Showing papers in "Plant Cell Tissue and Organ Culture in 1999"
TL;DR: The soybean transformation procedure using the Agrobacterium-cotyledonary node transformation system and the bar gene as the selectable marker coupled with glufosinate as a selective agent is described.
Abstract: The soybean transformation procedure using the Agrobacterium-cotyledonary node transformation system and the bar gene as the selectable marker coupled with glufosinate as a selective agent is described. Soybean cotyledonary explants were derived from 5 day old seedlings and co-cultivated with Agrobacterium tumefaciens for 3 days. Explants were cultured on Gamborg's B5 medium supplemented with 1.67 mg l-1 BAP and glufosinate at levels of 3.3 mg l-1 or 5.0 mg l-1 for 4 weeks. After 4 weeks explants were subcultured to medium containing MS major and minor salts and B5 vitamins (MS/B5) supplemented with 1.0 mg l-1 zeatin-riboside, 0.5 mg l-1 GA3 and 0.1 mg l-1 IAA amended with 1.7 mg l-1 or 2.0 mg l-1 glufosinate. Elongated shoots were rooted on a MS/B5 rooting medium supplemented with 0.5 mg l-1 NAA without further glufosinate selection. Plantlets were transplanted to soil and grown to maturity and set seed in the greenhouse. Primary transformants and their progeny were characterized by Southern blot analysis and a leaf paint assay.
216 citations
TL;DR: Hypocotyls, cotyledonary nodes and primary leaves were used as explants to establish a regeneration protocol for Eucalyptus grandis × E. urophylla and plantlets were successfully transplanted to greenhouse conditions.
Abstract: Hypocotyls, cotyledons, cotyledonary nodes and primary leaves were used as explants to establish a regeneration protocol for Eucalyptus grandis × E. urophylla. These seedling-derived explants were incubated on a modified MS medium (SP medium), supplemented with 2.0 μM TDZ. After 1 month, the calluses obtained were transferred to SP medium containing different concentrations of BA and NAA or zeatin and NAA. Shoots were induced from these calluses at a high frequency. Shoot elongation was then stimulated on SP medium supplemented with BA, NAA and GA3 for 20 to 30 days. For rooting, 50 mm long shoots were cultivated on root induction medium containing IBA (2.5 μM) for different periods and then transferred to the same medium but without auxin, for 30 days. Plantlets were then successfully transplanted to greenhouse conditions.
108 citations
TL;DR: An Agrobacterium-mediated transformation protocol for Japonica rice, using conventional genetic vectors and explants pretreated with antinecrotic compounds, allowing satisfactory transformation performance without the need of super-binary vectors and hyperinfective A. tumefaciens strains.
Abstract: An Agrobacterium-mediated transformation protocol for Japonica rice (cv R321), using conventional genetic vectors and explants pretreated with antinecrotic compounds is presented We evaluated the effect of two compounds with known antioxidant activity (ascorbic acid and cysteine) and silver nitrate on the viability of stem sections taken from in vitro rice plantlets, and on their interaction with Agrobacterium tumefaciens (At 2260) containing a shuttle vector bearing the gusand bar genes After co-culture, calli formed on the callus-induction medium were supplemented with phosphinotricin and cefotaxime; putative transgenic plants were recovered on the regeneration medium after three months All recovered plants were challenged with the herbicide BASTA under greenhouse conditions, and some resistant individuals were analyzed using PCR and a histochemical GUS test Southern blot analysis of several R1 transgenic plants indicated the presence of at least two intact bar gene copies per genome Inheritance of the bar gene at the R2 generation was confirmed Antinecrotic pretreatment of the explants provides an adequate environment for the interaction of A tumefacienswith the plant cells, thus allowing satisfactory transformation performance without the need of super-binary vectors and hyperinfective A tumefaciens strains
99 citations
TL;DR: Nodal segments from micropropagated plants were used to evaluate the effect of growth regulators on the in vitro shoot proliferation and rooting of Lavandula vera DC, exhibiting a normal development, with high uniformity and no evidences of somaclonal variation.
