Shoot Primordia Research Articles

Brief Note : Plant regeneration after long term callus culture in clones of Asparagus officinalis L.

Callus growth and plant regeneration from long-term callus cultures were studied in two elite clones of Asparagus officinalis cv. Argenteuil, to establish a suitable protocol for a prospective in vitro selection program. Callus initiation and growth was evaluated on MS medium with 3% sucrose, 0.9% agar, 1 mg x l(-1) kinetin, and three levels of 2,4-D. The highest callus relative growth was obtained on medium with 1.5 mg x l(-1) 2,4-D and 1 mg x l(-1) kinetin. Shoot primordia (SP) induction from > 18-months-old calluses was evaluated on several media; the highest percentage of SP induction (89%) and average number of SP per callus (8.6) were obtained with clone "265" on MS medium with 5 mg x l(-1) 2iP, 1 mg x l(-1) IAA, 3% sucrose and 0.9% agar. The highest percentage of root induction (100%) was achieved with clone '265' on MS medium with 0.1 mg x l(-1) kinetin, 0.1 mg x l(-1) NAA, 1.32 mg x l(-1) ancymidol, 7% glucose and 0.8% agar. Important medium x genotype interactions were detected, pointing to the need of adjusting this and other in vitro protocols for specific asparagus genotypes.

Light quality influences the polyamine content of lettuce (Lactuca sativa L.) cotyledon explants during shoot production in vitro

Light quality has previously been shown to influence morphogenesis in lettuce cotyledon explants, with white or red light promoting adventitious shoot production, and blue light inhibiting it. Endogenous polyamine (PA) concentrations were compared between explants cultured under different light qualities. Explants cultured under white or red light accumulated PAs during shoot primordia production, with a 5.6-fold increase compared to initial concentrations under white light, and 6.7-fold increase under red light. These results suggest polyamines are involved in the formation of shoot primordia. After 18 days in culture PA concentrations decreased under white light, and to a lesser extent under red light, signaling a shift in polyamine metabolism that correlates with shoot expansion, which occurs more readily under white light. Explants cultured under blue light accumulated polyamines for the first 7 days, to a level 1.3 times greater than initial values, followed by a gradual decline during the remainder of the culture period. Explants cultured under blue light also contained a greater proportion of PCA-insoluble conjugated PAs, compared to explants under white or red light, which contained greater proportions of free or PCA-soluble conjugated polyamines. The ratio of putrescine to spermidine was also different with a lower Put:Spd ratio being associated with shoot production under white or red light, and higher Put:Spd ratio being associated with culture under blue light.

Methyl jasmonate disrupts shoot formation in tobacco thin cell layers by over-inducing mitotic activity and cell expansion.

The aim of the present study was to determine early cyto-histological events associated with the reduced number of shoots formed at the end of culture in tobacco (Nicotiana tabacum L.) thin cell layers treated with methyl jasmonate (MJ) [S. Biondi et al. (2001) J Exp Bot 52:1-12]. The results show that 0.1-10 microM MJ strongly stimulated mitotic activity early in culture relative to untreated controls. Treatment with MJ also induced anomalous mitoses. Enhanced proliferative growth was confirmed by northern analysis and in situ hybridisation using cDNA probes of the G1/S phase-specific genes ubiquitin carboxyl-extension protein (ubi-CEP), topoisomerase 1 (top1) and ribonucleotide reductase (RNR). The formation of meristematic cell clusters on day 5 was also enhanced by 1 muM MJ, but subsequent development of these cell clusters into meristemoids and shoot primordia was reduced by all MJ concentrations in a dose-dependent manner. Cell expansion was stimulated by MJ concentrations ranging from 0.001 to 10 microM; expanded cells frequently occurred around and within meristemoids and shoot primordia, and displayed thickened and suberised cell walls; cell wall thickness increased with increasing MJ concentration. These cytological events caused alterations in the tunica and stem differentiation of the shoot dome. The apparently paradoxical role of MJ, which deregulates shoot formation through a stimulation of growth events, i.e., mitotic activity and cell expansion, is discussed.

