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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

The effect of entire and bisected pistils on embryogenic pollen development in the recalcitrant cv. “Golden Promise.” Isolated microspores at an initial density of 5000 per mL medium were co-cultivated with either entire or bisected wheat pistils as compared to a negative control without nurse tissue. The microspores were kept in Millicell inserts to facilitate the observation of their response without being obscured by nurse tissue. The inserts were positioned in 3.5 cm Petri dishes containing a total volume of 2 mL medium, while the nurse tissue was kept in the medium portion outside the inserts. Three entire pistils or parts thereof were used per mL medium. (A) The formation of embryogenic calli after 4 weeks of pollen culture. Bar size = 2 mm. (B) Numbers of embryogenic calli obtained in three size classes (data shown in the form mean ± SD) and plantlet regeneration (data shown for total and green regenerants as single values from three different biological replicates).
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Figure 4: The effect of entire and bisected pistils on embryogenic pollen development in the recalcitrant cv. “Golden Promise.” Isolated microspores at an initial density of 5000 per mL medium were co-cultivated with either entire or bisected wheat pistils as compared to a negative control without nurse tissue. The microspores were kept in Millicell inserts to facilitate the observation of their response without being obscured by nurse tissue. The inserts were positioned in 3.5 cm Petri dishes containing a total volume of 2 mL medium, while the nurse tissue was kept in the medium portion outside the inserts. Three entire pistils or parts thereof were used per mL medium. (A) The formation of embryogenic calli after 4 weeks of pollen culture. Bar size = 2 mm. (B) Numbers of embryogenic calli obtained in three size classes (data shown in the form mean ± SD) and plantlet regeneration (data shown for total and green regenerants as single values from three different biological replicates).

Mentions: Barley cv. “Golden Promise” is recalcitrant with respect to pollen embryogenesis (Coronado et al., 2005). When microspores isolated from cold-treated spikes were cultivated at a density of 5000 per mL without any nurse tissue, no callus formation ensued, but in the presence of uncut pistils, a small number of embryogenic calli of diameter ≥500 μm did form (Figure 4A). In all (across three biological replicates), 10 plantlets (0/4/6) were regenerated, of which only one (10%) was non-albino. When the nurse tissue consisted of longitudinally bisected pistils, the number of calli of diameter ≥100 μm formed was 229 ± 12 (six-fold increased), of which over half had a diameter of ≥500 μm. This treatment yielded a mean of 10–11 plantlets per replicate, of which 15.6% were green. The latter were successfully established in soil, constituting a five-fold improvement over the use of uncut pistils (Figure 4B).


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)

The effect of entire and bisected pistils on embryogenic pollen development in the recalcitrant cv. “Golden Promise.” Isolated microspores at an initial density of 5000 per mL medium were co-cultivated with either entire or bisected wheat pistils as compared to a negative control without nurse tissue. The microspores were kept in Millicell inserts to facilitate the observation of their response without being obscured by nurse tissue. The inserts were positioned in 3.5 cm Petri dishes containing a total volume of 2 mL medium, while the nurse tissue was kept in the medium portion outside the inserts. Three entire pistils or parts thereof were used per mL medium. (A) The formation of embryogenic calli after 4 weeks of pollen culture. Bar size = 2 mm. (B) Numbers of embryogenic calli obtained in three size classes (data shown in the form mean ± SD) and plantlet regeneration (data shown for total and green regenerants as single values from three different biological replicates).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: The effect of entire and bisected pistils on embryogenic pollen development in the recalcitrant cv. “Golden Promise.” Isolated microspores at an initial density of 5000 per mL medium were co-cultivated with either entire or bisected wheat pistils as compared to a negative control without nurse tissue. The microspores were kept in Millicell inserts to facilitate the observation of their response without being obscured by nurse tissue. The inserts were positioned in 3.5 cm Petri dishes containing a total volume of 2 mL medium, while the nurse tissue was kept in the medium portion outside the inserts. Three entire pistils or parts thereof were used per mL medium. (A) The formation of embryogenic calli after 4 weeks of pollen culture. Bar size = 2 mm. (B) Numbers of embryogenic calli obtained in three size classes (data shown in the form mean ± SD) and plantlet regeneration (data shown for total and green regenerants as single values from three different biological replicates).
Mentions: Barley cv. “Golden Promise” is recalcitrant with respect to pollen embryogenesis (Coronado et al., 2005). When microspores isolated from cold-treated spikes were cultivated at a density of 5000 per mL without any nurse tissue, no callus formation ensued, but in the presence of uncut pistils, a small number of embryogenic calli of diameter ≥500 μm did form (Figure 4A). In all (across three biological replicates), 10 plantlets (0/4/6) were regenerated, of which only one (10%) was non-albino. When the nurse tissue consisted of longitudinally bisected pistils, the number of calli of diameter ≥100 μm formed was 229 ± 12 (six-fold increased), of which over half had a diameter of ≥500 μm. This treatment yielded a mean of 10–11 plantlets per replicate, of which 15.6% were green. The latter were successfully established in soil, constituting a five-fold improvement over the use of uncut pistils (Figure 4B).

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