Regeneration of somatic embryos and roots from quince leaves cultured on media with different macroelement composition

The effects of different macroelement combinations on somatic embryogenesis of quince (Cydonia oblonga Mill.) were tested. Leaves were excised from shoot cultures of quince clones and cultured on macroelement combinations of 8 different growth media. Callus production varied depending on the medium and the clone combinations. The influence of genotype and macronutrient combination on somatic embryo and root regeneration was also observed. Clone BA 29 showed the highest embryogenic properties and Murashige and Skoog-based medium appeared to be the most favourable for somatic embryo formation. Root regeneration was higher on Woody Plant Medium and Schenck and Hildebrandt-based media. Interactive effects between genotypes and macroelement combinations were also detected both for embryo and root formation. In all treatments, somatic embryos underwent early developmental arrest and failed to convert into plants. Differences in embryo and root regeneration observed among macroelement combinations may be ascribable to different levels of medium nitrogen and probably to the ratio between nitrate and ammonium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Increasing NaCl and CaCl<Subscript>2</Subscript> concentrations in the growth medium of quince leaves: I. Effects on somatic embryo and root regeneration

Summary The effects of increasing concentrations of NaCl and CaCl₂ on quince (Cydonia oblonga Mill. BA 29 clone) somatic embryogenesis and adventitious root regeneration were investigated. Leaves collected from in vitro-grown shoots were used as explants and induced for 2d in liquid Murashige and Skoog medium containing 11.3 μM 2,4-dichlorophenoxyacetic acid. Explants were then cultured on semisolid Murashige and Skoog medium enriched with 4.7 μM kinetin and 0.5 μM naphthaleneacetic acid under red light for 25 d and under white light for another 25 d. Two experiments were performed: in the first, NaCl was used at 0,25, 50, 100, and 200 mM in factorial combination with CaCl₂ at 3, 9, and 27 mM; in the second, NaCl was applied at 0, 5, 10, 20, 40, and 80 mM in combination with CaCl₂ at 0.3, 1.0, and 3.0 mM. Quince leaves revealed the capacity to regenerate somatic embryos and/or adventitious roots. Quantitative and qualitative regeneration from leaves was affected by NaCl treatments: increasing NaCl concentrations, in combination with CaCl₂ at 1 mM, led to an increase in the proportion of leaves producing somatic embryos only, and to a decrease of both leaves regenerating roots only and leaves simultaneously producing somatic embryos and adventitious roots. This suggests a beneficial effect of salt stress on the embryogenic process. The regeneration response decreased with increasing salt concentrations and was almost totally inhibited above 50 mM NaCl and 9 mM CaCl₂. The presence of CaCl₂ in the culture medium apparently mitigated the effects of salt stress, but only when NaCl was applied at 40 mM. NaCl at 5 mM, in the presence of 0.3 or 1 mM CaCl₂, was favorable both to somatic embryo and root production. No value of the ratio Na⁺/Ca²⁺ was found to be optimal for the regeneration processes.     

Effects of leaf soluble sugars content and net photosynthetic rate of quince donor shoots on subsequent morphogenesis in leaf explants

The effects of different growth conditions (ventilated and closed vessels, medium with 0, 15 and 30 g dm⁻³ sucrose) during proliferation of donor quince (Cydonia oblonga Mill.) shoots (stage I) on net photosynthetic rate and soluble sugars content were evaluated. In order to assess the influence of these physiological parameters on morphogenesis, leaf explants harvested from donor shoots were induced to form somatic embryos and adventitious roots under ventilated and closed Petri dishes (stage II). Natural ventilation and low sucrose contents (0–15 g dm⁻³) promoted the photosynthetic rate of quince shoots whereas biomass accumulation was the highest in those shoots cultured with 30 g dm⁻³ sucrose in both vessel types and 15 g dm⁻³ sucrose under natural ventilation. Increasing sucrose content in the medium induced greater accumulation of sucrose in leaf tissues of donor shoots. The content of reducing sugars was higher than that of sucrose, and it appeared to be higher in shoots cultured under natural ventilation compared to those in closed vessels. Somatic embryogenesis and root regeneration were influenced by stage I and II treatments. A significant correlation between sucrose content in the leaves of donor shoots and the number of somatic embryos regenerated was found, suggesting that identification of biochemical and physiological characteristics of donor shoots associated with increased regeneration ability might be helpful for improving morphogenesis in plant tissue culture.     