Abstract: Nodal segments from micropropagated plants were used to evaluate the effect of growth regulators on the in vitro shoot proliferation and rooting of Lavandula vera DC. The highest multiplication rate was obtained using MS medium supplemented with 1.0 mg l-1 of TDZ (2.25 μM) or BA (2 μM). Hyperhydricity occurred at high concentrations of these growth regulators. Rooting of the plantlets was obtained in all the media evaluated. However, rooting rates and root growth increased with increased concentrations of NAA and the reduction of the salt strength of the media. The plantlets were successfully transferred to soil and grown to maturity, exhibiting a normal development, with high uniformity and no evidences of somaclonal variation.
88 citations
TL;DR: A temporary immersion system for potato microtuber production using 4-l vessels was designed in this article, which showed several advantages compared to solid cultures: three fold increase in shoot length, more internodes per plant and improved vigor.
Abstract: A temporary immersion system for potato microtuber production was designed using 4-l vessels. This culture technique showed several advantages compared to solid cultures: i.e., three fold increase in shoot length, more internodes per plant and improved vigor. In the tuber induction stage, microtubers can be induced at all plant nodes, indicating that the tuberization is not restricted to specific regions. For both cultivars tested, Desiree and Atlantic, an average of 3.1 and 2.8 tubers per single node cutting was achieved after 9 weeks in culture. The size and weight of the tubers were higher than on solid media. Scale up was performed with cv. Atlantic in 10-l polycarbonate flasks and 12 units were mounted containing 150 single nodal cuttings each. An average of 2.6 tubers per inoculated cutting was obtained, with 1.3 g fresh weight per microtuber. Temporary immersion is a valuable option for potato microtuber production, as well as for shoot production during the planting season.
87 citations
TL;DR: Callus cultures were initiated from axillary leaves, axillary shoots, hypocotyls, and root segments on Murashige and Skoog medium supplemented with 2,4-D and KN to establish axillary shoot base callus on MS medium containing BA.
Abstract: Callus cultures were initiated from axillary leaves, axillary shoots, hypocotyls, and root segments on Murashige and Skoog (MS) (1962) medium supplemented with 2,4-D (2 mg l−1) and KN (0.2 mg l−1). Shoots differentiated best from axillary shoot base callus on MS medium containing BA (2 mg l−1). Regenerated shoots rooted best on MS medium containing IBA (2 mg l−1) alone, and IBA (2 mg l−1) with IAA (2 mg l−1). Plantlets were transferred to pots containing sand and soil mixture, acclimatized in a culture room and afterwards transferred to the glasshouse.
85 citations
TL;DR: Number of leaves of plants exposed to 5 and 7 Gy was increased when compared with the non-irradiated control and shoot length of ‘Helwani’ and ‘Cabernet Franc’ was increased by 7 Gy irradiation.
Abstract: Shoot tips and single node explants of two rootstocks (R.99 and 3309) and two varieties (‘Helwani’ and ‘Cabernet Franc’) of grapevine cultured on DSD1 media for a period of 60 days, were irradiated with 0, 2, 5 and 7 Gy doses of gamma irradiation. Shoot length of ‘Helwani’ and ‘Cabernet Franc’ was increased by 7 Gy irradiation. The 5 Gy dose increased the number of roots in plants of the two rootstocks and ‘Helwani’. Root length of ‘Helwani’ and ‘Cabernet Franc’ at the 2 and 7 Gy doses were significantly higher than those of the non-irradiated control. A similar effect was noticed on R.99 rootstock subjected to 5 Gy. Five Gy also increased the dry weight of the R.99 rootstock, whereas 2 and 7 Gy had a similar effect on ‘Helwani’ and ‘Cabernet Franc’. Number of leaves of plants exposed to 5 and 7 Gy was increased when compared with the non-irradiated control.
79 citations
TL;DR: Embryogenic suspension cultures of oil palm allow mass propagation of somatic embryos; however regeneration rates are low and Histological observations have revealed that shoot development might be limited by the absence of a caulinary meristem.
Abstract: Embryogenic suspension cultures of oil palm ( Elaeis guineensis Jacq.) allow mass propagation of somatic embryos; however regeneration rates are low. Histological observations have revealed that shoot development might be limited by the absence of a caulinary meristem. The addition of 6-benzyladenine during development was found to induce shoot apex differentiation and thus increased germination rates, by up to 70%. However, multiple shoot formation was a consequence of a longer period of cytokinin supply during the development of the embryo. In contrast, a short period of culture on medium with 6-benzyladenine at the begining of embryo development was found to result in single shoot production.
