Shoot Proliferation, Callus Induction And Plant Regeneration In Tripsacum Laxum Nash

The peduncles of Tripsacum laxum Nash were used as explants to induce axillary shoots. Multiple shoots were 13 proliferated on Murashige and Skoog (MS) medium to establish, for the first time, efficient shoot proliferation 14 and plant in vitro regeneration systems. Optimal shoot proliferation medium was MS with 3.0 mg/L 15 6-benzyladenine (BA) and 0.2 mg/L α -naphthaleneacetic acid (NAA), resulting in a shoot proliferation 16 coefficient of 11.0 within 45 d. Optimal rooting medium was MS with 0.1 mg/L NAA and/or 0.1 mg/L 17 indole-3-butyric acid (IBA), inducing 100% root formation from shoots within 30 d. When young roots, leaf 18 sheaths and shoot bases were used as explants, MS medium with 1.0 mg/L thidiazuron (TDZ) and 0.2 mg/L 19 BA induced most shoots, with the least callus. Shoot bases induced beige-white callus and shoots directly on 20 MS medium with 1.0 mg/L TDZ and 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), while leaf sheaths 21 induced beige-white callus and shoots directly on MS medium with 1.0 mg/L TDZ and 0.2 mg/L BA. Rooted plantlets showed 99.3% survival when transplanted into a substrate of vermiculite: peat soil (1:3, v/v ).


Introduction 31
The genus Tripsacum (Maydeae tribe, Panicoideae, Gramineae) includes 16 species that grow in many 32 ecologically distinct niches and habitats that are typically distributed in tropical and subtropical regions (Gray 33 1974; Wet et al. 1985). Since Tripsacum has a common ancestor with maize and teosinte, it may be important 34 to better understand the origin and evolution of maize. Tripsacum is a perennial warm-season C4 type of grass 35 that is often used to produce high-quality forage and biomass energy, and control soil erosion (Zhao et al. 36 2020). Tripsacum laxum Nash (Guatemala grass) is widely used globally as a forage crop (Guyadeen 1951). 37 Its strong roots and long period of growth and utilization allow it to be harvested for many years, and given its 38 high yield, high nutritional value, and good taste, it is suitable for cutting green feed or process into silage 39 material, and can thus be used as feed for cattle, ducks, geese and pigs (Guyadeen 1951;Boonman 1993). The 40 roots of T. laxum develop well, and when tilled into soil and used as organic matter, this improves the physical 41 and chemical structure of the soil, so it is often used as a multi-year cover crop (Shem et al. 1995). After T. 42 laxum was introduced to China, it is now a major source of forage feed (Jiang et al. 2002;Zhong et al. 2011). 43 The diploid chromosome number of T. laxum is 2n = 72 (Dodds & Simmonds 1946;Zhong et al. 2011). 44 Although most chromosomes are bivalents, there multiple chromosomal irregularities, ultimately resulting in 45 male sterility (Dodds & Simmonds 1946). T. laxum is rarely propagated by stem cuttings because stems tend 46 to shrink (Guyadeen 1951) and are prone to bacterial infections (Tuley 1961;Schieber 1975). To resolve 47 limitations associated with proliferation and to overcome disease-related problems, the establishment of an in 48 vitro regeneration system would allow this plant to be mass propagated and to create a platform that would 49 allow for its genetic improvement through transgenic strategies. To our knowledge, there are no studies on the 50 tissue culture or related biotechnologies of T. laxum. In this study, for the first time, we employed the 51 peduncles of T. laxum as explants to induce axillary shoots that were then proliferated to establish an efficient 52 in vitro regeneration system. T. laxum plants growing on a farm in Guigang city, Guangxi province with taxonomy ID: 47471, were brought 57 back to Guangzhou in 2010. All the studies comply with relevant institutional, national, and international 58 guidelines and legislation. It has been specified under the appropriate permissions and licenses for the 59 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 61 produced (Fig. 1a). Stems were cut into 30 cm long cuttings, planted in a field and allowed to grow naturally. 62 Plants were identified by Dr. Liu Qing, a botanist in South China Botanical Garden. When the plants began to 63 flower, between March and April of 2016, young inflorescences of T. laxum were removed with a surgical 64 knife (Fig. 1b). Segments (5 cm long) were first surface disinfected with 75% ethanol using cotton balls, 65 dipped into 0.1% (w/v) mercuric chloride solution (HgCl2) for 10 min, then washed three times with sterile 66 distilled water. Surface-disinfected explants (2-3 cm long peduncles) were inoculated into Murashige and

Effects of plant growth regulators on axillary shoot proliferation 77
Using a similar technique as was employed for Scaevola sericea (Liang et al., 2020), axillary shoot clusters 78 were cut into smaller clusters, each with three shoots. These were inoculated onto MS medium containing 79 different combinations and concentrations of plant growth regulators (PGRs) for axillary shoot proliferation 80 (Table 1). For each treatment, 10 jars were used. Each jar contained three shoot clusters. After culture for 45 d,

