Multi-omics analysis reveals the ontogenesis of basal chichi in Ginkgo biloba L.

Chichi is a unique biological phenomenon observed in Ginkgo biloba L.. In this study, multi-omics analysis was used to compare basal chichi (C) with roots (R) and stems (S) to explore the regulatory mechanisms of basal chichi ontogenesis. The results showed that compared with roots and stems, the tZ, SA and ABA contents in basal chichi were the highest, and the ratio of IAA/tZ was the lowest. Nucleotides and their derivatives in basal chichi were upregulated, and phenylpropane metabolites were downregulated. Some differentially expressed genes (DEGs) strongly correlated to plant hormones were screened. We speculate that auxin and cytokinin are involved in the morphogenesis of basal chichi and that cytokinin plays a major role. The ontogenesis of basal chichi is closely related to environmental stress, and it may be a coping strategy of G. biloba in the face of environmental stress.     

Competence for regeneration of cucumber cotyledons is restricted to specific developmental stages

The total duration of the plant regeneration process from cucumber (Cucumis sativus L.) cotyledonary explants was only six weeks, which included the induction of buds and their development into plants. Regeneration of shoots from cotyledons from three to five day-old seedlings ranged up to 100%. The regenerated plants were morphologically normal, flowered and set seed. The regeneration capacity of cotyledons from seven days-old and older seedlings was lowered dramatically. Most of those regenerated plants were polyploid/mixoploid and had an abnormal morphology. During seedling development (3 to 13 days), the DNA content of cotyledonary cells changed from 2C to 4C and more. The results show that the decrease in regeneration competence correspond with the change in DNA content of the cotyledonary cells.     

An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

Summary An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA₃) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.     

Influence des racines sur le développement végétatif ou floral des bourgeons cotylédonaires chez le Scrofularia arguta: role possible des cytokinines

Influence of roots on the vegetative or floral development of cotyledonary buds of Scrofularia arguta Sol.: A possible cytokinin role. This study shows that the presence of “nonabsorbing roots” insures a vegetative development of cotyledonary buds cultured in vitro whereas buds growing without roots produce flowers early. In the same way, roots suppress floral expression of axillary meristems of the same cotyledonary buds and induce these buds to vegetative functioning. Various trophic modifications in the culture medium are ineffective on non-rooted buds as also are gibberellin As and adenine. On the contrary, several cytokinins (kinetin, benzyladenine and zeatin) exert the same influence as roots. These results suggest that roots regulate meristematic functioning through cytokinins.     

Establishment of efficient and rapid regeneration system for some diploid wild species of <Emphasis Type="Italic">Arachis</Emphasis>

Though peanut tissue culture has advanced to a considerable extent using a number of explants with the subsequent production of transgenic plants, wild Arachis species appeared to be recalcitrant to using similar explants. In this study, the use of cotyledonary nodes as explants prepared from 7-day old seedlings resulted in the development of a simple and rapid regeneration protocol for five diploid wild species including A. diogoi, A. stenosperma, A. duranensis, A. cardenasii and A. correntina belonging to the genus Arachis for producing multiple shoots. Shoot bud initiation was observed 10 to 15 days after culture initiation. Responding cotyledonary nodes with shoot buds were subcultured to lower levels of cytokinin and finally to MS basal medium for further shoot development and elongation. Production of multiple shoots was observed in all the five diploid species with a maximum of 9 to 16 shoots were obtained per explant in the primary cultures. The number of shoot buds increased significantly with repeated explant subculturing with recovery up to 45 shoots from responding explants. These shoots were rooted efficiently on MS medium supplemented with 1 mg l⁻¹ naphthalene acetic acid and the time taken from explanting to the transfer of shoots to potting mixture was about 12 weeks. All rooted shoots were successfully established in soil in glass house and further transferred to field. These plants survived to maturity and set seed.     