73 citations
TL;DR: The effect of different carbohydrates was tested on early somatic embryogenesis of Hevea brasiliensis: maltose, fructose, glucose or glucose.
Abstract: The effect of different carbohydrates was tested on early somatic embryogenesis of Hevea brasiliensis. Sucrose was replaced with maltose, fructose or glucose. Somatic embryo production was significantly higher with maltose. With maltose, the initial yellow colour of the calli turned orange, and dry matter production after 28 days' culture was half that obtained with sucrose. Maltose also reduced the soluble sugar content by around 60%, whereas in the presence of sucrose, the soluble sugar content of calli increased. On maltose medium, embryonic cells were initiated after 2 weeks, whereas calli obtained on media containing sucrose mostly consisted of parenchymatous cells. The embryogenic cells rapidly formed into globular embryos. The origin of the `maltose' effect is discussed: is it carbohydrate stress or a signal molecule? The effect of maltose was checked on 6 Hevea callus lines maintained in the laboratory.
66 citations
TL;DR: There were significant differences among cultivars for potential of regeneration from immature embryo, and ‘Shahivandi’ a native durum wheat cultivar originating from western Iran was superior among the cultivars tested.
Abstract: Response of twenty eight cultivars of durum wheat (Triticum turgidum var durum) to immature embryo culture, callus production and in vitro salt tolerance was evaluated For assessment of cultivars to salt tolerance, growing morphogenic calli were exposed to different concentrations of NaCl (0, 03, 06, 09, 12, 15, 18 and 21% w/v) added to the culture medium during two subsequent subcultures (4 weeks each) Comparison of cultivars for callus induction from immature embryo was based on callus induction frequency and fresh weight growth of callus (FWG) While, for salt tolerance, the relative fresh weight growth (RFWG) and necrosis percent of callus were used There were significant differences among cultivars for potential of regeneration from immature embryo, and ‘Shahivandi’ a native durum wheat cultivar originating from western Iran was superior among the cultivars tested The FWG distinguished cultivars more than callus induction frequency did for callus induction evaluation Hence, a range of FWG from 123 to 1465 g was observed in ‘Mexical-75’ and ‘Omrabi-5’ cultivars, respectively Growing calli derived from cultivars ‘PI 40100’ and ‘Dipper-6’ showed superiority for tolerating salinity under in vitro conditions
66 citations
TL;DR: A transgenic system was developed for Artemisia annua L. via Agrobacterium rhizogenes-mediated transformation and found that the foreign FDS gene was expressed at the transcriptional level in three hairy root lines, which grew faster than the control hairy root line.
Abstract: A transgenic system was developed for Artemisia annua L. via Agrobacterium rhizogenes-mediated transformation. Using this system a cDNA encoding farnesyl diphosphate synthase (FDS) placed under a CaMV 35S promoter was transferred into Artemisia annua using Agrobacterium rhizogenes strain ATCC15834. Among the 150 hairy root lines established, 16 lines showed resistance to kanamycin (20 mg l-1). The intergration of FDS gene was confirmed by PCR and Southern blot analysis, and analysis of Northern blot revealed that the foreign FDS gene was expressed at the transcriptional level in three hairy root lines (F-1, F-24 and F-26 root line). F-1, F-24 and F-26 root lines grew faster than the control hairy root line. However, on the MS medium growth of F-26 root line was abnormal in that callus frequently formed. Analysis of artemisinin demonstrated that about 2–3 mg g-1 DW of artemisinin were then detected in the three root lines, which is about 3–4 times higher than that in the control hairy roots.
TL;DR: The economically important citrus crop plant, grapefruit, was genetically transformed, which has not been previously reported, by histochemical staining, PCR and Southern hybridization.
Abstract: The economically important citrus crop plant, grapefruit, was genetically transformed, which has not been previously reported. Seedling epicotyl tissue was infected with Agrobacterium tumefaciens carrying a T-DNA vector plasmid containing genes for the scorable marker, β-glucuronidase (GUS) and the selectable marker NPTII. Shoots that regenerated from epicotyl segments from nucellar seedlings in the presence of kanamycin were analyzed. Histochemical staining verified expression of the GUS reporter gene in 43.5% of the regenerated shoots examined; however, the majority were chimeric for GUS expression while 11.9% stained solid blue. Transgenic plants were recovered from regenerated shoots by direct rooting using a simple protocol. The presence of the transgene was confirmed in 25 regenerated grapefruit plants by histochemical staining, PCR and Southern hybridization.
