Anther Culture of Rice for Haploidy Induction and Accelerated Development of Striga Resistant Germplasm

Abstract

Doubled haploidy is the fastest way of achieving completely homozygous plants in one generation. It shortens the time required for the development of mutant varieties allowing rapid response against threats to sustainable food production caused by changing climate and stresses due to emerging pests like Striga. In this chapter, we describe step-by-step protocols to obtain doubled haploid rice with fixed novel mutant variants by anther culture from gamma ray irradiated M1 populations to accelerate mutation breeding for resistance to Striga in rice.

Introduction

The development of resistance in host species remains one of the most efficient and cost-effective ways to control infestations of parasitic plants (Press et al. 1995). With limited natural alleles conferring Striga resistance among cereal crops, particularly rice and maize (see Chapter “Striga as a Constraint to Cereal Production in Sub-Saharan Africa and the Role of Host Plant Resistance”), physical mutagenesis is an attractive means by which to generate new sources of host plant resistance (Ulukapi and Nasircilar 2018). As we have seen in previous chapters (Chapters “Screening for Resistance to Striga Hermonthica in Mutagenized Sorghum and Upland Rice in Burkina FASO”–“Phenotyping for Resistance to Striga Asiatica in Rice and Maize Mutant Populations in Madagascar” and “Rapid Cycling and Generation Advancement for Accelerated Mutation Breeding in Sorghum”), achieving true-breeding Striga resistant lines from mutagenized cereals takes several generations. The single seed descent (SSD) method is one of the conventional ways used for generating homozygous lines in cereal breeding. However, six generations of inbreeding are required to obtain about 98% homozygosity in the SSD method (Khound et al. 2013). Screening for Striga resistance cannot begin until the M2 and verification of gained resistance is usually not accomplished until the M4 or M5. Advancement to new Striga resistant varieties suitable for public release also involves several generations of multi-location field trials. Anther/microspore cultures are of tissue origin, based on the method to produce completely homozygous (doubled haploid; DH) plants from immature pollen grains. Doubled haploidy is the fastest way of achieving completely homozygous plants (in one generation). It shortens the time required for development of novel plant genotypes than the conventional methods that require at least 6–7 generations of inbreeding and selection. This process is the reason for the time taken, often 11–13, years between making crosses to releasing cultivars (Germana 2011). Progress in haploid/doubled haploid production, such as the rapid generation of large microspore-derived haploid populations via androgenesis, may be exploited in mutation breeding. For example, genome-fixed novel mutant variants obtained by anther culture from the irradiated M1 population can be rapidly incorporated into breeding/selection programs such as developing novel variants resistant to Striga. The aim of this study was to develop and apply haploid/doubled protocols via androgenesis in rice for production of mutant haploid/doubled haploid populations.

Protocols

Rice (Oryza sativa L. cvs. Taipei 309, Osmancık-97, TG1) seeds were irradiated at 100, 200 and 300 Gy doses by exposure to a ¹³⁷Cs gamma ray source in the Nordion Gamma Cell 3000 at the Elan irradiation facility of the Istanbul School of Medicine Blood Bank Center, Istanbul University, Istanbul, Turkey.

Panicle collection and pre-culture treatment. Sow the irradiated rice seeds in the field or in greenhouse pots under optimal growing conditions for rice. Collect panicles from primary tillers when the auricle distance of the flag leaf to the next leaf is 7–10 cm (Fig. 1). Remove outer leaf sheaths and ensure that the panicles are wrapped with the inner leaf sheath. Wash the boots or panicles in tap water and stand them in a plastic water bottle. Cold treat panicles at 4 °C for 8–10 days in a refrigerator or a cold room.

Fig. 1

Photos show the optimum stage for rice panicle collection indicated by white arrow (a), panicles with the outer leaf sheath removed (b), and cold treating panicles in a refrigerator (c)

Determination of microspore viability and pollen stage. Crush anthers in buffer and filter them through 40 µm steel mesh. Centrifuge the homogenate at 2000 rpm for 2 min. Remove supernatant and use the pellet for the squash preparation. Add 0.1 µL of FDA (fluorescein diacetate) stock solution (5 mg of FDA in 1 mL of acetone) to 1 mL of the microspore suspension. Remove a drop to a glass microscope slide and add a coverslip. Examine slides under a fluorescent microscope using UV-light and the appropriate filter set. Viable microspores glow bright green (Fig. 2a).

Fig. 2

2% Aceto-carmine (a), FDA (b) and DAPI (c) staining: black arrows indicated that uni-nucleated microspore cells

Determination of microspore developmental stages. Crush anthers in 2% aceto-carmine stain. Cover them with a glass slip. Observe microspore stage under a light microscope (Fig. 2b).

