Medium Lacking Plant Growth Regulators Research Articles

Regeneration of transgenic plants by Agrobacterium-mediated transformation of Quercus ilex L. somatic embryos with the gene CsTL1

Here we describe for the first time a protocol for the genetic transformation of the holm oak Quercus ilex. Holm oak populations are seriously affected by a disorder known as oak decline syndrome. However, tolerant plants can be obtained by overexpression of pathogenesis-related (PR) proteins. The aim of the present study was to define a protocol for transforming somatic embryos (SEs) of holm oak with the gene CsTL1 encoding a chestnut thaumatin-like protein (PR protein). Lethal concentrations of kanamycin (kan) were determined in previous studies, in which SE formation was found to be inhibited by kan concentrations of 100 mg/L and above. Genetic transformation was only achieved when target explants were pre-cultured for one or 2 weeks, with a transformation efficiency of 2%. Transformation was also affected by genotype and was only successful in 2 of the 3 embryogenic lines evaluated. A total of 11 transformed lines (10 corresponding to line Q8 and 1 to line Q10–16) were obtained, all of which were maintained by secondary embryogenesis on proliferation medium (Schenk and Hildebrandt medium lacking plant growth regulators). The transgenic embryogenic lines were successfully cryopreserved by a vitrification-based procedure, thus enabling the lines to be preserved while tolerance tests were performed. The presence of CsTL1 in genomic DNA was confirmed by PCR, and expression of the gene was determined by qPCR. CsTL1 expression was up to 5.93 times higher in transgenic lines than in the corresponding untransformed line. Transgenic plants were produced from transformed SEs, with embryo conversion rates ranging from 2.8 to 66.7%.

Evaluation of plant regeneration ability of different explants and establishment of an efficient regeneration system using immature seeds in Actinidia kolomikta, a cold-hardy kiwifruit relative

Efficient protocols for plant regeneration systems are useful for genetically improving Actinidia species using biotechnological approaches such as somatic hybridization, gene transfer, and in vitro chromosome doubling. In this study, we used diploid Actinidia kolomikta (Maxim. & Rupr.) Maxim., a cold-hardy kiwifruit relative, to establish a plant regeneration system. Immature seeds, leaves, petioles, stems, and roots were examined to evaluate callus induction and plant regeneration ability. As a result, all explants on Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin exhibited callus formation at different frequencies varying from 42.4% to 100%. Among them, root-derived and immature seed-derived calli exhibited high shoot regeneration ability at 41.7% and 9.7%, respectively. Immature seed-derived callus produced a higher number of shoots and showed vigorous growth. Therefore, we used immature seed-derived callus to characterize the regeneration process and evaluated the regenerated plants. Cultured immature seeds exhibited two types of calli: compact (white, compact, and unable to regenerate) and soft (white, soft, composed of easily separable mixed granular and nodular structures). When the soft callus was transferred to MS medium with zeatin, the nodular structures developed adventitious shoots, and these shoots developed many adventitious roots on half-strength MS medium lacking plant growth regulators. Flow cytometric analysis revealed that all plantlets obtained were diploid, and thus, appeared to be of embryonic origin. These plantlets were easily acclimatized and were without any morphological abnormalities. The immature seed-derived soft callus lines, thus, may be valuable as an efficient plant regeneration system of this species.

Endosperm-derived triploid plant regeneration in diploid Actinidia kolomikta, a cold-hardy kiwifruit relative

Endosperm culture is a unique technique for producing a triploid plant from diploid plants. In the present study, endosperm of a diploid cold-hardy kiwifruit relative, Actinidia kolomikta (Maxim. & Rupr.) Maxim., was cultured to regenerate triploid plants and expand ploidy variation in this mostly diploid species. Endosperm derived from fruits harvested 6–10 weeks after flowering (WAF) was used for the initial explants to examine the effect of plant growth regulators on callus formation and organogenesis. The endosperm-derived calli induced in MS medium with above 1.0mgL−1 of 2,4-dichlorophenoxyacetic acid (2,4-D) were soft, small, and translucent, and no morphogenic response was observed. Conversely, the endosperm-derived yellow-green calli induced in the medium with 2,4-D (0.1mgL−1) plus kinetin (1.0, 2.0, and 5.0mgL−1) were hard, compact, fast growing, and showed adventitious shoot primordia. These primordia elongated into shoots when the calli were transferred to MS medium supplemented with zeatin (1.0, 2.0, and 4.0mgL−1) under continuous light conditions. The shoots developed roots on half-strength MS medium lacking plant growth regulators. In total, 14 plants were obtained. Flow cytometry and chromosomal analysis confirmed that the regenerated plants were triploid, suggesting that the plant tissue originated from the endosperm. This study reveals that the endosperm of A. kolomikta has plant regeneration abilities and that the regenerated plants have the same ploidy level as the endosperm (triploid). These results may be of use for ploidy manipulation or polyploidy breeding in other species in the genus Actinidia.

