Improved somatic embryo maturation in loblolly pine by monitoring ABA-responsive gene expression

During loblolly pine zygotic embryo development, increases in mRNAs for three ABA-responsive LEA-like genes coincided with the two developmental stage-specific peaks of endogenous ABA accumulation (Kapik et al. 1995). These ABA concentration profiles from zygotic embryo development were used to develop several tissue culture approaches that altered the exposure of somatic embryos to exogenous ABA. Elevating exogenous ABA at a time corresponding to mid-maturation improved the germination and resulted in more zygotic-like expression of selected genes in somatic embryos. Extending the time on maturation medium for a fourth month increased embryo yield, dry weight, and germination in high-and low-yield genotypes. Optimizing the amounts of embryogenic suspension, plated and exogenous ABA concentration increased from 22 to 66% in the early-stage bipolar embryos that developed to the cotyledonary stage.     

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.     

Callus concentration regulates somatic embryo production in orchardgrass suspension cultures

Summary The effects of callus inoculation concentration and culture duration on somatic embryogenesis of orchardgrass,Dactylis glomerata L., were evaluated in suspension cultures of an embryogenic genotype Embryogen-P. Somatic embryo formation was induced in liquid SH medium containing 30 μM dicamba (SH-30 and 1.5% casein hydrolysate; embryo development was in liquid SH medium without plant growth regulators (SH-0); and embryo maturation and germination occurred on solid SH-0 medium. Callus proliferation in SH-30 suspension cultures was greatest when callus was inoculated into the liquid medium at a relatively high concentration of 4% (4 g callus/100 ml medium), but the induction of somatic embryos was highest in this medium if the callus was inoculated at a lower concentration (<2%). In a second experiment, somatic embryo yield was highest when SH-0 development medium was inoculated with suspension culture callus at 0.1% concentration and declined markedly as inoculation concentration increased. Cell concentration is a critical factor in regulating the somatic embryogenesis response in orchardgrass suspension cultures.     

Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata

Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don . During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no significant change in putrescine. This led to a several-fold increase in spermidine/putrescine ratios during development of both types of embryos. Cell cultures of plant-forming and non-plant-forming lines derived from the same clone and growing on proliferation (maintenance) medium differed significantly in their polyamine levels. Mature, cotyledonary stage somatic embryos capable of germination and formation of plants could be distinguished by their higher spermidine/putrescine ratios from abnormal cotyledonary stage somatic embryos which were incapable of forming plants.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Somatic embryogenesis,encapsulation, and plant regeneration of <Emphasis Type="Italic">Rotula aquatica</Emphasis> lour., a rare rhoeophytic woody medicinal plant

Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.     

Effect of medium salt concentration on differentiation and maturation of somatic embryos of cassava (Manihot esculenta Crantz)

Culture of cassava somatic embryos on media with an altered macro- and micro-nutrient salt concentration affected embryo development and germination capability. In the tests, quarter-, half-, full- or double-strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half- or full-strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full-strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half- and full-strength MS medium yielded 8.3 and 8.6 germinants g(-1) NEC tissue, respectively. For this important but often disregarded culture factor, either half- or full-strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination.     

Role of enzymes and identification of stage-specific proteins in developing somatic embryos of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Accumulation of proline, activities of peroxidase (POX), catalase (CAT), phenylalanine ammonia lyase (PAL) and malate dehydrogenase (MDH) were studied during different developmental stages of somatic embryos in chickpea. Callus cultures that did not form somatic embryos served as control. While increased levels of proline and POX activity were noticed in globular stages of embryos, CAT activity increased during early and late heart-shaped embryo formation indicating tissue-specific activation of these enzymes. The activity of PAL reached a peak during torpedo and cotyledonary stages of embryo development. On the other hand, MDH activity enhanced during the germination of somatic embryos inferring more requirement of energy during this stage. Electrophoretic (sodium dodecyl sulfate polyacrylamide gel electrophoresis) pattern of proteins revealed that ten bands are associated with non-embryogenic tissues, whereas 11 bands with globular, heart, torpedo and cotyledonary stages of embryo development and nine bands during the germination stage of embryos. Two extra stage-specific protein bands with molecular masses of 16 and 18 kDa appeared during globular, heart, torpedo, and cotyledonary stages. But, these bands disappeared during germination of embryos and are absent in non-embryogenic cultures. This study thus may help in the identification of proteins and the role of above enzymes during different developmental stages of somatic embryo induction and their maturation in a recalcitrant leguminous crop plant chickpea.     

