Shoot Primordia Research Articles

In vitro Organogenesis in Ginger (Zingiber officinale Rosc.)

ABSTRACT Efficient plant regeneration in Zingiber officinale Rose, was achieved using callus derived from shoot primordia and grown on MS media. Organogenesis was maximum on media supplemented with 5.0 mg/liter 6-benzyladenine, 1.0 mg/liter indole acetic acid, 100 mg/liter adenine sulfate and 3 percent (w/v) sucrose. D-glucose had intermediary effect on rooting, but fructose, maltose, and mannitol had no effect. Of the conditions tested, shoot bud regeneration was highest at pH 5.7 or 5.8 and under 24 h illumination. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Isolated shoots could be rooted on media containing half-strength MS mineral salts supplemented with 1.0 mg/liter indole-3-butyric acid or in-dole-3-acetic acid and 2 percent (w/v) sucrose. Liquid media (agitated or static) were less effective than a solid (agar-gelled) media for root development. The in vitro derived plantlets could be hardened in the green house and successfully established in soil.

Agrobacterium-mediated Introduction of Athb-7, Water Stress Gene, into Solanum spp.

A genetic transformation in Solanum spp. was performed using Agrobacterium tumefaciens: C58:pGV2260:Athb-7. Athb-7 gene, known to be related to water stress and ABA level, one of Arabidopsis thaliana homeobox genes was inserted into pBin-Hyg-Tx. Explants were placed on callus induction medium for 14 days, and then transferred on shoot induction medium. Shoot primordium appeared on callus surface after 2 weeks of culture. About 6 weeks later, 100 putatively transgenic plants were obtained, and DNA was extracted from each plant for PCR analysis. Twenty out of 100 putatively transgenic plantlets turned to be positive, having a band of 800 bp in M.W. corresponding to the hygromycin gene. Both PCR and genomic Southern hybridizations using HPTII and Athb-7 genes as probes showed that these genes were inserted into plant genome.

Adventitious Shoot Formation and Plant Regeneration from Bell Pepper (Capsicum annuum L.) Cultivars and Dihaploid Lines

Adventitious shoots and viable plants were regenerated from bell pepper (Capsicum annuum L.) cultivars and dihaploid lines (DHLs) obtained from F1 hybrids via androgenesis (Dolcet-Sanjuan et al., in press). Hypocotil and cotyledon sections from in vitro-germinated seeds were used as explants. A modified MS medium (Murashige and Skoog, 1962) supplemented with IAA (0 to 3.2 μM) and BAP (0 to 100 μM) was used in a 3-week-long shoot primordia induction phase. Shoot elongation was best performed in the same basal medium, but supplemented with silver thiosulfate and GA3. Shoots were regenerated from eight selected DHLs (`C213', `C215', `C218', `C2123', `C2125', `C3111', `C3113', and `P493') and two cultivars (`Padrón' and `Yolo Wonder'). The percentage of cotyledon sections with shoot primordia after the induction phase was not genotype-dependent and always higher than with hypocotil sections (93.4% and 17.9%, respectively). The number of shoot primordia per responsive cotyledon section was also higher than with hypocotil sections (3.3 and 1.7, respectively). The genotype had a significant effect on the number of shoots regenerated per responsive cotyledon (1.1 to 5.5) or hypocotil (0.5 to 3.5) section. All adventitiously regenerated plants were fertile. This adventitious shoot regeneration protocol is being used to obtain transgenic plants from sweet bell pepper genotypes.

Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures.

