Season, Environment Stress and Refrigerated Storage Affect Genomic DNA Isolation of Tung Tree

Many metabolites in leaf tissue disturbed plant genomic DNA isolation and always varied when leaves was harvested from different environments. Objective of this study was to investigate whether season, environment stress and refrigerated storage affect genomic DNA isolation of tung tree leaves. Five types of young leaves and two DNA isolation protocols, the recycling CTAB protocol I and II, were adopted to carry out the experiment. Our results showed that both leaf type and protocol affected DNA isolation of tung tree. Using the recycling CTAB protocol II, though little DNA were obtained from three types of young leaves, the other two have satisfying results. Whereas the recycling CTAB protocol I could produce high yield genomic DNA from all the five types of young leaves. All the detectable DNA samples in agarose gel electrophoresis were good templates for PCR reaction. Season, environment stress and refrigerated storage had a big effect on genomic DNA isolation of tung tree. The recycling CTAB protocol I was proved to be an effective and universal protocol for DNA isolation of tung tree. Five types of young leaves could all act as the tissue for isolation of genomic DNA, but the summer healthy young leaves without long-time refrigerated storage are the best. The optimal leaf tissue will benefit DNA isolation of plant species.

Exogenous Vitamin K3 and Peroxides Can Alleviate Hypoxia in Bean Seedlings (<i>Phaseolus vulgaris</i>L.)

Oxygen limiting conditions are a common occurrence in root zones of most crop plants and can adversely affect nearly all aspects of plant growth and development including its survival. The objective of this study was to determine the effectiveness of a novel redox cycling agent, vitamin K3, and various peroxides including hydrogen peroxide, calcium peroxide and magnesium peroxide in alleviating the effects of hypoxia in bean seedlings grown in nutrient culture. All the anti-hypoxic agents including vitamin K3 had a positive impact on the overall growth of bean seedlings under hypoxic conditions, but their responses were variable depending on the concentration. With regard to shoot growth, vitamin K3 (5 μM) increased the leaf area significantly, by more than 58% over the hypoxic control plants and produced the highest stem fresh weight similar to calcium peroxide (20 μM) and magnesium peroxide (10 μM). In addition, the use of vitamin K3 resulted in the highest accumulation of chlorophyll (chla + chlb) in the leaves, an increase of nearly two-fold over the hypoxic control plants. Furthermore under hypoxia, calcium peroxide (20 μM) and magnesium peroxide (10 μM) produced the highest leaf biomass (FW) followed by vitamin K3. Vitamin K3 (1 μM) also favored root growth in bean seedlings under hypoxia;it produced the largest increase in root length and root biomass (DW) similar to calcium peroxide and magnesium peroxide. Based on the overall shoot and root growth response of bean seedlings to various anti-hypoxic substances under hypoxic conditions, calcium peroxide, magnesium peroxide and vitamin K3 performed better than hydrogen peroxide. These findings show that vitamin K3 and peroxide salts are effective in alleviating hypoxic stress in bean seedlings and also, further highlight their potential for dealing with hypoxia in wide ranging situations.

Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses

Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system has been widely used for precise gene editing in plants.However,simultaneous gene editing of multiple homoeoalleles remains challenging,especially in self-incompatible polyploid plants.Here,we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue,using both CRISPR/Cas9 and LbCas12a(LbCpf1)systems.Loss-of-function mutants of FaPDS exhibited albino leaves,while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue.Moreover,these mutations were inheritable.Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.