoot organogenesis rates were obtained by supplementing the medium with 2 and 1 mg l−1 AgNO3 in KU50 and Hanatee, respectively, while cultivar T5 showed an optimum response at 4 mg l−1 The shoots regenerated from explants cultured on a medium containing AgNO3 were more elongated than those cultured on a medium without AgNO3 The application of AgNO3 did not change the dose response of shoot organogenesis for the selective agents hygromycin and mannose
TL;DR: Overall, 3% sorbitol was found to be the best of the osmotic supplements tested, resulting in 51 ± 5% conversion frequency (mean ± SE), as compared to 4 ± 1% in the control.
Abstract: Polyethylene glycol 4000, mannitol and sorbitol were tested as supplements to a liquid Finer and Nagasawa medium-based histodifferentiation/maturation medium, FNL0S3, for soybean (Glycine max L. Merrill) somatic embryos of ‘Jack’ and F138 or ‘Fayette’. Significant differences were found among types and levels of osmotica for their influence on quantity of mature embryos recovered, and on germination (root and shoot emergence) and conversion of embryos into plants. Supplementation of FNL0S3 with 5% polyethylene glycol or 1.5% sorbitol improved germination frequencies without limiting embryo histodifferentiation. Supplementation with 3% sorbitol resulted in a 9-fold increase in germination frequencies and a 13-fold increase in conversion frequencies of ‘Fayette’ and ‘Jack’ embryos. However, these improvements were accompanied by a significant, 22% reduction in fresh weight of mature embryos. Overall, 3% sorbitol was found to be the best of the osmotic supplements tested, resulting in 51 5% conversion frequency (mean SE), as compared to 4 1% in the control. Supplementation of FNL0S3 with 3% mannitol did not improve embryo maturation. Abbreviations: FN ‐ Finer and Nagasawa (1988); H/M ‐ histodifferentiation/maturation; MS ‐ Murashige and Skoog (1962); PEG ‐ polyethylene glycol
TL;DR: The results taken as a whole suggest that 300 μmol m−2 s−1 is the upper threshold for acclimatization of chestnut although grapevine showed a better response than chestnut to an increase in light.
Abstract: This study reports the effects of light availability during the acclimatization phase on photosynthetic characteristics of micropropagated plantlets of grapevine (Vitis vinifera L.) and of a chestnut hybrid (Castanea sativa × C. crenata). The plantlets were acclimatized for 4 weeks (grapevine) or 6 weeks (chestnut), under two irradiance treatments, 150 and 300 μmol m−2 s−1 after in vitro phases at 50 μmol m−2 s−1. For both treatments and both species, leaves formed during acclimatization (so-called `new leaves') showed higher photosynthetic capacity than the leaves formed in vitro either under heterotrophic or during acclimatization (so-called `persistent leaves'), although lower than leaves of young potted plants (so-called `greenhouse leaves'). In grapevine, unlike chestnut, net photosynthesis and biomass production increased significantly with increased light availability. Several parameters associated with chlorophyll a fluorescence indicated photoinhibition symptoms in chestnut leaves growing at 300 μmol m−2 s−1. The results taken as a whole suggest that 300 μmol m−2 s−1 is the upper threshold for acclimatization of chestnut although grapevine showed a better response than chestnut to an increase in light.
TL;DR: A high frequency shoot organogenesis and plant establishment protocol has been developed for Coleus forskohlii from leaf derived callus and should be useful for conservation as well as mass propagation of this plant.
Abstract: A high frequency shoot organogenesis and plant establishment protocol has been developed for Coleus forskohlii from leaf derived callus. Optimal callus was developed from mature leaves on Murashige and Skoog (MS) medium supplemented with 2.4 μM kinetin alone. Shoots were regenerated from the callus on MS medium supplemented with 4.6 μM kinetin and 0.54 μM 1-naphthalene acetic acid. The highest rate of shoot multiplication was achieved at the sixth subculture and more than 150 shoots were produced per callus clump. Regenerated shootlets were rooted spontaneously on half-strength MS medium devoid of growth regulators. The in vitro raised plants were established successfully in soil. The amount of forskolin in in vitroraised plants and wild plants was estimated and found that they produce comparable quantity of forskolin. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this plant.
