Rapid Cycling and Generation Advancement for Accelerated Mutation Breeding in Sorghum

Abstract

Rapid cycling techniques have become preferred methods in recent years to speed up the plant breeding process, especially for plant varieties recalcitrant to in vitro tissue culture. Manipulation of plant growth management of components such as pot size, irrigation, nutrition and light/day length can substantially accelerate the plant growth cycle from seed to harvest. In this chapter, we describe how to shorten the generation time of sorghum to have 4–6 generations per year to speed the plant breeding process including mutation breeding for resistance to Striga.

Introduction

Sorghum (Sorghum bicolor (L.) Moench), is among the global top five grain crops, widely consumed as food in many parts of the world, especially Africa. In contrast to maize and rice, sorghum can remain productive under low input agriculture in areas of limited irrigation and soil fertility. Recent abrupt climate changes and spread of the parasitic weed Striga spp. have constrained crop production in sub-Saharan Africa and semi-arid tropical regions of Asia particularly. This situation has compelled plant breeders to produce climate change-resilient crops, which can withstand broad-spectrum biotic and abiotic stresses. New sources of genetic variation are vital for crop improvement and achieving sustainable yield increases. However, limited variation for traits that mitigate the impact of these productivity challenges within some sorghum populations make the breeding gains difficult (Atera et al. 2012; Weltzien et al. 2018). Mutagenesis can expand the genetic base for crop resilience to environmental stresses. This was the basis for the Coordinated Research Project (D25005) launched by the IAEA in 2016 to optimize protocols for mutation breeding and efficiency enhancing technologies for resistance to Striga spp. in cereals for food security.

Aside from genetic resource erosion or scarcity in certain crop populations, long growing seasons can also limit rapid development and delivery of improved varieties. A number of the intervention technologies in crop management can shorten the delivery time of improved varieties. One such technology is doubled haploids (DH) which increases the efficiency of selection through accelerating fixation of desirable alleles to homozygosity thereby avoiding multiple generations of inbreeding. Doubled haploid techniques have been successfully deployed in maize (Chaikam et al. 2019) and rice (see following chapter) and therefore could streamline the process of deploying traits like Striga resistance gained through mutagenesis in these crops. However, for crops recalcitrant to tissue culture like sorghum, employing DH technology is severely restricted.

As an alternative to DH mitigated fixation of mutant alleles that improve Striga resistance in sorghum varieties, rapid cycling of selfing generations can be employed. Inbreeding in sorghum is fairly easy since it is a self-pollinated crop. However, many varieties, particularly those native to Striga endemic agroecologies, are day-length sensitive, requiring a short photoperiod (12 h or less) to flower and/or very long growing seasons (> 180d). Recent developments enable acceleration of sorghum growth cycles through the use of glasshouses and growth chambers with controled temperature, photoperiod, pot size, and irrigation combined with in vitro embryo rescue techniques. Perhaps owing to sorghum’s ability to produce grain in stressful environments, horticultural management that limits rooting space and other resources generally speeds generation time (seeds sown to seeds harvested). Rapid cycling techniques can be adapted to varieties with both neutral and short-day photoperiods. They can greatly reduce the conventional mutation breeding time to improved variety delivery of 10–12 years (Forster et al. 2014; Ghanim et al. 2014; Yan et al. 2017; dan Anisiyah 2020). This chapter describes such rapid cycling protocols for sorghum combining growth management with embryo rescue for accelerating mutation breeding for Striga resistance.

Protocols

Plant materials. Nine different sorghum varieties with varying growth season lengths were originally collected from Sudan Agricultural Research Corporation germplasm. These are named Abu 80, Arfagadamak, Arossarimal, Bashair, Botana, Tabat, Mugud, Wad Ahmed and Yruasha. Table 1 shows days to flowering, days to maturity and a typical number of seeds/panicle for each of these varieties under normal agricultural condition in Sudan (Eltahir et al. personal communication).

Table 1 Days to flowering, days to maturity, number seeds per head and sensitivity to photoperiod of the tested varieties under the normal growing condition at different region in Sudan

Plant growth conditions. Germinate sorghum seeds of selected genotypes/varieties in petri dishes and transplant germinated seeds after 5–7 days. Recently harvested seeds can be sown directly in soil in containers such as plastic boxes, trays with small cells (5 × 5 × 5 cm), small pots (9 × 9 × 5 cm) containing the soil mixture: one part sand to one part peat moss (Fig. 1).

Fig. 1

Representative photos for seedlings of sorghum in small trays (a) and round small pots (b) for rapid generation cycling (c) shows plants at harvesting stage and representative bigger (left) and smaller (right) heads harvested from round small pots and trays, respectively

Initially, set growth chamber or glasshouse temperature to 25 °C with a 24 h photoperiod (use supplemental light in glasshouse), at 60% relative humidity (RH) until seedlings reach the 4–6 leaf stage. Note that these conditions depend on the sorghum genotype and the control capacity of the growing facility. Night time temperatures should not fall below 22 °C. Keep the seedlings regularly irrigated with tap water in 2–3 day intervals or when needed and fertilize weekly with full strength Hoagland’s solution (Hoagland and Arnon 1950). Ensure that the plants remain free from pests and diseases. After about four weeks from planting, change the day length to shorter than 12 h to stimulate flowering in short-day varieties. For photoperiod sensitive varieties, growth chambered plants will need 12 h days from approximately four weeks until they are six weeks old to induce flowering. If photoperiod sensitive plants are grown in a glasshouse, shading during this developmental period that completely excludes light (e.g., putting a box over the tray or black cloth over a bench) are required to achieve 12 h day length in the months between the Spring and Winter equinox to induce flowering. Cover flowering heads with an appropriate sized paper bag to ensure self-pollination. Record flowering dates and physiological maturity to estimate the generation time. Collect pre-mature heads (10, 15, 20 days after anthesis) and proceed to in vitro laboratory for embryo rescue for testing the best time of collection for immature embryos as an option to further shorten the generation times. Keep the plants until physiological maturity and harvest the seeds for planting directly in the next cycle if there is no in vitro culture facility for embryo rescue (Fig. 1). Repeat the above cycle consecutively until sufficient inbreeding (mutant alleles fixed) is achieved.

