Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.     

Somatic embryogenesis,organogenesis, and regeneration from leaf callus culture of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., an endangered shrub

Summary A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N⁶-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.     

Do stress-related phytohormones,abscisic acid and jasmonic acid play a role in the regulation of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. somatic embryogenesis?

This study examined the role of endogenous abscisic acid (ABA) and jasmonic acid (JA) in indirect somatic embryogenesis of Medicago sativa L. A multiplex GC-MS/MS technique allowed quantitative single-run analyses of ABA, JA, 12-oxophytodienoic acid (OPDA) and indole-3-acetic acid (IAA). The preparation of initial explants led to a strong accumulation of ABA, JA and OPDA but not of IAA. Substantially higher levels of ABA, JA and OPDA were detected in developing somatic embryos than in callus or embryogenic suspension. Fluridone (FLD) decreased ABA, JA and OPDA levels. Indoprofen (INP) appeared to be a specific inhibitor of octadecanoid biosynthesis. Somatic embryo production and development were negatively affected by FLD or INP. Only INP (0.5 μM) applied during proliferation phase increased the number of cotyledonary embryos. The results strongly indicate the involvement of ABA and JA in somatic embryogenesis of M. sativa. Surprisingly, low IAA contents in comparison to stress-related compounds (ABA, JA and OPDA) were detected in explants, embryogenic tissues and somatic embryos.     

Changes in jasmonates and 12-oxophytodienoic acid contents of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. during somatic embryogenesis

Jasmonic acid (JA), its methyl ester (MeJA) and the biosynthetic precursor 12-oxophytodienoic acid (OPDA) were detected quantitatively during somatic embryogenesis of Medicago sativa L. Using GC-MS analysis, these compounds were found in initial explants, in calli and in somatic embryos in the nanogram range per gram of fresh weight. In distinct stages of somatic embryogenesis, JA and 12-OPDA accumulated preferentially in cotyledonary embryos. Initial explants exhibited about five-fold higher JA content than OPDA content, whereas in other stages OPDA accumulated predominantly. These data suggest that also in embryogenic tissues OPDA and JA may have individual signalling properties.     

Endogenous Abscisic Acid and Indole-3-Acetic Acid and Somatic Embryogenesis in Cultured Leaf Explants of Pennisetum purpureum Schum. : Effects in Vivo and in Vitro of Glyphosate, Fluridone, and Paclobutrazol

Effects of application in vivo of glyphosate, fluridone, and paclobutrazol to glasshouse-grown donor plants of Pennisetum purpureum Schum. on endogenous levels of abscisic acid (ABA) and indole-3-acetic acid (IAA) in young leaves and on somatic embryogenesis in cultured leaf explants were studied. Treatment of plants with glyphosate (100 milligrams per liter) resulted in elevated levels of endogenous ABA and IAA in young leaves. In contrast, paclobutrazol (50% active ingredient; 200 milligrams per liter) did not alter the endogenous levels of ABA and IAA. Fluridone (100 milligrams per liter) markedly inhibited synthesis of ABA and leaf explants from fluridone-treated plants lost the capacity for somatic embryogenesis. Explants from glyphosate- or paclobutrazol-treated plants did not show any reduction in embryogenic capacity when compared with untreated control plants. Glyphosate and fluridone were also incorporated into the culture media at various concentrations (0 to 20 milligrams per liter) to study their effects in vitro on somatic embryogenesis in leaf explants from untreated, field-grown plants. Glyphosate was inhibitory to somatic embryogenesis but only at concentrations above 5 milligrams per liter. Fluridone inhibited somatic embryogenesis at all concentrations tested. Inhibition of somatic embryogenesis by fluridone, by either in vivo or in vitro application, could be overcome partially by (±)-ABA added to the culture medium. Exogenous application of (±)-ABA enhanced somatic embryogenesis and reduced the formation of nonembryogenic callus. Application of IAA or gibberellic acid (GA₃; >5 milligrams per liter) was inhibitory to somatic embryogenesis. These results indicate that endogenous ABA is one of the important factors controlling the embryogenic capacity of leaf explants in Napier grass.     

Somatic embryogenesis in leaf callus from a mature Quercus suber L. Tree

Summary Somatic embryos were obtained from a 60-yr-old Quercus suber L. tree. Leaf explants were cultivated on Murashige and Skoog medium with 30 gl⁻¹ sucrose, 3 gl⁻¹ gelrite, pH adjusted to 5.8, and different growth regulator combinations. Callus induction took place at 24±1°C in the dark during the first 3 wk. After 3 mo, calluses that showed embryogenic structures were transferred to the same medium without growth regulators. Somatic embryogenesis was only observed in calluses induced on E3 medium (supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid and 9.0 μM zeatin). On average, 7.5% of the initial explants formed embryogenic calluses in this medium. Somatic embryo proliferation was high due to secondary embryogenesis. On average, 10% of the somatic embryos germinated and 40% of these germinated embryos converted into plants. Plants were elongated on the same medium without growth regulators and acclimated to greenhouse conditions.     

Somatic embryogenesis from leaf cultures of potato

An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and -naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA. Treatment with zeatin (22.8 M) and BA (10.0 M) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.     