Shoot primordia induced inArmoracia rusticana Gaertn. Mey. et Scherb. (horseradish) hairy root cultures were successfully cryopreserved by two cryogenic procedures. Encapsulated shoot primordia were precultured on solidified Murashige-Skoog medium supplemented with 0.5M sucrose for 1 day and then dehydrated with a highly concentrated vitrification solution (PVS2) for 4 h at 0°C prior to a plunge into liquid nitrogen. The survival rate of encapsulated vitrified primordia amounted to 69%. In a revised encapsulation-dehydration technique, the encapsulated shoot primordia were precultured with a mixture of 0.5M sucrose and 1M or 1.5M glycerol for 1 day to induce dehydration tolerance and then subjected to air-drying prior to a plunge into liquid nitrogen. The survival rate of encapsulated dried primordia was more than 90%, and the revived primordia produced shoots within 2 weeks after plating. A long-term preservation of shoot primordia was also achieved by the technique. Thus, this revised encapsulation-dehydration technique appears promising as a routine method for the cryopreservation of shoot primordia of hairy roots.

Induction of In Vitro Cultured Masses of Shoot Primordia of Hybrid Statice and Its Cryopreservation by Vitrification

Bulbous structures consisting of meristematic clumps (designated “shoot primordia”) were induced from a meristematic culture of a hybrid statice (Limonium altaica Mill. × L. caspium Mill., cv. Blue Symphonet). The shoot tips were cultured in 25 mL of liquid 1/2 Murashige & Skoog (MS) medium supplemented with 0.44 μm BA and 0.054 μm NAA and 3% sucrose at pH 5.8 by vertically shaking at 2 rpm on rotating stages (1 m in diameter) at 25 °C. One month after inoculation of shoot tips, numerous small globular structures were formed and propagated vegetatively at a high rate following subculture. Segments of shoot primordia had developed into plantlets 2 weeks after transfer to solidified 1/2 MS medium supplemented with 0.44 μm BA and 1% sucrose. Plantlets successfully acclimated and grew into normal plants in a greenhouse. Cold-hardened, precultured small segments of shoot primordia were successfully cryopreserved in liquid N by vitrification. Vitrified and warmed segments plated on solidified 1/2 MS medium produced shoots about 21 d after plating. Cultured masses of shoot primordia appear promising for large-scale production and cryopreservation of annual and biennial statice. Chemical names used: 6-benzyladenine (BA); 1-naphthaleneacetic acid (NAA).

Vegetative propagation and the tissues involved in lateral spread of Lythrum salicaria

The potential for vegetative propagation of Lythrum salicaria L. (Lythraceae) from stem and root sections, and the tissues involved in the lateral spread of L. salicaria were assessed. Only stem sections consisting of one or more nodes produced shoots and established a new root system. Internodal sections formed adventitious roots and rarely, one occurrence of 36, a new shoot. Neither woody nor non-woody root pieces formed shoot primordia. Entire root systems from which the stem was severed failed to produce new shoots, while root systems in which the stems were severed above the first node formed several new shoots arising from the remaining stem tissue. Plants excavated from field sites revealed that lateral spread of L. salicaria resulted from elongation of lateral meristems on horizontal stems, although the presence of adventitious roots on the stems and changes resulting from secondary growth of both the horizontal stems and adventitious roots gave the appearance of shoot primordia forming on roots. It is concluded that lateral spread and perennation of L. salicaria is facilitated by lateral shoot meristems in the leaf axils and not shoot primordia developing on roots.

Long-term ploidy stability of shoot primordium cultures and produced plants of melon

The chromosome number of cells in the shoot primordium aggregates and produced plants of melon [Cucumis melo L. 'Prince' (2n=2x=24)] was examined. Shoot primordium aggregates were induced from shoot-tips cultured in liquid medium and shaken at low speed (2 rpm). They were maintained by subculturing small pieces (5mm<) every 4 weeks. Shoot primordium aggregates just after induction contained about 97 diploid and 3 tetraploid cells, which was similar to those maintained in shoot primordium cultures for 6 years. This indicates that the ploidy level was maintained stably. On the other hand, plants produced from the shoot primordium aggregates just after induction were either diploid, tetraploid or mixoploid with both diploid and tetraploid cells. These ploidies were again observed among plants produced from shoot primordium cultures that were 2, 3 or 4 years old. A majority of produced plants were diploid while the total frequency of tetraploids and mixoploids was less than 8 of plants produced from all ages. Therefore, the frequency of somaclonal variation with respect to ploidy among plants produced from shoot primordium aggregates is likely to be stable at a low level over the long term.

