Desiccation Tolerance In Somatic Embryos Research Articles

Induction of desiccation-tolerance in somatic embryos of Cyclamen persicum Mill.

SummaryAs a first step in the development of synthetic seed of Cyclamen persicum Mill. various procedures for induction of desiccation-tolerance in somatic embryos of cyclamen have been tested. Most important proved to be the careful selection of mature embryos by morphological characteristics. Torpedo-shaped somatic embryos with a size between 700 and 1000 µm exhibited the highest germination rate after desiccation to only 0.2 g H2O g–1 DW. Addition of 75 g l–1 polyethylene glycol (PEG 4000) and 10 mg l–1 abscisic acid to the maturation medium increased the germination rate of somatic embryos after desiccation to 28% compared to 4% in the control without these supplements.

Improving tolerance of somatic emrbyos of Picea glauca to flash desiccation with a cold treatment (desiccation after cold acclimation)

Controlled mild desiccation of mature white spruce somatic embryos prior to germination improves the quality of the germinated embryos. More severe desiccation results in increased injury and death but is desirable for long-term storage of embryos and production of desiccated artificial seed. A method was developed to improve desiccation tolerance in somatic embryos using a temperature treatment. Culture plates with embryos at four stages of development were subjected to temperatures of 1, 5, 10, or 20°C for periods of 0, 1, 2, 4, or 8 wk duration. After the temperature treatment, the embryos were harvested and air-dried for 2h under a laminar flow hood. Dried embryos were placed directly on germination medium and the quality of the germinants was assessed after 4 wk. The initial maturation stage of the embryo and the temperature and duration of the treatment had a significant effect on the quality of the germinants. Most treatments caused marked differential survival of organs. The optimal response was obtained with embryos that had been grown for 51 d (cotyledonary stage) on maturation medium and that were subsequently exposed to a temperature of 5°C for 8 wk prior to air drying. This treatment produced 58% undamaged germinants with normal cotyledons, hypocotyls, and roots. Only 1% of the untreated air-dried embryos germinated normally.

Membrane permeability, morphology, and desiccation tolerance ofDaucus carota somatic embryos as influenced by drying rate

Carrot (Daucus carota) somatic embryos that were treated with abscisic acid during their development have been shown to acquire complete desiccation tolerance when slowly dried, but fail to do so when rapidly dried. We studied plasma membrane permeability by a spin probe technique. On rehydration, the plasma membranes of rapidly dried somatic embryos became permanently leaky, whereas those of slowly dried embryos retained permeability levels as low as for fresh embryos. The leakage was associated with extensive deesterification of the phospholipids. Using low-temperature scanning electron microscopy (LTSEM), we examined (re)hydrated and dry somatic and zygotic embryos. No differences in surface morphology were detected between dry tolerant and intolerant somatic embryos that were obtained by slow and rapid drying, respectively. However, on rehydration, the nonviable somatic embryos had lost turgor and intracellular organization, as revealed by LTSEM images of fractured embryos, whereas the viable somatic embryos had turgescent cells with intact cell structure. In 3-day-imbibed, viable embryos, cell extension was visible. These findings are consistent with the electron paramagnetic resonance data. Somatic embryos always had reduced and abnormal cotyledons, mostly fused, and the surface was irregular. The surface of dry somatic embryo cells was considerably more wrinkled than that of dry zygotic embryo cells. Maturation in polyethylene glycol-containing medium increased the percent dry matter in the fresh somatic embryos and reduced the extent of wrinkling after drying. Wrinkling of the dried embryo cells apparently depends on the filling with dry matter in the hydrated state. We suggest that wrinkling per se does not limit desiccation tolerance of somatic embryos.

Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16-26 days after pollination (DAP) and desiccation tolerance was induced after 20-26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.

Manipulating the desiccation tolerance and vigor of dry somatic embryos of Medicago sativa L. with sucrose, heat shock and abscisic acid

The effects of sucrose concentration in the maturation medium in combination with a heat shock treatment at 36°C were investigated in an attempt to improve the vigor of seedlings grown from dry somatic embryos of alfalfa (Medicago sativa L.). Callus was formed from petiole expiants and dispersed in liquid suspension medium in the presence of 5 μM 2,4-D. The cell suspension was sieved to synchronize embryo development. The 200 - 500 μm fraction was plated on embryo development medium without 2,4-D, grown for 14 days, and transferred to maturation medium. With 3% sucrose in the maturation medium, the somatic embryos germinated precociously and were unable to survive desiccation. At higher sucrose concentrations, germination was delayed and the embryos continued to accumulate dry matter. After 13 days on 6% sucrose medium (27 days after sieving), the somatic embryos were tolerant of drying to 12% moisture without exposure to exogenous ABA. Heat shock, which presumably stimulates endogenous ABA synthesis, improved the desiccation tolerance of somatic embryos if applied prior to day 27 after sieving, but its effects were minimal after day 27. High sucrose concentrations up to 9% in the maturation medium were optimal during the first 8 days on maturation medium (days 14 to 22 after sieving), but a lower concentration (6%) was optimal during the later stages of embryo maturation (days 22 to 30 after sieving). The inclusion of 10(-5) M ABA in the maturation medium with 6% sucrose further improved embryo quality if applied approximately 20 days after sieving.

FACTORS INDUCING DESICCATION TOLERANCE IN CELERY SOMATIC EMBRYOS

Desiccation tolerance of somatic embryos is a key factor for production of dry synthetic seeds. In celery (Apium graveolens L.) desiccation tolerance can be enhanced by optimization of culture duration, ABA application, or sucrose concentration in the embryo production medium. Morphologically mature embryos cultured for 10 days have shown higher desiccation tolerance then those cultured for 8 days, indicating that biochemical changes occur without any noticeable morphological changes. Application of ABA (1 μM) for the last two days of the embryo production cycle was critical for inducing desiccation tolerance; ABA application for the last four days had some additional beneficial effect. Desiccation tolerance was further enhanced by increasing the sucrose concentration of the embryo production media from 3% to 7% for the last two days. Increased desiccation tolerance achieved with optimal harvest timing and ABA application were associated with increased endogenous proline and aminobutyrate, and reduced glutamine.

Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.