Induction of somatic embryogenesis and adventitious shoots from immature leaves of cassava

Direct somatic embryogenesis was successfully achieved from immature leaves of cassava (Manihot esculenta Crantz) cultured on induction medium containing 2,4-dichlorophenoxyacetic acid or naphthaleneacetic acid. Changing the duration of induction or changing plant growth regulators resulted in differences in regeneration of somatic embryos or adventitious shoots. The results showed that auxin was a key factor for inducing embryogenic cells. The embryogenic cells were mainly induced within 4–12 days. Only if the embryogenic cells were induced, the auxin enhanced formation of somatic embryo whereas 6-benzylaminopurine stimulated development of adventitious shoots. Histological examinations supported the conclusion.     

Somatic embryogenesis and adventitious shoot formation in Burma reed (<Emphasis Type="Italic">Neyraudia arundinacea</Emphasis> Henr.)

Burma reed (Neyraudia arundinacea Henr.) is a C₄ grass native to Southeast Asia and Indomalaya that grows quickly, exhibits strong resistance to environmental stresses, and is extremely adaptable. It can be widely utilized as a bioenergy crop for biomass conversion. In vitro multiple shoots were first established from axillary buds and then subcultured on propagation medium containing 10 μM 6-benzylaminopurine (BA) and 2.0 μM naphthaleneacetic acid (NAA). Multishoot clumps were used as explants to induce somatic embryogenesis and adventitious shoot formation. The results showed that auxin 2,4-dichlorophenoxyacetic acid or NAA play a key role for the induction of somatic embryogenesis and adventitious shoot formation, whereas cytokinin BA or kineatin enhance shoot proliferation and plant regeneration from callus and somatic embryos. Efficient somatic embryogenesis, mass propagation, and plant regeneration systems in Burma reed were established.     

Shoot regeneration and somatic embryogenesis from needles of redwood (Sequoia sempervirens (D.Don.) Endl.)

A rapid and effective system of somatic embryogenesis and organogenesis from the in vitro needles of redwood (Sequoia sempervirens (D.Don.) Endl.) had been established. The influences of plant growth regulators (PGRs) and days of seedlings in vitro on adventitious bud regeneration and somatic embryogenesis were studied. The process of somatic embryo formation was also observed. The results showed that embryogenic callus was induced and proliferated on Schenk and Hildebrandt (SH) medium with BA (0.5 mg/l), KT (0.5 mg/l) and IBA (1.0 mg/l). SH medium containing BA (0.5 mg/l), KT (0.2 mg/l) and IBA (0.2 mg/l) effectively promoted adventitious bud regeneration. The highest frequency (66.3%) of direct somatic embryogenesis was obtained in the combination of BA (0.5 mg/l) and IBA (0.5 mg/l). The optimal days of seedling in vitro for adventitious bud and somatic embryogenesis were 30 days and 30–40 days, respectively. The developments of somatic embryos were similar to that of zygotic embryogenesis. The result of histocytological studies indicated that proteins were gradually accumulated in the process of somatic embryo formation and there were two peaks of starch grains accumulation that one was in the embryogenic callus and the other was in the globular embryos. These results indicated that starch and protein were closely related with the energy supply and the molecular base of somatic embryogenesis, respectively.     

Endogenous ethylene in indirect somatic embryogenesis of <Emphasis Type="Italic">Medicago sativa</Emphasis> L.

Ethylene biosynthesis during different phases of somatic embryogenesis in Medicago sativa L. cv. Rangelander using two regeneration protocols, RPI and RPII, was studied. The highest ethylene production was detected during callus growth on induction medium in both regeneration protocols. Significantly less ethylene was produced by embryogenic suspension than by callus (RPII). Developing embryos synthesized higher amounts of ethylene than mature embryos. Production of ethylene was strongly limited by the availability of 1-aminocyclopropane-1-carboxylic acid and also by ACC-oxidase activity. However, removal of ethylene from culture vessels’ atmosphere using KMnO₄ or HgClO₄ had no significant effect on callus growth, somatic embryo induction and development. Reducing of ethylene biosynthesis by aminoethoxyvinylglycine substantially decreased somatic embryo production and adversely affected their development, indicating ethylene requirement during proliferation and differentiation but not induction.     