TL;DR: Quantitative changes in the major monoterpene components (1,8-cineole, fenchol, borneol and camphor) and sesquiterpene content of plantlet oil, were observed in response to the effect of varying growth regulator concentration in the culture medium.
Abstract: Micropropagation of L. dentata L. through shoot-tips (ca. 5 mm) was achieved successfully. Micropropagated plantlets were cultured without plant regulators in the culture medium (control) or in media containing 0.1 mg l−1 of 6-benzyladenine (BA) and/or indole-3-butyric acid (IBA). These plantlets were examined for their essential oil content and composition in relation to growth rate and density and secretory or postsecretory stage of glandular hairs at five weeks of culture. The growth rates of these plantlets were not always correlated with their essential oil content (0.26%–0.65% v/w). However, all cultured plantlets showed a positive correlation between oil accumulation and the percentage of glandular hairs in secretory stage. Quantitative changes in the major monoterpene components (1,8-cineole, fenchol, borneol and camphor) and sesquiterpene content of plantlet oil, were also observed in response to the effect of varying growth regulator concentration in the culture medium. The influence of this effect on HMG-CoA reductase activity of cultured plantlets is also discussed.
TL;DR: In this paper, gametophytes of several species of ferns were mechanically triturated and the resulting homogenates cultured in vitro for propagation purposes, and differences in the time period from spore culture to sporophyte development were perceived between species.
Abstract: Gametophytes of several species of ferns were mechanically triturated and the resulting homogenates cultured in vitro for propagation purposes. Differences in the time period from spore culture to sporophyte development were perceivable between species. For those species with a fast life cycle and high sporophyte production such as Woodwardia virginica and Dryopteris affinis sp. affinis, homogenization of gametophytes can be considered to be excellent method for propagation, yielding hundreds of sporophytes in a short period of time. Sporophyte formation was inhibited in O. regalis by the succesive application of homogenization to gametophytes regenerated by this technique. The effect of the culture medium composition on fern production was also studied in O. regalis and P. ensiformis gametophytes. In these species, sporophyte formation increased when the gametophytes were cultured in a medium containing water+0.7% agar. Addition of sucrose inhibited gametophyte development and induced their necrosis. The 1/2 dilution of Murashige and Skoog basal medium, without sucrose, favoured leaf expansion in P. ensiformis sporophytes.
TL;DR: None of the tested carbon sources could considerably improve plant regeneration from M. `Giganteus' callus, but a higher number of plants tended to be regenerated per callus piece from filter-sterilized carbon sources.
Abstract: Different carbon sources, sterilized by autoclaving or filter-sterilization, were tested during induction, maintenance, and plant regeneration of embryogenic Miscanthus x ogiformis Honda `Giganteus' callus, derived from various explant types. Explants from small immature inflorescences, between 2.5 and 8 mm, produced more embryogenic callus than explants from shorter or longer inflorescences, shoot apices or leaf explants. On medium containing mannitol or sorbitol, only small amounts of callus were induced and no embryogenic callus was formed. Callus induction and embryogenic callus formation on shoot apices and immature inflorescences did not differ significantly between media containing sucrose, glucose, fructose, maltose or a mixture of glucose and fructose. However, callus induction and embryogenic callus formation from leaf explants were best on glucose. A higher percentage of leaf explants formed callus on autoclaved sucrose, as opposed to the other carbon sources where filter-sterilization in general resulted in a higher callus percentage. The growth rate of embryogenic callus was influenced both by carbon source and sterilization method when less than 1 g of callus was inoculated. None of the tested carbon sources could considerably improve plant regeneration from M. `Giganteus' callus, but a higher number of plants tended to be regenerated per callus piece from filter-sterilized carbon sources.
TL;DR: The shoot apices of both cotton and kenaf developed successfully without intervening callus formation, and no significant differences among cultivars were found.