Regeneration of Pelargonium × hederaefolium ‘Bonete’ from petiole explants

The adventitious shoot regeneration from petiole explants of Pelargonium × hederaefolium ‘Bonete’ was achieved via a mixed pathway i.e. organogenesis and somatic embryogenesis. The histological study of regenerated structures revealed the presence of both shoot primordia and embryo-like structures. The initial growth in petiole explants occurred on media with BAP + auxin or TDZ alone. However, the most effective regeneration (24 structures/explant) was noted in the presence of TDZ (2 mg l−1) and IBA (0.1 or 0.2 mg l−1). Moreover, the application of TDZ in the induction phase reduced the time needed for the formation of adventitious structures and positively influenced the further shoot development on the medium containing m-topolin and IBA.

The Effect of Different Plant Growth Regulators on Adventitious Shoot Formation from Virginia Pine (Pinus virginiana) Zygotic Embryo Explants

The effects of different plant growth regulators on in vitro adventitious shoot formation in Virginia pine (Pinus virginiana Mill.) zygotic embryo explants were quantitatively evaluated. Using Tang and Ouyang (1999) (TE) basal medium supplemented with 11.4 µM indole-3-acetic acid (IAA) and 2.2 µM N6-benzyladenine (BA), callus was observed after 3–6 weeks of culture. Calluses were transferred to TE basal medium supplemented with 0.49 µM indole-3-butyric acid (IBA) and 8.8 µM BA for 6–9 weeks, where they produced numerous small shoot primordia. They were then transferred to TE basal medium supplemented with 0.49 µM IBA and 4.4 µM BA to promote growth and elongation of adventitious shoots. After elongated shoots were transferred to TE medium containing 0.05 µM α-naphthaleneacetic acid (NAA) for 6 weeks, adventitious roots were formed. Regenerated plantlets were established in soil in greenhouse.

The homeobox genes ATHB12 and ATHB7encode potential regulators of growth in response to water deficit in Arabidopsis

The Arabidopsis thaliana homeodomain leucine-zipper gene ATHB7 , which is active specifically under water deficit conditions, is proposed to act as a negative regulator of growth (Soderman et al ., 1996, Plant J. 10: 375 381; Hjellstrom et al ., 2003, Plant Cell Environ 26: 1127 1136). In this report we demonstrate that the paralogous gene, ATHB12 , has a similar expression pattern and function. ATHB12 ,like ATHB7 ,was up-regulated during water deficit conditions, the up-regulation being dependent on abscisic acid (ABA) and on the activity of the Ser/Thr phosphatases ABI1 and ABI2. Plants that are mutant for ATHB12 , as a result of T-DNA insertions in the ATHB12 gene, showed a reduced sensitivity to ABA in root elongation assays, whereas transgenic Arabidopsis plants expressing ATHB12 and/or ATHB7 as driven by the CaMV 35S promoter were hypersensitive in this response compared to wild-type. High-level expression of either gene also resulted in a delay in inflorescence stem elongation growth and caused plants to develop rosette leaves with a more rounded shape, shorter petioles, and increased branching of the inflorescence stem. Transgenic Arabidopsis plants expressing the reporter gene uidA under the control of the ATHB12 promoter showed marker gene activity in axillary shoot primordia, lateral root primordia, inflorescence stems and in flower organs. Treatment of plants with ABA or water deficit conditions caused the activity of ATHB12 to increase in the inflorescence stem, the flower organs and the leaves, and to expand into the vasculature of roots and the differentiation/elongation zone of root tips. Taken together, these results indicate that ATHB12 and ATHB7 act to mediate a growth response to water deficit by similar mechanisms.

The delay in hormonal treatment modulates the expression of LESK1, a gene encoding a putative serine-threonine kinase, marker of in vitro caulogenesis in tomato ( Lycopersicon esculentum Mill.)