In vitro culture ofBellevalia romana (L.) Rchb. I. Plant regeneration through adventitious shoots and somatic embryos

Summary Callus induction, adventitious shoot and root formation, and somatic embryogenesis were investigated in root, cotyledon and mesocotyl cultures ofBellevalia romana (L.) Rchb. grown on a synthetic nutrient medium containing different plant hormones. The combination of naphtaleneacetic acid plus benzylaminopurine was very effective in causing callus growth and plant regeneration from mesocotyl explants. On the contrary 2,4-dichlorophenoxyacetic acid caused suppression of shoot bud development in the same type of callus. Both cotyledon and root derived calli showed a low growth rate and did not regenerate shoots but only roots. Differentiation of somatic embryos which eventually developed into plantlets was promoted by 2,4-dichlorophenoxyacetic acid in suspension cultures. The results are discussed in relation to studies on nuclear behaviour during different morphogenetic pathways.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

Response of stem explants to screening and explant source as a basis for methodical advancing of regeneration protocols for chrysanthemum

The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration, length of induction period and light quality on leaf regeneration of quince clone BA 29 were investigated. After 2, 4 or 6 days of induction with 2.5 mg l⁻¹ or 5.0 mg l⁻¹ 2,4-D, leaves were cultured under red, blue, red+blue, far-red+blue, white, far-red light or darkness conditions. Leaves thereby treated showed different responses, with respect to somatic embryogenesis, callus, red-nodular structures or roots. Callus production increased with increasing 2,4-D concentration and induction period, although it was not influenced by light quality; the only exception was far-red+blue light, which reduced callusing response. This result suggested involvement of the blue-absorbing photoreceptor system in the callus formation processes. A high regeneration of red-nodular structures with a meristematic appearance was also observed; from some histological characterizations, we presumed they were adventitious buds that were arrested at an early developmental stage. Red-nodular structures increased with decreasing 2,4-D concentration and induction period. In the regeneration of such structures, the blue-absorbing photoreceptor system appeared to have a negative effect but only at a low photoequilibrium value. In contrast, light quality which activated phytochrome induced an increment in regeneration, but the response did not vary for photoequilibrium values ranging from 0.43 to 0.86. For root regeneration, phytochrome seemed to be the only photoreceptor involved. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration via direct somatic embryogenesis in Panax japonicus

Panax japonicus is one of the important medicinal plants. Here, we established the protocol for plant regeneration of P. japonicus via direct somatic embryogenesis. Somatic embryos were directly obtained from the segments of zygotic embryos on MS medium with 4.4 μM 2,4-D. Thereafter, somatic embryos were produced by repetitive secondary somatic embryogenesis. The secondary somatic embryo formation was enhanced by plasmolyzing pretreatment (1.0 M mannitol for 10 h). Frequency of secondary somatic embryo formation from cotyledon segments was lowered by plasmolyzing pretreatment, but the number of somatic embryos per explants was greatly increased. Plasmolyzing pretreatment resulted in retardation of embryo growth and required subculture to fresh medium for further growth of embryos into cotyledonary stage. Without plasmolyzing pretreatment, cotyledonary embryos were obtained after 8 weeks of culture. All the cotyledonary somatic embryos germinated by 5 μM GA₃ treatment, but only 15.3% were germinated on hormone-free medium. After 2 months of culture on 1/2 strength WPM medium, plantlets produced flowers spontaneously. In the anthers of in vitro flowers, microsporogenesis occurred normally with low number of pollen grains.     

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or -naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA₃), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.     

In vitro morphogenesis in zygotic embryo cultures of neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.)