Abstract: Cotton (Gossypium hirsutum L) and kenaf ( Hibiscus cannabinus L) belong to the Malvaceae family, and both are used as sources of fibers Shoot apices from vigorous seedlings aseptically germinated from 3 different cultivars of both cotton and kenaf were used in this study The cotton and kenaf shoot apex size was between 2–3 mm containing the meristem, unexpanded leaves, and a small portion of the cotyledon Shoot apices were placed on 18 different media comparing full and 1/2 strength Murashige and Skoog (1962) plus vitamins, and combinations of 0, 01, and 1 mg l-1 of naphthaleneacetic acid and 6-benzyladenine (BA) The shoot apices of both crops developed successfully without intervening callus formation, and no significant differences among cultivars were found An average of 58% of the cotton shoot apices initiated shoot and rooted in full strength Murashige and Skoog (1962) plus vitamins in 6 weeks For kenaf, an average of 92% of shoot apices initiated shoot and rooted in full strength Murashige and Skoog plus vitamins and 01 mg l-1 BA in 3 weeks All regenerated plants of both crops were phenotypically normal and set seeds
TL;DR: In this paper, three cultivars (cvs) of Gladiolus hybridus Hort, namely "Her Majesty", "Aldebaran", and "Bright Eye", were successfully micro-propagated.
Abstract: Three cultivars (cvs.) of Gladiolus hybridus Hort., namely ‘Her Majesty’, ‘Aldebaran’ and ‘Bright Eye’ were successfully micropropagated. The cultures were established using intact cormels or segments of cormels and inflorescence axes on Murashige and Skoog (1962; MS) medium. The response depended on media supplements; both callus formation or direct induction of shoot buds was observed. Shoot differentiation from callus could be obtained on MS medium containing 1.0 μM BA (6–benzyladenine) and 10.0 μM NAA (α-naphthalene acetic acid) in all three cultivars. The same could be achieved by giving a heat shock (HS; 50 °C, 1h) to callus cultures (in case of ‘Her Majesty’ and ‘Aldebaran’ only) maintained on the basal medium. In these two cultivars, high sucrose concentration (0.232, 0.290 or 0.348 M) also favoured growth and proliferation of shoot cultures on a plant growth regulator-free medium at 20 °C in comparison to the cultures kept at 25 °C. On the other hand, shoot cultures maintained on the basal medium at 25 °C containing normal (0.058 M, i.e., 2.0%, w/v) sucrose concentration responded similar to those maintained at 20 °C on a high sucrose medium; reduced response was observed on normal sucrose containing medium at 20 °C. Heat shock enhanced shoot proliferation in the cultures maintained on basal medium, but induced prolific rooting in shoot cultures, within 5 days of HS, on high sucrose (optimum 0.232 M) medium. While the number of roots increased at higher sucrose concentrations in the medium in case of cvs. ‘Her Majesty’ and ‘Aldebaran’, the same was found to be independent of sucrose concentration in cv. ‘Bright Eye’. Generally the rooted plants produced on high sucrose (0.232 M) medium in comparison to medium with normal sucrose concentration showed better survival (ca. 90% as against 40%) in the soil.
TL;DR: Photosynthetic ability of coffee plantlets in vitro was also significantly increased when grown on sugar-free medium with the high number of air exchanges and Florialite as a supporting material.
Abstract: Growth of coffee (Coffea arabusta) plantlets cultured in vitroas affected by sugar, types of supporting material and number of air exchanges of the vessel was investigated. Single node cuttings of in vitro coffee plantlets were cultured on half strength MS medium with or without 20 g l−1 sucrose. Two types of supporting material, agar and Florialite, and two levels of air exchange expressed by number of air exchanges per vessel, 0.2 and 2.3 h−1, were studied. At the end of a 40-day culture period, fresh weight, shoot length, root length and leaf area of plantlets when cultured on Florialite soaked in sugar-free medium and under the higher number of air exchanges were greater than those in sugar containing medium. Callus was observed at the shoot base of plantlets grown on agar medium containing sucrose. Photosynthetic ability of coffee plantlets in vitro was also significantly increased when grown on sugar-free medium with the high number of air exchanges and Florialite as a supporting material.
TL;DR: The results suggest that high-light increases carbon assimilation and growth although with a low investment in the photosynthetic apparatus.