Efficient in vitro direct shoot organogenesis from seedling derived split node explants of maize (Zea mays L.).

Maize is one of the important cereal crops around the world. An efficient and reproducible regeneration protocol via direct organogenesis has been established using split nodes as ex-plants derived from 7 to 10 day old in vitro grown seedlings. Surface sterilized maize seeds were germinated on MS medium lacking plant growth regulators. Nodal sections of 7–10 day old seedlings were isolated, split longitudinally into two halves and cultured on regeneration medium containing different concentrations of 6-benzyladenine (2.20, 4.40, 6.60, 8.80, 11.0 and 13.2 μM) or kinetin (2.32, 4.65, 6.97, 9.29, 11.6 and 13.9 μM). Inclusion of 8.80 μM BA into MS supplemented medium triggered a high frequency of regeneration response from split node explants with a maximum number of shoots (12.0 ± 1.15) and the highest shoot length (3.0 ± 0.73) was obtained directly (without an intervening callus phase) within 4 weeks of culture. Further shoot elongation was achieved on medium containing 4.40 μM BA. The elongated micro shoots were rooted on MS medium fortified with 1.97 μM indole-3-butyric acid. The regenerated plantlets with roots were successfully hardened on earthen pots after proper acclimatization under greenhouse conditions. This new efficient regeneration method provides a solid foundation for genetic manipulation of maize for biotic and abiotic stresses and to enhance the nutritional values.

Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees.

A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor.

How Do Rare Earth Elements (Lanthanoids) Affect Root Development and Protocorm-Like Body Formation in Hybrid CYMBIDIUM?

Abstract Only few studies in the plant tissue culture literature have examined the impact of lanthanoids, or rare earth elements, on in vitro plant organogenesis. In this study, using a model plant, hybrid Cymbidium Twilight Moon ‘Day Light’, the impact of six lanthanoids (lanthanum (III) nitrate hexahydrate (La(NO3)3 · 6H2O), cerium (III) nitrate hexahydrate (Ce(NO3)3 · 6H2O), neodymium (III) nitrate hexahydrate (Nd(NO3)3 · 6H2O), praseodymium (III) nitrate hexahydrate (Pr(NO3)3 · 6H2O), samarium (III) nitrate hexahydrate (Sm(NO3)3 · 6H2O), gadolinium (III) nitrate hexahydrate (Gd(NO3)3 · 6H2O) on new protocorm-like body (neo-PLB) formation on Teixeira Cymbidium (TC) medium was examined. 0 (control), 1, 2, 4 and 8 mg·dm-3 of each lanthanoid was tested. All lanthanoids could produce more neo-PLBs and neo-PLB fresh weight than TC medium lacking plant growth regulators (PGRs), suggesting some PGR-like ability of lanthanoids, although PLB-related traits (percentage of half-PLBs forming neo-PLBs; number of neo-PLBs formed per half-PLB; fresh weight of half-PLB + neo-PLBs) was always significantly lower than TC medium containing PGRs. Except for Gd, all other lanthanoids had no negative impact on the number of new leaves from neo-PLB-derived shoots, but all lanthanoids showed a significantly lower plant height, shoot fresh weight and shoot dry weight and, in most cases, SPAD (chlorophyll content) value. In addition, using the same concentration of the six lanthanoids, the ability to fortify root formation of neo-PLB-derived plantlets was also assessed. Except for Sm, all other lanthanoids significantly increased the number of roots, root fresh and dry weight.