Growth regulators play a multiple role in somatic organogenesis, affecting the phases of competence acquisition, determination and organ differentiation. The establishment of these phases is conceivably determined by a differential gene expression. We have recently found that the expression of LESK1, a gene that encodes a putative serine/threonine kinase marks an elevated intrinsic caulogenetic attitude in tomato hypocotyl, that does not require the presence of exogenous growth regulators. In tomato cotyledon explants, caulogenesis is instead induced supplying the culture medium with growth regulators. In these explants, LESK1 expression peak precedes the induction phase and marks the acquisition of caulogenic competence. The removal of growth regulators just after the reaching of the peak prevents the achievement of an optimal shoot production. A delay in hormonal treatment greatly reduced caulogenesis, affecting both competence acquisition, as suggested by the reduced LESK1 expression, and consequently the proper induction, driving to a sharp decrease in shoot primordia per explant. We hypothesize that LESK1 kinase is activated during the transduction of the hormonal signal driving to caulogenesis and required for the fulfillment of the inductive phase. LESK1 expression is differently modulated in tomato horticultural varieties showing different caulogenic attitude. As all the examined cultivars have one copy of this gene, a different regulation of the hormonal signal transduction pathway involving LESK1, is conceivably responsible for the diverse morphogenetic outcomes in these varieties.

Direct adventitious shoot formation on seedling radicles in seed cultures of strawberry

Mature seeds of strawberry (Fragaria x ananassa) were placed on Murashige and Skoog medium supplemented with 2.22 μM 6-benzyladenine. After four weeks of culture, and without an intervening callus phase, approximately 36% of the resulting seedling radicles had formed numerous adventitious buds near their tips. A few buds on each radicle developed into shoots, while others formed disorganized calli. Consequently, the seedlings exhibited shoot apices at both ends of the axis of polarity. Our overall results suggest that a considerable level of plasticity in organ determination occurs even in higher plants, and that exogenous growth regulators can cause a root primordium in the radicle to be converted to a shoot primordium.

Effect of thidiazuron in germinating tamarind seedlings

Abstract Tamarind, a multipurpose tropical tree species, is economically important for sustainable development of wasteland due to its hardy nature and adaptability to various agroclimatic conditions. Reports on in vitro morphogenesis in this species are limited, due to its recalcitrant and callogenic nature. To overcome these limitations, an attempt was made to induce meristematic activity in seedling explants. Seedlings were germinated in medium with or without thidiazuron (4.54, 9.08, 13.12, 18.16 μM). This growth regulator restricted the differentiation of the apical meristem to form shoots. It triggered proliferation of the meristematic tissue at the cotyledonary node and a large number of meristematic buds appeared in a radial pattern around the node. The meristematic activity extended to the junction of the epicotyl and hypocotyl, giving rise to buds in the form of protuberances in all sides of the junction. These buds differentiated to form shoot primordia and subsequently to shoots in medium devo...

Thidiazuron Induced Multiple Shoot Induction and Plant Regeneration from Cotyledonary Explants of Mulberry

A rapid micropropagation protocol through induced multiple shoots from the cotyledonary explant of mulberry (Morus alba L) is described. The highest number of shoots (20.3) was obtained when explants from 14-d-old embryos were cultured on Murashige and Skoog (MS) medium supplemented with 7 μM thidiazuron for 45 d. Of the three cultivars used, cv. S-36 was the best followed by cv. K-2 and S-1. The shoots were transferred to MS medium supplemented with 5 μM 6-benzylaminopurine for elongation. The elongated shoots were rooted on half strength MS medium containing 1 - 7 μM indole 3-butyric acid or 1-naphthalene acetic acid. The rooted plants were transplanted to soil with 90 % success. The emerged shoot primordia probably initiated from the pre-existing meristems since the shoot bud show definite vascular connection to the major vascular tissue.

Isolation and localization of new germination-related sequences from wheat embryos.

