Influences of medium consistency and shoot density on in vitro shoot proliferation of Populus alba × P. grandidentata

The in vitro shoot proliferation of Populus alba × P. grandidentata was affected by the medium consistency and shoot density, but not by three sizes of vessels. After 4 weeks of culture, the fresh weight and number of shoots per explant on liquid medium were significantly greater than those on agar-solidified medium. In particular, 3.2 shoots, 7 mm or longer per explant, were produced on liquid medium compared with 1.6 shoots per explant or agar-solidified medium. The fresh weight per explant after 4 weeks of culture on liquid medium and agar-solidified medium were 0.68 and 0.25 g, respectively. Increasing the number of shoots per vessel slowed the growth of the explants as measured by fresh weight and the number of shoots produced. There was little difference in the number of shoots produced between vessels with 1 or 2 shoots per vessel, but there were many fewer shoots produced when 3 shoots were placed in each vessel.Journal Paper No. J-11977 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2210.     

Effects of NO3– and BAP on organogenesis in tissue-cultured shoot primordia induced from shoot apices of Utricularia praelonga St. Hil.

The effects of NO₃ ^– and BAP on organogenesis in shoot primordia of Utricularia praelonga subcultured in B5 liquid medium were studied. In B5 liquid basal medium supplemented with 24.73 mM KNO₃ and 2.0 mg/l BAP the subcultured shoot primordia continuously multiplied into numerous small, globular masses, while with dilution of the KNO₃ to 3 mM organogenesis was promoted. Pulse treatment of the shoot primordia with 3 mM KNO₃ in B5 liquid medium for 72 h and then transplantation to the B5 basal liquid-medium induced meristemoids in this tissue. When the shoot primordia regenerated meristemoids, they never reverted back into the proliferation cycle. The addition of BAP in the B5 liquid medium with 3 mM KNO₃ regulated the differentiation rate of the stems and leaves in the meristemoids induced in the masses of shoot primordia. The control produced 3 parts stems to 1 part leaves; medium with 0.02 mg/l BAP regenerated approximately 2 parts stems and 1 part leaves; that of 0.20 mg/l BAP 1 part stems and 2 parts leaves; and medium with 2.00 mg/l BAP regenerated leaves only. Received: 17 September 1997 / Revision received: 30 October 1997 / Accepted: 18 November 1997     

Histocytological analysis of yam (Dioscorea alata) shoot tips cryopreserved by encapsulation–dehydration.

In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation–dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1–3) and seven to nine (L7–9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1–3 and L7–9 cells. Nuclear protein content increased, particularly in L1 and L1–3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7–9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1–3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1–3 and L7–9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.     

In vitro shoot growth of Brugmansia × candida Pers

The objective of this study was to improve the growth of in vitro shoot cultures of Brugmansia × candida ‘Creamsickle’. Several mineral nutrient experiments were conducted to determine the effect of NH₄⁺, NO₃⁻, K⁺, FeSO₄/EDTA, ZnSO₄, MnSO₄, and CuSO₄ on quality, leaf width and length, size and weight of shoot mass, and shoot number. The experiment to determine the levels of NH₄⁺, NO₃⁻, and K⁺, was conducted as a 2-component NH₄⁺: K⁺ mixture crossed by [NO₃⁻] and resulted in an experimental design free of ion confounding and capable of separating the effects of proportion and concentration. The results of the NH₄⁺-K⁺-NO₃⁻ experiment revealed a region in the design space where growth was significantly improved; the region generally had lower total nitrogen and lower NH₄⁺:K⁺ ratios than MS medium. The experiments to determine the appropriate levels of Fe, Zn, Mn, and Cu were conducted at six log levels ranging from 0 to 1 mM. Of the four metal salts tested, MnSO₄ had the least effect on in vitro shoot growth and its concentration was reduced from 0.1 mM (MS level) to 0.001 mM. CuSO₄ had large effects on in vitro shoot growth and was increased from 0.0001 mM to 0.001 mM. A 2-level factorial of NH₄⁺-K⁺-NO₃⁻, FeSO₄/EDTA, and ZnSO₄ was conducted and several formulations identified for their improvements of quality and growth. In addition to the changes to MnSO₄ and CuSO₄, these formulations were characterized by lower levels of NH₄⁺, K⁺, NO₃⁻ and Zn, and higher levels of FeSO₄/EDTA. Overall, several nutrient formulations were identified as superior to MS medium for growth of in vitro shoot cultures of B. ‘Creamsickle’.     