Rooting and shooting: dual function of thidiazuron in in vitro regeneration of soybean (<Emphasis Type="Italic">Glycine max.</Emphasis> L)

Thidiazuron (TDZ), primarily a cotton defoliant, has been later accepted as a plant growth regulator. In spite of extensive studies, the physiological function of TDZ is still uncertain. The aim of the present experiment was to study the activity of TDZ in in vitro regeneration of soybean. The seeds of soybean were cultured separately on MS and B5 medium supplemented with TDZ. The hypocotyls, cotyledons, cotyledonary nodes without axillary buds and cotyledonary nodes with axillary buds were used as explants and their capacity to direct regeneration was tested on both MS and B5 media containing TDZ (0.9–5.4 μM). Shoot formation was observed only on cotyledonary nodes with axillary buds cultured on MS and B5 basal media with TDZ (0.9–5.4 μM). All tested explants cultured on B5 medium with TDZ produced roots. Root formation was not observed on MS basal media supplemented with TDZ. Results show that TDZ functions as cytokinin (to produce the shoots) and auxin (to produce the roots) on various explants depending on the basal medium used.     

LIGNOTUBER ONTOGENY IN THE CORK-OAK (QUERCUS SUBER; FAGACEAE) I. LATE EMBRYO

Changes at the cotyledonary node of the cork-oak (Quercus suber L.) were examined during the embryo maturation phase using light microscopy and scanning electron microscopy techniques. During the maturation phase the embryo axis elongates by diffuse growth, the apical meristem forms the first leaf primordia, and the radicle meristem remains inactive. The primary axis of the embryo bears, axillary to the cotyledons, in the range of five to seven pairs of lateral buds at differing stages of development. Two or three pairs of these buds are visible, occurring on the upper unfused portion of the embryonic axis, while the remaining buds are hidden by the fused cotyledonary tissues. Lateral buds develop from clusters of cells in the peripheral meristem forming a shell zone delimiting the bud meristem. Lateral buds do not undergo much development until germination begins. The results are discussed with reference to the possible role of the cotyledonary node as the source of the lignotuber in the cork-oak.     

ONTOGENY OF THE COTYLEDONARY REGION OF CHAMAESYCE MACULATA (EUPHORBIACEAE)

Development of the cotyledonary region in Chamaesyce maculata is described from germination of the seed through formation of the dense mat of branches which characterize this common weed. The cotyledonary node is trilacunar with split-lateral traces. Epicotyl development is limited to a pair of leaves (“V-leaves”) inserted directly above and decussate to the cotyledons. The two V-leaves are also vascularized by three traces and insertion of these traces relative to the vasculature at the immediately subjacent cotyledonary node is asymmetrical; four of the six V-leaf traces arise on one side of the intercotyledonary plane and two arise on the opposite side. Further shoot development is limited to lateral branches that develop sequentially from cotyledonary axillary buds, and then from de novo buds which arise at bases of previously developed lateral branches. The first axillary bud to develop is located on that half of the seedling which supplies the V-leaves with four traces. Development or insertion of the third and fourth branches (first and second de novo branches) relative to the first and second (cotyledonary) branches occurs in two patterns, termed cis and trans. Subsequent de novo branches generally develop between the two most recently developed branches on that half of the seedling, gradually forming a large branch plexus at the cotyledonary region. In mature robust specimens, however, the sequence of lateral branch development may become irregular. Structure of the cotyledonary region of C. maculata does not readily support widely held concepts of homology between the pleiochasium of Euphorbia subgenus Agaloma and the lateral branch system of Chamaesyce.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

Maturation,germination, and conversion of norway spruce (Picea abies L.) somatic embryos to plants

Summary Quantitative data are presented on the efficiency of three stages of plant regeneration from somatic embryos of Norway spruce (Picea abies L.): 1) Maturation, the development of immature embryos to the cotyledonary stage; 2) Germination, primary root growth; and 3) Conversion, plantlet survival and continued growth in nonaxenic conditions. Maturation frequency was calculated relative to the number of immature somatic embryos induced to develop on the basal medium of von Arnold and Eriksson (1981). The average number of immature somatic embryos was 700 per gram of embryogenic callus, on medium supplemented with ABA and IBA (1 μM each). Maturation was the least efficient stage of regeneration; an average of 3% of the embryos induced to develop reached the cotyledonary stage. Mean germination frequencies were improved on treatments which avoided immersion of the radicle in medium solidified with agar. Whereas, 27% of the somatic embryos germinated when radicles were immersed in agar medium, 45% germinated when placed on the surface of the medium, and 56% germinated when cotyledons were immersed in agar medium and the culture vessel inverted. Twenty-nine percent of the somatic embryos germinatedin vitro were converted to plants. Under greenhouse conditions these plants set dormant buds, subsequently survived overwintering (to −5°C), and renewed vegetative growth synchronously with seedlings grown under the same conditions. Our results verified long-term (2 year) growth and development potential of conifer somatic embryo plants.     