Subtractive library hybridization was used to isolate the cDNA clones that corresponded to the transcripts that were specifically up-regulated during wheat embryo germination. The clones with numbers 5, 6, 7, 8, 24, and 26 appeared to be more abundant in germinating wheat embryos. Among the isolated clones, we identified four new members of the wheat "germin" gene family. We also identified two novel sequences which exhibited distinct germination up-regulation, and displayed characteristic spatial patterns of expression. One of these, represented by clone pSB10, was principally expressed in the root tissue of germinating embryos. The second was represented by the pSB7 clone and was expressed in both the root and shoot primordia of the embryonic axis, as well as within the coleoptile.

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Mature and immature cotyledon explants of cowpea were cultured on Murashige and Skoog's (MS) medium supplemented with 0.1 - 2.0 mg dm-3 benzyladenine (BA). Shoot organogenesis was observed from the minimal greenish calli formed at proximal cutting edges of the immature cotyledon explants after 15 - 20 d of culture. Among whole immature zygotic embryo and seven explant types we tested, single whole cotyledon was suitable for shoot organogenesis. Nearly 67.5 % of the explant types produced adventitious shoots on MS medium containing with 1 mg dm-3 BA, and the shoot number (10.1) per explant was higher than other explant types. From the histological studies, the shoot primordia originated from the procambial strands of immature cotyledon explants. When the shoots were transferred to 1/2 MS basal medium, formed roots within 20 d of culture. The rooted plants were subsequently transferred to the pots and to the field.

Competence, determination, and meristemoid plasticity in tobacco organogenesis in vitro

Tobacco leaf explants can produce both shoots and roots depending on the phytohormones in the medium. These arise directly via meristemoids (meristematic centers), which form distinct primordia and then organs. In this study it was found that shoot primordia arose from the palisade mesophyll cells at the adaxial surface, while root primordia arose from the rib parenchyma cells, near the existing vascular bundles. In studies on competency and determination, it was found that the tobacco leaf explants required 4–6 days in culture on a shoot-inducing medium (SIM) to become determined for shoot formation, while the explants were competent for rooting at excision and needed only 1 day on the root-inducing medium (RIM) to become determined for root formation. Transfer of explants from SIM or RIM to basal medium (BM without phytohormones) and vice versa supported the above findings. Transfer of explants from SIM to RIM and vice versa, delayed the timing of root and shoot formation, but not the position in the explant from which the organs arose. On transfer from SIM to RIM or vice versa, meristemoids that were already determined for shoot or root formation continued to develop, while those not yet determined were inhibited and (or) reverted to parenchymatous tissue. Thus under our culture conditions meristemoids in tobacco leaf explants are not plastic.Key words: competence, determination, meristemoid plasticity, organogenesis, tobacco.

Effect of Genotype and Explant Type on Shoot Regeneration in Tomato (Lycopersicon esculentum Mill.) in vitro

The regeneration capacity of six types of explants (segments from hypocotyl, cotyledons, epicotyl, leaf, internodes and petiole) was compared in 13 cultivars of tomato (Lycopersicon esculentum Mill). Explants were cultured on a regeneration medium containing 1 mg/l zeatin and 0.1 mg/l indole-3-acetic acid. The number of shoot primordia and shoots with 1 or more fully developed leaves was evaluated after 6 weeks. The regeneration capacity was significantly influenced by cultivars and explant types. The total number of shoot primordia produced in all types of explants was highest in the cultivars Hana and Premium and lowest in UC 82 and Money Marker. Cv. Hana also produced the highest number of shoots. The most responsive explants in most cultivars were hypocotyls and epicotyls with up to 100% regeneration and mean production of 6.3 and 6.5 shoot primordia per explant, respectively.  

