Androgenesis

Abstract

Haploid plants, characterized genetically by the presence of gametic number of chromosomes in their cells (i.e., half the number of chromosomes as in somatic cells), are of considerable importance in genetics and plant breeding programmes. However, there has been no reliable and reproducible method to produce haploids under field conditions. Therefore, the full potential of haploids could not be exploited in agriculture. Since the first report of androgenic haploid production in anther cultures of Datura innoxia by Guha and Maheshwari (1964, 1966), considerable success has been achieved in developing protocols for androgenic haploid production in anther/pollen culture of over 200 species, including several major crop plants. The technique is being routinely used in crop improvement programmes and has aided development of several improved varieties.

Appendix

• 1. Anther culture to produce androgenic haploids of Nicotiana tabacum. (after Touraev et al. 2003).

  • (i) Grow the plants of Nicotiana tabacum in a glasshouse at 20–25 °C under 16 h photoperiod with a light intensity of 219–270 μmol m⁻² s⁻¹ provided by sodium lamps.

  • (ii) Harvest the flower buds from the first flush of flowers and bring them to the laboratory in a nonsterile Petri dish.

  • (iii) Classify the buds according to their corolla length. Excise anthers from one of the buds of each category and crush them in acetocarmine to determine the stage of pollen development. Identify and select the buds (ca 10 mm) with pollen just before, at, and immediately after first pollen mitosis.

  • (iv) Incubate the buds at 7–8 °C for 12 days in a pre-sterilized Petri plate sealed with parafilm.

  • (v) Surface sterilize the chilled buds with a suitable sterilant (0.1 % mercuric chloride for 10 min or 5 % sodium hypochlorite for 10 min).

  • (vi) Rinse the buds 3–4 times in sterile, double distilled water in a laminar air flow cabinet.

  • (vii) Using forceps and a needle, flame-sterilized and cooled, tease out the buds, and excise the anthers in a pre-sterilized Petri dish. Carefully detach the filament and place the anthers on MS medium (Table 3.1), supplemented with 2 % (w/v) sucrose and 1 % activated charcoal in Petri dishes (five anthers from a bud per 50 × 18 mm dish containing 5 ml of medium). Seal the Petri dishes with Parafilm and incubate the cultures at 25 °C in dim light (10–15 μmol m⁻² s⁻¹).

  • (viii) After 3–4 weeks, when the anthers have burst to release the pollen-derived embryos, transfer the culture to 16 h photoperiod and light intensity of 50 μmol m⁻² s⁻¹ provided by cool white fluorescent tubes. At this stage, if the responding anthers are crushed in acetocarmine (0.5–1.0 %) and observed under the microscope, different stages of pollen embryogenesis can be seen which is asynchronous.

  • (ix) Complete green plants will develop after 4–5 weeks of culture.

  • (x) Isolate the plantlets emerging from the anthers and transfer them to MS basal medium with 1 % (w/v) sucrose and 1 % (w/v) activated charcoal to allow root development. During this period, incubate the cultures under continuous light (3.6 μmol m⁻² s⁻¹) from white fluorescent tubes.

  • (xi) When the plants attain a height of about 5 cm, transfer them to potting mix in small pots or polythene bags and maintain under high humidity. Gradually reduce the humidity and transfer the plants to field.

• 2. Pollen culture to produce androgenic haploids of Nicotiana tabacum (after Touraev et al. 2003)

  • (i) Follow steps 1–6 as in Protocol 8.8.1.

  • (ii) Squeeze the anthers from 10 buds in a glass vial (17 ml) with about 3 ml of medium B (Kyo and Harada 1986), containing (in mg L⁻¹) KCl (149), CaCl₂.2H₂O (147), MgSO₄.7H₂O (250), KH₂PO₄ (136) and mannitol (54,700).

  • (iii) Place a magnetic bar in the vial and stir for 2–3 min at maximum speed until the medium becomes milky.

  • (iv) Collect the suspension of pollen and debris using a Pasteur pipette and filter it through a 40–60 μm pore size metallic or nylon sieve.

  • (v) Centrifuge the filtrate for 2–3 min at 250 g. Discard the supernatant along with the upper green pellet using a 200 or 1,000 μl pipette.

  • (vi) Suspend the lower whitish pellet in 2–10 ml of medium B and centrifuge again. Repeat the fifth step 2–3 times until there is no green layer above the white pellet.

  • (vii) Suspend the white pellet, comprised of purified pollen, in the B-medium and plate the suspension in a pre-sterilized Petri dish. Seal the Petri dish with Parafilm and incubate in dark at 33 °C for 5–6 days. The induction of androgenesis occurs during this starvation stress treatment.