Adventitious shoot regeneration from cotyledonary explants of rapid-cycling fast plants of <Emphasis Type="Italic">Brassica rapa</Emphasis> L

Rapid-cycling fast plants (Brassica rapa; RCBr) is also known as Wisconsin Fast Plant and is widely used in K-12 and undergraduate studies. RCBr has a short generation time (seed-to-seed in 30–60 days), which allows for the completion of experiments in a semester. Previous studies have shown that cotyledonary explants with attached petioles are capable of generating shoots. However, there is no published adventitious shoot regeneration protocol to date. Sterile cotyledonary explants were excised; all edges and petioles were removed. Five-day-old cotyledonary explants produced shoots on a Murashige and Skoog medium containing 1.5 mg/L thiadiazuron (TDZ) and 0.5 mg/L 1-naphthaleneacetic acid (NAA) (FPM I) at a mean rate of 8.8%. This rate increased to 14.8% in explants placed on FPM I medium supplemented with 5.0 mg/L silver nitrate (AgNO₃) (SRM 2). The rate increased to 32.5% when 5-day-old explants, excised from the part of the cotyledon nearest to the petiole, were placed adaxial side up on SRM 2 medium. The shoot regeneration rate increased to 44.5% using 4-day-old cotyledonary explants. A shoot regeneration rate of 23% was observed among 9-day-old leaf explants. Shoots from cotyledonary explants were elongated on basal medium with 0.5 mg/L NAA, rooted on basal medium, and later acclimatized. This is the first report of shoot regeneration from cotyledonary explants of rapid-cycling Brassica rapa without pre-existing meristematic tissues.     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

Influence des corrélations entre organes sur la croissance et le développement floral des bourgeons cotylédonaires chez leScrofularia arguta Sol.

The importance of various correlative influences on growth and vegetative or floral development of cotyledonary buds inScrofularia arguta Sol. is shown. The terminal bud, on the one hand, inhibits growth of cotyledonary buds and, on the other hand, induces their early flowering. The cotyledon stimulates growth of its axillary bud, but has no action on its floral development. Leaves above the cotyledonary node have the same effect as the cotyledon. Finally, roots stimulate vegetative growth of cotyledonary buds and suppress floral expression, but only when apical dominance has been removed at an early stage of development.     

Shoot regeneration from seedling explants of Acacia mangium Willd

Summary Regeneration of adventitious shoots was obtained in over 80% of explants, consisting of wounded cotyledonary nodes of Acacia mangium, by culturing germinated seedlings on DKW medium with combinations of N⁶-benzyladenine and either thidiazuron or N-(2-chloro-4-pyridyl)-N-phenylurea. Electron microscopy showed the presence of adventitious buds arising from wound tissue of the cotyledons and cotyledonary nodes. Shoot regeneration was also obtained at lower frequency in isolated cotyledon explants cultured with 6% sucrose alone (10%), or with 3% sucrose and 30.0 mg l⁻¹ (0.1 μM) 2–4-dichlorophenoxyacetic acid (2,4-D; 16%). With 2,4-D,>60% of explants produced organized structures but these did not develop into shoots or somatic embryos. Shoot formation was not induced in either hypocotyl or root explants.     

In vitro regeneration of plantlets from immature zygotic embryos of Picea chihuahuana Martínez, an endemic mexican endangered species

Summary Adventitious buds were induced from immature embryos of Picea chihuahuana, a species from the north of Mexico. The medium was Schenk and Hildebrandt supplemented with benzyladenine and kinetin, alone or in combination with naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid. Adventitious buds were obtained through a wide range of growth regulator concentrations, mainly from the cotyledonary region. Kinetin was more effective than benzyladenine in shoot induction. The optimum shoot induction medium contained 23.0 μM kinetin, on which 11.2 adventitious buds per embryo were obtained. Shoot development and elongation were achieved on basal SH medium at 50% concentration (SH 50) without growth regulators and improved with the reduction of sucrose concentration to 10 g l⁻¹. Differential response appeared to be associated with the provenance of the seeds, relative to the percentage of explants that responded as well as the number of adventitious buds produced per embryo. A low percentage of shoot rooting was obtained with a 24-h pulse on a medium with 16.1 or 26.85 μM of naphthaleneacetic acid. In the process of micropropagation of Picea chihuahuana, rooting is still the limiting factor.     