Abstract: Grapevine plantlets multiplied in vitro were acclimatized at 40 or 90 μmol m−2 s−1 photon flux density for 12 or 16 h per day, respectively In the high-light regime a decrease in total chlorophyll and an increase in chlorophyll a/chlorophyll b ratio occurred However, at high-light intensity lower photosynthetic capacities and higher apparent photosynthesis were measured than at the low-light regime In leaves expanded during acclimatization, the light compensation point was higher in plantlets under high-light while quantum yield was higher in low-light conditions High-light also gave rise to an increase in carbohydrate concentration As a whole, the results suggest that high-light increases carbon assimilation and growth although with a low investment in the photosynthetic apparatus
TL;DR: In this article, a shoot regeneration was obtained from leaves of in vitro cultures of wild pear genotypes using leaves wounded by three cuts transversely to the mid-rib, a Quoirin and Lepoivre macro-salt composition, 250 mg l-1 cefotaxime and maintaining the explants in darkness for the first 30 days (induction phase), then transferring them to an auxin-free medium in light.
Abstract: Shoot regeneration was obtained from leaves of in vitro cultures of wild pear genotypes. The highest regeneration rates, ranging from 40% to 64%, depending on the genotype, were obtained using leaves wounded by three cuts transversely to the mid-rib, a Quoirin and Lepoivre macro-salt composition, 250 mg l-1 cefotaxime and maintaining the explants in darkness for the first 30 days (induction phase), then transferring them to an auxin-free medium in light (expression phase). A concentration of 8.8 μM BA induced the highest number of explants to produce adventitious shoots. TDZ was less effective than BA and induced hyperhydricity in regenerated shoots. The histological studies revealed that the regenerated shoots originated mainly from callus formed by epidermal and sub-epidermal cells and by cells of the vascular tissue. The regenerated shoots were micropropagated, rooted and transplanted to the soil.
TL;DR: Results showed that growth of cacti can be considerably accelerated by in vitro culture, and growth of Coryphantha minima grew 7-fold larger than plants cultured under similar ex vitro conditions.
Abstract: Unlike C-3 plants, cacti possess a crassulacean acid metabolism (CAM) physiology that can alter the pattern of carbon uptake and affect plant growth under artificial environmental conditions, especially in tissue culture In vitro-derived plantlets of Coryphantha minima grew 7-fold larger than plants cultured under similar ex vitro conditions Growth regulators incorporated into the culture media during shoot proliferation stage of micropropagation had a strong influence on this increased growth Other important factors that contributed to increased growth under in vitro conditions were high relative humidity and sugar in the culture medium An analysis of gas exchange and daily fluctuations of malic acid levels revealed an increase in net photosynthetic rate, in terms of carbon assimilation, by in vitro plants compared with that of ex vitro plants This stimulated photosynthesis in the presence of an external carbon source was unexpected but apparently true for cacti exhibiting CAM physiology Unlike CAM plants grown in ex vitro conditions, net CO2 uptake by in vitro-cultured cacti occurred continuously in the light as well as the dark Once regenerated, cacti were transferred to ex vitro conditions where the normal CAM pathway resumed with a concomitant reduction in growth and CO2 uptake These results showed that growth of cacti can be considerably accelerated by in vitro culture
TL;DR: Somatic embryos of myrtle were induced from mature zygotic embryos cultured in MS medium supplemented with several concentrations of 2,4-D or Picloram and germinating somatic embryos produced adventitious shoot buds which contributed to an increase in plantlet regeneration.
Abstract: Somatic embryos of myrtle (Myrtus communis L.) were induced from mature zygotic embryos cultured in MS medium supplemented with several concentrations of 2,4-D (2.26 μM – 18.98 μM) or Picloram (2.07 μM – 16.5 μM) combined with 0.087 M or 0.23 M sucrose. For all the concentrations of 2,4-D or Picloram tested, 0.087 M sucrose proved to be more effective than 0.23 M. The best frequencies of induction were obtained in a medium containing 2.26 μM 2,4-D in which 97.3% of the explants produced somatic embryos. Although most embryos were produced from the adaxial side of the cotyledons, some of them differentiated from the hypocotyl. Secondary somatic embryos were often seen arising from the periphery of the former somatic embryos. Somatic embryo development was not synchronous but practically all the embryos germinated well after being transferred to media containing GA3 (0.29, 0.58 and 1.44 μM) alone. When benzyladenine was combined with gibberellic acid, germinating somatic embryos produced adventitious shoot buds which contributed to an increase in plantlet regeneration. Histological observations suggested that somatic embryos arise from the upper surface of the cotyledons probably from peripheral cells. Polyphenol-rich cells were usually seen in association with meristematic-like cells from which somatic embryos originate or with earlier steps of somatic embryo differentiation. Regenerated plants were phenotypically normal, showing a diploid (2n = 22) set of chromosomes.