High irradiance increases organogenesis in friable callus of Caustis blakei Kük. (Cyperaceae)

Caustis blakei is an attractive cut foliage plant harvested from the wild in Australia and marketed under the name of koala fern. Previous attempts to propagate large numbers of this plant have been unsuccessful. The effect of four light irradiances on organogenesis from compact and friable callus of C. blakei was studied for 21 wk. Both callus types produced numerous primordial shoots but many failed to develop into green plantlets. However, significantly more primordial shoots and green plantlets developed on the friable callus than on the compact callus, and significantly more green plantlets were regenerated under the higher photon irradiances of 200 and 300 μmol m−2s−1 than under the lower irradiances of 100 and 150 μmol m−2s−1. The compact callus produced its maximum number of green plantlets early in the experiment (after 9 wk), while the friable callus continued to produce primordial shoots and green plantelets throughout the period of the experiment, and reached its maximum production of green plantlets at 21 wk under the irradiance of 300 μmol m−2s−1. Organogenesis from friable callus under high irradiance (300 μmol m−2s−1) offers an efficient propagation method for C. blakei.

RAPD analysis of mutants obtained by ion beam irradiation to hinoki cypress shoot primordia

Mutants were induced by irradiation of the shoot primordia of Hinoki cypress with 50 MeV 4He 2+ heavy ion beam. Fresh shoot primordia on the CD medium in the plastic Petri dish (35 × 10 mm) were irradiated. Xanta mutants were induced from 38 to 266 Gy irradiation. Waxy mutants were induced from 76 to 266 Gy irradiation. Xanta, waxy and control type of regenerated Hinoki cypress in vitro were checked for their DNA level difference using RAPD analysis. Among 81 primers used, 23 primers produced the 68 bands. Among them stable 44 bands produced by 15 primers were compared between mutants and control plant. So far, there is no variation among the RAPD analysis band patterns of those mutants. Bigger test size may detect the gene variation specific for mutants.

Effect of genotype, growth regulators, carbon source, and pH on shoot induction and plant regeneration in tomoto

Ten cultivars of tomato were screened for their ability to produce shoots and shoot primordia on media containing a range of 6-benzyladenine (BA) concentrations (0.0–4.0 mgl−1; 0.0–17.7 μM) in combination with indole-3-acetic acid (IAA; 0.0, 0.1, 0.2 mgl−1; 0.0, 0.57, 1.14 μM, respectively). Both genotypes and growth regulator levels showed significant differences at α=0.05. However, their interaction was not significantly different. A comparison of the mean number of shoots produced six cultivar classes, with UC82, UC97-3, Castlerock, Red Rock, and Peto86 producing the highest means. There were four cultiver frequency classes, the highest including cultivars Peto Rock (93.7%), Peto86 (92.3%), and Strain B (90.7%). Growth regulator mean comparisons produced eight classes, the highest included three different BA/IAA combinations, with media containing 2.0 mgl−1 (8.87 μM) BA and 0.2 mgl−1 (1.14. μM) IAA giving the highest mean and frequency (94.1%). The effect of carbon source was studied using glucose, fructose, maltose, and sucrose at 3.0% concentration. Three high regenerating genotypes and four media were used in combination with each sugar. Sugars were significantly different with maltose giving the highest number of shoots (31.6) followed by glucose (20.9). Growth regulator means, sugar-growth regulator interaction, and cultivar-sugar interaction were significant. A three-way interaction was nonsignificant. The effect of maltose and sucrose concentration (1.5–9.0%) showed the positive effect of maltose over sucrose in inducing shoots and in reducing loss rates of shoots on the regeneration media. Maltose at 1.5, 3.0, and 4.5% was the most effective. Six pH values (4.0–6.5) were used to test their effect on shoot induction in three cultivars (Peto86, UC97-3 and Castlerock). pH effects and cultivar-pH interaction were not significant.

Liquid Culture and Transformation of Hinoki Cypress (Chamaecyparis obtusa Sieb. et Zucc.)