  • (viii) After the pretreatment, transfer the suspension to a screw capped centrifuge tube and pellet by centrifugation at 250 g for 5 min.

  • (ix) Discard the supernatant and suspend the pellet in AT-3 medium (for composition see Table 8.1) and plate back in the original dishes (1 ml per dish). Seal the dishes with Parafilm and incubate the cultures in dark at 25 °C for 6–8 weeks.

  • (x) After 4–5 weeks, when fully differentiated pollen embryos have developed, transfer the culture dishes to 16 h photoperiod and light intensity of 50 μE m⁻² s⁻¹.

  • (xi) After another week, transfer individual embryos to culture tubes or jars containing MS basal medium with 1 % sucrose and 1 % activated charcoal for germination and full plant development. During this period incubate the cultures in light as above.

  • (xii) After the plants attain a height of about 5 cm transfer them to potting mix in small pots or polythene bags and maintain them under high humidity. Gradually reduce the humidity, and finally transfer the plants to field.

• 3. Pollen culture to produce androgenic haploids of Brassica juncea. (after Channa et al. 2005)

  • (i) Sow the seeds in 20 cm pots containing an artificial potting mixture, such as Agropeat PV, and maintain them at 25 °C under natural light.

  • (ii) At the bolting stage, move the plants to a growth chamber at 10 °C/5 °C day/night temperatures with 16 h photoperiod and 150–200 μE m⁻² s⁻¹ light intensity from cool white fluorescent tubes.

  • (iii) After 2 weeks, when 2–3 flowers have opened, collect the young green inflorescences and transfer them to the laboratory in nonsterile Petri dishes.

  • (vi) Classify the buds into 2–4 categories on the basis of their length (2.7–2.9 mm, 3.0–3.1 mm, 3.2–3.3 mm and 3.4–3.5 mm) with the help of a Vernier Caliper.

  • (v) Determine the stage of pollen development in the buds of the different categories by staining with DAPI (4, 6-diamino-2-phenylindole) and observing under UV light using a fluorescence microscope. Select the buds at the late uninucleate stage, when the nucleus has migrated to one side, for culture. Hereafter, all operations must be performed under aseptic conditions in a sterile air flow cabinet.

  • (vi) Transfer the selected buds to a tea egg and immerse in 0.1 % (w/v) mercuric chloride or 2 % (w/v) sodium hypochlorite solution, with a drop of Tween or Teepol for 10–12 min with continual shaking.

  • (vii) After three rinses each of 5 min in cold sterile distilled water, transfer the buds (maximum 20) to an autoclaved 25 ml beaker containing 7 ml of cold liquid B₅ medium with the salts reduced to half strength and 13 % (w/v) sucrose (½ B₅-13; Table 8.1).

  • (viii) Homogenize the buds with the aid of an injection piston, applying turning pressure movement to release the pollen. Wash the piston with ½ B₅-13 medium.

  • (xi) Filter the pollen suspension through a double layer of nylon sieve (Nytex 63 μm pore size top and 44 μm bottom) in a 15 ml sterilized, screw cap centrifuge tube. Rinse the nylon sieve with 2 ml of ½ B₅-13 medium and adjust the volume to 10 ml with medium.

  • (x) Wash the pollen twice with ½B₅-13 medium by pelleting at 100 g for 3 min in a refrigerated centrifuge pre-cooled to 4 °C.

  • (xi) Wash the pollen in NLN-13 medium containing 0.83 mg L⁻¹ KI (NLN-13-KI; Table 8.1).

  • (xii) Suspend the pellet in 1 ml of NLN-13-KI medium and determine the density of pollen using a hemocytometer. Adjust the density to 1 × 10⁴ pollen grains ml⁻¹ using NLN-13-KI medium.

  • (xiii) Dispense the suspension into sterilized Petri dishes (3 ml per 60 mm dishes) as thin layers. Seal the dishes with Parafilm and incubate at 32 or 35 °C in the dark.

  • (xiv) After 3 days, transfer the culture dishes to 25 °C in dark.

  • (xv) After another 3 weeks of culture, transfer individual embryos to B₅ medium with 2 % (w/v) sucrose for germination. Place the dishes in a culture room with 16 h photoperiod (50–100 μE m⁻² s⁻¹ provided by cool fluorescent tubes) at 25 °C. If necessary, after 4–5 days reorientate the embryos in a vertical plane to facilitate their germination.