Embryogenic suspensions of adult cork oak: the first step towards mass propagation

Protocols have been established to clone adult cork oak trees by somatic embryogenesis using semisolid medium. However, for economically viable mass propagation, embryogenic cultures in liquid medium need to be developed. In this study, suspension cultures were initiated from embryo clusters obtained by secondary embryogenesis on a gelled medium lacking plant growth regulators. After 6 days of culture, these embryo clusters generated high cell density suspensions that also contained small organized structures (embryos and embryogenic clumps). As the culture duration increased, tissue necrosis and fewer embryogenic structures were observed and the establishment of suspension cultures failed. An alternative method was found adequate for initiation of embryogenic suspensions: embryo clusters from gelled medium were briefly shaken in liquid medium and detached cells and embryogenic masses of 41–800 μm were used as inoculum. Maintenance of embryogenic suspensions was achieved using a low-density inoculum (43 mg l−1) by subculturing four embryogenic clumps of 0.8–1.2 mm per 70 ml of medium. Proliferation ability was maintained for almost 1 year through ten consecutive subcultures. The initiation and maintenance protocols first developed for a single genotype were effective when tested on 11 cork oak genotypes.

Regeneration of daylily (Hemerocallis) from young leaf segments

Calli were initiated from leaf segments (~0.5 × 0.5 cm) of daylily incubated on Murashige and Skoog (MS) (1962) medium supplemented with 2,4-dichlorophenoxyacetic acid (2.4-D), and either benzyladenine (BA) or thidiazuron (TDZ). The highest frequency of callus induction was observed on medium with 6.79 μM 2,4-D plus either 4.55 or 6.81 μm TDZ. A period of callus maintenance on medium containing 5.37 μM naphthaleneacetic acid (NAA) plus 2.22 or 4.44 μM BA was necessary following induction to improve the quality of the callus, and significantly increase the frequency of embryogenic-like callus formation and shoot regeneration once calli were transferred to light. Over 70% of the regenerated shoots produced roots on ½ strength MS medium lacking plant growth regulators. The regenerated plantlets were successfully transferred into soil and acclimatized in growth rooms. This is the first report showing that leaf segments can be used for daylily regeneration.

Efficient genetic transformation of Impatiens hawkerii Bull. (Balsamiaceae) using agrobacterium rhizogenes

Transformation of Impatiens hawkerii Bull. mediated by Agrobacterium rhizogenes strain A4M70GUS was studied. Hairy roots developed 10 days after inoculation were excised from the shoot explants and transferred onto Murashige and Skoog's (MS) basal medium lacking plant growth regulators. More than 20 hairy root clones were established and eight of them were further analyzed. Each clone differed significantly from the others in growth capacity and lateral branching. Clone C2 showed the highest biomass (20.6 g L-1) as well as the highest number of lateral roots (37 ? 2.2). The transgenic nature of the established hairy root clones was confirmed by GUS assay and PCR analysis. In conclusion, hairy roots were developed for the first time in I. hawkerii Bull., and transgenic hairy root clones showed a distinct morphological nature and growth patterns.

Embryogenic cell lines of Juniperus communis; easy establishment and embryo maturation, limited germination

Several coniferous species belonging to the Pinaceae family can be propagated via somatic embryogenesis, while species belonging to the Cupressaceae family cannot. The aim of this study was to identify possibilities and limitations with somatic embryogenesis in Cupressaceae. Juniperus communis was chosen as model species. We show that a high initiation frequency of embryogenic cell lines can be established from intact megagametophytes at the time when intensive cleavage polyembryogeny takes place. The embryogenic cell lines proliferate fast on medium lacking plant growth regulators. Early somatic embryos develop after transfer to medium with decreased content of nitrogen and calcium. The early embryos mature after exposure to abscisic acid. Mature cotyledonary embryos germinate after partial desiccation. A high proportion, over 40%, of the germinating embryos retain the embryogenic potential in the basal part, resulting in development of new embryogenic tissue.

Enhancement of banana plant regeneration by incorporating a liquid-based embryo development medium for embryogenic cell suspension

SummaryRecovering plants from embryogenic cell suspensions is a complex process starting with somatic embryo development which is synchronised on medium lacking plant growth regulators, followed by differentiation and regeneration of plants. An improved method for high frequency recovery of banana plants was investigated by incorporating a liquid-based, embryo-development medium. The highest regeneration rate (approx. 32,000 plants ml–1 settled cell volume) was obtained using this liquid protocol. This is one of the highest scores recorded among published data on plant recovery in Musa spp. A detailed, comparative histological study has been carried out on somatic embryos developed in semi-solid or liquid-based media. Most mature embryos developed from the liquid protocol enabled differentiation and regeneration within 4 months. Over 3,000 plants are currently under field observation to establish the rate of somaclonal variation.