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.     

Shoot apex explants for induction of somatic embryogenesis in mature Quercus robur L. trees

A procedure for inducing somatic embryos in shoot apex explants (2 mm) excised from shoot proliferation cultures established from adult oak trees (Quercus robur) was investigated. Embryogenesis was induced in shoot tip as well as leaf explants in three out of the five genotypes evaluated. Somatic embryos were formed by culture in induction medium supplemented with 21.48 μM naphthalene acetic acid and 2.22 μM benzyladenine for 8 weeks, and successive transfer of explants to expression media with a low concentration of growth regulators and without them. Both types of explants formed callus tissue from which somatic embryos developed, indicating indirect embryogenesis. Although the embryogenic frequencies were lower than 12%, it did not prevent the establishment of clonal embryogenic lines maintained by repetitive embryogenesis. Histological study confirmed an indirect somatic embryogenesis process from shoot tip explants, in which leaf primordia and the corresponding axial zones were involved in generating callus, whereas the apical meristem itself did not proliferate. The origin of embryogenic cells appeared to be associated with dedifferentiation of certain parenchymal cells in callus regions after transfer of explants to expression media without auxin. Division of embryogenic cells gave rise to proembryo aggregates of unicellular origin, although a multicellular origin from bulging embryogenic areas would also seem possible. Further development led to the formation of cotyledonary-stage somatic embryos and nodular embryogenic structures that may be considered as anomalous embryos with no clear bipolarity. Inducement of somatic embryos from explants isolated from shoot cultures ensures plant material all year round, thus providing a significant advantage over the use of leaf explants from field-grown trees.     

Somatic embryogenesis in <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill.

Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established in the field.     

Improved somatic embryogenesis from Cocos nucifera (L.) plumule explants

Summary Coconut is one of the most recalcitrant species to regenerate in vitro. Although previous research efforts using plumule explants have resulted in reproducible somatic embryogenesis, efficiency is only 4 or 10 somatic embryos per plumule without or with a brassinolide treatment, respectively. In order to increase the efficiency of somatic embryogenesis in coconut, two different approaches were evaluated and reported here: secondary somatic embryogenesis and multiplication of embryogenic callus. Primary somatic embryos obtained from plumule explants were used as explants and formed both embryogenic callus and secondary somatic embryos. The embrogenic calluses obtained after three multiplication cycles were capable of producing somatic embryos. The efficiency of the system was evaluated in a stepwise process beginning with an initial step for inducing primary somatic embryogenesis followed by three steps for inducing secondary somatic embryogenesis followed by three steps for embryogenenis callus multiplication, and finally production of somatic embryos from callus. The total calculated yield from one plumule was 98 000 somatic embryos. Comparing this to the yield obtained from primary somatic embryogenesis results in about a 50 000-fold increase. When compared to the yield previously reported in the literature with the use of a brassinolide treatment, it is about a 10 000-fold increase in yield. The present protocol represents important progress in improvement in the efficiency of coconut somatic embryo production.     

Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogaea): Endogenous growth regulator levels and significance of cotyledons

The regulatory role of thidiazuron (TDZ) and explant factors in imparting somatic embryogenic potential was assessed in relation to endogenous growth regulator levels in peanut ( Arachis hypogaea L. cv. Tango). TDZ induced somatic embryogenesis over a range of concentrations (0.5 to 10 μ M ). Culture of seedlings for just 2 days on TDZ-supplemented medium was sufficient to induce somatic embryogenesis. Seedling age and retention or removal of cotyledons during culture influenced morphogenic potential significantly. The younger the seedlings, the better was the embryogenic response to TDZ treatment. The embryogenic potential of seedlings was limited for explants with no cotyledons and they did not respond to increasing levels of TDZ. In contrast, retention of at least one or both the cotyledons resulted in increased response to TDZ. Endogenous levels of cytokinins, auxins and abscisic acid were influenced by TDZ treatment, while TDZ itself was detected only in the cotyledons. The cytokinin N⁶-(2-iso-pentenyl)adenine (2iP) fluctuated on a 10-day cycle in the cotyledons; TDZ treatment suppressed this change and caused an overall decrease in the pool of available 2iP in the tissue. It appeared that TDZ induced somatic embryogenesis in peanut by influencing endogenous levels of both auxin and cytokinins.     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

Somatic embryogenesis,organogenesis and callus growth kinetics of flax

The effects of plant growth regulators (PGR) on calli induction, morphogenesis and somatic embryogenesis of flax were studied. The organogenic and callus formation capacity were assessed for different types of source explants. Root and shoot explants were equally good material for calli production but the former produced calli without shoot regeneration capacity. Under the experimental conditions tested, 2,4-dichlorophenoxyacetic acid (2,4-D) + zeatin was the most efficient PGR combination on calli induction and biomass production. The calli were green but with no rhizogenic capacity. In contrast, and at similar concentrations, indole-3-butyric acid (IBA) + kinetin induced white or pale green friable calli with a good root regeneration capacity (60%). A factorial experiment with different combinations of 2,4-D + zeatin + gibberellic acid (GA₃) levels revealed that the direction of explant differentiation was determined by specific PGR interactions and concentrations. The results from these experiments revealed that the morphogenetic pathway (shoot versus root differentiation) can be manipulated on flax explants by raising the 2,4-D level from 0.05 to 3.2 mg l^?1 in the induction medium. The induction and development of somatic embryos from flax explants was possible in a range of 2,4-D + zeatin concentrations surrounding 0.4 mg l^?1 2,4-D and 1.6 mg l^?1 zeatin, the most efficient growth regulator combination.     