Rapid multiple shoot production from cotyledonary node explants of pea (Pisum sativum L.)

Summary Multiple shoots were produced, from cotyledonary node explants of pea (Pisum sativum L.) cultured on MS medium containing 1 mg 1⁻¹ BAP. The number of buds formed could be increased by scraping the nodes before culture or by increasing the cytokinin concentation. However, cytokinin levels over 5 mg 1⁻¹ increasingly produced shoots that were vitrified and dificult to root. With all the genotypes tested, developing buds were visibile as soon as 5 days after culture and elongated shoots could be removed after 21 days., Histological studies indicated, that the buds and shoots were formed from superficial layers of tissue. The efficiency of this regeneration system compared favorably with previosly published methods. The rapid, genotype-independent, high frequency system described here may be of use in the production of transgenic pea plants.     

Somatic embryogenesis and shoot regeneration from intact seedlings of Phaseolus acutifolius A., P. aureus (L.) Wilczek,P. coccineus L., and P. wrightii L.

A rapid, one-step procedure has been developed for inducing direct organogenesis and somatic embryogenesis in cultures of Phaseolus coccineus L., P. acutifolius A., P. aureus L. [Vigna radiata L. Wilczek] and P. wrightii L. Development of somatic embryos and shoot buds occurred within 6–8 weeks of culture from intact seedlings raised on MS (Murashige and Skoog 1962) medium supplemented with N⁶-benzylaminopurine (BAP). Shoot buds or embryoids originated from subepidermal tissue of the regions adjacent to the shoot apex, hypocotyl and cotyledonary axils. While P. acutifolius and P. aureus were regenerated via shoot formation and P. wrightii by somatic embryogenesis, both embryogenesis and shoot regeneration were observed in P. coccineus. Relatively higher levels of BAP, 50–80 M, were found to be optimal for inducing regeneration while lower concentrations were ineffective. About 40–70 shoots and 70–250 somatic embryos were produced per responding seedling. Regenerated shoots and somatic embryos developed into whole plants on a basal medium or the one supplemented with 1 M naphthaleneacetic acid.     

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

The morphogenetic responses of seedling explants of mung bean (Vigna radiata L. Wilczek cv ML-5) were studied in vitro. Direct induction of shoots/plants was possible from shoot tip, cotyledon and cotyledonary node explants. Dedifferentiation of the explants viz; Shoot tip, cotyledons, cotyledonary node, primordial leaves and roots was obtained on basal medium supplemented with auxin and cytokinin. Shoot regeneration was limited to primary calli while rhizogenesis was of common occurrence in established calli. In addition to differences in hormonal requirements, the various explants showed preferential growth in different basal media.     

Vegetative regeneration of biennial Oenothera species after disturbance: Field observations and experiment

It has been recently shown that some annual and biennial species of man-made habitats cope with severe disturbance by resprouting (vegetative regeneration) from their bud bank and do not only rely on regeneration from seeds. Nevertheless, information on the ecology of this phenomenon is rare. In a field study, we answered the question how frequent is resprouting from root buds in populations of the ruderal biennial herb Oenothera biennis, and how it is affected by habitat conditions. In an experiment, we tested the hypothesis that higher severity of injury and later life-cycle phase of the injured plants suppress resprouting from both axillary and root buds in O. biennis and also in its closely related congeners O. fallax and O. glazioviana.In 25 out of 29 studied ruderal populations of O. biennis severely injured individuals were found; however, only half of these populations included injured individuals that resprouted from roots. Among these populations, the number of root-sprouting individuals varied highly (from 3% to 67% of injured individuals). The largest populations and the highest percentage of root-resprouting individuals were found in urban habitats with sandy/gravelly substrate, a low vegetation cover, and a high frequency of disturbance.In the experiment with three Oenothera congeners, removal of aboveground biomass with all axillary buds largely led to the death of plants of all three species. When a portion of the basal axillary buds remained intact, individuals of O. biennis mostly failed to regenerate, whereas individuals of O. fallax and O. glazioviana survived and formed seeds. A higher severity of injury suppressed resprouting in Oenothera congeners in this experiment. However, the relationship between life-cycle phase and the ability to resprout remains unclear in Oenothera species.This study showed that resprouting after severe injury is an important feature of Oenothera individuals occurring in man-made habitats and may represent an alternative strategy to regeneration from the seed bank under disturbance conditions.