DAPI staining. Crush anthers in buffer and filter them through 40 µm steel mesh. Centrifuge homogenate at 2000 rpm for 2 min. Remove supernatant and use the pellet for the squash preparation. Fix 100µL of cell debris in ethanol:acetic acid (3:1) for 15 min. Centrifuge homogenate at 2000 rpm for 2 min. Remove supernatant, wash the pellet once (or twice) in 50% ethanol, and then centrifuge homogenate again between each of the washing steps. Remove supernatant and add 10 µL DAPI (4,4-diamidino-2-phenylindole) working solution [stock solution (5 mg of DAPI in 1 mL of 50% ethanol) diluted in 1 ml of 50% ethanol] and a drop of glycerol. Examine slides under the fluorescent microscope using UV-light and the appropriate filter set (Fig. 2c). Finally, according to 2% aceto-carmine and DAPI staining, select anthers having microspores at mid-uninucleate to early binucleate stage of development, as these are the most responsive to anther culture.

Surface sterilization of panicles. Surface-sterilize cold treated boots by immersion in 70% (v/v) ethanol for 2 min followed by three times wash with sterilized distilled water. Put in 40% Clorox (2.1% NaOCl) for 25 min and then wash by dipping three to four times in a series of vials filled with sterilized distilled water until the bleach smell dissipates.

Anther culture and callus induction. Place panicles on sterilized filter papers. Remove anthers aseptically with a pair of forceps and gently place in a 35 × 15 mm disposable petri dish containing 3 mL of androgenic calli induction medium [N6 (Chu et al. 1975) contains 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), gum arabic, and 40 g/L maltose, pH at 5.7–6.0 (modified by Afza et al. 2000)]. Up to 50 anthers are cultured in each disposable petri dish with a few ovaries. Firmly seal Petri dish with Parafilm M^® barrier film and incubate them at 27 ± 1 °C with 12/12 h light/dark period. Observe for callus formation in three to four weeks after anther planting (Fig. 3a–d).

Fig. 3

Androgenic callus formation in induction media. Pictured from inside of phytotron (a), image of callus formation from anthers under a binocular microscope (b), image of calli under a binocular microscope (c), callus formation in induction medium (d)

Media preparation for androgenic calli induction and sterilization. Dissolve 4 g/L N6 (Chu et al. 1975) basal salt mixture (Sigma C1416), 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 40 g/L maltose in 1L distilled water at room temperature. Adjust the pH to 5.7–6.0 using 1N NaOH/KOH or 1N HCl. The media should be autoclaved for 20 min at 121 °C under 1.25Atm. Filter-sterilize gum arabic and then add it to the autoclaved medium cooled to 40–50 °C. Store medium at 8–10 °C.

Plantlet induction from androgenic calli. Pick up androgenic calli with a sterile pair of forceps. Transfer them into semisolid regeneration medium [MS (Murashige and Skoog 1962) containing 1 mg/L 1-naphthaleneacetic acid (NAA), 4 mg/L kinetin, 100 mg/L myo-inositol, 100 mg/L casein hydrolysate, gum arabic, 1 mL/L 1000 × MS vitamin stock solution (Sigma M3900), 30 g/L sucrose as a source of carbon and 6.5 g/L gum agar into media to solidify, pH 5.7–6.0]. Firmly seal the Petri dish with Parafilm M^® barrier film. Maintain calli at 27 ± 1 °C, with 12/12 h of light/dark period up to plantlet regeneration (Fig. 4a, b).

Fig. 4

Healthy plantlets in regeneration medium showing root (a) and shoot (b) formation

Media preparation for seedling regenerations and sterilization. Dissolve 4.3 g/L MS (Murashige and Skoog 1962) basal salt mixture (Sigma M5524) containing 1 mg/L naphthaleneacetic acid (NAA), 4 mg/L kinetin, 100 mg/L myo-inositol and 30 g/L sucrose in 1 L of distilled water at room temperature. Adjust the pH at 5.7–6.0 using 1N NaOH/KOH or 1N HCl. Put 6.5 g gum agar into media to solidify. The media should be autoclaved for 20 min at 121 °C under 1.25 Atm. Put 1 mL/L 1000 × MS vitamin stock solution (Sigma M3900) to the autoclaved medium cooled to 40–50 °C. Store medium preferably at 8–10 °C.

Determination of ploidy level of the regenerants. Chop equal amounts of putative haploid/doubled haploid rice regenerants obtained from anther cultures and tomato (Solanum lycopersicum L.) leaf samples, as a standard, together for 30–60 s in 500 µL of ice-cold nucleic extraction buffer. Since the c value of the tomato genome is one of the plants closest to rice, it is often preferred as a standard when analyzing rice in flow cytometry. Add 2 mL of CyStain^® PI Absolate P nucleic staining solution (Sysmex 05-5022) on the crude mixture. Pass the suspension through a 30 µm CellTrics^® filter (Sysmex) to eliminate cell debris. Collect nuclei into the pre-chilled tubes. Add 2 mL of dye solution previously prepared into the tube. Incubate tubes in the dark for about 60 min. Analyze the ploidy level of samples by a flow cytometer. We used the Sysmex Partec flow cytometer at Tekirdağ Namık Kemal University, Field Crops Department, Plant Genetics and Cytogenetics Laboratory equipped with a 488 nm Argon laser to excite the PI fluorochrome, and a FL-2 detector with a 585/42 band pass filter. Samples were run on low pressure and 104 nuclei were counted within the double gate (Fig. 5).

