Spruce Somatic Embryo Maturation Research Articles

The anti-actin drugs latrunculin and cytochalasin affect the maturation of spruce somatic embryos in different ways

The role of the actin cytoskeleton in somatic embryo development was investigated using latrunculin B and cytochalasin D. Brief treatments (1h) with either drug at the start of maturation fragmented the actin in suspensor cells and/or depolymerized actin filaments in meristematic cells. The drugs targeted different cells: latB primarily affected the suspensor cells, but cchD damaged both suspensor and meristematic cells. Lethal damage to the meristematic and suspensor cells was observed when the drugs were applied throughout the maturation period, although the severity of this effect depended on their concentrations.The drugs’ effects on the yield of mature somatic embryos were investigated by applying them to embryo cultures throughout the maturation period or for one week at three different points in the maturation process: immediately prior to the start of maturation, during the first week of maturation, and during the fourth week of maturation. The strongest effects were observed when the drugs were applied at the start of maturation. Under these conditions, latB destroyed the suspensors, eliminating the underdeveloped embryos that depend on them. This accelerated the development of embryos that were capable of separating from the suspensors. Thus, while the total number of embryos at the end of the maturation period was lower than in untreated control cultures, the surviving mature embryos were of high quality. cchD treatment at the start of maturation strongly inhibited embryo development. Drug treatment at the end of the maturation period did not significantly affect embryo development: latB caused no change in the yield of somatic embryos, but cchD treatment increased the number of malformed embryos compared to untreated controls.

The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos.

The present study was conducted to understand the role of sucrose in the medium on the maturation of black spruce and white spruce somatic embryos. A maturation medium containing 6% sucrose, which hydrolyzed into glucose and fructose, gave significantly more embryos than a medium containing 3.16% of each glucose and fructose. Preventing the complete sucrose hydrolysis by a daily transfer of the tissues onto fresh medium significantly decreased the yield of somatic embryos compared to when sucrose was allowed to complete its hydrolysis. This reduction was not due to the manipulation of the tissues during the transfer, since a daily in situ transfer did not affect embryo production. To verify if the better embryo production observed on a medium containing 6% sucrose was due to the increasing osmotic pressure of the medium, this increasing osmotic pressure was simulated with a sequence of media containing different concentrations of glucose and fructose. Unexpectedly and for both species, this simulation did not improve somatic embryo production, which stayed similar to the one obtained on constant osmotic pressure. To understand these results, embryos produced on the different treatments were analyzed in terms of sucrose, glucose, fructose and starch levels and protein contents. The embryo carbohydrate content was independent from the carbohydrate used in the maturation medium. However, embryos matured on 6% sucrose allowed to hydrolyze during the maturation period contained significantly more soluble and insoluble proteins than embryos matured on any other treatment. Furthermore, embryos with a higher protein content also exhibited a higher epicotyl appearance frequency. The role of sucrose as a regulatory factor during the maturation of spruce somatic embryos is discussed.

Ascorbic acid metabolism during white spruce somatic embryo maturation and germination

Changes in ascorbic acid (AA) metabolism were investigated in 3 different cell lines of white spruce; a non‐embryogenic line (NE)WS and two embryogenic lines with low (E)WS1 and high (E)WS2 percentage of embryo germination. During embryo maturation, a high AA/dehydroascorbate (DHA) ratio was observed in the two embryogenic cultures. The increment of this ratio was mainly ascribed to the high ability of the (E)WS1 and (E)WS2 lines to generate AA from its last precursor, l‐galactono‐γ‐lactone, since no increase in the activities of the AA‐recycling enzymes, i.e., AA free‐radical reductase (AFRR) and DHA reductase (DHAR), was observed as the embryos developed. During the partial drying treatment needed for successful embryo germination, the ability to de novo generate AA continued to increase in the embryos of the two embryogenic lines, although the total ascorbate pool (AA+DHA) sharply decreased. A reactivation of the overall AA metabolism was observed at the inception of germination, especially in the (E)WS2 embryos. These results suggest that (1) the ability to metabolically alter the AA/DHA ratio may be a fundamental requirement for somatic embryo maturation and (2) the restoration of the AA metabolism during the first days of germination may be necessary for successful embryo germination.

Endochitinase and beta-1,3-glucanase genes are developmentally regulated during somatic embryogenesis in Picea glauca.