TL;DR: Calli were induced from five types of explants (embryo axes and cotyledons of mature zygotic embryos as well as leaflets, petioles and stems of seedlings) of A. mangium to achieve plant regeneration in callus cultures and plantlets grew up well in the green house.
Abstract: Plant regeneration of Acacia mangium was achieved through organogenesis in callus cultures. Calli were induced from five types of explants (embryo axes and cotyledons of mature zygotic embryos as well as leaflets, petioles and stems of seedlings) of A. mangium on MS (Murashige and Skoog, 1962) basal medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 13.95 μM kinetin (KT). Green or green purple compact nodules containing clusters of meristematic centers were induced in these calli after transfer to MS basal medium containing 1.14–22.75 μM thidiazuron (TDZ) and 1.43–2.86 μM indole-3-acetic acid (IAA). A combination of 4.55 μM TDZ and 1.43 μM IAA promoted the highest percentage of calli to form nodules, in 8–11% of calli derived from cotyledons, embryo axes, leaflets or petiole and in 4% of calli derived from stems. Twenty-two percent of the nodules formed adventitious shoots on MS basal medium containing 0.045 μM TDZ. Shoots were elongated on MS medium containing 0.045 μM TDZ supplemented with 7.22 μM gibberellic acid. The medium containing 10.75 μM NAA and 2.33 μM KT promoted rooting of 10% of the elongated shoots. Plantlets grew up well in the green house.
TL;DR: An effective protocol for regeneration from transformed calli resulted in a relatively high transformation frequency and co-culture and selection on regeneration medium was found to be better than callus medium and led to minimal media manipulations.
Abstract: The present study describes a simple and efficient protocol for plant regeneration from scutellar-derived embryogenic calli of an elite basmati indica rice (Oryza sativa L., cv Pusa Basmati 1) transformed with Agrobacterium. A supervirulent plasmid pTOK233 as well as a non-supervirulent plasmid pJB90GI containing β-glucuronidase (gus) and hygromycin phosphotransferase (hpt) chimeric genes were used to assess transformation and regeneration efficiency. The effects of some factors like the bacterial density and inclusion of sorbitol in the medium on the co-culture and transformation have been evaluated; the procedure for selection and regeneration from transformed calli was found to be critical. Furthermore, co-culture and selection on regeneration medium was found to be better than callus medium and led to minimal media manipulations. Regeneration medium supplemented with 3% maltose was found to be better for regeneration as compared to 3% sucrose. The transformed calli were subjected to three cycles of regeneration, thus converting a higher number of transformation events into regenerants. The selected calli as well as leaf sections and roots of the transformants were GUS positive. The stable integration of the transgene was confirmed by polymerase chain reaction and Southern blot analysis of the transformants. Interestingly, the presence of three additional vir genes in supervirulent plasmid pTOK233 was not required for transformation as transformation was successful with non-supervirulent plasmid pJB90GI, although the transformation and regeneration frequency was higher with the former. This effective protocol for regeneration from transformed calli resulted in a relatively high transformation frequency.
TL;DR: 20 ml static liquid medium with subculture periods at an interval six to eight weeks seems to be a cost and labour effective process as compared to the existing protocols involving solid media with sub culture periods at 4 weeks interval.
Abstract: The efficiency of thidiazuron in promoting tea shoot proliferation in liquid medium was evaluated. As compared to 6-benzyl adenine which induced hyperhydricity in the proliferated shoots in liquid medium, a progressive increase in the multiplication rate together with healthy shoot growth was achieved when thidiazuron (2.5 to 5.0 μM) was used instead of 6-benzyl adenine. Of the different liquid volumes compared in 250 ml Erlenmyer flasks, 20 ml was the most effective. While an increase in the multiplication rate coupled with normal but healthy shoots was observed under static and agitated conditions at this volume of liquid medium, hyperhydricity was induced in 50 ml liquid medium. Therefore, 20 ml static liquid medium with subculture periods at an interval six to eight weeks seems to be a cost and labour effective process as compared to the existing protocols involving solid media with subculture periods at 4 weeks interval.