Calorespirometric studies of in vitro-grown carnation (Dianthus caryophyllus L. var. ‘Improved White Sim’) shoot tips

This study characterizes a potential model system for the use of calorespirometry to make rapid and non-destructive estimations of in vitro responses of carnation. Determinations of steady-state heat production rates, long-term heat rate stability, base trap limitations, effects of wounding and the predictions of dry mass accumulation using calorespirometric measurements were undertaken. Carnation shoot tips grown in vitro provide stable and adequately large heat production rates that vary linearly depending on dry mass. Wounding of tissues had inconsistent effects on R_{CO 2}, but greatly increased q. Linear relationships were found between dry mass and both q and R_{CO 2}. This revised version was published online in June 2006 with corrections to the Cover Date.     

TDZ induces shoot regeneration in various Kalanchoë blossfeldiana Poelln. cultivars in the absence of auxin

In order to optimize shoot regeneration in Kalancho? blossfeldiana, leaf and internode explants of seven cultivars including one inter-specific were studied. The effects of various combinations of α-naphthalene acetic acid (NAA) (0, 0.57 M) and thidiazuron (TDZ) (0, 0.45, 4.5, 22.5, 67.5 μM) on MS medium were examined. In all cultivars shoot regeneration frequency and number of shoots per explant were enhanced by increasing TDZ concentration. Supplementing the media with NAA did not improve shoot regeneration. Maximum regeneration frequency and optimum concentration of TDZ for shoot regeneration depended significantly on the cultivar. Internode explants, but not leaf explants, of some cultivars, were able to produce adventitious shoots without treatment with growth regulator.     

High-frequency adventitious shoot bud induction and shoot elongation of Chile pepper (Capsicúm annuum L.)

Summary In vitro plantlet regeneration was obtained from cultured cotyledon and young leaf explants of five Indian chile pepper cultivars (Capsicum annuum L. evs. Gujarat-1, Gujarat-2, Guntur-4, Selection-49, and Jwala). Adventitious shoot buds (ASB) were regenerated directly from cotyledon and young leaf explants in all the five cultivars on media containing benzyladenine (BA) alone or in combination with 1-naphthaleneacetic acid (NAA). Regeneration frequency was highly influenced by cultivar explant type, media combination and their interactions, except the interaction between cultivar and explant, for number of ASB per explant. Percent contribution of individual source suggested that selection of explant type followed by medium combination and cultivars was essential for obtaining high-frequency ASB induction. Across different cultivars the young leaf explant was found to be the most responsive explant, while Murashige and Skoog (MS) medium containing BA alone (17.8, 26.6, and 35.5 μM) was found to be the best medium for the production of maximum number of ASB. Between the two explants, shoot elongation was observed with ASB obtained from young leaf explants on MS medium containing BA (2.2 and 4.4 μM) and gibberellie acid (GA₃) (1.4, 2.9, 4.3 and 5.8 μM). The MS medium fortified with 4.4 μM BA+2.9μM GA₃ was optimum for shoot elongation. Elongated shoots were rooted on liquid MS medium supplemented with 2.9 μM indole-3-acetic acid (IAA) and successfully established ex vitro.     

Auxin–cytokinin interactions in the control of shoot branching

In many plant species, the intact main shoot apex grows predominantly and axillary bud outgrowth is inhibited. This phenomenon is called apical dominance, and has been analyzed for over 70 years. Decapitation of the shoot apex releases the axillary buds from their dormancy and they begin to grow out. Auxin derived from an intact shoot apex suppresses axillary bud outgrowth, whereas cytokinin induced by decapitation of the shoot apex stimulates axillary bud outgrowth. Here we describe the molecular mechanisms of the interactions between auxin and cytokinin in the control of shoot branching.     

Somatic embryogenesis and shoot regeneration from leaf derived callus of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh

Abstract

The development of in vitro regeneration systems for Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh, a medicinal plant used for treating neurological disorders, is described. For the first time in this variety, somatic embryogenesis and shoot regeneration were induced from leaf-derived callus. Well-formed plantlets were obtained through both shoot regeneration and somatic embryogenesis, with separate morphogenetic programmes. Proembryogenic masses were obtained in liquid MS and B5 media supplemented with 5.8 μM 2,4-D, 1.34 μM NAA, and 1.16 μM Kin; after being transferred onto hormone-free medium, they formed whitish and spherical structures that subsequently developed into the heart and torpedo stages.