Two cDNAs isolated from white spruce [Picea glauca (Moench) Voss] somatic embryos, are predicted to encode a basic class IV chitinase and a beta-1,3-glucanase, respectively corresponding to genes PgChi-1 and PgGlu-1. Each represents a multigene family in spruce. Transcripts homologous to PgChi-1 or PgGlu-1 genes were highly abundant in embryogenic tissues and gradually decreased after tissues were placed on abscisic acid-containing maturation medium, with lowest abundance in globular embryos. Transcripts related to PgGlu-1 became highly abundant again in early cotyledonary embryos but decreased thereafter, whereas transcripts related to PgChi-1 were also highly abundant in late cotyledonary embryos and plantlets in vitro; transcripts were either low (PgChi-1) or were not detectable (PgGlu-1) in needles. Wounding, drying and flooding stresses enhanced PgChi-1- and PgGlu-1-related gene expression. Fungal cell wall suspension enhanced PgGlu-1-related transcript accumulation, but reduced PgChi-1-related transcript abundance within 24 h. PgChi-1 and PgGlu-1 and their homologues may have roles in plant defense, and possibly developmental roles during spruce somatic embryo maturation.

Abscisic acid [(+)-ABA] content in white spruce somatic embryo tissues related to concentration of fed ABA

Summary The availability of ABA and tissue content of (+)-ABA were investigated, when using different starting concentrations of (±)-ABA to stimulate white spruce somatic embryo maturation. ELISA analysis of tissue (+)-ABA in white spruce somatic embryos during maturation showed that tissue content of (+)-ABA rose proportionally to the concentration of fed ABA (between 12 and 60μmol·L −1 (±)-ABA). Irrespective of the starting concentration of fed (±)-ABA, peak values in tissue (+)-ABA were obtained after 3–7d. Following this there was a steady decline from approximately 12 to 28 d, after which tissue (+)-ABA remained at relatively constant low levels. The levels of tissue (+)-ABA under each starting concentration of (±)-ABA were very similar to one another by 28 d. There was no significant difference in tissue (+)-ABA content between embryos and embryo-free tissue fractions. Metabolism of fed (±)-ABA followed a similar pattern to (+)-ABA content, being proportional to the initial concentration. Metabolites of (+)-ABA were present in the medium in the first 6d, metabolism of (+)-ABA is likely to have depleted it from the medium between 12 to 28 d, irrespective of the initial concentration of (±)-ABA in the medium. These data indicate that the high levels of exogenous (±)-ABA that are needed to stimulate white spruce somatic embryo maturation, most likely have their major stimulatory effect on maturation during the first 12 d of the 6 to 8 wk culture period, there being no substantive difference among the concentrations in the period of (+)-ABA availability. To improve the yield of mature somatic embryos it will either be necessary to increase the abundance of responsive immature somatic embryos during this 12 d period, or to extend the period of availability of (+)-ABA to the embryo culture through periodic addition of (±)-ABA.

Effects of ethylene and ethylene inhibitors on white spruce somatic embryo maturation

The influence of ethylene on somatic embryo maturation of white spruce ( Picea glauca (Moench) Voss) was studied through applications of ethephon, silver nitrate (AgNO 3), cobalt chloride (CoCl 2) and aminoethoxyvinylglycine (AVG) in the presence or absence of abscisic acid (ABA). Ethylene was produced by the embryogenic tissue during embryo maturation. After an initial rise in ethylene production 1 day after transferring from the maintenance to maturation medium, the level of ethylene decreased and this was followed by a gradual rise in the latter half of the culture period. ABA, AgNO 3, and CoCl 2 were effective in suppressing ethylene production during the first 2 weeks in the maturation medium. AgNO 3 and CoCl 2 were effective to a lesser extent than ABA in promoting embryo production. AVG was effective in suppressing ethylene production through the entire culture period. However, when applied alone in the absence of ABA, AVG could not stimulate embryo formation. The presence of AVG or AgNO 3 improved the quality of the embryo by preventing large intercellular space formation in the shoot pole of the somatic embryo. Ethephon inhibited embryo production and enhanced formation of the intercellular spaces. It is concluded that ethylene had a negative effect on white spruce somatic embryo development. An alternate role for AgNO 3 is also discussed.

Racemic abscisic acid and abscisyl alcohol promote maturation of white spruce ( Picea glauca) somatic embryos

Racemic abscisic acid [(±)-ABA] and racemic abscisyl alcohol were used to obtain maturation of white spruce ( Picea glauca (Moench.) Voss) immature (stage 1) somatic embryos. Data for somatic embryo development were collected between 21 and 70 days after initiation of maturation treatments. The culture period for the maximum production of cotyledonary (stage 3) somatic embryos was approx. 42 days, with either 46 μM (±)-ABA or 41 μM abscisyl alcohol during the first 35 days. Two additional abscisic acid analogues, an acetylenic aldehyde and a dihydroacetylenic alcohol were less effective in promoting somatic embryo maturation. A comparison of the molecular structures of compounds indicated that the presence of an ABA-like side chain with two double bonds was most effective in white spruce somatic embryo maturation. Another factor that influenced the maturation process was the solvent used to dissolve (±)-ABA and the analogues. Somatic embryo development could be arrested at stage 3 by extending exposure to 60 μM (±)-ABA for up to 9 weeks. Germination resulted after removal of (±)-ABA.