Effect Of Single-wall Carbon Nanotube On Soybean Regeneration From Mature Cotyledonary Node Explants

Soybean is an important grain and economic crops in the world which is rich in edible protein, oiland other healthy ingredients. Soybean is recognized as a recalcitrant crop for genetic transformation;the transformation frequency is low and can repeat badly. Therefore a high efficiency regenerationsystem is needed for the transformation. Soybean Zhonghuang35is a fine variety with high yield, highoil, early ripening and wide adaptability, however, urgently, it is quite necessary to increase itsresistance to diseases. Genetic engineering breeding is a direct and effective way for soybean molecularbreeding, but there is not yet a study about genetic transformation on Zhonghuang35nowadays. In thispaper, we conducted: effect of single-wall carbon nanotube （NM） on soybean regeneration from maturecotyledonary node explants and the effects of NM on cell physical activity of callus derived fromsoybean immature embryo. The main results were as follows:1. Effect of NM on soybean regeneration from mature cotyledonary node explants.NM improved soybean regeneration frequency of cotyledonary node explants. Explants werecultured with4concentrations of NM for differentiation and regeneration.40mg/L NM supplemented inshoot initiation medium was found to be the optimal concentration with shoots regeneration frequencysignificantly increased by21.5%compared with the control treatment （56.9%）. Faster elongation androoting of adventitious shoots was observed in shoot elongation medium with40mg/L NM. Soybeanplantlet formation frequency within the limited4weeks treated with40mg/L NM reached48.4%whilethe other three treatments:4mg/L,400mg/L and control,0were observed.Explants were cultured in shoot initiation medium with0-160mg/L NM.40mg/L NM was found tobe the optimal concentration that has8.7%-15.3%higher shoot regeneration frequency compared withthe control treatment during the period of shoot initiation and development. Soluble protein content,physical indexes of reactive oxygen species （ROS）, ROS scavenge system, and concentrations of sixkinds of main plant endogenous hormones of different treatments were measured, we found:40mg/L NM treatment has the highest protein content compared with the control and othertreatments.40mg/L NM treatment has lower MDA and H₂O₂content, lower protective enzyme activityand VC content before the16thday compared with control, which made a balance between ROS andROS scavenge system. At the later stage with adversity stress induced by lack of nutrient, the amplitudeof MDA and H₂O₂elevation is much smaller in40mg/L NM treatment than in control;40mg/L NMtreatment reduced the ROS damage to the shoots mainly by increase the CAT content.IAA/CKs for40mg/L NM treatment is lower than control on the7thday while it is higher thancontrol from10th-19thdays; BR content of40mg/L NM treatment is significantly higher than controlbefore the13thday and its peak value appeared earlier;40mg/L NM treatment has lower ABA contentthan control during1st-10thdays;40mg/L NM treatment has two iPA peak, each with a GA peakfollowed, but control treatment has only one iPA content peak.Based on these results we concluded that NM improved shoots regeneration and growth byregulate soluble protein content, physical indexes of reactive oxygen species （ROS）, ROS scavenge system, and concentrations of plant endogenous hormones.2. Effects of NM on cell physical activity of soybean callus. Mitochondria activity of soybeancallus cells was measured by MTT method, and mitochondria activity of40mg/L NM treatment has nodifference with control; necrocytosis rate and cell apoptosis rate of40mg/L NM treatment is lower thancontrol at5thday, no differences at other time measured by propidium iodide （PI） staining and4’,6-diamidino-2-phenylindole （DAPI） staining.Our study demonstrated that40mg/L NM can improve regeneration frequency of soybeancotyledonary node explants, and40mg/L NM treatment has no negative effect on cell physical activity.