Hinoki cypress (Chamaecyparis obtusa) is one of the most important timber resource forest trees in Japan. Because seed production from a seed orchard of hinoki cypress is not constant every year, micropropagation from a limited amount of material is useful. Up to now, the conventional tissue culture method using solid medium has been used. Here a new method using liquid culture in tubes rotated vertically is described. Shoot primordium of hinoki cypress was inoculated in Campbell and Durzan's (CD) liquid medium containing different cytokinins (6-benzylaminopurine (BAP), Zeatin, thidiazurone (TDZ)), and the container tubes were rotated vertically around the axis at 2 times / min. Culture room temperature was 25°C and light condition was 16 h photoperiod per day of fluorescent lamps. Zeatin at 1 μM concentration was the best for maintaining the shoot primordium production and TDZ induced callus on the surface of the shoot primordia. After shoot primordium multiplication in the liquid culture, they were transplanted to agar medium for shoot elongation. A high concentration of agar (up to 16 g/L) or AVF (anti vitrification factor from Dr. Nairn, 1995) was effective to prevent vitrification of the shoots. Transformation of shoot primordium was done using particle bombardment with vectors containing β-glucuronidase (GUS) gene or herbicide resistance gene (bar). Positive result for transient transformation was observed with the histo-chemical study for transformation with GUS. Integration of a useful herbicide bar gene into the shoot primordium culture system was also tried and stably transformed plants were obtained. This is the first report of stable transformation of Japanese conifer using practically useful gene.

Comparison of adventitious shoot formation from node and leaf explants of various carnation (Dianthus caryophyllus L.) cultivars

SummaryShoot regeneration of leaf explants from in vitro plants of 35 carnation cultivars, and node explants from greenhouse plants of 38 cultivars was compared. First to third node explants cultured on medium containing 1.0 mg l–1 TDZ for 10.d and subcultured onto medium contain 1.0 mg l–1 BA showed a high shoot regeneration percentage, while the upper node showed higher shoot regeneration ability than the lower node. In contrast, there was no difference in regeneration ability among the first to sixth in vitro leaves (cultured on medium with 0.5 mg l–1 IBA and 0.22 mg l–1 TDZ). In both node and leaf explants regeneration ability was different depending on the cultivar, and there was no difference between standard and spray types. The correlation coefficients between regeneration percentage and the number of shoots per regenerating node and leaf explants were significantly positive (r=0.536** and 0.720**, respectively P<0.01). The correlation coefficient between shoot regeneration percentage of node and leaf explants was significantly positive (r=0.213*; P<0.05). Histological observations on adventitious shoot organogenesis by SEM and light microscopy showed that in both node and leaf explants, shoot primordia were regenerated from the transition zone between the leaf base and the stem. In both node and leaf explants shoot primordia regenerated in the cortex tissue near the wound surface.

PkMADS1 is a novel MADS box gene regulating adventitious shoot induction and vegetative shoot development in Paulownia kawakamii

Direct regeneration of shoot buds in vitro is an important technique in plant genetic manipulation. We describe the isolation and functional characterization of a novel MADS box cDNA (PkMADS1) from Paulownia kawakamii leaf explants undergoing adventitious shoot regeneration. mRNA gel blot analysis confirmed the expression of PkMADS1 in the shoot-forming cultures, but no signal was observed in the callus-forming cultures. PkMADS1 transcripts were also detected in shoot apices, but not in root apices, initial leaf explants or the flower. In situ hybridization revealed that its expression was restricted to developing shoot primordia in the excised leaf cultures, suggesting a role for this gene in adventitious shoot formation. Transgenic Paulownia plants over-expressing the PkMADS1 gene showed some changes in phenotype, such as axillary shoot formation. In the antisense transformants, shoots were stunted and had altered phyllotaxy, and, in some lines, the shoot apical meristem appeared to have been used up early during shoot development. Leaf explants from the antisense transgenic plants showed a tenfold decrease in shoot regeneration compared with explants from sense transformants or wild-type. Our results show that PkMADS1 is a regulator of shoot morphogenesis.