Embryogenic cultures of the leguminous tree Albizia julibrissin and recovery of plants

A somatic embryogenic system was developed and plants regenerated in mimosa (Albizia julibrissin Durazz). Development of somatic embryos in the species has not previously been reported. Immature seeds, embryo cotyledons and embryo axes (cotyledons removed) at defined developmental stages were placed on induction media with different concentrations of 2,4-D. Two distinct embryogenic responses occurred: either proembryo masses or cotyledonary-stage embryos. Twenty five percent of all embryo axes cultured on basal medium produced cotyledonary somatic embryos. Six percent of in ovulo immature seed explants generated proembryo masses. These masses proliferated in liquid culture in the dark. Proembryos developed further when transferred to a growth-regulator-free semisolid medium in the light. Somatic embryos derived from either proembryo suspensions or cotyledonary embryo cultures on semisolid medium germinated to form plants that continued to grow vigorously following transfer to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Disturbance of ethylene biosynthesis and perception during somatic embryogenesis in <Emphasis Type="Italic">Medicago sativa</Emphasis> L. reduces embryos’ ability to regenerate

Effects of non-specific ethylene biosynthesis inhibitors: salicylic acid (SA) and aminoethoxyvinylglycine (AVG), and of specific inhibitors of ethylene binding to receptors: 1-methylcyclopropene (1-MCP) and 2,5-norbornadiene (NBD) applied during proliferation and differentiation phases of indirect somatic embryogenesis (SE) of Medicago sativa L. cv. Rangelander on embryogenic suspension growth, embryo production, development, and ability to germinate and convert were studied. Application of SA and AVG alone or together at concentrations from 1 to 500 μM in B₅g liquid medium during the proliferation phase had an inhibitory effect on ethylene production and embryogenic suspension growth. Additionally, it caused a drastic reduction in production of embryos and their development on BOi2Y solid differentiation medium. The inhibitory effect of SA was more visible than that of AVG. In addition, disturbance of ethylene biosynthesis during the proliferation phase of SE resulted in diminished lateral germination and conversion of cotyledonary embryos on MS solid medium. Moreover, blocking of ethylene receptors by 1-MCP during the proliferation phase also inhibited ethylene production and embryogenic suspension growth and reduced embryo production during differentiation. MCP almost completely inhibited development of cotyledonary embryos. At the same time, development of more embryos was arrested at the globular stage, and the number of abnormal embryos almost doubled. Similarly, addition of 1-MCP or NBD to the ambient atmosphere during the differentiation phase evidently arrested the development of embryos and, consequently, their ability to germinate and convert on MS regeneration medium. All the results presented above demonstrated that not only ethylene biosynthesis, but also ethylene action is involved in the control of individual phases of SE in Medicago sativa L. cv. Rangelander. And what is more, disturbance of these processes during distinct phases of SE adversely affects vigor of the somatic embryos obtained.     

Methyl laurate and 6-benzyladenine promote the germination of somatic embryos of a hybrid rose

Globular stage somatic embryos were induced in callus cultures of Rosa Heritage 2 Alister Stella Gray on medium containing 13.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and developed to the cotyledonary stage on medium containing 9 µM 2,4-D. Cotyledonary-stage embryos were transferred to germination media with or without 1.5 µM 6-benzyladenine (BA) and with or without 44 µM methyl laurate (Mela). BA and Mela both promoted the development of shoots and roots and increased the frequency of bipolar germinations. An average of 56.5% (SEdž.1%) embryos on medium containing both BA and Mela underwent bipolar germinations compared with less than 20% in treatments where either or both were excluded. The effectiveness of BA and Mela was reduced if Mela was included in the development medium or if the concentration of salts and vitamins in the germination media was sub-optimal. There was evidence that growth at one pole of the somatic embryo promoted development at the other.     

Embryogenic suspension cultures of Pinus nigra Arn.: growth parameters and maturation ability

Suspension cultures have been established from embryogenic tissues of Pinus nigra initiated from immature zygotic embryos. The growth of tissues in liquid medium has been influenced by initial tissue weight used for the establishment of the cultures as well as by genotype. In most of the cases initial tissue weight 0.5 g was insufficient and the cultures showed poor growth and later degeneration. Higher amount of initial tissues (1 or 2.5 g) was more efficient for the establishment and proliferation of somatic embryos in liquid medium. The growth of suspension cultures was also cell line dependent. Somatic embryo maturation in liquid medium was very limited and no plantlet regeneration occurred. Cotyledonary somatic embryos developed and produced emblings when the suspension was plated on filter paper discs and cultured on solid maturation medium. Based on our experiments we can state that the embryogenic tissues are able to grow and proliferate in liquid medium but somatic embryo maturation and plantlet regeneration occur only on solid medium.     

