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The low molecular weight fraction of compounds released from immature wheat pistils supports barley pollen embryogenesis.

Lippmann R, Friedel S, Mock HP, Kumlehn J - Front Plant Sci (2015)

Bottom Line: Here, a micro-culture system was established to enable the culturing of populations of barley pollen at a density too low to allow unaided embryogenesis to occur, and this was then exploited to assess the effect of using various parts of the pistil as nurse tissue.The differential effect of various size classes of compounds present in the pistil-conditioned medium showed that the relevant molecule(s) was of molecular weight below 3 kDa.This work narrows down possible feeder molecules to lower molecular weight compounds and showed that the cellular origin of the active compound(s) is not specific to any tested part of the pistil.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.

ABSTRACT
Pollen embryogenesis provides a useful means of generating haploid plants for plant breeding and basic research. Although it is well-established that the efficacy of the process can be enhanced by the provision of immature pistils as a nurse tissue, the origin and compound class of the signal molecule(s) involved is still elusive. Here, a micro-culture system was established to enable the culturing of populations of barley pollen at a density too low to allow unaided embryogenesis to occur, and this was then exploited to assess the effect of using various parts of the pistil as nurse tissue. A five-fold increase in the number of embryogenic calli formed was obtained by simply cutting the pistils in half. The effectiveness of the pistil-conditioned medium was transitory, since it needed replacement at least every 4 days to measurably ensure embryogenic development. The differential effect of various size classes of compounds present in the pistil-conditioned medium showed that the relevant molecule(s) was of molecular weight below 3 kDa. This work narrows down possible feeder molecules to lower molecular weight compounds and showed that the cellular origin of the active compound(s) is not specific to any tested part of the pistil. Furthermore, the increased recovery of calli during treatment with cut pistils may provide a useful tool for plant breeders and researchers using haploid technology in barley and other plant species.

No MeSH data available.


Related in: MedlinePlus

Threshold of self-feeder effect in embryogenic pollen culture. Isolated microspores of cv. “Igri” were cultured at various densities (numbers per mL are given on the individual photographs recorded after 4 weeks of culture) in the absence of nurse tissue to determine the minimum culture density required to permit embryogenic development. The microspores were kept in Millicell inserts to facilitate the observation of their response at low culture density, while the inserts were positioned in 3.5 cm Petri dishes containing a total volume of 2 mL medium. Callus formation was completely inhibited at a culture density below the threshold level of ca. 1000 microspores per mL medium, whereas embryogenic calli developed only in cultures with a density of 3200 mL or higher. Bar size = 1.5 mm.
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Figure 1: Threshold of self-feeder effect in embryogenic pollen culture. Isolated microspores of cv. “Igri” were cultured at various densities (numbers per mL are given on the individual photographs recorded after 4 weeks of culture) in the absence of nurse tissue to determine the minimum culture density required to permit embryogenic development. The microspores were kept in Millicell inserts to facilitate the observation of their response at low culture density, while the inserts were positioned in 3.5 cm Petri dishes containing a total volume of 2 mL medium. Callus formation was completely inhibited at a culture density below the threshold level of ca. 1000 microspores per mL medium, whereas embryogenic calli developed only in cultures with a density of 3200 mL or higher. Bar size = 1.5 mm.

Mentions: The minimum culture density of immature pollen grains of cv. “Igri” required to permit embryogenic development without the need of any nurse tissue was 3200 per mL (Figure 1); lower densities than this supported only a few rounds of cell division, insufficient to produce any regenerable callus. In assessing the stimulatory effect of the nurse tissue therefore, the culture density was reduced by more than one order of magnitude to 75 grains per mL. The nurse tissues tested consisted of either entire or specific parts of wheat pistils (Figure 2), and all of these induced the formation of small calli (100–500 μm) to a similar extent (Figure 2A), while the negative control (no nurse tissue) did not develop any callus (Supplemental Table S1). Isolated ovules (both longitudinally bisected and uncut; lOvu, Ovu) had much the weakest stimulatory effect on callus growth, which was diagnosable already after 7 days of culture due to the comparatively low proportion of enlarged pollen (Supplemental Figure S1). After 2 weeks of culture, the pollen co-cultivated with stigmas (St), ovules, whole ovaries (Ovr; pistil with the stigma being cut off) or the basal, micropylar section thereof (mOvr) remarkably lagged behind in development as compared to those supported by the other tested nurse materials that had already stimulated the formation of larger individuals reminiscent of immature embryos (Supplemental Figure S1). A callus diameter of at least 500 μm is generally considered as being large enough to be capable of whole plant regeneration. Supporting the test pollen with an EPC at a density of 50,000 per mL induced the formation of only a small number of calli ≥500 μm; nonetheless, this positive control treatment was significantly superior to the co-cultivation of ovules or to pollen cultured without nurse tissue (Figure 2B, red line). While the stigma or the micropylar section of the ovary could satisfactorily substitute for the whole pistil (P) as a nurse tissue, longitudinally bisected pistils (lP) were superior to perpendicularly (cross-)bisected ones (cP), which were in turn superior to the chalazal part of the ovary (cOvr); and the latter was superior to the whole ovary (Figures 2B,C). Taken together, bisected pistils were consistently superior to other tested nurse tissues in supporting the formation of embryogenic calli ≥500 μm, whereas the behavior of longitudinally bisected pistils, either with (lP) and without (lP−Ovu) ovular tissue attached, showed that the presence of gametophytic tissue made no positive contribution to the stimulatory effect of the pistil. The relevant raw data are presented as Supplemental Table S1, and a representative image set during the cultivation of embryogenic pollen is given as Supplemental Figure S1.