  • (xvi) After 2 weeks, transfer the plants to culture tubes with their roots immersed in 1–2 ml colchicine solution (0.1–0.2 %) and leave overnight. Wash the roots with sterile distilled water and transplant them to a 1:1(v/v) mixture of Agropeat and soil in Hycotrays (Sigma) and maintain in a glasshouse under high humidity. Gradually move the plants to the areas of decreasing humidity. The plants should be ready after another 3 weeks for transfer to the field.

• 4. Anther culture to produce androgenic haploids of Oryza sativa (after Zapata-Arias 2003).

  • (i) Collect, at 8–9 a.m., the tillers from glasshouse-grown plants with the central florets at the middle to late uninucleate stage of the pollen.

  • (ii) Wipe dry and wrap the spikes in aluminum foil and store at 8–10 °C for 8 days.

  • (iii) Rinse the spikes in 70 % (v/v) ethanol for 30 s before surface sterilizing them with 2 % (v/v) Chlorax (a commercial bleach with 5.2 % NaOCl₂) containing a drop of Teepol for 20 min. Carry out all further steps in a sterile air flow cabinet.

  • (iv) Rinse the spikes three times in sterile distilled water.

  • (v) To excise and culture the anthers, cut the base of the florets just below the anthers with sharp scissors. Pick the floret at the tip with forceps and tap on the rim of the Petri dish, so that the anthers fall on N₆ medium (Table 8.1) supplemented with 5 % (w/v) sucrose, 0.5–2.0 mg L⁻¹ 2,4-D (callus induction medium). About 60 anthers may be cultured in a 55 mm diameter Petri dish containing 6 ml of callus induction medium.

  • (vi) Incubate the cultures at 25 °C in the dark.

  • (vii) After 4–5 weeks, transfer the pollen-derived calli (each 2–3 mm in diameter) to MS-based regeneration medium with 0.5–4.0 mg L⁻¹ kinetin and incubate the cultures in light (12 h photoperiod with 50–100 μE m⁻² s⁻¹ of illumination) at 25 °C.

  • (viii) Transfer the regenerated shoots to MS-based rooting medium lacking growth regulators.

• 5. Pollen culture to produce androgenic haploids of rice (after Raina and Irfan 1998).

  • (i) Follow steps (i), (iii), and (iv) as in Protocol 8.8.4.

  • (ii) Collect 150 aseptic anthers in a 6 cm diameter Petri dish containing 0.4 M mannitol solution, following the procedure described in step (v) of protocol 4. Incubate them in dark at 33 °C.

  • (iii) Simultaneously, isolate the unfertilized ovaries from the same batch of florets and culture in Petri dishes containing 3 ml of M-019 medium (Table 8.1) to condition the medium for pollen culture. Culture 30 ovaries per plate and incubate the plates in dark at 25 °C.

  • (iv) After 4 days of osmotic stress in the mannitol solution, some of the pollen grains will be liberated from the anthers into the pretreatment medium. Transfer the pollen suspension with the pretreated anthers to a small beaker and stir at slow speed for 2–3 min using a Teflon-coated magnetic bar to release the remaining pollen.

  • (v) Filter the above suspension through a nylon/metallic sieve (40–60 μm pore size), pipette out the filtrate, transfer it to a screw cap centrifuge tube and centrifuge at 500 rpm for 2–3 min. Discard the supernatant and suspend the pellet in fresh mannitol solution and wash again by centrifugation. Give the final wash in M-019 medium. Finally, suspend the pollen in M-019 medium conditioned by the cultivation of unfertilized ovaries (1 ml suspension per 3.5 cm dish) for 4 days. Transfer 10 ovaries into each dish. Seal the Petri dish with Parafilm and incubate the cultures in dark, at 25 °C.

  • (vi) After 4 weeks of initiation of the pollen cultures, transfer the embryo-like structures (ELS) or calli, each measuring 2–3 mm in size, to semi-solid MS-based regeneration medium (Table 8.1) supplemented with BAP (2.0 mg L⁻¹), Kinetin (1.0 mg L⁻¹) and NAA (0.5 mg L⁻¹) and gelled with 6 % (w/v) agarose (Sigma). Incubate the cultures under 12 h photoperiod and 50–100 μE m⁻² s⁻¹ illumination. After 7–10 days, more ELS/calli from the induction medium may be transferred to regeneration medium.

  • (vii) Transfer the regenerated plants to hormone-free ¼ strength MS-based medium containing 2 % (w/v) sucrose and gelled with 0.25 % (w/v) Phytogel in culture tubes.

  • (viii) When the plantlets attain a height of about 15 cm, transfer them to liquid 1/10 strength MS-based medium without sucrose, vitamins or hormones for hardening before transfer to pots.