AGROBACTERIUM-MEDIATED PRODUCTION OF TRANSGENIC PLANTS IN TRICYRTIS HIRTA(LILIACEAE)

A system for producing transgenic plants has been developed for the Liliaceous geophyte Tricyrtis hirta via Agrobacterium-mediated transformation. Tepal-derived embryogenic calluses were co-cultivated with A. tumefaciens EHA101/pIG121Hm, which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing β-glucuronidase (GUS-intron) genes in the T-DNA region. The duration of co-cultivation and acetosyringone (AS) treatment during co-cultivation affected the frequency of transient expression of the GUS gene: the best result was obtained when embryogenic calluses were co-cultivated for 7 days in the presence of 50 mg l -1 AS. Following co-cultivation, the calluses were transferred to a medium lacking plant growth regulators (PGRs) but containing 40 mg l -1 hygromycin and 300 mg l -1 cefotaxime, on which several hygromycin-resistant (Hyg r ) somatic embryos were produced 8 weeks after transfer. These embryos were transferred to the same medium but containing lower concentrations of antibiotics (20 mg l -1 hygromycin and 100 mg l -1 cefotaxime) to promote germination. Finally, Hyg r embryo-derived plantlets were established on a medium without both PGRs and antibiotics. Most of them were verified to be stable transformants by GUS histochemical assay and PCR analysis.

Cytokinin-Mediated Axillary Shoot Formation in Pinus heldreichii

Shoot formation from seedling explants of Pinus heldreichii was induced by pulse treatment with benzyladenine at different concentration, followed by culture on medium lacking plant growth regulators. After treatment with 222 μM benzyladenine (BA) an average of 4.6 shoots per explant was obtained. Shoots, detached from explants, rooted with a frequency of about 10 %, and rooted plantlets were successfully transferred to ex vitro conditions.

In vitro regeneration of Ensete ventricosum from zygotic embryos of stored seeds

Plant regeneration through in vitro germination and the production of adventitious shoots from zygotic embryos was investigated using stored seeds of two enset genotypes (Mariya and Oniya), as germination of intact seeds could not be achieved. The seeds were decontaminated before and after water pretreatment and the embryos cultured on MS medium. Germination was studied with vertical and horizontal embryo orientations, on three media compositions: MS medium, MS with 0.5 mg l − 1 BA + 0.2 mg l − 1 lAAand MS with 0.5 mg l − 1 BA + 0.2 mg l − 1 2,4-D. Treating seeds of the Oniya clone with distilled water before decontamination and excision of embryos caused extensive contamination of cultured embryos. Decontamination without water pretreatment showed only about 5% contamination. Contamination was low with Mariya but no germination occurred. Horizontal embryo orientation resulted in better germination for Oniya. Supplementing MS medium with BA and IAA improved germination of the excised embryos (44%) and promoted formation of multiple shoots and simultaneous initiation of callus in some embryos. Subculturing the callus on the same medium composition (MS with BA + IAA) resulted in more and earlier initiation and proliferation of organogenic callus in the dark than other media compositions. Adventitious shoots were regenerated in the light on MS medium lacking plant growth regulators. Proliferation of a watery and friable callus was better when 2,4-D (0.2 mg l − 1 ) was added in the presence of BA and IAA during subculturing.

Horticultural characterization of Angelonia salicariifolia plants transformed with wild-type strains of Agrobacterium rhizogenes.

Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines. All of these selected lines produced mikimopine. Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs. The transgenic nature of regenerated plants was confirmed by Southern hybridization. Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse. Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation. These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves. There were no apparent alterations observed in the number, shape, and size of the flowers. Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining. These results indicate the possibility of applying A. rhizogenes-mediated transformation for introducing a dwarf trait into angelonia.

Efficient plant regeneration from cell cultures of ornamental statice, Limonium sinuatum mill.

A plant regeneration system from cell suspension cultures was established in an important ornamental crop, Limonium sinuatum Mill. cv. ‘Early Rose’. Friable callus was initially induced from leaf segments of in vitro-cultured seedlings on 0.25% gellan gum-solidified half-strength Murashige and Skoog [1/2MS] medium containing 1.0 mg l−1 (4.14 μM) picloram. These calluses were maintained as cell suspension cultures, which showed high proliferation ability with about 80 times increase in fresh weight during the 2-wk interval of subculture. Shoot regeneration from these cell cultures was achieved by cytokinins, especially zeatin, which was the most effective in producing normal shoots with reduced hyperhydration when used in combination with 0.5% gellan gum. Shoot regeneration ability was different among the cell lines originated from each different seedling. Shoot formation was observed at different frequencies on four of five cell lines whereas one cell line showed no shoot differentiation. Regenerated shoots detached from callus readily rooted 1 mo. after the transfer onto 0.5% gellan gum-solidified 1/2MS medium lacking plant growth regulators. The plantets were successfully transferred to the greenhouse after acclimatization. No ploidy changes were observed in the callus induced or in the regenerated plantlets. The regenerated plantlets that were transferred to the greenhouse after acclimatization grew normally and did not any morphological signs of somaclonal variation.

