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Embryogenesis from isolated microspores of chickpea and field pea – Progress towards a doubled haploid protocol as a tool for crop improvement

Janine Croser1, Monika Lulsdorf2, Bifang Cheng2, Kara Allen2, Julia Wilson1, Tim Dament2, Kadambot Siddique1, Tom Warkentin2 and Albert Vandenberg2

1 Centre for Legumes in Mediterranean Agriculture, The University of Western Australia, 35 Stirling Hwy, Crawley, 6009 WA
www.clima.uwa.edu.au
Email jcroser@agric.uwa.edu.au
2
The Crop Development Centre, The University of Saskatchewan, 51 Campus Drive, S7N5A8, Saskatoon, Saskatchewan,
Canada www.usask.ca/agriculture/plantsci/cdc.html Email lulsdorf@skyway.usask.ca

Abstract

The ability to produce doubled haploid plants is an effective tool for accelerating homozygosity in many broad-acre crop species. The leguminous species are generally recalcitrant to this technique and there are only isolated reports of successful embryogenesis or plant regeneration. Collaboration between Australian and Canadian researchers has led to the development of protocols for the development to early stage microspore-derived embryos in chickpea and field pea. Stress pretreatments are used to induce the switch from gametic to sporophytic development. Immature chickpea flower buds are pretreated for 16 h at 32.5C, and field pea buds at 4C for 48 h. The microspores are cultured on AT3-1 or ML6 media to induce cellular division and embryogenesis. DAPI staining is used to enable analysis of cellular divisions which proceed via a symmetrical division of the vegetative nucleus and then continued division to a synctium prior to cellularisation. Experiments are underway to induce embryo maturation and plant regeneration.

Media summary

Australian and Canadian researchers are developing a protocol for doubled haploid production in chickpea and field pea which will reduce breeding time and enable better exploitation of molecular markers.

Key Words

Haploid, isolated microspore culture, chickpea, field pea, embryogenesis, tissue culture.

Introduction

The development of protocols for doubled haploid plant production offers breeders a tool for the rapid production of homozygous breeding lines. These homozygous lines are multiplied and released as cultivars, or more commonly, used as recombinant inbred lines for molecular mapping or as parents in breeding programs. Embryogenesis from immature pollen (microspores) is widely regarded as the most efficient system for the production of doubled haploid plants and is routinely used in many of the broad-acre crops e.g. wheat, barley, rice and canola. The cool-season pulse species are recalcitrant to this technique and protocols have yet to be developed for the target species of our research, chickpea and field pea.

There are a number of steps in the development of an isolated microspore culture protocol.

  • Responsive genotypes and appropriate growing conditions need to be identified. These genotypes can then be used to optimise the conditions for the protocol, prior to widening its application to other genotypes.
  • Microspores must be switched from the gametic to the sporophytic pathway. Stress factors such as cold pretreatment of immature flower buds, carbon starvation pretreatments or heat pretreatment of immature buds or isolated microspores have been shown to act as triggers for this switch (Touraev et al., 1997).
  • Microspore culture conditions require optimisation. Embryo induction, maturation and plantlet regeneration are affected by factors such as the medium composition and osmolarity, microspore culture density, incubation temperature, light intensity, and culture container (Obert et al., 2000, Castillo et al., 2000).
  • A chromosome doubling protocol using an anti-mitotic chemical such as colchicine is required if there is a low level of spontaneous doubling of haploid material.

Australian and Canadian pulse researchers have joined forces to overcome the paucity of information regarding isolated microspore culture in the cool-season grain legumes and develop protocols for doubled haploid production in field pea and chickpea. We reason that optimisation of the donor plant growth conditions, coupled with an appropriate stress pretreatment and culture regime will lead to a switch from gametic to sporophytic development and the initiation of embryogenesis in these species.

Methods

Donor Plant Growth

Donor plants were grown in 30 cm plastic pots at 19C day/14C night for chickpea and 18C day/13C night for field pea and a light intensity >450 E m-1 s-2 for 16 h. Pots were filled with a composted pine bark/cocco peat/river sand soil mix (Australia) or Redi-earth mix (Canada). The seedlings were fertilised with 3 g/pot of 20:20:20 N:P:K soluble fertiliser two weeks after planting and fortnightly thereafter.

Bud Harvest, Pretreatment and Sterilisation

Buds were harvested when the microspores reached the uninucleate stage. Chickpea buds were treated at 32.5C for 16 h and field pea buds at 4C for 48 h before culture. Buds were sterilised with stirring in 1% w/v NaOCl for 18 minutes.

Microspore Isolation and Culture

The microspore isolation procedure was modified from protocols for barley (Hunter, 1988) and canola (Chuong and Beversdorf, 1985). Microspores were isolated by hand maceration of buds in 0.3 M mannitol. The resulting microspore slurry was passed through a cheesecloth filter, followed by a 40 m then a 5 m nylon filter (chickpea) or a 45 m then a 15 m nylon filter (field pea). The microspores were floated off the smaller diameter filters, transferred to a 15 ml centrifuge tube and centrifuged for 5 min at 1000 rpm. The supernatant was discarded and the pellet re-suspended in 10 ml of 0.3 M mannitol and re-centrifuged. The supernatant was poured off and the microspore pellet re-suspended in 1 ml liquid culture medium. Density was determined using a Hausser Scientific haemocytometer and microspores were plated at 1 ml/well with a density of 105/ml in an IWAKI 24-well suspension plate. Microspore cultures were incubated in the dark at 27C. Chickpea microspores were cultured in modified AT3-1 medium (Hofer et al., 1999) with 9% sucrose and field pea microspores were cultured in modified ML6 medium with 9% sucrose (Kumar et al., 1988). All media had a pH of 5.8 and were filter sterilised.