Factors influencing induction and maintenance of <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. embryogenic cultures

Unopened leaves, petioles and fully opened leaves from micropropagation cultures of five Vitis rotundifolia Michx. varieties were cultured on induction medium to study their embryogenic response. Among the various explants tested, the maximum number of varieties produced embryogenic cultures from unopened leaves followed by fully opened leaves and petioles. Based on morphological differences, two types of embryogenic cultures were identified. Friable cultures typically arose as proembryonic masses (PEM) on induction medium, whereas somatic embryo production without an intervening PEM stage was observed in compact cultures. Of the five varieties tested, the highest frequency of embryogenic response was observed from fully opened leaves of ‘Supreme’ and unopened leaves and petioles of ‘Delicious’. Attempts to initiate suspension cultures from varieties resulted in proliferation and maintenance of ‘Alachua’ and ‘Carlos’ cultures in liquid medium for 16 weeks. Embryogenic potential of varieties was studied on cultures growing on embryo development medium. The maximum number of cotyledonary stage somatic embryos from 0.2 g proembryonic masses were observed in ‘Carlos’ (379.3) followed by ‘Alachua’ (350.0) and ‘Delicious’ (305.0). Cotyledonary stage somatic embryos germinated when cultured on Murashige and Skoog medium containing 1 μM Benzyladenine (BA). Although high embryo germination rates (80–100%) were observed in the varieties tested, plant recovery from germinated somatic embryos ranged from 6–47%. Embryogenic cultures could be maintained on X6 medium and used in genetic engineering studies.     

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.     

Effects of ABA on secondary embryogenesis from somatic embryos induced from inflorescence culture ofAralia cordata Thunb

In order to investigate the effect of ABA on secondary embryogenesis from somatic embryos inAralia cordata Thunb., embryogenic callus and somatic embryos were induced from inflorescence on solid MS basal medium supplemented with 1.5 mg/L 2,4-D after eight weeks without subculture. For mass production of somatic embryos, embryogenic cell clumps were maintained in liquid MS medium supplemented with 1.0 mg/L 2,4-D, and then transferred to 2, 4-D-free medium. When developing embryos at various stages were cultured separately in liquid medium with ABA (0 to 2.0 mg/L) for three weeks, and then cultured in ABA-free liquid medium for two weeks, torpedo-shaped embryos exhibited secondary embryogenesis of 65.9% in only 0.2 mg/L ABA pretreatment. Cotyledonary embryos in cultures by 0.2, 0.5 and 1.0 mg/L ABA pretreatment also exhibited secondary embryogenesis (73%, 9.4% and 6.0%, respectively). However, globular and heart-shaped somatic embryos treated with ABA did not form secondary embryos on their hypocotyl surfaces. When cotyledonary embryos were cultured in ABA-free medium or 0.2 mg/L ABA treated medium for three weeks, and then in ABA-free liquid medium for 6 weeks, the germination frequency was lower in medium with 0.2 mg/L ABA (45.9%) than in hormone-free medium (56.8%). This result seems to be related to the high frequency of secondary embryogenesis. It is suggested that secondary embryogenesis by ABA application depends upon the stage of embryo cultured and the ABA concentration.     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

Enhancement of American chestnut somatic seedling production

Somatic embryogenesis holds promise for mass propagation of American chestnut trees bred or genetically engineered for resistance to chestnut blight. However, low germination frequency of chestnut somatic embryos has limited somatic seedling production for this forest tree. We tested the effects of culture regime (semi-solid versus liquid), cold treatment, AC and somatic embryo morphology (i.e., cotyledon number) on germination and conversion of the somatic embryos. Cold treatment for 12 weeks was critical for conversion of chestnut somatic embryos to somatic seedlings, raising conversion frequencies for one line to 47%, compared to 7% with no cold treatment. AC improved germination and conversion frequency for one line to 77% and 59%, respectively, and kept roots from darkening. For two lines that produced embryos with one, two or three-plus cotyledons, cotyledon number did not affect germination or conversion frequency. We also established embryogenic American chestnut suspension cultures and adapted a fractionation/plating system that allowed us to produce populations of relatively synchronous somatic embryos for multiple lines. Embryos derived from suspension cultures of two lines tested had higher conversion frequencies (46% and 48%) than those from cultures maintained on semi-solid medium (7% and 30%). The improvements in manipulation of American chestnut embryogenic cultures described in this study have allowed over a 100-fold increase in somatic seedling production efficiency over what we reported previously and thus constitute a substantial advance toward the application of somatic embryogenesis for mass clonal propagation of the tree.     