TL;DR: The data point to the possibility of the use of A. rhizogenes, combined with regenerating Lycopersicon genotypes, in a very simple protocol, based on genetic capacity instead of special procedures for regeneration, to produce transgenic tomato plants expressing rol genes, as well as, genes present in binary vectors.
Abstract: The organogenetic competence of roots and Agrobacterium rhizogenes-induced hairy roots of twelve Lycopersicon genotypes was investigated. Both roots and hairy roots of L. peruvianum, L. chilense, L. hirsutum and two L. peruvianum-derived genotypes regenerated shoots after 2–4 weeks of incubation on zeatin-contained medium. Anatomical analysis showed that shoot regeneration in roots could be direct or indirect, depending on the genotype considered. Hairy roots showed considerable differences in their morphogenetic responses, when compared to the corresponding non-transgenic roots. The differences observed may reflect the influence of the introduced rol genes on hormonal metabolism/sensitivity. Hairy root-derived T0 plants had shortened internodes, wrinkled leaves and abundant root initiation, and most produced flowers and fruits with viable seeds. The hairy root syndrome was detected early in germinating T1 seedlings as a strong reduction in the hypocotyl length. Our data point to the possibility of the use of A. rhizogenes, combined with regenerating Lycopersicon genotypes, in a very simple protocol, based on genetic capacity instead of special procedures for regeneration, to produce transgenic tomato plants expressing rol genes, as well as, genes present in binary vectors. Furthermore, the regeneration differences observed in each Lycopersicon genotype and in transgenic materials expressing rol genes open the possibility for their use in the analysis of both the biochemical and the genetic background of organogenetic competence.
TL;DR: When analysing the endogenous hormone concentrations in the various callus types generated in each genotype, it was found that only differences in the free IAA concentrations were associated with variations in the morphogenic properties of the calluses.
Abstract: Immature zygotic embryos of two wheat (Triticum aestivum L.) genotypes, known for their different ability to generate embryogenic callus, were used as initial explants to establish callus cultures. Embryogenic and non-embryogenic calluses were obtained from the competent genotype (`Combi'), while only non-embryogenic callus was produced by the incompetent one (`Devon'). The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. The endogenous hormone concentrations (free indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins 1, 3 and 20 [GAs], zeatin/zeatin riboside [Z/ZR] and N6[Δ2-isopentenyl] adenine/ N6[Δ2-isopentenyl] adenosine; [iP/iPA]) of the initial explants were determined by means of radio-immunoassay and showed that the only difference was the higher concentration of ABA found in the embryos of the most competent genotype; whose embryos showed a reduced rate of precocious germination. When analysing the endogenous hormone concentrations in the various callus types generated in each genotype, it was found that only differences in the free IAA concentrations were associated with variations in the morphogenic properties of the calluses. Higher concentrations of endogenous free IAA were typical of embryogenic callus cultures. It was also observed that a loss in the embryogenic competence of the calluses, due to a prolonged time of culture, occurred concomitantly with a reduction in free IAA concentrations, practically to the concentrations found in the non-embryogenic calluses.
TL;DR: Processes expected to be automated for somatic embryo production are evaluation of embryogenic cultures, embryo development, harvesting, post-harvesting (pre-delivery) processes for enhancing conversion and preparing for delivery.
Abstract: Automation could enhance the use of somatic embryogenesis for micropropagation in two ways: as effective tools for research on somatic embryogenesis, and for improving the efficiency of embryo production by reducing labor costs. Processes expected to be automated for somatic embryo production are: (1) evaluation of embryogenic cultures, (2) embryo development, (3) harvesting, (4) post-harvesting (pre-delivery) processes for enhancing conversion and preparing for delivery. In this review, the techniques related to the automation of each process are introduced and discussed.
TL;DR: The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.
Abstract: Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.
TL;DR: An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv.
Abstract: An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and α-naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA3) and BA. Treatment with zeatin (22.8 μM) and BA (10.0 μM) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.
