The genus Tripsacum (Maydeae tribe, Panicoideae, Gramineae) includes 16 species that grow in many ecologically distinct niches and habitats that are typically distributed in tropical and subtropical regions (Gray, 1974; Wet et al., 1985). Since Tripsacum has a common ancestor with maize and teosinte, it may be important to better understand the origin and evolution of maize. Tripsacum is a perennial warm-season C₄  type of grass that is often used to produce high-quality forage and biomass energy, and control soil erosion (Zhao et al., 2020). Tripsacum laxum Nash (Guatemala grass) is widely used globally as a forage crop (Munyasi et al., 2015; Maleko et al., 2018; Klapwijk et al., 2020). Its strong roots and long period of growth and utilization allow it to be harvested for many years, and given its high yield,  high  nutritional value, and good taste, it is suitable for cutting green feed or process into silage material, and can thus be used as feed for cattle, ducks, geese and pigs in the limestone soils and a seasonally dry habitat (Wilkes, 1972; Boonman, 1993; Boschini-Figueroa and Vargas-Rodríguez, 2018). The roots of T. laxum develop well, and when tilled into soil and used as organic matter, this improves the physical and chemical structure of the soil, so it is often used as a multi-year cover crop (Shem et al., 1995). After T. laxum was introduced to China, it is now a major source of forage feed (Jiang et al., 2002; Zhong et al., 2011). Recently, it has been completed in chloroplast genome and evolutionary relationship analysis showed that it is more closely related to Tripsacum dactyloides (Luo et al., 2021).

Establishment of in vitro tissue culture

T. laxum plants growing on a farm in Guigang city, Guangxi province were brought back to Guangzhou in 2010. All the studies comply with relevant institutional, national, and international guidelines and legislation. It has been specified under the appropriate permissions and licenses for the collection of plant specimens. Plants were propagated by cutting and grown in a test field of South China Botanical Garden, Guangzhou, Guangdong Province. The plants flowered every year but no seed were produced (Figure 1a). Stems were cut into 30 cm long cuttings, planted in a field and allowed to grow naturally. Plants were identified by Dr. Qing Liu, a botanist in South China Botanical Garden. When the plants began to flower, between March and April of 2016, young inflorescences of T. laxum were removed with a surgical knife (Figure 1b). The peduncles segments (5 cm long) were the first surface disinfected with 75% ethanol using cotton balls, dipped into 0.1% (w/v) mercuric chloride solution (HgCl₂) for 10 min, then washed three times with sterile distilled water. Surface-disinfected explants (2-3 cm long peduncles) were inoculated into Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962) containing 1.0 mg/L 6-benzyladenine (BA) and 30 g/L sucrose. Medium pH  was  adjusted to 6.0 before being solidified with 0.7% (w/v) agar (Sigma-Aldrich, St. Louis, MO, USA), then autoclaved at 121°C for 20 min. Culture jars (height = 10 cm; diameter = 8 cm) were placed in an air-conditioned culture room at 25 ± 2°C with a 12-h photoperiod and 100 μM m⁻² s⁻¹ fluorescent light (Philips, Tianjin, China). Tissue culture conditions were identical to those used for another grass Lepturus repens (Xiong et al., 2021). After 15 days in culture, some axillary shoots buds (Figure 1c) were induced from peduncle internodes. Axillary shoots were subcultured on the same medium every 45 days. When sufficient stock was proliferated, experiments were initiated.

Effects of plant growth regulators on axillary shoot proliferation

Using a similar technique as was employed for Scaevola sericea (Liang et al., 2020), axillary shoot clusters were cut into smaller clusters, each with three shoots. These were “inoculated onto MS medium containing different combinations and concentrations of plant growth regulators (PGRs) for axillary shoot proliferation (Table 1). For each treatment, 10 jars were used. Each jar contained three shoot clusters. After culture for 45 days, axillary shoot proliferation coefficient (SPC) was assessed as: number of axillary shoots after proliferation for 45 days/number of axillary shoots before proliferation” (Liang et al., 2020).

Adventitious root formation

Adventitious root formation used a similar method as was employed for S. sericea (Liang et al., 2020). Axillary shoots were separated and cultured on rooting medium (½MS) “supplemented with different concentrations and combinations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) (Table 2). In each treatment, 10 jars were inoculated and each jar contained three shoots. PGR-free ½MS medium was used as the control. After 15 and 30 days of culture, rooting percentage was observed and assessed, as follows (Liang et al., 2020):

(number of buds that rooted after 30 days/number of inoculated buds) × 100%.

Effects of plant growth regulators on callus induction from three explant types

Young roots, young leaf sheaths and shoot bases were used as explants. Roots were derived from 15 day-old plantlets that had been rooted in ½MS medium with 0.1 mg/L NAA. Roots were cut into 1.0 cm long explants. The young leaf sheaths and shoot bases were derived from shoots that had been proliferated on MS medium with 1.0 mg/L BA for 45 days. These tissues were cut into explants 0.5 cm² in size and inoculated onto MS-based media with different plant growth regulators (PGRs) to induce callus and observe differentiation after 30 days (Tables 3 to 5).

Acclimatization and transplantation

An acclimatization protocol, similar to that which was used for S. sericea (Liang et al., 2020), was employed. Culture jars with shoots that were rooted in ½MS medium with 1.0 mg/L IBA for 30 days were transferred to natural light for 7 days. Using tap water, agar was gently rinsed off roots. Rooted plantlets were transplanted into plastic pots (height and diameter = 10 cm) containing yellow mud and peat soil (1:1, v/v), or peat and vermiculite (3:1, v/v). A single plantlet was planted in each plastic pot, and each treatment had 30 plantlets. Plants were watered every morning with tap water. After 30 days, plantlet height was determined. Survival percentage of transplanted plantlets was assessed as (Liang et al., 2020):

(number of living plantlets before transplanting / number of living plantlets after transplanting for 30 d) × 100%.

Shoot proliferation on different media

BA induced shoots more effectively than KIN, as assessed by SPC, but not when its concentration exceeded 3.0 mg/L  (Table 1). When 6-benzyladenine (BA) was supplemented with 0.2 mg/L NAA, axillary shoot number increased significantly (Table 1 and Figure 1d), with 3.0 mg/L BA and 0.2 mg/L NAA assessed as the optimal medium for shoot proliferation (Figure 3a). When culture period was extended to 45 day, some shoots formed roots at their base (Figure 3b), suggesting that rooting was easy.

Root formation

After 15 days, 67 to 75% of shoots induced roots when medium contained 0.1 mg/L NAA or IBA, or 100% if 0.1 mg/L of both these auxins were employed (Figure 3c and Table 2). Control (no auxins) shoots did not induce roots within 15 days. However, after 30 days, 100% of shoots on any medium with an auxin formed roots (85% in the control) (Table 2).

Callus induction and adventitious shoot induced from root explants

When BA, 2,4-D and NAA were used alone, almost no callus was induced from root explants, and only TDZ induced some expansion of the root explant and the induction  of some callus. In all cases, adventitious shoot buds developed (Figure 2a). When thidiazuron (TDZ) and NAA were combined, the percentage of explants inducing callus increased to 13.3%, ultimately forming 2.2 adventitious shoot buds per explant after 30 days. Callus induction percentage (20% of explants) and number of adventitious shoot buds/explant (3.8) were largest on MS medium with 1.0 mg/L TDZ and 0.2 mg/L BA (Figure 2b and Table 3).