Premature harvesting of heads and embryo rescue. Harvest immature heads after 10, 15 and 20 days from anthesis and remove immature sorghum seeds (Fig. 2). Inside a laminar flow hood, surface sterilize the harvested immature seeds with 70% ethanol for two minutes and 40% Chlorox (2.1% NaOCl) for five minutes. Rinse the seeds after each of the disinfection steps three times with sterile distillated water. Remove immature embryos from the caryopses with a sterile lancet and forceps under aseptic conditions (Fig. 2). Place the scutellum up on the surface of the growth medium in a petri dish with the embryo-axis in contact with the culture medium (see details below).

Fig. 2

Representative photos showing in vitro set up and sterilization solutions for embryo rescue (a), collected immature head and removed seeds (b), and aseptically removed embryos placed on culture medium for germination (c)

Embryo rescue and culture conditions. Culture the collected immature embryos in petri dishes containing regeneration medium with half strength MS salts (Murashige and Skoog 1962), 10 gL⁻¹ sucrose, 0.8% (w/v) agar and 1% (w/v) charcoal (see preparation instructions below). Firmly seal the culture plates with Parafilm M^®. Incubate these cultures in a growth chamber/incubator under 16 h light/8 h dark photoperiod, light of 500 μmol m⁻² s⁻¹ photon flux density and temperature of 25 °C for 4–5 days or until proper germination. Transfer germinated sorghum seedlings in small trays/pot containing 1:1 peat:soil mixture (Fig. 3). Incubate transplanted seedlings in greenhouse conditions similar to those described for rapid cycling of mature seeds. Repeat to new cycle when the plants reach the optimum time for immature seed collection for embryo rescue or the physiological maturity for seed collection.

Fig. 3

Representative photos showing the transfer of germinated seedling from immature embryos (petri-dish inset of A) into trays with cells filled with peat:soil mixture (a), covering of the transplanted seedlings with a nylon cover for about 7–10 days for acclimatization (b), and removal of the nylon cover from acclimated seedlings for continued growth in the open environment of the glasshouse (c)

Media preparation for seedling regeneration and sterilization. Dissolve 2.15 g/L MS (Murashige and Skoog 1962) Basal Salt Mixture (Sigma M5524) and 10 g/L sucrose in 1L distillated water at room temperature. Adjust the pH to 5.7–6.0 using 1N NaOH/KOH or 1N HCl. Add 10 g/L charcoal and 8 g agar into media to solidify. Autoclave for 20 min at 121 °C at 15 psi. Distribute the autoclaved media after cooled to 50–60 °C into 90 mm diameter disposable sterile petri-dishes. Store the media at room temperature or 4 °C for prolonged storage.

Validation of the protocol. Among the nine tested sorghum varieties, the flowering time ranged between 36–47 days when grown in trays, and 42–57 days in round small pots (Table 2). Days to seed physiological maturity ranged between 65–75 days in the trays, and between 75–92 days in the small pots. The number of seeds harvested per head ranged between 25–76 seeds in trays and between 85–295 seeds in the small pots. These results indicate that in two months one could harvest sufficient seeds to proceed to the next cycle using the trays (5 × 5 × 5 cm) or small round pots (9 × 9 × 5 cm). This would allow up to 4–5 crop cycles per year. Seed germination tests at different dates from flowering indicated that within a month from harvesting, germination is more than 50% compared to 100% after 45 days from flowering (Table 3). This suggests that further shortening is possible in the time needed for the crop cycle with early harvesting and immediate planting as only a few (theoretically just one) plants are needed to proceed from each parental plant to the next cycle. Furthermore, with intervention by embryo rescue from immature seeds at 15 days from flowering, 70–100% of the immature seeds germinate. This would further shorten the crop cycle by two weeks (Table 4).

Table 2 Effect of different pot type and management on days to flowering, days to maturity and number of seeds per head in the different sorghum varieties

Table 3 Germination percent of freshly harvested seeds after 30, 35 and 45 days from flowering among the nine tested sorghum varieties across trays and small round pots

Table 4 Frequency of regeneration of plantlets from immature embryos rescued at 10, 15 and 20 days post-anthesis from nine sorghum varieties

Conclusion

Induced mutations in sorghum by physical mutagens such as gamma and X-rays coupled with rapid cycling of generations offer promising opportunity to accelerate delivery of mutant varieties with induced resistance to the parasitic weed Striga. In this study, the protocol is optimized for horticultural factors such as pot size, temperature, irrigation, photoperiod and use of immature embryo rescue to accelerate propagation and time needed to complete the growth cycle (Fig. 4). The optimized protocol was validated on nine different sorghum varieties. By using small rooting areas and intervention with embryo rescue, the time needed to grow and harvest seeds/embryos was reduced from 5 to 6 months under natural conditions in the field to 2–3 months under the optimized protocol (Fig. 4). This means with the optimized protocol we can advance four to six generations per year and thus fix the mutant trait in two years and quickly advance to multi-location evaluation and varietal registration. The entire process takes up to 10–12 years under a conventional mutation breeding approach.

Fig. 4

Summarizes the optimized scheme for rapid generation cycling of sorghum