Acetylsalicylic acid enhances and synchronizes thidiazuron-induced somatic embryogenesis in geranium (Pelargonium x hortorum Bailey) tissue cultures

Summary Thidiazuron (TDZ) effectively induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey) during only a 3-day period of induction. The presence of acetylsalicylic acid (ASA) during this period caused a two-fold increase in the number of somatic embryos and enhanced synchronization of embryo development compared to the TDZ treatment alone. Salicylic acid was ineffective in modulating similar embryogenic responses as ASA. The ASA-induced enhancement and synchronization of somatic embryogenesis could possibly be used as an experimental system to study the interplay of growth regulators in somatic embryogenesis.Abbreviations ASA acetylsalicylic acid - SA salicylic acid     

Somatic embryogenesis of holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.): ethylene production and polyamine content

The production of ethylene and the endogenous content of polyamines (PAs) have been recorded during the early development, maturation and germination of holm oak (Quercus ilex L.) somatic embryos. Ethylene production was high in embryogenic callus, immature somatic embryos and in explants showing secondary embryogenesis, while it was lower in mature and germinating somatic embryos. A higher ethylene production was also associated to the process of secondary embryogenesis. The exogenous application of 1-amino-1-cyclohexane carboxylic acid was not significantly effective on the production of ethylene by holm oak somatic embryos. Total PAs were more abundant in embryogenic callus and in both somatic and zygotic immature embryos, decreasing later on in the mature and germination phases. Immature somatic embryos of holm oak and immature zygotic embryos contain high levels of spermidine (Spd), which decreased during maturation and germination. Spermine (Spm) concentration was lower than that of Spd. Spm was more abundant in embryogenic callus and immature zygotic embryos than in mature embryos. Ethylene production did not seem to interfere with PA metabolism.     

In vitro morphogenic response in cotyledon explants of Semecarpus anacardium L.

Three different morphogenic responses??caulogenesis, direct somatic embryogenesis, and callusing??were noted in cotyledon explants of Semecarpus anacardium L. cultured in woody plant medium (WPM) containing thidiazuron (TDZ). Thidiazuron, at all concentrations tested, induced organogenic as well as embryogenic responses. The organogenic buds differentiated to shoots and the embryogenic mass (EM) gave rise to globular embryos which differentiated up to cotyledon-stage embryos on repeated culture in growth regulator (GR)-free WPM medium containing 0.2% activated charcoal after the removal of TDZ. The organogenic and embryogenic responses were optimal in 9.08???M TDZ after the removal of TDZ. Elongated shoots rooted in half-strength liquid WPM medium with 2.46???M indole butyric acid. Plants were successfully acclimatized and transferred to soil. Histological studies confirmed the direct origin of the organogenic buds from the cotyledon explants. The EMs produced somatic embryos on repeated culture in charcoal incorporated GR-free medium. Morphogenic callus formation from the cotyledon explants was also noted. This callus on repeated culture in WPM medium with charcoal differentiated into somatic embryos. Repetitive somatic embryogenesis was evident from direct and indirectly formed primary embryos. The somatic embryos did not convert into plantlets, though sporadic germination of embryos was observed through the emergence of roots.     

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.     

Somatic embryogenesis and plant regeneration through cell suspension in diploid (AB) banana cultivar Elakki Bale (syn Neypoovan)

An efficient protocol was developed using cell suspensions for somatic embryogenesis and plantlet regeneration in a most popular diploid AB banana (M.accuminata X M.bulbisiana hybrid) cv. Elakki Bale (syn Neypoovan) known for its taste and keeping quality in southern India. Floral primodia from position 8–16 of male inflorescence which were more responsive for embryogenesis were used as explants for the embryogenic callus production in MS media supplemented with different concentration of 2,4-D. A concentration of 18.1 μM 2, 4-D produced maximum embryogenic calli in 1 % of the explants inoculated. Embryogenic calli on repeated sub culturing on MA2 media produced good embryogenic cell suspensions (ECS). Microscopic examination of ECS showed globular, smaller with dense cytoplasm filled with starchy granules characteristic of embryogenic cells. Highest number of somatic embryos (189) was produced on modified MA3 media. A germination percentage of 31 % were observed in BAP 22.19 μM concentration. Regenerated plants with normal shoot and root were hardened in soilrite. Direct somatic embryogenesis and plant regeneration was also noticed in embryogenic calli which did not pass through the ECS stage. The protocol optimized for somatic embryogenesis through cell suspension and also direct embryogenesis leading to plantlet regeneration can be used for the micropropagation and genetic manipulation.