Immature zygotic embryo cultures of neem yielded highly regenerative cultures, with the response varying with the embryo stage at culture. Early dicotyledonous stage embryos were the most responsive followed by torpedo stage embryos. The embryo cultures differentiated three types of regenerants: somatic embryos (SEs), shoot buds and neomorphs. SEs exhibited morphological abnormalities such as pluricotyledony, fusion of cotyledons and absence of cotyledons. Although these SEs showed secondary embryogenesis, the occurrence of normal dicotyledonous embryos was extremely rare. On MS basal medium 3% of SEs developed a long tap root but a plumular shoot did not appear. However, it was possible to regenerate plantlets from immature zygotic embryo cultures of neem via neomorph formation and adventitious shoot bud formation. The transplantation survival of these plants was more than 80%.Abbreviations BAP 6-Benzylamino purine - CH Casein hydrolysate - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - SE Somatic embryoCommunicated by W. Harwood     

Somatic embryogenesis from immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.     

Regeneration of <Emphasis Type="Italic">Pelargonium × hederaefolium</Emphasis> ‘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.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

We established a plant regeneration system for Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis. Embryogenic tissues were successfully induced on three kinds of Smith media from megagametophyte explants containing pre-cotyledonary embryos of C. obtusa plus-trees. Factors affecting somatic embryo maturation were examined. The concentration of polyethylene glycol 4000 in the medium was a critical factor for embryo maturation and its effective concentration was 150 g/l. The addition of 30 g/l maltose to the medium had a positive effect on embryo maturation, but sucrose was ineffective. The mature somatic embryos germinated at a germination frequency of approximately 60%, and the presence of activated charcoal was effective in stimulating plantlet growth. The plantlets acclimatized successfully in a greenhouse. To our knowledge, this is first report describing details of a plant regeneration method for C. obtusa via somatic embryogenesis.Abbreviations ABA Abscisic acid - PEG Polyethylene glycol 4000 - SM1 Smith Standard Embryonic Tissue Capture Medium - SM2 Smith Standard Embryogenesis Medium - SM3 Smith Embryo Develop Medium     

Changes of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

Somatic embryos and embryogenic callus were initiated from immature zygotic embryos of ginseng (Panax ginseng C.A. Meyer). These somatic embryos were multiplied by adventitious (secondary and tertiary) embryogenesis and their growth and development were dependent on growth hormones in the medium. Auxins, 2,4-d, NAA, and IAA at 1.0 mg l^-1 were effective in inducing secondary and tertiary somatic embryos, which proliferated directly from the apical or cotyledonary portions of the primary somatic embryos. Single somatic embryos or clusters or embryos developed from the explanted primary embryos. Cytokinin (Kn, BA) inhibited adventitious embryogenesis. Secondary somatic embryos developed to maturation and later regenerated into plantlets in two stage process; firstly elongation of the shoot axes on MS +1.0 mg l^-1 Kn, secondly formation of root on 1.0 mg l^-1 Kn+1.0 mg^-1 GA₃ medium.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA in-doleacetic acid - Kn kinetin - BA benzylaminopurine - PSE primary somatic embryo - SSE secondary somatic embryo - TSE tertiary somatic embryo     

Somatic embryogenesis from mesocotyl and leaf-base segments of <Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L., a minor millet

Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N₆) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N₆ medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N₆ regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N₆ medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.     

Embryo induction and regeneration from root explants of Medicago truncatula after osmotic pre-treatment

Embryo induction and regeneration from suspension culture of two Medicago truncatula cvs. (cv. R 108 1 and cv. Jemalong) have been studied. The influence of osmotic pre-treatment (1 M solution of sucrose for 48 h and 72 h) of roots as an initial explant, on embryogenic efficiency of the suspension culture was assessed. In comparison to the control, the level of abscisic acid (ABA) increased significantly after osmotic stress. The increased ABA level did not correlate with the induction of embryogenesis neither with the improved embryogenic potential of cv. R 108 1. The shortest regeneration period and the highest percent of conversion to plants were found in cv. R 108 1 after 72-h pre-treatment of roots. The efficiency of somatic embryo conversion was less after 48-h pre-treatment and much less for the untreated control. Osmotic stress did not positively affect the process of embryogenesis from root explants of cv. Jemalong, confirming its cultivar dependence. A single cell suspension fraction was produced in both Medicago trunacatula cvs. during the somatic embryo maturation stage. A higher embryogenic potential than the initial suspension culture was established only for the cell suspension originating from 72-h pre-treated roots of cv. R 108 1. The data confirms that the process of somatic embryo induction and embryo conversion from root explants of cv. R 108 1 could be promoted by osmotic stress pre-treatment.