Studies On Tissue Culture And Physiological And Biochemical Basis Of Freezing Tolerance In Saussurea.Laniceps Hand.-Mazz.

Alpine environments are characterized by low temperature,intensive radiation ,strong wind,excessive evaporation,drought atmosphere and short growing seasons.The most well adapted ones are preserved and are expanding along the direction which is favorable to their survival under hard condition.To elucidate the freezing tolerant mechanism of alpine plants , Cottonhead Windhairdaisy (Saussurea laniceps Hand.-Mazz.) was selected as a plant model. It is a perennial herbal plant distributed in alpine regions of Yunnan and Tibet of China.lt can survive on mountains with elevation over 4000m.Wild S. aniceps is only propagated by seed in the alpine areas , establishment of an efficient method for micropropagation was a desirable method to get enough seedlings for research.Seedlings ,plantlets and calli were used to reveal the adaptation of the morpho-structures to the ecological environments and the physiological and biochemical basis of freezing tolerance in S. aniceps. under controlled conditions. Main results and conclusions are as follows.1 .Morpho-structures adapted to the alpine ecological environmentsThe wild Saussurea laniceps plant grown in Tibet was short and covered by long erion.Seedlings germinated from seed also has special structural characteristics,such as small leaf with multicellular long hairs. The cuticle and 1-2 layer palisade tissues are tight. Much aerenchyma distributed in spongy mesophyll tissues and cortex of petiole medial vein,Ther are many stomata in leaf cuticle.The morphology and the anatomical structure characteristics of plantlets are similar to those of the seedlings.2. Micropropagation and callus inductionSeedlings were formed from germinated seeds in 7-10 days . Then leaf explants were transplanted to MS medium supplemented with 1-naphthalene-acetic acid (NAA)/2.4-Dand benzyladenine (BA)/KT and callus was achieved 10 days after transfer. Shoot bud regeneration occurred from callus cultured on MS medium supplemented with different growth regulators 20 d after culturing.The regeneration percentages varied with the different components of plant growth regulators. The percent regeneration from callus pretreated at low tempertature of 5°C was significantly increased as compared with those incubated at 23/20°C directively. Optimal regeneration was observed with explants on media supplemented with 1.5mg/l BA plus O.2mg/1NAA. In the presence of 0.2 mg/1 NAA. in half-strength MS, 78% of the shoots formed roots. Plantlets from explants showed 63% survival after acclimatization.3. Cold-hardening increased the freezing tolerance of sprout, plantlets and callus After acclimation at 2°C for 15 days, significant freezing tolerance was attained insprout and seedlings.As for callus , significant freezing tolerance was achieved after acclimation at 0°C for 15 days.4. Antioxidative enzymes and profile of isoenzymic zymogramThe activities of SOD, peroxidase ,and catalase in cold acclimated plantlets and calli were increased.New isoenzyme bands appeared after cold acclimation. Interestingly,deacclimation was slow; within 1-5 days after plantlets returned to normal growh temperatue (21-23°C) , higher enzyme activities remained.5.Protein changes during cold acclimationProtein contents of plantlets and calli increased after acclimation at subzero temperatures. SDS-PAGE of soluble proteins revealed that specific new polypeptides of the 17.1KD,32.3KD and 54.8KD were accumulated while two other bands of 38.2KD and 29.5KD became richer.The three specific new polypeptides did not decreased after three-day deacclimation,but the 32.3KD band disappeared after six-day deacclimation,indicating the 32.3KD polypeptide was very related to freezing tolerance of S. aniceps.6.Antioxidants associated with freezing toleranceAn increase of proline content was observed in the planlets,calli and sprouts after cold acclimation. Whereas solute carbohydrate changed little. Polyphenol content and activity of polyphenol oxidase were also increased after cold-hardening. Isoenzymic zymogram profile indicated new bands of polyphen...