TL;DR: It is indicated that parthenogenesis induced in vitro by triploid pollen can be an alternative method to obtain haploid plantlet regeneration through gynogenesis in monoembryonic cultivars of Citrus.
Abstract: This study reports haploid plantlet regeneration through gynogenesis in Citrus clementina Hort ex Tan, cv Nules, induced by in vitro pollination with pollen grains of Oroblanco, a triploid cultivar of grapefruit It indicates that parthenogenesis induced in vitro by triploid pollen can be an alternative method to obtain haploids in monoembryonic cultivars of Citrus Actually, despite considerable efforts, androgenesis has not been yet successful in many genotypes of Citrus Pollination and mature stage of pistils was necessary for gynogenic embryo regeneration Fourteen haploid gynogenic embryos of Nules clementine were obtained Embryo conversion was high (857%) and embryos vigorously germinated producing twelve plantlets Chromosome counting, performed in the roots of obtained embryos, showed the haploid level (n=x=9) Isozyme analyses confirmed the expected homozygous state of embryos and plantlets
TL;DR: The effects of six microelements on the production of betalains and the growth of suspension cultures of Beta vulgaris were studied and no effects on cell growth and ratio of betacyanines to betaxanthines were observed.
Abstract: The effects of six microelements (Cu2+, Mn2+, Fe2+, Mo2+, Zn2+, Co2+) on the production of betalains and the growth of suspension cultures of Beta vulgaris were studied. The increase of Co2+ from 1–5 μM resulted in a 60% increment on the production of betalains. A positive effect of this divalent ion was only accomplished when it was added at the beginning of the culture. This was related to a doubling in the specific betalains production rate compared to B5 control medium. No effects on cell growth and ratio of betacyanines to betaxanthines were observed. Mo2+, Fe2+ and Cu2+ presented a positive but less marked effect, while the increase of Mn2+ did not show effects on the production of betalains compared to B5 control medium.
TL;DR: In vitro mutagenesis of multicellular meristems of Musa spp.
Abstract: In vitro mutagenesis of multicellular meristems of Musa spp. leads to a high degree of chimerism. Repeated vegetative propagation must be carried out to dissociate chimeras but the minimum number of cycles required is unknown. In general, mutated cells are difficult to monitor but mutations which result in a change in genome number are an exception. We simulated this case by colchicine treatment, followed by flow cytometric analysis. Colchicine treatment induced ploidy chimerism (mixoploidy), and chimera dissociation was assessed using three different propagation systems (shoot-tip culture technique - ST, multi-apexing culture technique - MA and corm slice culture technique - CS). The average percentage of cytochimeras was reduced from 100% to 36% after three subcultures using shoot-tip culture, from 100% to 24% when propagating by the corm slice culture technique and from 100% to 8% after the same number of subcultures using the multi-apexing technique. All propagation systems failed to eliminate chimerism completely. Factors that may influence chimera dissociation in vitro are discussed.
TL;DR: Optimize techniques of meristem culture have supplemented the culture indexing methods in commercial greenhouse production resulting in availability of large-scale pathogen indexed planting material, thereby providing a valuable resource to the horticultural industry.
Abstract: Recent advances in the development of protocols for in vitro culture and genetic manipulation have provided new avenues for the development of novel varieties of Pelargonium and for use as model systems for investigating the factors controlling plant morphogenesis. Optimized techniques of meristem culture have supplemented the culture indexing methods in commercial greenhouse production resulting in availability of large-scale pathogen indexed planting material. Currently, technologies are available for the mass in vitro propagation of F1 hybrid Pelargonium through both organogenesis and somatic embryogenesis. The somatic embryogenesis model system has allowed researchers to identify critical factors controlling plant morphogenesis in vitro such as regulation of regeneration by growth regulators, choice of explant and characterization of induction and expression phases of morphogenesis in Pelargonium. Also, optimization of technologies for genetic transformation of Pelargonium opened up the possibilities for developing genotypes with novel characters, including resistance to some of the major diseases. Finally, the development of regeneration systems for Pelargonium spp. has facilitated conventional crop improvement programs, thereby providing a valuable resource to the horticultural industry.