Cryopreservation of shoot primordia cultures of melon using a slow prefreezing procedure

Tissue-cultured shoot primordia of melon (Cucumis melo L. cv. prince melon) were successfully cryopreserved in liquid nitrogen (LN) using a slow prefreezing method. The highest survival and recovery were obtained with the following procedure. Three week-old shoot primordia clumps were dissected into pieces of 2-3 mm of diameter and precultured in standard medium for 3 days. They were directly soaked in CSP1 cryoprotective solution (10%w/v sucrose, 10%w/v dimethylsulfoxide and 5%w/v glycerol) and incubated at room temperature for 30 min. Samples were ice-inoculated at -8 °C and cooled at a rate of between 0.3 and 1 °C min−1 with a programmable freezer to -30 °C for prefreezing. They were then plunged into LN for storage. After rapid thawing in 40 °C water, the cryoprotective solution was slowly diluted 5 fold in a dropwise manner with 3% sucrose and the shoot primordia were transferred onto regeneration medium. Under optimal conditions, more than 80% of cryopreserved shoot primordia were viable and 50 to 80% regenerated shoots after one month of reculture. Cryopreserved shoot primordia could be used both for reproducing a shoot primordia culture and for regenerating plants.

Micropropagation of Actinidia polygama from fruit galls

Callus was induced from the fruit galls of Actinidia polygama on the B5 medium supplemented with NAA, BA and kinetin (1 mg l−1 each). When the callus was subcultured on the same medium 3 times, shoot primordia and adventitious roots appeared on the surface of the callus. Shoots subcultured on growth regulator free MS medium or supplemented with NAA (1 mg l−1) formed roots. Chromosome numbers in the root tip of regenerated plantlets were determined as 2n=58, which was the same as that of the mother plants.

Effects of genotype, explant source and growth regulators on organogenesis in carnation callus

For callus induction, shoot tips and nodal or internodal stem segments of carnation cultivars (Coral, Jaguar, Salome and Sarinah) were grown on MS basal medium with 2,4-dichlorophenoxyacetic acid and kinetin. To achieve organogenesis, calli were transferred onto MS medium without or with growth regulators (indoleacetic acid, naphthaleneacetic acid, indolebutyric acid, kinetin, benzyladenine) in different combinations. Shoot primordia emerged from the subsurface meristemoids of calli, roots developed from the inner callus cells. The effects of genotype, explant source and growth regulators on callus-mediated organogenesis differently manifested themselves in caulogenesis and rhizogenesis, respectively. The number of root-forming calli most of all depended on genotype and least of all on explant source. Unlike rhizogenesis, caulogenesis essentially depended on explant source: internodal calli of all the tested cultivars practically missed the shoot formation ability. The number of caulogenetic calli from apical-nodal segments significantly depended on genotype, but was also affected by growth regulators.

New Physiological Effects of 5-Aminolevulinic Acid in Plants: The Increase of Photosynthesis, Chlorophyll Content, and Plant Growth

5-Aminolevulinic acid (ALA) promoted the growth and yield of several crops and vegetables at concentrations lower than those eliciting herbicidal responses, i.e., less than 1.8 mm by foliar spray and 60 μm by root soaking. To evaluate the physiological action of ALA, the effects of ALA on plants were examined by several bioassay systems at 0.0006-600 μm. ALA at 0.06-6 μm by root soaking increased the growth of rice seedlings in light, but did not affect this in darkness. In horseradish shoot primordia, promotion by ALA was not proportional among total chlorophyll content, chlorophyll concentration, and fresh weight. In the test using pothos, ALA at 0.06 μm elicited the accumulation of chlorophyll, but the photosynthesis of the plants was promoted by treatment together with ALA and nutrients. These results suggest that ALA have a variety of plant physiological effects on chlorophyll synthesis, photosynthesis, and plant growth, and ALA acts as a growth regulator in plants at low concentrations. These effects of ALA were also assumed to be linked to light irradiation and an uptake of fertilizer by plants. However, excess ALA suppressed these effects.