Effects of plant growth regulators on callus induction from three explant types 92
Young roots, young leaf sheaths and shoot bases were used as explants. Roots were derived from 15 d-old 93 plantlets that had been rooted in ½MS medium with 0.1 mg/L NAA. Roots were cut into 1.0 cm long explants. 94 The young leaf sheaths and shoot bases were derived from shoots that had been proliferated on MS medium 95 with 1.0 mg/L BA for 45 d. These tissues were cut into explants 0.5 cm 2 in size and inoculated onto MS-based 96 media with different PGRs to induce callus and observe differentiation after 30 d (Tables 3-5).

Statistical analyses 108
All experiments were repeated three times within one week. Data are reported as mean ± SD (standard 109 deviation). Means were statistically analyzed by one-way analysis of variance (ANOVA). Treatment means 110 were considered to be significantly different from controls after applying Duncan's multiple range test 111 (P ≤ 0.05) using SPSS v. 19.0 (IBM, New York, NY, USA).  (Table 1). When BA was supplemented with 0.2 mg/L NAA, axillary shoot number increased 117 significantly (Table 1; Fig 1d), with 3.0 mg/L BA and 0.2 mg/L NAA assessed as the optimal medium for 118 shoot proliferation (Fig. 3a). When culture period was extended to 45 d, some shoots formed roots at their 119 base (Fig. 3b), suggesting that rooting was easy. 120 121

Root formation 122
After 15 d, 67-75% of shoots induced roots when medium contained 0.1 mg/L NAA or IBA, or 100% if 0.1 123 mg/L of both these auxins were employed ( Fig. 3c; Table 2). Control (no auxins) shoots did not induce roots 124 within 15 d. However, after 30 d, 100% of shoots on any medium with an auxin formed roots (85% in the 125 control) ( Table 2).

Callus inducing from shoot basal meristem explants 143
When TDZ or 2,4-D were used alone, some hyperhydric pink callus was induced, but it was unable to 144 differentiate, and eventually turned brown and died. BA did not induced callus, instead inducing adventitious 145 shoots from callus. When 2,4-D was combined with BA and TDZ, they induced a low frequency of callus in 146 1-2% of explants after 30 d (Table 5). Milky white or yellow granular callus possessed a strong ability to 147 develop adventitious shoot buds directly, especially the combination of 1.0 mg/L TDZ and 0.2 mg/L 2,4-D 148 (9.2 adventitious shoot buds/explant) (Fig. 2e, 2f), followed by 1.0 mg/L TDZ and 0.2 mg/L BA (5.3 149 adventitious shoot buds/explant) (Table 5). Axillary shoot proliferation (i.e., SPC) was enhanced in the presence of a cytokinin and NAA (Table 1), 165 similar to the tissue culture of Lepturus repens, another Gramineae plant (Xiong et al. 2021). In T. dactyloides, 166 mature zygotic embryos were used to induce embryogenic callus cultures on MS medium with dicamba (10 or 167 20 μM) and sucrose (3 or 6%), while plantlets were regenerated on PGR-free MS medium containing 2% 168 sucrose (Furini and Jewell 1991). In our study on T. laxum, only TDZ was able to induce callus from root 169 explants, while the further addition of BA also stimulated shoot formation (Table 3) The base of leaf sheaths were used as explants to induce callus, although only TDZ combined with BA 174 successfully induced callus, which differentiated into adventitious buds (Table 4). Transverse sections of 175 young leaf spindle rolls in sugarcane oriented distal end into medium were critical for shoot regeneration, 176 which was observed within 3 weeks on MS medium with 10-60 µM NAA and 4-8 µ M BA (Lakshmanan et al. 177 2006). Sugarcane explants cultured in the dark, and exposed to 4.5 μM 2,4-D, induced callus from stem 178 parenchyma while pre-embryogenic masses formed from immature leaves (Garcia et al. 2007). 179 In Sorghum bicolor, callus was induced from thin seedling-derived root or epicotyl explants when KIN or BA were used (Gendy et al. 1996). The use of 3.0 mg/L BA and 1.0 mg/L TDZ in MS most efficiently induced 181 multiple shoots from immature seeds of S. bicolor (Liu et al. 2015). When S. bicolor leaf bases were used as 182 explants, callus was induced and ultimately plantlets could be regenerated on MS medium with 2.0 mg/L 183 2,4-D (Mishra et al. 2003). Also in S. bicolor, most callus was induced on MS medium containing honey and

Competing interests 195
The authors declare that they have no competing interests.      Values represent means ± SD. Different letters within a column indicate significant differences according to 304 Duncan's multiple range test (P ≤ 0.05). n = 30 per treatment.  Table 6). Bars = 1.0 cm. 334