Rice is one of the main food crops in the world and also one of the important model plants. Rice anther culture refers to the in vitro culture of rice anthers or pollen grains, inducing microspores to form callus tissue and then differentiate into complete rice plants. Anther culture has been widely used in the selection of hybrid rice restorer lines. In conventional rice breeding, anther culture has bred multiple varieties that are promoted in production. Anther culture can also be combined with other biotechnologies, such as anther culture for homozygous and disease-resistant breeding of target traits of transgenic rice.

Principle

The sophisticated practice of rice anther culture and subsequent plant regeneration stands as a pivotal facet of contemporary breeding methodologies. This technique involves the aseptic transfer of rice anthers onto synthetic media, prompting dedifferentiation to generate callus, which then undergo differentiation into haploid plants. Through chromosome doubling—typically accomplished via colchicine treatment—diploid plants emerge. This approach confers numerous advantages to rice breeding, including accelerated breeding cycles, heightened selection efficiency, and expedited breeding operations.

Determinants Influencing Rice Anther Culture

Medium Composition

The intricate composition of the culture medium markedly influences callus formation and the subsequent plant regeneration potential. For instance, the N6 medium, enriched with maltose, agar, and vitamins, has demonstrated superior efficacy in inducing callus formation and facilitating plant regeneration in rice anthers.

Hormone Concentration

Hormonal concentrations, particularly of agents like 6-BA, NAA, and colchicine, play a crucial role in dictating both the formation and the regenerative efficiency of callus. Research indicates that the integration of colchicine into differentiation mediums significantly enhances green seedling differentiation and regeneration.

Radiation Treatment

The application of γ-ray radiation has emerged as a catalyst for callus formation and green seedling regeneration.

Genetic Background

The genetic backdrop of rice varieties dictates the efficiency of anther culture. Notably, japonica rice varieties generally exhibit a lower regeneration rate compared to their indica counterparts.

Fig. 1. Anther culture-derived plantlet production in rice. (a) Rice plant as source of explants, (b) boots, (c) spikelets, (d) anther, (e) callus induction from anthers, (f) regeneration of plantlet, (g) plant establishment in pot mixture, and (h) field grown plants at panicle stage. (Tripathy, et al., 2019)

Applications of Rice Anther Culture

Accelerated Breeding

Anther culture techniques enable rapid purification of breeding material, curtailing the breeding duration. Consequently, rice variants exhibiting distinct superior traits can be swiftly developed using this method.

Genetic Enhancement

Through chromosome doubling, haploid plants transition into diploid organisms, streamlining the genetic improvement of rice. This technology facilitates the selection of rice cultivars that are drought-resistant or disease-resistant.

Genetic Engineering Breeding

The amalgamation of anther culture with genetic engineering permits precise gene editing and enhancement within rice genomes, paving the way for targeted genetic advancements.

Procedures

1. Anthers are taken from rice plants growing normally in the field. According to years of experience, the best age for anther culture is when pollen develops to the middle stage of mononuclear, not only because the rate of inducing green seedlings is high, but also because diploid tissues are inhibited and cannot form seedlings. The field reference standard for this anther age is generally: the leaf sheath of the second leaf at the top is about 3 cm away from the flag leaf, but each variety and different growing seasons of the same variety must be corrected by microscopic examination.

2. The collected rice ears should be immediately brought back to the laboratory and placed in the refrigerator and stored at a low temperature of 5-10°C. Experiments on low-temperature pretreatment show that low-temperature treatment at 8°C for 12 days is best, but attention must be paid to the preservation (moisture retention) of rice ears during the treatment. For this purpose, the plastic film bag that packages the ears must be tied tightly.

3. After the collected rice ears are pretreated at low temperature, the young ear inflorescence is peeled off, and the anthers are peeled off from the upper, middle and lower parts of the inflorescence in turn. After staining with 0.1% I-KI solution and microscopic examination, the anthers with the nucleus at the periphery are the mononuclear middle and late anthers.

4. After taking the ears out of the bag, it is best to soak them in disinfectant first, and immediately put them into the clean bench that has been turned on for 15 min. Peel the ears in the clean bench, put them into a 50 mL large test tube according to the serial number, and then inject 0.1% mercuric chloride solution for disinfection for 10 min, and rinse with sterile water 3 times.

5. Cut the spikelets on the dried ears one by one into a pre-sterilized culture dish lined with clean filter paper, then hold the spikelet base stalk in the left hand and the pre-sterilized sharp scissors in the right hand, and cut the husk with anthers from the top of its filaments into the filter paper of the culture dish. Then use sterilized tweezers to clamp the top of the husk, so that the opening is facing the tube, and gently knock it into a test tube or a small triangular bottle filled with callus induction medium (N6 + 2 mg/L 2,4-D + 1.0 mg/L NAA＋0.5% LH), and place it in a 26-27°C constant temperature box to culture in the dark. Inoculate 20 anthers in a large test tube and 50 anthers in a 25 mL triangular bottle, and plug it with cotton plugs.

6. Differentiation of buds. The callus tissue was transferred to the differentiation medium [(N6＋(1-2) mg/L KT＋(0.25-0.5)mg/L NAA＋3% maltose, pH 5.8-6.0)] for differentiation culture. The temperature during differentiation was 23-25°C, and the light intensity was maintained at 30 μmol/(m²·s) for 9-11 h.

7. Seedling hardening and transplanting. The differentiated green seedlings were transferred to the modified White medium for seedling hardening culture. Alternatively, the seedlings could be first cultured in sand with culture solution and then transplanted.

Alkaloids List

Plant Growth Regulators List

Related Services & Products

• Plant Breeding
• Plant Tissue Culture
• Rice anther In Vitro Culture
• Plant Suspension Cell Culture Process Development
• Adventitious Root Propagation of Woody Plants
• Somatic Embryogenesis and Plant Regeneration
• Tissue Culture in Forestry
• Plant Genetic Engineering
• Plant Genetic Transformation
• Rice Transformation
• Auxins
• Cytokinins
• Plant Growth Regulators
• Tissue Culture Media & Supplements

References

1. Tripathy, S. K., et al. (2019). Exploring factors affecting anther culture in rice (Oryza sativa L.). Journal of Applied Biology and Biotechnology, 7(02), 87-92.
2. El-Maksoud, A., et al. (2007). Genetical analysis of anther culture response in rice under drought stress. Journal of Plant Production, 32(4), 2499-2509.