Fig. 5

Ploidy detecting using a flow cytometer. Haploid plantlet (a) and diploid plantlet (b). First pick related to plantlet obtained from rice anther culture and, for comparison, second pick from a standard plant (Solanum lycopersicum L.)

Colchicine treatment of haploid plantlets to induce chromosome doubling. Take the haploid seedlings based on the result of the flow cytometer analysis, from regeneration culture (Fig. 6). Wash the roots with tap water and trim back to about 2–3 cm. Immerse trimmed seedlings in 0.2% colchicine with dimethyl sulfoxide (DMSO) and few drops of Tween 20 for 4 h at room temperature. Take treated seedlings out of the colchicine and wash under running tap water overnight. Transplant treated plants into pots containing 2:1:1 soil:sand:peat-moss mixture. Check ploidy of new tillers to confirm chromosome doubling. Doubled haploid tillers will likely have seeds after heading and successful pollination.

Fig. 6

Colchicine application to rice seedlings found to be haploid as a result of flow cytometer analysis (a and b)

Growing the haploid/doubled haloid plantlets. Place the newly transplanted plantlets in a plastic house or growth chamber and cover with plastic bags for a few days for acclimatization (Fig. 7a). Incubate seedlings at 21/18 °C night/day under 16/8 h of day/night photoperiod (Fig. 7b). Irrigate seedlings with Yoshida’s solution (Yoshida et al. 1976) or any other similar nutrient solution.

Fig. 7

Plantlet in growth chamber, transferred plantlets into soil for acclimatization (a) and plantlets in a growth chamber (b)

Validation of the protocols on some rice varieties and M1 population. The optimized protocols were tested on three rice varieties and their M1 populations. M1 seeds were produced by irradiation with 0, 100, 200 and 300 Gy doses of gamma rays. As can be seen from Table 1, an equal number of anthers for each population were planted in the culture medium. In all three cultivars, a decreasing trend is observed in the callus induction percentage, except for the population of Osmancık 97 irradiated with 200 Gy, with increasing radiation dose. It was determined that the percentage of callus induction rate ranged from 17.9 to 19.8% in Taipei 309, 16.2 to 17.3% in Osmancık 97, and 14.4 to 16.6% in TG1. Generally, regenerability of androgenic calli decreased with increasing radiation dosage. In all three cultivars, only albino plants were obtained at the highest radiation dose of 300 Gy. The percentage of green plants obtained in the study also decreases with increasing radiation dose in all three cultivars, except for the 200 Gy gamma rays irradiated population of Taipei 309. The percentage of green plants regenerated in cultures established from the populations obtained by 100 Gy gamma rays doses was 27.3% in Taipei 309 and 25% in both Osmancık 97 and TG1. In cultures established from mutant populations obtained with 200 Gy, green plants regenerated at a rate of 56.7% in Taipei 309, 16.7% in Osmancık 97 and 20% in TG1. TG1 seems the most sensitive rice variety to gamma radiation both in the callus induction stage and plant regeneration stage.

Table 1 Effect of gamma irradiation dose on anther culture ability of rice cultivars

Conclusions

This study was conducted to investigate the potential of obtaining haploid/doubled haploid plants from M1 populations derived from rice seeds irradiated with different doses of gamma radiation. This reflects the potential of introducing novel germplasm resources with new genetic background that can be used for different purposes in breeding programs, such as developing new plant variants resistant/tolerant to Striga spp. Regenerability of androgenic callus is particularly sensitive to gamma rays. One-third of the green plantlets obtained were determined as haploid by flow cytometry. We also found that with applying cold pretreatment times over ten days, gamma ray application reduced the rate of obtained healthy plants and increased the rate of obtaining albino plants. As a result of the radio-sensitivity analysis, the GR₅₀ value was determined between 250–400 Gy (Brunner 1995; see Chapter “Physical Mutagenesis in Cereal Crops”), depending on the source of gamma radiation and the rice variety. Considering these values, the radiation doses of haploid/doubled haploid rice plants obtained in this study approximately correspond to the GR₃₀ and GR₄₀ values. Progress in haploid/doubled haploid production, such as the rapid generation of large microspore-derived haploid populations via androgenesis, may be exploited in mutation breeding. For example, genome-fixed novel mutant variants obtained by anther culture from the irradiated M1 population can be rapidly incorporated into breeding/selection programs to develop novel variants resistant to Striga infestation.