The effect of collection date and frozen storage on the formation of embryo-like structures and elongating shoots from explants from mature Larix decidua and L. x eurolepis

Branches were collected in the winter and early spring from a 37-year-old Larix decidua and a L. x eurolepis tree. Part of these were used immediately and the rest stored at −5 °C and −10 °C for 3 months. Primordial shoot explants were excised from stored and non-stored material and cultured on half-strength Litvay medium free of growth regulators. Storage at −5 °C promoted formation of embryo-like structures which formed shoots with elongated stems (long-shoots). The highest number of these was obtained with material collected shortly before bud break. De-needled stem sections, cut from elongating stems in early summer, produced axillary short-shoots instead of embryo-like structures.

Evidence that cytokinin controls bud size and branch form in Norway spruce

Shoot elongation in many coniferous species is predetermined during bud formation the year before the shoot extends. This implies that formation of the primordial shoot within the bud is the primary event in annual shoot growth. Hormonal factors regulating bud formation are consequently of utmost importance. We followed the levels of the endogenous cytokinins zeatin riboside (ZR) and isopentenyladenosine (iPA) in terminal buds, whorl buds and lower lateral buds of the uppermost current‐year whorl shoots of 15‐ to 20‐year‐old trees of Norway spruce [Picea abies (L.) Karst.] from June to September. Cytokinins were isolated with affinity chromatography columns, purified by high performance liquid chromatography, and quantified by ELISA. The level of ZR was low in June but increased gradually in all buds until September. Throughout the measurement period, the ZR level was highest in terminal buds and lowest in the scattered lateral, buds, with the whorl buds intermediate. The level of iPA peaked in July and decreased later without any consistent differences among the three classes of buds. The development of different kinds of buds was followed by scanning electron microscopy. We found that bud growth was greatest during August and September. The final size of primordial shoots within the buds varied considerably and the weight of the terminal bud was three times that of the whorl buds and more than five times that of the other lateral buds.We conclude that the increase in ZR level during the period of active bud development is indicative of the importance of cytokinin for this process. Furthermore, the positive correlation between the level of ZR and bud growth during the period of predetermination of next year's branch growth suggests that this hormone indirectly controls the form of single branches in the spruce tree.

Cryopreservation of tissue-cultured shoot primordia from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation

A cryopreservation protocol including preculture with ABA followed by desiccation was developed for tissue-cultured shoot primordia of Vanda pumila (Orchidaceae). After a 3-day-preculture period in B5 liquid medium supplemented with 1.0 mg/l ABA, shoot primordia were desiccated under 40–45% relative humidity down to ∼24% relative water content, then cryopreserved in liquid nitrogen (LN). After 10 days of recovery in B5 liquid medium supplemented with 0.02 mg/l BAP, cryopreserved shoot primordia showed a survival rate of 65.0 ± 7.5%. The regrown shoot primordia from cryopreservation displayed no abnormalities in chromosome number or cell structure, and could induce new meristematic tissues like those of the non-cryopreserved controls.

Shoot regeneration from mature endosperm of Passiflora foetida

Murashige and Skoog (1962) medium supplemented with 2 μM 6-benzylaminopurine (BA) induced adventitious shoots on mature endosperm explants, whilst gibberellic acid (GA3) and casein hydrolysate stimulated growth and development of these shoot primordia. Plantlets were successfully weaned in vivo. These plants were found to be triploid and flowered, although fruit set was not observed.

Frozen storage stimulates the formation of embryo-like structures and elongating shoots in explants from mature Larix decidua and L. x eurolepsis trees

Branches were collected from a Larix decidua and a L. x eurolepis tree, both 36 years old, in mid-winter. These branches were placed in freezers at −5°C, −10°C, and −18°C. Primordial shoot explants were excised after 2 to 9 months of frozen storage. The material remained viable at all three temperatures for at least 9 months. The frozen storage stimulated formation of embryo-like structures that were capable of forming shoots with elongated stems.

