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Anther and microspore culture • Suggested reading: Vasil ch. 7, Bhojwani ch.

7 • Importance of anther/microspore culture
– completely homozygous inbred lines • no segregation in progeny (doubled haploid lines) • uniform inbred lines in one generation • inbred lines for F1 hybrid cultivars (e.g., oilseed brassicas) – in polyploids (e.g., Medicago & potato), used for making DHs for classical breeding & for somatic hybridization

Anther and microspore culture • Importance of anther/microspore culture – genome mapping (easier ID of markers) • Ontogeny of androgenesis – normal sequence (review) • mmc (2n) proceeds thru meiosis • tetrad formation and microspore (1n) liberation • 1st pollen mitosis yields 1 veg. & 1 gen cell • 2nd pollen mitosis (of gen cell) yields 2 sperm nuclei .

and (2) both cells divide to give a multicellular structure . cell divides w/o participation by veg. cell begins a series of mitoses ends in a multicellular structure. growth proceeds thru typical stages of embryogenesis (androgenesis) • alternative pathways: (1) gen.Anther and microspore culture • Ontogeny of androgenesis – Redirected sequences (via anther/microspore culture) • uninucleate cell goes thru 1st pollen mitosis • tobacco pathway: veg. under approp. cell. conditions.

Anther and microspore culture • Culturing methods – anther culture – easiest and simplest – pollen (microspore) culture – advantages • less competition among microspores • no diploid anther walls • greater potential haploid plant production – protocol for tobacco anther culture • (aseptically) detach anther from tobacco filament • float anther on a liquid (MS-type) culture medium .

. 40 μm for Brassicas) • centrifuge at 50-100g for ca.Anther and microspore culture • Culturing methods – protocol for Brassica microspores (see Bhojwani.g. 7-4 (done aseptically) • squeeze out microspores into liquid medium • filter through nylon screen of approp. pore size (e. 5 min. • resuspend and load onto a 24%/32%/40% Percoll gradient solution and spin . fig.

pellet and adjust plating density of pollen to 2-5 x 104 microspores per ml • plate suspensions as a thin layer in petri dishes and incubate at 32° C in the dark 3-5 days. resuspend. then at 25° C . fig.Anther and microspore culture • Culturing methods – protocol for Brassica microspores (see Bhojwani. 7-4 (done aseptically) • separate upper 2 layers and pipet out into fresh medium • spin.

"staging") .Anther and microspore culture • Culturing methods – protocol for Brassica microspores (see Bhojwani. 7-4 (done aseptically) • transfer embryos to liquid medium in flasks and shake at 60 rpm at 32° C • transfer embryos to solid medium w/2% sucrose and incubate at the lower temp • Factors affecting the response in vitro – stage of pollen development at the time of culture (aka. fig.

buds harvested just before 1st mitosis are most responsive (in general) – pretreatments • cold (7°-9° C for 72 h) – Nicotiana • heat (35°-40° C for 12-24 h) – Brassicas • how do pretreatments work? – research suggests cold or heat acts as a shock treatment causing a 90° shift in division plane of microspore causing a symmetrical division .Anther and microspore culture • Factors affecting the response in vitro – for staging.

incl. maltose) • PGRs – direct (no intermediary callus) – no PGRs – indirect – high auxin conc. auxin deleted to regenerate . to induce. a carbon source (sucrose.Anther and microspore culture • Factors affecting the response in vitro – culture medium • standard inorganics and organics.

Anther and microspore culture • Factors affecting the response in vitro – donor plants • growth conditions – light. nutrition. • genotype – some are more responsive than others • Ploidy status of regenerants – ploidy determined by chromosome counts • root-tip squashes • DNA content analysis (contact ISU Cell Facility) . effect of season. temp. etc.

.g. rapeseed and rye • colchicine (whole plants or anthers) • callus culture from explant of haploid plant • Uses in plant breeding – group 1 (asparagus. rubber) .Anther and microspore culture Ploidy status of regenerants – doubling methods • spontaneous doubling (e. potato. maize. over 70% of regenerants double spontaneously in barley.

g. maize. of DHs can be produced • goals – genetic maps. melons. large no.Anther and microspore culture • Uses in plant breeding – group 1 (asparagus..asparagus all male F1 hybrids • maize DHs are screened like other inbreds – group 2 (wheat. separating & IDing disease resistance genes (e. peppers) • rel. CMV resistance in pepper) . rubber) • DHs produced with low efficiency • goals .

rice) – DH lines easy • rice – lines used to develop varieties.Anther and microspore culture • Uses in plant breeding – group 3 (rapeseed. esp. barley. japonica subspecies • rapeseed – pedigree selection programs and parental lines for F1 hybrid varieties • barley – winter barley varieties use microspore culture. spring barley varieties use the bulbosum method (to be discussed later) .