Cytology

A 100 l microspore sample was taken for cytological analysis at extraction and 5 d after culture. The sample was fixed in 500 l Carnoy’s fixative (3:1 Ethanol:Glacial acetic acid) for ca. 24 h. The fixative was removed and the microspore pellet was suspended in 30 l of 0.3 M pH 4 mannitol with 1 l of 0.1 mg/l of the DNA specific fluorochrome DAPI (Sigma D-8417). After 10 min in the dark, the sample was transferred to a slide and microspore development examined with a Zeiss Axioscope microscope.

Results

Our research has identified genotype, stress pre-treatments and medium composition as important to the induction of sporophytic development in the microspores of both chickpea and field pea.

A wide range of genotypes were screened for responsiveness of the isolated microspores to induction of symmetrical division. The highest induction frequency was obtained from the field pea cultivar Highlight (Swedish) and chickpea cultivars Bumper (Australian), CDC Cabri (Canadian) and breeding line WACPE 2095 (Australian).

Heat stress applied to the chickpea buds at 32.5C for 16 h prior to culture was the most successful pretreatment for the induction of symmetrical division with the exception of the breeding line WACPE 2095, which underwent induction in ca. 15% of the microspores without any stress pretreatment. For field pea, cold stress applied to the buds at 4C for 48 h prior to culture was the most successful pretreatment for the induction of symmetrical division.

Symmetrical microspore nuclear division was observed in chickpea (Fig. 1A) when cultured on modified AT3-1, a medium developed for apple microspore culture. A similar response was seen in field pea (Fig. 1B) following culture on the legume-specific ML6 medium, also with an increased sucrose concentration. Fructose, maltose and glucose were trialed as alternative carbohydrate sources, but microspore survival and development were not improved or reduced compared with sucrose. The optimal concentration of sucrose in the medium was 9% for both species. On the same media, division continued in the symmetrically divided nuclei leading to the formation of multinucleate synctiums in both chickpea (Fig. 1C) and field pea (Fig. 1D). These synctiums underwent cellularisation to form early stage globular embryos in chickpea (Fig. 1E & 1F), and less often in field pea (Fig. 1G). In rare instances, early stage embryos were observed to continue development in chickpea (Fig. 1H) and in field pea (Fig. 1I).

A

B

C

D

E

F

G

H

I

Figure 1. Cytological development of male gametophyte in chickpea and field pea.

(A) Symmetrical division of the vegetative nucleus in a chickpea microspore stained with DAPI (200x). (B) Symmetrical division of the vegetative nucleus in a field pea microspore stained with DAPI (400x). (C) Chickpea microspore-derived multinucleate synctium stained with DAPI (200x). (D) Field pea microspore-derived multinucleate synctium stained with DAPI (400x). (E) Phase contrast micrograph showing cellularisation of an early stage embryo in chickpea (200x). (F) Inverted microscope micrograph of a chickpea microspore-derived early stage embryo following cellularisation (200x). (G) Cellularisation of a field pea microspore-derived early stage embryo stained with DAPI (200x). (H) Chickpea microspore-derived globular embryo with suspensor cells stained with 2% acetocarmine (100x). (I) Field pea microspore-derived torpedo shaped embryo (60x).

Conclusion

By optimising the donor-plant growing conditions, genotype, pretreatment regime and culture conditions we have developed a protocol for the routine induction of microspore embryogenesis in both chickpea and field pea. The results of this research compare favourably with early research in crops where this technique is now used routinely, such as barley and canola. More research is required to overcome barriers to further embryo development. Current experiments are focusing on increasing induced microspore frequency and establishing protocols for routine embryo maturation and plantlet regeneration.

References

Castillo AM, Valles MP, Cistue L (2000) Comparison of anther and isolated microspore cultures in barley. Effects of culture density and regeneration medium. Euphytica 113, 1-8.

Chuong PV and Beversdorf WD (1985) High frequency embryogenesis through isolated microspore culture in Brassica napus L. and B. carinata Braun. Plant Science 39, 219-226.

Hofer M, Touraev A, Herberle-Bors E (1999) Induction of embryogenesis from isolated apple microspores. Plant Cell Reports 18, 1012-1017.

Hunter CP (1988) Plant regeneration from microspores of barley, Hordeum vulgare L. PhD thesis, Wye College, University of London.

Kumar AS, Gamborg OL, Nabors MW (1988) Plant regeneration from cell suspension cultures of Vigna aconitifolia. Plant Cell Reports 7, 138-141.

Obert B, Pretova A, Butler B, Schmid JE (2000) Effect of different saccharides on viability of isolated microspores and androgenic induction in Zea mays. Biol Planta 43,125-128.

Touraev A, Vincente O, Herberle-Bors E (1997) Initiation of microspore embryogenesis by stress. Trends in Plant Sciences 2, 297-302.

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