Cumin regeneration from seedling derived embryogenic callus in response to amended kinetin

Callus was induced from hypocotyl and primary leaf explants of cumin (Cuminum cyminum L.) seedlings on a medium with 4 μM 2,4-D alone or plus 2 or 4 μM kinetin. An embryogenic callus developed within 2 weeks after transferring the callus to medium lacking plant growth regulators (PGR). The presence of kinetin in the callus induction medium with 2,4-D enhanced both the callus proliferation and the subsequent differentiation of the embryoids on the PGR-free medium. Plumules with or without simultaneously developed roots were observed 3–4 weeks after subculturing the embryogenic callus on medium containing 0.5 or 1.0 μM kinetin. Subsequently, they were transferred onto half-strength medium supplemented with 1 μM indole-3-butyric acid (IBA) and 2% polyethylene glycol (PEG, 6000) for root induction and/or proliferation, and in vitro hardening of the regenerated plants. The survival rate ex vitro was 70%. No plants developed from the embryogenic callus continuously incubated on medium lacking kinetin. We concluded that kinetin is crucial for plant regeneration from the induced embryoids of cumin.

Adventitious shoot proliferation from hypocotyl and internodal stem explants of cumin

A novel in vitro protocol was developed for regeneration of adventitious cumin shoots from explants of hypocotyl and stem internodal segments. The best response towards shoot induction was obtained from explant tissues of stem internodes when placed slightly embedded (3–4 mm) in Murashige and Skoog (MS) solid medium containing 2.5 μM benzyladenine (BA). No shoot initiation was observed on medium lacking BA. The induced shoot initials on BA-containing medium, proliferated and elongated during secondary culture on the medium lacking plant growth regulators (PGR). Up to 30 shoots per explant were harvested on this medium. Although, these shoots readily formed roots (95%) on PGR-free medium, only 30% of them survived after the acclimatization to the greenhouse conditions. The plant survival rate ex vitro was enhanced (73%) when the excised shoots were cultured for rooting in the medium with 2% polyethylene glycol (PEG 6000). Adding 1 μM indole-3-butyric acid (IBA) to the PEG-containing medium increased the percentage of rooting from 75 to 85%.

Induction of embryogenesis in cultured mature zygotic embryos of Abies fraseri (Pursh) Poir

Embryo suspensor masses were induced by culture of isolated mature zygotic embryos of Fraser fir (Abies fraseri [Pursh] Poir.). Maximum induction frequencies were observed after 10 weeks culture on one-half strength Murashige and Skoog medium containing 10 µM thidiazuron and on one-half strength Verhagen and Wann medium containing 10 µM cytokinin [6-(dimethylallylamino)purine, 6-benzyladenine, or thidiazuron). Proliferation of embryo suspensor masses occurred on one-half strength Verhagen and Wann medium supplemented with 10 µM cytokinin. When embryo suspensor masses were transferred to media containing 5-80 µM abscisic acid, cotyledonary-stage embryos were formed. Somatic embryos germinated on medium lacking plant growth regulators, but abnormal cotyledonary-stage somatic embryos with stunted cotyledons and reduced embryo axes were also observed.

Cytokinin pulse-mediated shoot organogenesis from cotyledons of Sitka spruce [Picea sitchensis (Bong.) Carr.] and high frequency in vitro rooting of shoots

Shoot formation from cotyledons of Sitka spruce was induced by a 2 h incubation in an aqueous solution of 400 µM benzyladenine, followed by culture on medium lacking plant growth regulators. Several basal media were assessed. Murashige and Skoog (1962) based medium was found to be optimal on which, after 25 weeks, 62.5% of cotyledons produced shoots with a mean of 10.7 shoots per cotyledon. Shoots, detached from cotyledons and propagated individually by axillary bud proliferation, rooted with a frequency of 84% following culture in half-strength MS-based medium lacking growth regulators under low illumination (1.5 µmol m−2s−1). Rooted plants were successfully transferred to ex vitro conditions.