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Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1-2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4-5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA3, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.

The purpose of this study was to determine whether Cercis chinensis seeds contain endogenous germination inhibitors, and if so, to clarify the dynamic changes to the inhibitors during dormancy release. A cabbage seed germination test was conducted to assess the seed extract activities during dormancy release. The endogenous inhibitor components were analyzed by gas chromatography–mass spectrometry and the dynamic changes to the endogenous inhibitors were analyzed by high-performance liquid chromatography. The analyses revealed 1,2,3-benzenetriol (phenolic compound) in the seedcoat is a water-soluble endogenous inhibitor, and the IC50 (the concentration at which 1,2,3-benzenetriol inhibited radicle growth of cabbage seeds by 50%) of 1,2,3-benzenetriol was 51.2 µg⋅mL–1. During dormancy release, the seed 1,2,3-benzenetriol content decreased to 17.7 µg⋅mL–1 (stratified for 60 days) from 561.4 µg⋅mL–1 (control). The abscisic acid (ABA) content exhibited the same tendency, decreasing from 5.6 ng⋅mL–1 to 0.5 ng⋅mL–1 after 60-day stratification. Exogenous ABA was highly inhibitory toward cabbage seed germination, with an IC50 of 1.5 ng⋅mL–1. These results indicate that ABA and 1,2,3-benzenetriol are important endogenous inhibitors in C. chinensis seeds, wherein they regulate seed dormancy, even at low concentrations.

The seeds of Cercis chinensis are important for reproduction and propagation, but strong dormancy controls their germination. To elucidate the causes of seed dormancy in C. chinensis, we investigated the permeability of the hard seed coat, the contribution of the endosperm to physical dormancy, and we examined the effect of extracts from the seed coat and endosperm. In addition, the effectiveness of scarification methods to break seed dormancy were compared. C. chinensis seeds exhibited physical and physiological dormancy. The hard seed coat played an important role in limiting water uptake, and the endosperm acted as a physical barrier that restricted embryo development in imbibed seeds. Germination percentage of Chinese cabbage seeds was reduced from 98.0% (control) to 28.3% and 56.7% with a seed-coat extract and an endosperm extract, respectively. This demonstrated that both the seed coat and endosperm contained endogenous inhibitors, but the seed coat extract resulted in stronger inhibition. Mechanical scarification, thermal scarification, and chemical scarification had positive effects on C. chinensis seed germination. Soaking non-scarified seeds in Gibberellic acid (GA3) solution did not promote germination, however, treatment with exogenous GA3 following scarification significantly improved germination. The optimal method for promoting C. chinensis seed germination was soaking scarified seeds in 500 mg·L−1 GA3 for 24 h followed by cold stratification at 5 °C for 2 months.