TL;DR: Cytogenetic analysis of the transformed root cultures of D. stramonium reported here shows a great chromosomic stability, which is not the case with normal root cultures.
Abstract: The karyotypic status of three root lines, transformed with Agrobacterium rhizogenes, versus a non-transformed root line of Datura stramonium, as well as grown plants (control), was investigated. In the transformed cultures, the karyotype as well as the number of chromosomes (2n = 24) remained the same as in the control plants. In the normal root line there were clear differences at a morphological level despite showing the same chromosomic number (2n = 24) at a high frequency. The existence of aneuploids (2n = 2n ± x) and tetraploids (2n = 4x = 48) was also observed in the latter. The frequency of chromosomic alterations was very low in all the cultures. Cytogenetic analysis of the transformed root cultures of D. stramonium reported here shows a great chromosomic stability, which is not the case with normal root cultures.
TL;DR: Rapid plant regeneration was achieved in callus cultures derived from leaf and stem explants of Plumbago zeylanica Linn and selection of variants that may be induced to widen the genetic base of the genus.
Abstract: Rapid plant regeneration was achieved in callus cultures derived from leaf and stem explants of Plumbago zeylanica Linn. on MS basal medium supplemented with 4.44 μM 6-BA, 1.42 μM IAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of growth regulators in the nutrient media. The leaf explants were more responsive (82.3%) than the stem explants on medium containing 1.42μM IAA in combination with 4.44 μM BA. The rate of regeneration was found to maintain the same level for 12 months without loss of vigour. Rooting of the differentiated shoots was achieved in media having 0.57 μM IAA with 2% (w/v) sucrose within 10 days of culture. Regenerated plantlets were transferred to soil which grew normally with a survival rate of 90%. This protocol may help in the conservation of the species and selection of variants that may be induced to widen the genetic base of the genus.
TL;DR: A method for the micropropagation of Lippia junelliana (Mold.) Tronc from shoot tips or nodal segments was developed and the shoot cultures showed a lower essential oil accumulation in comparison with parent plants.
Abstract: A method for the micropropagation of Lippia junelliana (Mold.) Tronc. from shoot tips or nodal segments was developed. Proliferating microshoot cultures were obtained by placing shoot tips or nodal segments on full strength Murashige and Skoog medium (MS) supplemented with 4.4 μM benzyladenine (BA) or 0.04 μM indolebutyric acid- (IBA) plus 4.4 μM BA. The rooting of shoots was better on full-strength MS medium without growth regulators. Rooted plantlets were successfully acclimatized to soil. The shoot cultures showed a lower essential oil accumulation in comparison with parent plants. Essential oil accumulation is closely related with growth and shows a negative correlation with shoot proliferation.
TL;DR: Embryogenic suspension cultures became increasingly disorganized over time, and this was associated with progressive loss of embryogenic potential.
Abstract: Embryogenic cultures were induced from immature avocado zygotic embryos representing different botanical races and complex hybrids. The optimum induction medium consisted of B5 major salts, MS minor salts, 0.4 mg l−1 thiamine HCl, 100 mg l−1 myo-inositol, 30 g l−1 sucrose, 0.41 μM picloram and 8 g l−1 TC agar. Somatic embryogenesis occurred directly from the explants on induction medium, and secondary embryos and proembryonic masses proliferated in liquid and on semisolid maintenance medium. Embryogenic culture maintainance was optimized in liquid, filter-sterilized MS medium, supplemented with 30–50 mg l−1 sucrose, 4 mg l−1 thiamine HCl and 0.41 μM picloram. Two types of embryogenic cultures were recognized: –genotypes that proliferated as proembryonic masses in the presence of auxin (PEM-type) and; –genotypes in which the heart stage and later stages of somatic embryos developed in the presence of auxin(SE-type). Embryogenic suspension cultures became increasingly disorganized over time, and this was associated with progressive loss of embryogenic potential.
TL;DR: Direct adventitious shoot regeneration from ovary explants of Crocus sativus L. was influenced by media components, incubation conditions, and age of the explant.