On MS agarized medium containing thidiazuron (TDZ) at different concentrations (3, 6, 9, 12 μM) combined with 2 μM IBA, only shoot regeneration, and not somatic embryogenesis, was obtained. The mean number of shoots increased significantly when the concentration of TDZ was 3 μM.     

Does shoot water status limit leaf expansion of nitrogen-deprived barley?

The role of shoot water status in mediating the decline in leaf elongation rate of nitrogen (N)-deprived barley plants was assessed. Plants were grown at two levels of N supply, with or without the application of pneumatic pressure to the roots. Applying enough pressure (balancing pressure) to keep xylem sap continuously bleeding from the cut surface of a leaf allowed the plants to remain at full turgor throughout the experiments. Plants from which N was withheld required a greater balancing pressure during both day and night. This difference in balancing pressure was greater at high (2.0 kPa) than low (1.2 kPa) atmospheric vapour pressure deficit (VPD). Pressurizing the roots did not prevent the decline in leaf elongation rate induced by withholding N at either high or low VPD. Thus low shoot water status did not limit leaf growth of N-deprived plants.     

Somatic embryogenesis and shoot regeneration from excised adventitious roots of the rootstock Rosa hybrida L. ‘Moneyway’

Plants were regenerated from excised adventitious roots of the rose rootstock Moneyway via a three step procedure: callus induction, induction of somatic embryos and shoot development. Callus was induced on excised roots after incubation for 4 weeks in the dark on SH-medium (Schenk and Hildebrandt) containing 50 M 2,4-dichlorophenoxyacetic acid. For embryo induction, calluses were transferred to hormone-free SH-medium and incubated for 8 weeks. The use of Gelrite instead of agar during callus induction stimulated somatic embryogenesis (up to 16% of the explants formed organized structures), whereas the presence of 6-benzylaminopurine in this phase inhibited subsequent regeneration. Yellow solid calluses with embryo-like cotyledons or primordia and friable calluses with embryos were selected, and upon incubation in the light shoots developed. Shoot development was faster and more frequent on solid callus than on friable callus (64% and 21% of the calluses finally formed one or more shoots, respectively). Eleven out of thirteen regenerants developed similarly to control shoots. Finally this regeneration method is compared with other systems for somatic embryogenesis and opportunities for the production of transgenic rose rootstocks and rose cultivare are discussed.Abbreviations BAP 6-benzylaminopurine - BM basal medium - BM+ enriched basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI-4,6 diamidino-2-phenylindole - FeEDDHA ferric ethylenediamine di(ohydroxyphenylacetate) - FeEDTA ferric ethylenediamine tetraacetate - IBA indole-3-butyric acid     

Adventitious shoot regeneration from ovaries of Hosta ‘Golden Scepter’

Summary The objective of this study was to evaluate the ability ofHosta Golden Scepter (GS) ovary explants to generate adventitious shootsin vitro. Ovaries were transversely cut into halves and transferred to petri dishes containingHosta initiation medium supplemented with naphthaleneacetic acid (NAA) at 2.5 μM and N⁶-benzyladenine (BA) at 10 μM. GS produced adventitious shoots from the ovary base via organogenesis. The number of adventitious shoots regenerated from callus increased linearly with repeated subculturing on Murashige and Skoog (MS) medium supplemented with 2.5 μM NAA and 10 μM BA. The number of multiple shoots developing from callus (15.8), shoot tip (8.4), leaf (6.7), and root (4.3) occurred on MS medium supplemented with 2.5 μM NAA and 20–30 μM BA. There were significant differences in the number of shoots regenerated from shoot tips and callus on MS medium with 50 and 100 mgmyo-inositol per l. Similarly, there were significant differences in the number of axillary shoots and adventitious shoots produced with 20 g/l sucrose treatment.     

Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana?×?C. citriodora

A protocol was developed for short-term preservation and distribution of the plantation eucalypt, Corymbia torelliana × C. citriodora, using alginate-encapsulated shoot tips and nodes as synthetic seeds. Effects of sowing medium, auxin concentration, storage temperature and planting substrate on shoot regrowth or conversion into plantlets were assessed for four different clones. High frequencies of shoot regrowth (76–100%) from encapsulated explants were consistently obtained in hormone-free half- and full-strength Murashige and Skoog (MS) sowing media. Conversion into plantlets from synthetic seeds was achieved on half-strength MS medium by treating shoot tips or nodes with 4.9–78.4 μM IBA prior to encapsulation. Pre-treatment with 19.6 μM IBA provided 62–100% conversion, and 95–100% of plantlets survived after acclimatisation under nursery conditions. Synthetic seeds containing explants pre-treated with IBA were stored for 8 weeks much more effectively at 25°C than at 4°C, with regrowth frequencies of 50–84% at 25°C compared with 0–4% at 4°C. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile planting substrates. Highest frequencies (46–90%) of plantlet formation from pre-converted synthetic seeds were obtained by transferring shoot tip-derived synthetic seeds onto an organic compost substrate. These plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds.     

Storability, post-storage conversion and genetic stability assessment of alginate-encapsulated shoot tips of monopodial orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl.

An efficient short-term storage system of synthetic seeds, produced using in vitro shoot tips of the monopodial orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl. (AV), was developed. In vitro shoot tips (3–4 mm) were successfully encapsulated, resulting in uniform spherical beads (capsules), using 3 % sodium alginate with 75 mM CaCl₂·2H₂O. Maximum (~100 %) conversion (into plantlets with shoot and root) of capsules (or synthetic seeds) was achieved on quarter-strength Murashige and Skoog regrowth medium, while full-strength MS medium was required for effective conversion of non-encapsulated shoot tips. The capsules showed distinct difference in their response to temperature during storage. The conversion efficiency declined upon storage duration at both 4 and 25 °C, with those stored at 25 °C being more tolerant to storage. Capsules stored at 4 °C had rapid deterioration and faced complete death within 160 days while those stored for 200 days at 25 °C showed relatively high conversion (71.6 %). An inter-simple sequence repeats fingerprinting approach, employed on indiscriminately chosen plantlets from converted capsules (following 4 and 25 °C of storage), ensured the post-storage genetic stability.     

Plasmolysis and recovery of different cell types in cryoprotected shoot tips of Mentha × piperita

Summary. Successful cryopreservation of plant shoot tips is dependent upon effective desiccation through osmotic or physical processes. Microscopy techniques were used to determine the extent of cellular damage and plasmolysis that occurs in peppermint (Mentha × piperita) shoot tips during the process of cryopreservation, using the cryoprotectant plant vitrification solution 2 (PVS2) (30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, 0.4 M sucrose) prior to liquid-nitrogen exposure. The meristem cells were the smallest and least plasmolyzed cell type of the shoot tips, while the large, older leaf and lower cortex cells were the most damaged. When treated with cryoprotectant solutions, meristem cells exhibited concave plasmolysis, suggesting that this cell type has a highly viscous protoplasm, and protoplasts have many cell wall attachment sites. Shoot tip cells were most severely plasmolyzed after PVS2 treatment, liquid-nitrogen exposure, and warming in 1.2 M sucrose. Successful recovery may be dependent upon surviving the plasmolytic conditions induced by warming and diluting treated shoot tips in 1.2 M sucrose solutions. In peppermint shoot tips, clumps of young meristem or young leaf cells survive the cryopreservation process and regenerate plants containing many shoots. Cryoprotective treatments that favor survival of small, meristematic cells and young leaf cells are most likely to produce high survival rates after liquid-nitrogen exposure. Correspondence and reprints: National Center for Genetic Resources Preservation, U.S. Department of Agriculture, 1111 S. Mason Street, Fort Collins, CO 80521, U.S.A.     