Mr 33 k oil body associated protein in cultured shoot primordia of Matricaria chamomilla

A major oil body associated protein (p33) was isolated from the cultured shoot primordia of Matricaria chamomilla. The M r of p33 was estimated to be 33 k by gel chromatography and SDS-PAGE. P33 has protease activity which liberates the carboxyl terminal amino acid residues from angiotensin I.

Callus cultures of Artemisia absinthium L.: initiation, growth optimization and organogenesis

Callus cultures were initiated from micropropagated Artemisia absinthium plantlets on MS basal medium supplemented with different concentrations of BA, Kn, NAA, IAA and 2,4-d in combination or singly. Supplementing the medium with low doses of both BA in combination with NAA, and Kn in combination with NAA enhanced the growth rate of callus cultures. However, cultures grew slowly following the second subculture and the majority turned brown and died within the next month. Initiation of root and shoot primordia occured directly from leaf explants cultured on 1.81 μM 2,4-d, while adventitious shoot formation from callus was observed occasionally when BA was added to the medium in combination with IAA. Furthermore, medium containing 2.22 μM BA and 2.69 μM NAA stimulated both callus growth and organogenesis on some callus cultures derived from leaves and stems of young stock material. The best results were obtained with leaf explants. Cytological analysis of root meristems revealed that all regenerants were diploid (2n=18), as expected.

Lipid constituents of oil bodies in the cultured shoot primordia of Matricaria chamomilla

The oil bodies in the cultured shoot primordia of Matricaria chamomilla comprised 80% triacylglycerol, 5% diacylglycerol, 6% monoacylglycerol and 9% free fatty acids. Free sterols and steryl esters were minor components of the oil bodies. The lipid components in the oil bodies from the cultured shoot primordia were similar to those from seeds, flowers and leaves in the intact plants. Linoleic acid (18:2) and linolenic acid (18:3) were major components of fatty acid residue in the triacylglycerols in the oil bodies from the shoot primordia, whereas the content of palmitic acid (16:0) and stearic acid (18:0) was lower than that in oil bodies from the seeds, flowers and leaves of M. chamomilla. The free sterol composition varied only slightly between the oil bodies from seed or shoot primordia, but a greater variation was observed in the composition of the steryl esters, in terms of both the sterol and fatty acid moieties. The seed oil bodies contained a higher proportion of precursor sterols than those of the shoot primordia. As in the acylglycerols, linoleic and linolenic acids were the major fatty acids in the esters from shoot primordia oil bodies, whereas the steryl esters of the seed oil bodies contained a much higher proportion of saturated fatty acids.

Efficient isolation of non-chimeric tetraploids artificially induced in a stable culture of Haplopappus gracilis

A method for reducing cytochimerism and inducing homogeneous tetraploids in Haplopappus gracilis (2n = 4) was developed in which masses of shoot primordia treated with 0.5 mg/ml of colcemid for 3 days were cut into small meristematic domes. All of the shoot primordia sampled just after the colcemid treatment were cytochimeras that were mixoploids of 2x, 4x and 8x cells. However, when they were allowed to recover in a colcemid-free medium, the frequency of 4x cells spontaneously increased in most of the shoot primordia. Thirty days after the recovery, chimeric masses containing shoot primordia, each of which consisted uniformly of 4x or 2x cells, were observed. In order to obtain a completely homogeneous tetraploid mass, we then cut these primordia into small pieces, each of which had approximately one meristematic dome. Subsequent to this homogeneous tetraploid masses were easily obtained. Tetraploid shoot primordia could propagate with chromosomal stability over a year, and plants regenerated from these tetraploid shoot primordia were also completely tetraploid. These results show that non-chrimeric masses can be easily isolated from artificially induced cytochimeras using masses of shoot primordia as material.