An optimised protocol for plant regeneration from embryogenic suspension cultures of date palm,Phoenix dactylifera L., cv. Deglet Nour

An improved protocol is described for the large-scale micropropagation of an elite date palm ( Phoenix dactylifera L.) cultivar, Deglet Nour. Clonal plants were regenerated from somatic embryos derived from highly proliferating suspension cultures. Friable embryogenic calli were initiated from both leaf and inflorescence explants. Suspension cultures consisting of pro-embryonic masses were established from calli showing a high competency for somatic embryogenesis. The subculture of suspensions in liquid medium enriched with low amounts of plant growth regulators (1 mg l(-1) 2,4-dichlorophenoxyacetic acid with 300 mg l(-1) charcoal) resulted in the differentiation of large numbers of somatic embryos. The productivity of the cultures increased 20-fold (from 10 to 200 embryos per month per 100 mg fresh weight of embryogenic callus) when embryogenic suspensions were used instead of standard cultures on solid media. The overall production of somatic embryos reached 10,000 units per litre per month. Partial desiccation of the mature somatic embryos, corresponding to a decrease in water content from 90% to 75%, significantly improved germination rates (from 25% to 80%). The cutting back of the cotyledonary leaf was also found to stimulate embryo germination. Flow cytometric analysis showed that the micropropagation protocol followed here did not affect the ploidy level of somatic embryo-derived plantlets.     

Development and characterization of embryogenic suspension cultures of pecan

A method for the establishment and proliferation of developmentally stable, embryogenic suspension cultures in pecan is described, and the growth and development of cultures characterized. Suspension cultures were generated from somatic embryos derived from zygotic embryo cotyledon explants induced on a solidified medium with naphthaleneacetic acid. Cultures were repetitively embryogenic and proliferated in growth-regulator-free medium. The suspensions consisted of a mixture of globular stage embryo-aggregates, freely suspended globular embryos and pre-globular stage embryo masses. Culture growth and proembryo production were evaluated with respect to several liquid media and pH conditions. Significant differences in growth and productivity were observed between cultures. Pre-globular stage embryo masses collected on filter paper and overlaid on solidified medium continued ontological development and converted into plants. Thus a method has been developed for pecan suspension culture, which presents a major improvement in embryogenic tissue culture within the Juglandaceae. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of plant growth regulators,carbon sources and iron on the cyclic secondary somatic embryogenesis and plant regeneration of transgenic cherry rootstock `Colt' (<Emphasis Type="Italic">Prunus avium</Emphasis> × <Emphasis Type="Italic">P. pseudocerasus</Emphasis>)

The frequency of long-term secondary somatic embryogenesis and shoot meristem development from embryogenic masses of the cherry rootstock `Colt' ( Prunus avium × P. pseudocerasus), differentiated from transgenic roots containing the T-DNA of Agrobacterium rhizogenes, has opened the way for genetic improvement by biotechnological techniques. Whole plants were produced by stimulating shoot meristem development from somatic embryos. The combination of 4 mg l^–1 of kinetin and 2% of maltose under illumination stimulated shoot development and, subsequently, whole plants have been recovered by applying 1.5 mg l^–1 kinetin to the rooting medium. Although numerous treatments have been tested involving both embryogenic masses and whole embryos, normal embryo germination was observed sporadically. Cold treatment was effective in stimulating secondary somatic embryogenesis with embryo development to the cotyledonary stage, but did not promote their germination. Similarly, a higher concentration (44–55 mg l^–1) of chelated iron than that commonly used in tissue culture media (36.7 mg l^–1) produced, after 3 weeks in culture, almost a 50% increase in the number of embryos at the cotyledonary stage per embryogenic mass. Among the cytokinins tested, 1 mg l^–1 of 6-benzylaminopurine and 0.1 mg l^–1 of thidiazuron were effective in inducing secondary somatic embryogenesis; however, each of them expressed highest efficiency with specific medium and environmental conditions. Furthermore, application of 1 mg l ^–1 thidiazuron reverted morphogenic callus to non-morphogenic callus, particularly in medium containing 2% sucrose. Finally, hormone free medium with 2% maltose enhanced maturation of the emb-ryos to the normal cotyledonary stage. This paper has improved knowledge of embryo culture and plant production in this important genotype, opening the way for genetic improvement by biotechnological techniques, mainly with the aim of modifying the growth pattern of the canopy of sweet cherry grafted on it.