Auxin’s role as an example of the mechanisms of shoot/root relations

The development of the shoot and the roots exhibits a dynamic balance that is characteristic of the plant and the local conditions. Consideration of all aspects of these organ relations, including plant regeneration, suggest that information about organ size, performance and developmental rates is communicated over considerable distances. The movement of substrates, sucrose and essential ions, could not suffice to account for common situations in which they originate in storage tissues, nor for the essential correlation between organ development and vascular differentiation. More generally, the temporal and spatial separation of developmental processes on the one hand and mature functions on the other means that the information exchanged must include the potential for future and not only immediate function. In contrast to substrates, special signals could be selected to depend on development and to elicit varied responses throughout the plant. An example is the concrete evidence that auxin originates in shoot tissues and is essential for root initiation. Further, this same auxin can influence and thus integrate different processes throughout the plant. Mature organs and tissues participate in auxin relations and influence development, with the result that responses to auxin are in the context of the organs present on the plant and missing structures are added as needed. The acquisition of essential substrates feeds back to hormones by limiting development and influencing auxin distribution. A possibility that requires further study is whether feedback could also be due to direct coupling between functional performance and auxin relations. In more general terms, the discussion demonstrates the importance of the mechanisms that integrate plant development and ways they could mask mutant expression.     

Gas Transport through the Root–shoot Transition Zone of Rice Tillers

Rice plants (Oryza sativa L.) are mainly cultivated in flooded paddy fields and are thus dependent on oxygen transport through the plant to maintain aerobic root metabolism. This gas transport is effectuated through the aerenchyma of roots and shoots. However, the efficiency of gas transport through the root–shoot transition zone is disputed and there are indications that the root–shoot transition zone may represent one of the largest resistances for gas transport. Therefore, we present gas conductance measurements of the root–shoot transition of individual rice tillers measured using SF₆. SF₆ was detected with a highly advanced laser based photoacoustic detection scheme allowing sensitive, high resolution measurements. In conjunction with these measurements, various plant morphological parameters were quantified. These measurements indeed indicate that the conductance at the root–shoot transition may be much smaller than the conductance of root and shoot aerenchyma within the rice plant. Conductance was strongly correlated to tiller transverse area. After elimination of tiller area from the conductance equation, the resulting permeance coefficient was still correlated to tiller area, but negatively and related to the process of radial tiller expansion. In addition, a decrease in the permeance coefficient was also observed for increasing distance from the plant centre. No correlation was found with tiller type or age of the mother tiller. Incorporation of estimates of the conductance of the root–shoot transition zone coupled to plant morphological parameters will allow considerable improvement of understanding and models on gas transport through plants.     

Shoot regeneration and cryopreservation of shoot tips of apple (Malus) by encapsulation–dehydration

Here, we report an efficient and widely applicable method for cryopreservation of Malus shoot tips by encapsulation–dehydration using adventitious shoots. Shoots were induced from leaf segments cultured on a shoot induction medium containing 2–3 mg L^?1 thidiazuron, depending on genotype, and 0.5 mg L^?1 indole-3-butyric acid. Shoot tips (3 mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h prior to direct immersion in liquid nitrogen. With our protocol, we obtained a mean organogenesis rate of 100%, a mean of 4.5 adventitious shoots per explant (leaf segment), and a mean shoot recovery of 57.0% from cryopreserved shoot tips in four Malus species. Inter-simple sequence repeat (ISSR) analysis did not reveal any polymorphic bands in regenerants recovered from either leaf segments or cryopreserved shoot tips of ‘Gala’. To the best of our knowledge, this is the first report on cryopreservation of Malus shoot tips using adventitious shoots derived from leaf segments and is the most widely applicable protocol so far reported for cryopreservation of Malus. Establishment of this protocol provides an alternative means for cryopreservation of Malus.     

Enhanced shoot organogenesis in Cassia angustifolia Vahl. — a difficult-to-root drought resistant medicinal shrub

In vitro regeneration was achieved through callus culture derived from cotyledon explants of Cassia angustifolia Vahl. on MS (Murashige and Skoog, 1962) medium. Calli were induced from cotyledon explants excised from aseptic 14?days old seedlings on MS medium containing 2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxy acetic acid) at different concentrations with 3% sucrose and 0.8% agar. Optimal growth of callus was obtained at 5.0???M 2,4-D, which was proved to be the best for shoot regeneration when sub cultured onto MS medium supplemented with cytokinins either alone or in combination with an auxin. Maximum number of shoots (23.2?±?1.4) were produced at 5.0???M 6-benzylaminopurine (BA) and 0.4???M ??-naphthalene acetic acid (NAA). Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 1.0???M indole-3-butyric acid (IBA) and 5.0???M phloroglucinol (PG). Rooted plantlets thus developed were hardened and successfully established in the soil. This protocol yielded an average of 23 plants per cotyledon explant over a period of 4?months.