The low molecular weight fraction of compounds released from immature wheat pistils supports barley pollen embryogenesis.

Lippmann R, Friedel S, Mock HP, Kumlehn J - Front Plant Sci (2015)

Threshold of self-feeder effect in embryogenic pollen culture. Isolated microspores of cv. “Igri” were cultured at various densities (numbers per mL are given on the individual photographs recorded after 4 weeks of culture) in the absence of nurse tissue to determine the minimum culture density required to permit embryogenic development. The microspores were kept in Millicell inserts to facilitate the observation of their response at low culture density, while the inserts were positioned in 3.5 cm Petri dishes containing a total volume of 2 mL medium. Callus formation was completely inhibited at a culture density below the threshold level of ca. 1000 microspores per mL medium, whereas embryogenic calli developed only in cultures with a density of 3200 mL or higher. Bar size = 1.5 mm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493395&req=5

Figure 1: Threshold of self-feeder effect in embryogenic pollen culture. Isolated microspores of cv. “Igri” were cultured at various densities (numbers per mL are given on the individual photographs recorded after 4 weeks of culture) in the absence of nurse tissue to determine the minimum culture density required to permit embryogenic development. The microspores were kept in Millicell inserts to facilitate the observation of their response at low culture density, while the inserts were positioned in 3.5 cm Petri dishes containing a total volume of 2 mL medium. Callus formation was completely inhibited at a culture density below the threshold level of ca. 1000 microspores per mL medium, whereas embryogenic calli developed only in cultures with a density of 3200 mL or higher. Bar size = 1.5 mm.
Mentions: The minimum culture density of immature pollen grains of cv. “Igri” required to permit embryogenic development without the need of any nurse tissue was 3200 per mL (Figure 1); lower densities than this supported only a few rounds of cell division, insufficient to produce any regenerable callus. In assessing the stimulatory effect of the nurse tissue therefore, the culture density was reduced by more than one order of magnitude to 75 grains per mL. The nurse tissues tested consisted of either entire or specific parts of wheat pistils (Figure 2), and all of these induced the formation of small calli (100–500 μm) to a similar extent (Figure 2A), while the negative control (no nurse tissue) did not develop any callus (Supplemental Table S1). Isolated ovules (both longitudinally bisected and uncut; lOvu, Ovu) had much the weakest stimulatory effect on callus growth, which was diagnosable already after 7 days of culture due to the comparatively low proportion of enlarged pollen (Supplemental Figure S1). After 2 weeks of culture, the pollen co-cultivated with stigmas (St), ovules, whole ovaries (Ovr; pistil with the stigma being cut off) or the basal, micropylar section thereof (mOvr) remarkably lagged behind in development as compared to those supported by the other tested nurse materials that had already stimulated the formation of larger individuals reminiscent of immature embryos (Supplemental Figure S1). A callus diameter of at least 500 μm is generally considered as being large enough to be capable of whole plant regeneration. Supporting the test pollen with an EPC at a density of 50,000 per mL induced the formation of only a small number of calli ≥500 μm; nonetheless, this positive control treatment was significantly superior to the co-cultivation of ovules or to pollen cultured without nurse tissue (Figure 2B, red line). While the stigma or the micropylar section of the ovary could satisfactorily substitute for the whole pistil (P) as a nurse tissue, longitudinally bisected pistils (lP) were superior to perpendicularly (cross-)bisected ones (cP), which were in turn superior to the chalazal part of the ovary (cOvr); and the latter was superior to the whole ovary (Figures 2B,C). Taken together, bisected pistils were consistently superior to other tested nurse tissues in supporting the formation of embryogenic calli ≥500 μm, whereas the behavior of longitudinally bisected pistils, either with (lP) and without (lP−Ovu) ovular tissue attached, showed that the presence of gametophytic tissue made no positive contribution to the stimulatory effect of the pistil. The relevant raw data are presented as Supplemental Table S1, and a representative image set during the cultivation of embryogenic pollen is given as Supplemental Figure S1.

Bottom Line: Here, a micro-culture system was established to enable the culturing of populations of barley pollen at a density too low to allow unaided embryogenesis to occur, and this was then exploited to assess the effect of using various parts of the pistil as nurse tissue.The differential effect of various size classes of compounds present in the pistil-conditioned medium showed that the relevant molecule(s) was of molecular weight below 3 kDa.This work narrows down possible feeder molecules to lower molecular weight compounds and showed that the cellular origin of the active compound(s) is not specific to any tested part of the pistil.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.

ABSTRACT
Pollen embryogenesis provides a useful means of generating haploid plants for plant breeding and basic research. Although it is well-established that the efficacy of the process can be enhanced by the provision of immature pistils as a nurse tissue, the origin and compound class of the signal molecule(s) involved is still elusive. Here, a micro-culture system was established to enable the culturing of populations of barley pollen at a density too low to allow unaided embryogenesis to occur, and this was then exploited to assess the effect of using various parts of the pistil as nurse tissue. A five-fold increase in the number of embryogenic calli formed was obtained by simply cutting the pistils in half. The effectiveness of the pistil-conditioned medium was transitory, since it needed replacement at least every 4 days to measurably ensure embryogenic development. The differential effect of various size classes of compounds present in the pistil-conditioned medium showed that the relevant molecule(s) was of molecular weight below 3 kDa. This work narrows down possible feeder molecules to lower molecular weight compounds and showed that the cellular origin of the active compound(s) is not specific to any tested part of the pistil. Furthermore, the increased recovery of calli during treatment with cut pistils may provide a useful tool for plant breeders and researchers using haploid technology in barley and other plant species.

No MeSH data available.


Related in: MedlinePlus