Plant material and root induction

We used the walnut cultivar ‘Zhongningsheng’ [ZNS (Juglans hindsii × Juglans regia)]. The stock tree was 23 years old, had exhibited steady reproductive growth and is located in the Luoning county, Henan province. Rejuvenated soft shoots of ZNS were obtained as follows: first, new ZNS shoots from the stock tree (scions) were grafted to rootstock (1-year-old seedlings). Then, 1-year-old grafted plants were brought into the greenhouse and placed horizontally under 4–5 cm of clean wet sand to ensure maximum shoot bud break. The humidity was held at 50%, the greenhouse used natural light, and the sand temperature was held at 15 ± 5 °C. After 60–70 days of culturing, the rejuvenated soft shoots were harvested. Mature soft shoots similar to the rejuvenated shoots were collected from the 23-year-old stock tree.

Both of the soft shoots bases were dipped in the rooting induction solution [9 mM IBA in 30% (v/v) ethanol in water] for 30 s and submerged 6–8 cm in the root development medium. The shoots were exposed to natural sunlight. The humidity was maintained at about 80% by automatic intermittent spraying. The temperature of the root medium was maintained at 23 ± 5 °C to allow rooting induction. The rooting rates were measured 5, 10, 15, and 20 days after rooting induction. A minimum of 30 soft shoots were collected at each time point to measure rooting rates. All results are expressed as means (± standard errors) of data from at least three replicate experiments. Adventitious roots were directly counted in 5–40 mm regions of basal shoots.