The present work was conducted at tissue culture Technique Laboratory, Central Labs, Ornamental Plants and Woody Trees Department, Agricultural and Biological Research Division, National Research Centre (NRC), Tissue Culture & Germplasm Conservation Research Lab., Horticulture Research Institute, Agricultural Research Center—Giza (ARC), and Agricultural Botany Department, Faculty of Agriculture, Cairo University, Egypt, during years the 2017 and 2018 to establish an efficient in vitro culture protocol for rapid micropropagation and bulblet formation of oriental lily “Starfighter.”
Explant source and surface sterilization
The scales of bulbs of Lilium (5–7 cm in diameter) were collected from a commercial nursery and gently excised from the points of attachment and surface sterilized in ethanol 70% (v/v) for 30 s, rinsed in Clorox 15% (sodium hypochlorite) for 7 min, then washed with sterilized distilled water three times. The scales were sterilized in 2% HgCl2 (MC) solution (w/v) for 10 min and finally rinsed three times in sterile water.
Culture medium
The sterilized bulb scales explants were cultured in jars containing 25 ml MS free of hormones (Murashige and Skoog 1962) supplemented with 3% sucrose and 0.7% agar. The pH of the medium was adjusted to 5.6–5.8 then autoclaved at 121 °C and 15 psi for 15 min.
Incubation conditions
The in vitro cultures during all stages were placed in the incubation room at 23 ± 2 °C under 16 h photoperiod and 1.5 k lux light intensity provided by cool, white, fluorescent lamps.
Shoot organogenesis
For direct and indirect shoots regeneration, the obtained leaves from culture starting were separated, sectioned, and transferred to MS medium supplemented with different plant growth regulators: 0.5 mg/l of thidiazuron (TDZ) and 6-γ,γ-dimethylallylamine purine riboside (2ip); 2,4-dichlorophenoxyacetic acid (2,4-D); and picloram at different concentrations (5 and 10 mg/l) for in vitro shoot multiplication. The formed shoots were used as secondary explants for further in vitro shoots propagation.
In vitro shoots proliferation
For this stage, 0.5 mg/l of both 6-benzyl amino purine (BA) and thidiazuron (TDZ) in combination or separated with α-naphthaleneacetic acid (NAA) at 0.1, 0.2, and 0.3 mg/l were tested. The characteristic features of regenerated plantlets were observed such as number of shootlets/explant, number of leaves/shootlet, and number of bulb scales/shootlet.
Bulblet formation
In this stage, shoot explants were transferred to culture media which contained different concentrations of sucrose (30, 60, 90, and 120 g L−1) only or combined with growth inhibitors (3 and 6 mg L−1 paclobutrazol) for bulblet formation of Lilium through three subcultures as well as the diameter of the obtained bulblets.
Hardening off
The bulblets were removed from culture jars and transferred to pots containing peat moss + sand (1:1), covered with transparent polyethylene pages for 2 weeks and gradually removed in the greenhouse.
Determination of total carbohydrates
Fresh samples of bulblets were used to determine total carbohydrates as described by Dubois et al. (1956).
Anatomical study
Shootlets and different stages of somatic embryos were chosen from survived in vitro cultures (aged 12 weeks old) as well as the acclimatized bulbs that were aged 8 weeks after transferring to the greenhouse. Samples were fixed in F.A.A. (10 ml formalin, 5 ml glacial acetic acid, and 85 ml ethyl alcohol 70%) and dehydrated in butyl alcohol series then embedded in paraffin wax of melting point 56 °C. Sections (20 μm) were cut with a rotary microtome. The sections were stained with crystal violet-erythrosine combination, cleared in xylene and mounted in Canada balsam (Willey 1971).
Statistical analysis
The average of recorded data for different parameters was statistically analyzed using randomized complete block design with ten replicates per treatment. LSD test at 5% for comparison among means was used according to methods of Steel and Torrie (1980).