2.1 Plant material and culture environment
The experiment used Faro, a variety of quinoa from Central Chile (34.65°latitude and 71.91° longitude), which was bred and preserved in the laboratory.
Except when otherwise stated, The basal medium consisted of twice-strength Murashige and Skoog (MS) (1962) along with 2 mg/L glycine, 0.5 mg/L nicotinic acid, 0.1 mg/l thiamine hydrochloride, and 0.5 g/l pyridoxine hydrochloride. All media were supplemented with 30 g/l sucrose, 100 mg/l inositol, 100 mg/l caseinhydrolysate, and solidified with 5.5 g/L agar. After the corresponding hormones were added to the medium, the PH was adjusted to 5.8 with 1M NaOH and autoclaved at 121℃ for 20 min.
Quinoa seeds were washed under running water for 2h, dipped in 75% ethanol for 1min, and then dipped in 0.1% mercury chloride solution for 10min. After disinfection, rinse with sterile distilled water five times to remove toxic substances and obtain sterile vaccine. The sterile seeds were inoculated into a half-strength MS medium, in which sucrose concentration was 20g/L and other media components remained unchanged. In addition, 1g/L activated carbon was added to the medium. After dark culture for 3 days and light culture for 7 days, sterile quinoa seedlings were obtained, and seedlings from different parts were cut as explants for experiment.
The experimental explants were inoculated in culture flask and cultured at 25±1℃ under the light intensity of 2000lux.
2.2 Screening the best cutting way of cotyledon
The obtained sterile seedlings were used as test materials, and four different explants were obtained by cutting different parts of the four seed leaves, namely, leaf, cotyledon without petiole, terminal bud with petiole (approx. 1cm length) and cotyledons with petiole (approx. 1cm length) (Fag.1).
The 4 groups of explants were inoculated on induction medium containing 3.0 mg/L 6-BA and 0.1 mg/L 2,4-D, respectively. Four explants were inoculated in each bottle of medium, and 10 bottles were inoculated in each group. Culture conditions were observed every week, and induction directions of different explants were observed after 25 days. The experiment was repeated three times.
2.3 Induction of axillary and adventitious bud
The axillary buds of quinoa were induced from cotyledons with 1cm hypocotyl and terminal bud as explants. The explants were inoculated with different concentrations of 6-BA (0.5mg/L, 1.0mg/L, 2.0mg/L, 3.0mg/L, 4.0mg/L) to induce axillary bud formation. Four explants were inoculated in each bottle of medium, and 10 bottles were inoculated in each group, and the experiment was replicated three times. After 25 days, the survival rate of plants, the number of axillary bud germination of each explants and the growth height of plants were counted.
The adventitious bud of quinoa were induced from cotyledons with terminal bud as explants. Explants were seeded in the media by the addition of 0.1 mg/L of NAA and various concentrations of 6-BA (1.0, 2.0, 3.0, 4.0mg/L). Four explants were inoculated in each bottle of medium, and 10 bottles were inoculated in each group, and the experiment was replicated three times. After 25 days, the survival rate, callus induction rate, germination rate, adventitious bud induction rate and plant growth were counted.
2.4 Multiplication of axillary and adventitious bud
The plants inducing axillary buds were cut at the third cotyl node, retaining the upper half. Plants with similar height and growth status were selected as explants for axillary bud multiplication experiments. The explants were inoculated into five media containing different concentrations of 6-BA (0.5, 1.0, 2.0, 3.0, 4.0mg/L). Four explants were inoculated in each bottle of medium, and 10 bottles were inoculated in each group, and the experiment was replicated three times. After 25 days, plant survival rate, number of axillary bud germination per explants and plant growth height were counted.
Adventitious bud multiplication experiments were performed using the induced adventitious buds as explants. The optimal hormone concentration was selected by adding different concentrations of 6-BA(0.5, 1.0, 2.0, 3.0, 4.0mg/L) to the culture medium. Four explants were inoculated in each bottle of medium, and 10 bottles were inoculated in each group, and the experiment was replicated three times. After 25days, the etiolation rate of plants, the average number of elongated plants per explants and the number of plants larger than 1cm were counted, and the most suitable concentration of 6-BA for the multiplication of adventitious buds was screened.
2.5 Rejuvenation of axillary and adventitious bud
The plants inducing axillary buds were cut at the third cotyl node, retaining the upper half. Plants with similar height and growth status were selected as explants for axillary bud rejuvenation experiments. The explants were inoculated into five media containing different concentrations of 6-BA (0, 0.1, 0.3, 0.5mg/L). Four explants were inoculated in each bottle of medium, and 10 bottles were inoculated in each group, and the experiment was replicated three times. After 25 days, the plant etiolation rate, the average number of total plants in each explants, the number of plants greater than 1cm and the percentage of the number of plants greater than 1cm were counted, and the most suitable concentration of 6-BA for the regeneration growth of in vitro seedlings induced by axillary buds was screened.
Adventitious bud rejuvenation experiments were performed using the induced adventitious buds as explants. The optimal hormone concentration was selected by adding different concentrations of 6-BA(0, 0.1, 0.3, 0.5mg/L) to the culture medium. Four explants were inoculated in each bottle of medium, and 10 bottles were inoculated in each group, and the experiment was replicated three times. After 25 days, the yellotization rate of plants, the average number of elongated plants per explants, the number of plants greater than 1cm and the percentage of the number of plants greater than 1cm were counted to screen the most suitable 6-BA concentration for the growth of strong vitro seedlings.
2.6 Rooting of quinoa in vitro
Quinoa plantlets in vitro were used as explants. The explants were inoculated into four kinds of medium containing different concentrations of IBA (0.5, 1.0, 2.0, 3.0mg/L). It is worth noting that the ion concentration in the rooting medium is low, which is single-strength MS, and 20g/L sucrose is added, while other components remain unchanged. Three explants were inoculated in each bottle of medium, and 10 bottles were inoculated in each group, and the experiment was replicated three times. After 25 days, the survival rate, etiolation rate, rooting rate, the number of induced primary roots and the number of roots greater than 1cm were counted, and the most suitable concentration of IBA for inducing rooting of quinoa in vitro seedlings was screened.
2.7 Data statistics and analysis
The statistical software IBM SPSS Statistics 22.0 was used for calculation, one-way anOVA and independent sample T test. The significance level was P<0.05, data were expressed as mean ± standard deviation (x±s).