Barley microspore in vitro culture is a true haploid cell culture. It has irreplaceable value compared to anther culture in academic and application, and therefore has attracted much attention from researchers.

Principle

The intricate procedure of barley microspore culture and subsequent plant regeneration is anchored in the fundamental concept of plant cell totipotency. Through meticulous isolation, cultivation, and induction of microspores, callus are formed, eventually giving rise to entire plants. This transformative journey unfolds across several crucial stages, each marked by distinct technical nuances, as delineated below:

Isolation and Pretreatment of Microspores

Microspores, the haploid progeny of meiotic division within pollen mother cells, are typically isolated from anthers via mechanical or chemical stratagems. In the case of six-rowed barley varieties like "Frances", microspore isolation employs an MS medium enhanced with MgSO₄, L-cysteine, and LBAP for optimal results. Moreover, administering low-temperature pretreatment, such as an exposure at 4°C, has been observed to bolster microspore viability and differentiation potential significantly.

Selection and Optimization of Culture Medium

The judicious selection of culture mediums is pivotal for inducing callus and fostering plant regeneration from microspores. Commonly adopted mediums include MS and IMI variants, where the strategic inclusion of phytohormones such as 6-BA, KT, and NAA catalyzes the formation and differentiation of healing tissues. Notably, enriching MS medium with 0.5 mg/L each of 6-BA and NAA significantly enhances tissue differentiation rates.

Induction and Differentiation of Callus

Subsequent to pretreatment, microspores are repositioned into hormone-infused mediums to induce callus formation. The differentiation into callus mandates precise hormone ratios and well-regulated culture conditions; for instance, low-temperature stressors (around 4°C) are known to facilitate this differentiation. Once differentiation is complete, continued culture fosters embryonic callus genesis, a critical juncture for plant regeneration.

Plant Regeneration and Chromosome Doubling

Upon reaching a developmental milestone within callus, they are transitioned to seedling-strengthening mediums to culminate in complete plant structures. Initially, regenerated plants may possess haploid chromosome counts (2n), yet chromosome doubling techniques, such as colchicine application, can elevate them to diploid status, ensuring genetic stability.

Fig. 1. Stress-induced embryogenesis in isolated barley microspore cultures and plantlet regeneration. (A) Isolated microspores at culture initiation. (B) Microspore-derived embryos after 28 days in culture. (C) Germination of microspore-derived embryos. (D) Regenerated plantlet in solid MS medium.(Rodríguez-Serrano, et al. 2012)

Genotype and Environmental Impact

The success of microspore cultivation and plant regeneration hinges significantly on the genotype and surrounding environmental parameters. For instance, barley genotypes display varying levels of salt and stress resilience throughout microspore culture. Artificial climate chambers emerge as vital tools in providing consistent environmental conditions, thereby amplifying culture success rates.

Applications and Advantages

Microspore culture technology distinguishes itself through its high efficiency and expedited processes, ideally suited for genetic enhancement and haploid plant breeding. This methodology allows for the rapid identification of mutants with heightened resistance. Furthermore, integrating this technique with chemical mutagens or radiation can further amplify plant regeneration efficiency and mutation incidence.

Procedures

1. Barley pollen culture donor plants were planted in a greenhouse. The greenhouse conditions were 16 h of light, light intensity of 350-400 μmol/(m²·s), and temperature of 20°C/15°C (day/night).

2. After staining with acetic acid carmine solution, the pollen with nuclei in the middle and late stages of mononuclear was suitable for pollen culture. Cut off the barley ears with pollen in the middle of the ear in the middle and late stages of mononuclear, insert them into a beaker filled with clean water, and store them in a 4°C refrigerator.

3. Disinfect the surface of the wheat ears in 3% sodium hypochlorite solution for 10-15 min, then rinse with sterile water 3-5 times, 5 min each time.

4. Put the wheat ears into a 9 cm diameter culture dish, pour 10-15 mL 0.3 mol/L mannitol into it, and pretreat it in a 4°C refrigerator for 3-5 d.

5. Cut the wheat ears into 2-3 cm small segments under sterile conditions, put them into the pre-cooled grinder cup, add 0.3 mol/L ice-cold mannitol to half the cup volume, and grind them at medium-low speed for 5-10 s. Too long grinding time and too high speed will damage the microspores and reduce their activity.

6. Filter the crushed mixture with a 100 μm nylon mesh and rinse it with 0.3 mol/L ice-cold mannitol 1-2 times. Pour the filtered microspores into a centrifuge tube and centrifuge them at 900 r/min for 5 min. To ensure the purity of the microspores, the centrifugation process can be repeated 1-2 times.

7. Suspend the pure microspores in the modified FHG induction medium, and use a pipette to transfer the microspore suspension into a 6 cm diameter disposable sterile culture dish, seal it with a double-layer sealing film, and the final volume of the modified FHG induction medium in the dish is 2-2.5 mL, and the microspore culture density is 100,000/mL. The microspore density can be measured by a hemocytometer. In addition, 200 g/mL Cefotaxime can be added to the modified FHG induction medium to control contamination.

8. The culture dish was placed in a dark environment at 25°C for 21-28 d. After that, some fast-growing embryoids (1-2 mm) can be directly transferred to MS regeneration medium. For most of the smaller embryoids, they were transferred to FHG differentiation medium for 1-2 weeks to allow them to continue to grow, and then transferred to MS regeneration medium. The culture conditions were 25°C, 8 h light, and the light intensity was 50 μmol/(m²·s).

9. The well-rooted seedlings (3-4 leaves) were transplanted into a nutrient pot containing peat/vermiculite. Pay attention to watering and moisturizing the newly transplanted seedlings. Most of the plants obtained by barley microspore culture have good fertility and generally do not require chromosome doubling.

Tissue Culture Media & Supplements List

Plant Growth Regulators List

Related Services & Products

• Plant Breeding
• Plant Tissue 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
• Barley Transformation
• Auxins
• Cytokinins
• Plant Growth Regulators
• Tissue Culture Media & Supplements

Reference

1. Rodríguez-Serrano, M., et al. (2012). NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley. Journal of experimental botany, 63(5), 2007-2024.