Abstract: Direct adventitious shoot regeneration from ovary explants of Crocus sativus L. was influenced by media components, incubation conditions, and age of the explant. The best response towards caulogenesis (28%) with highest shoot numbers per ovary was observed when full strength Murashige and Skoog (1962) medium was supplemented with naphthaleneacetic acid and benzyladenine. Incubation in the dark at 20 °C was beneficial for induction of shoot buds. Ovaries of different growth stages having stigmas of pale yellow, pale orange and bright orange regenerated a maximum mean number (3.8 – 4.2) of shoots per ovary. Further development of ovary-derived shoots was influenced by the composition of basal salts in the culture medium where full strength Murashige and Skoog salts gave the best response of those tested. Regenerated shoots produced normal photosynthetic leaves and corms.
TL;DR: Proembryogenic masses that were established in continuous culture and maintained on initiation medium for 17 months retained regenerability, though the embryo yield decreased over time.
Abstract: American chestnut (Castanea dentata (Marsh.) Borkh.) plants were regenerated from developing ovules through somatic embryogenesis. On an initiation medium containing 18.18 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine (BA), 25 out of 1,576 ovules were induced to form proembryogenic masses (PEMs). These PEMs were cultivated on a development medium for 4 weeks. Individual somatic embryos were then grown on a maturation medium for at least one month, until shoot meristems and radicles were developed. Both development and maturation media consisted of Gamborg's B-5 basal medium, 0.5 μM BA, and 0.5 μM α-naphthaleneacetic acid, but the former contained 20 g l−1 sucrose and the later contained 60 g l−1 sucrose. A range of 86 to 586 embryos per gram PEMs was observed beyond the cotyledonary stage. These embryos then germinated, resulting in plantlets with a 3.3% conversion rate. An additional 6.3% of the mature embryos produced shoots, which could also result in plantlets by rooting of microcuttings. Proembryogenic masses that were established in continuous culture and maintained on initiation medium for 17 months retained regenerability, though the embryo yield decreased over time. Twenty plantlets were acclimatized and grown in potting mix in a greenhouse. The largest 6 were transplanted, along with seedling controls, into a nursery bed in 1997. As of July, 1999, 4 out of the 6 were surviving.
TL;DR: Daidzein was demonstrated to be more concentrated in young parts (apexes) whereas coumestrol content was higher in older parts (brown tissues) compared to callus cultures from the same plant species, which displayed comparable concentrations.
Abstract: Eighteen transformed root cultures from 7 Psoralea plant species (Leguminosae) were established with the objective of producing daidzein and related flavonoids. All the 18 hairy root lines grew fast and had the same capacities for biomass production. Each of them produced daidzein as an intracellular secondary metabolite. The Lach5 hairy root line, obtained from P. lachnostachys, was a high producing line for daidzein and was further studied for biomass and flavonoid production. This root line showed exponential growth. Chitosan was used for elicitation purposes as well as for its permeabilizing effect. Little elicitation effect could be demonstrated and the metabolite release in the medium was weak (about 1%) and limited to the first 29 h after chitosan addition. Daidzein was demonstrated to be more concentrated in young parts (apexes) whereas coumestrol content was higher in older parts (brown tissues). Compared to callus cultures from the same plant species, hairy roots displayed comparable concentrations. However, high-producing lines were more frequently found with hairy roots (4 out of 18) than with callus cultures (4 out of 217)
TL;DR: A procedure has been developed to successfully micropropagate the IV-8 selection, an adult avocado rootstock, and survival rate during the acclimatization in the greenhouse was 70%.
Abstract: A procedure has been developed to successfully micropropagate the IV-8 selection, an adult avocado rootstock. Cultures were initiated from basal shoots obtained after pruning a tree back to ground level. Buds sprouted in Murashige and Skoog solid medium with macroelements at half strength and a 1.3 μM benzyladenine supplement. To induce proliferation, shoots were cultured for 2 weeks in liquid medium in a rollordrum with the Gamborg salt formulation and 1.3 μM benzyladenine, followed by 6 weeks in double phase conditions (solid medium with a layer of liquid medium on the top) using the same salt formulation and two different benzyladenine supplements, 2.8 μM in the solid phase and 0.4 μM in the liquid phase. Ninety percent of the shoots rooted after a 3-day culture in liquid medium in a rollordrum with MS macroelements at 0.3× and 4.9 μM indolebutyric acid, followed by transfer to solid medium in the absence of auxin but with 1 g l−1 activated charcoal. Survival rate during the acclimatization in the greenhouse was 70%.