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

Anther and microspore culture Ontogeny of androgenesis

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

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

Anther and microspore culture Culturing methods

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

Anther and microspore culture Culturing methods

protocol for Brassica microspores (see Bhojwani, fig. 7-4 (done aseptically) separate upper 2 layers and pipet out into fresh medium spin, resuspend, 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, then at 25 C

Anther and microspore culture Culturing methods

protocol for Brassica microspores (see Bhojwani, fig. 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, "staging")

Anther and microspore culture Factors affecting the response in vitro

for staging, 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

culture medium standard inorganics and organics, incl. a carbon source (sucrose, maltose) PGRs direct (no intermediary callus) no PGRs indirect high auxin conc. to induce, auxin deleted to regenerate

Anther and microspore culture Factors affecting the response in vitro

donor plants growth conditions light, temp, nutrition, effect of season, etc. 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)

Anther and microspore culture Ploidy status of regenerants

doubling methods spontaneous doubling (e.g., over 70% of regenerants double spontaneously in barley, potato, rapeseed and rye colchicine (whole plants or anthers) callus culture from explant of haploid plant

Uses in plant breeding

group 1 (asparagus, maize, rubber)

Anther and microspore culture Uses in plant breeding

group 1 (asparagus, maize, rubber) DHs produced with low efficiency goals - asparagus all male F1 hybrids maize DHs are screened like other inbreds group 2 (wheat, melons, peppers) rel. large no. of DHs can be produced goals genetic maps; separating & IDing disease resistance genes (e.g., CMV resistance in pepper)

Anther and microspore culture Uses in plant breeding

group 3 (rapeseed, barley, rice) DH lines easy rice lines used to develop varieties, esp. 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)