In Vitro Selection Of L-Hydroxyproline Resistant Variants Of Coronilla Varia L And Studies On Their Salt Tolerance

Three hydroxyproline (Hyp) resistant cell lines (marked as Xr1, Xr2 and Xr3) were selected from NaN₃ treatment calluses of Coronilla varia L by direct selection and culturing on the Hyp containing medium. A large amount of regenerated plantlets were obtained from the resistant variant calluses via somatic embryogenesis. The selected resistant calluses, regenerated plants and their progeny showed a higher tolerance to NaCl stress than the nonselected control.Physiological and biochemical analyses of calluses and their progeny were carried out. Compared to the control, the contents of free amino acids, especially, free proline in the variant lines significantly increased;the activity of Δ¹ - Pyrroline -5-carboxylase synthase (p5cs) enhanced, however, the activity of Proline-dehydrogenase (PDH) decreased;At the same time they had the characteristics of higher selective absorption of K⁺ and of maintaining a higher proportion of K⁺/N⁺;The over accumulation of proline and the higher proportion K⁺/N⁺ were closely related to the salt tolerance of the resistant lines. They might be the material foundation to improve the salt tolerance of the resistant lines.The marked improvement of the activity of SOD and POD, which could effectively scavenge hydroxyl radical, reduced the damage of oxidative stress, thus significantly decreased the MDA content and the permeability of membranes.The soluble protein electrophoresis showed that the resistant lines Xr2 and Xr3 lost 31.98KD and 32.71KD protein, but a new protein of 24KD appearred. RAPD analysis showed that some random primers could detect DNA polymorphism, which suggested that a certain variation at DNA level had taken place.The results were as follows:1. An efficient protocol for somatic embryogenesis and plant regeneration of Coronilla varia L was established. The friable calluses were induced from cotyledon segments of lOd old seedlings by culturing on MS medium with 2.0mg/L 2,4-D and 0.5mg/L KT. High frequency of somatic embryos was obtained on MSB mediumcontaining l.Omg/L 2,4-D, l.Omg/L NAA, 0.5mg/L KT and 300mg/L proline, the number of somatic embryos per gram fresh weight callus were 625. The plantlets were developed from somatic embryos on MS medium supplemented with l.Omg/L KT, O.lmg/L IB A and 300mg/L proline. The mean frequency of somatic embryos per gram fresh weight producing regenerating plantlets was 21 /g.2. The half lethal dosage for mutation of the calluses was 0.5% NaN3 (pH 5.4) treating calluses for lhr 45 min.3. Three variant calluses resistant to 50 mmol/L Hyp were isolated from 550 pieces of calluses by direct selection method. The select frequency was 0.545%. The variant calluses formed a great deal of somatic embryos and regenerated plantlets .4. The resistant calluses exhibited a higher tolerance to salt stress, and the resistant character could be expressed and transmitted in the Ml and their progeny. The relative growth rates of Xrl, Xr2 and Xr3 corresponded to 3.4, 4.01 and 5.3 times of the control line in the medium containing 1% NaCl;The relative growth rates of the calluses induced from progeny seedling were 3.7, 3.77 and 3.84 times higher as compared with the control's when cultured in 1.5% NaCl containing medium. Under 1% NaCl stress, the withered leaf rates of the regenerated plants of the Xrl, Xr2 and Xr3 were 1.97, 2.84, 3.41 times than that of the control's, respectively;the germination rate of Fi progeny seeds of Xrl, Xr2 and Xr3 were 1.13, 1.85 and 2.36 times higher than that of the control's.All of these characteristics indicated that the tolerance in the variants was strengthened when exposed to salt stress.5. Biochemical and physiological analyses indicated that the resistant calluses could over-accumulate proline. Under 1% NaCl stress, the contents of free proline in Xrl, Xr2 and Xr3 were 2.062, 2.970 and 4.131 times as high as that in the control's respectively. It suggested that the resistance to salt stress was remarkable positive correlation with the increase of free proline level (0.969582*). The character of proline accumulation in the resistant calluses could be transmitted through the sexual and asexual propagation.Proline metabolism enzyme activity analyses showed that either under NaCl stress or not, the activity of OAT—the key enzyme in ornithine synthetic pathway —had no remarkable difference between resistant lines and the control, while the activity of P5CS—the key enzyme in Glu synthetic pathway —was remarkably higherin the resistant lines than that in the control. This enzyme was sensitive to NaCl stress and easy to be induced. Its activity expressed significantly positive correlation with their proline contents(0.976439*). The above mentioned facts illustrated that over accumulation of free proline in resistant lines is closely related to P5CS activity, and Glu biosynthetic pathway is the main pathway in the resistant lines.The addition of exogenous proline could make free proline content decreas 33.93%, and the activity of P5CS 28.51% in the control, however in the resistant lines Xrl,Xr2 and Xr3 .free proline decreased only 21.91%, 6.11% and 3.98%, and the activity of P5CS declined only 12.85%, 5.35% and 5.46%. It suggested that P5CS show a relaxed feedback inhibition by proline and this might be the mechanism of the proline overproduction in the resistant cell lines.Since the contents of K+ in the resistant calluses were higher, the contents of Na+ were lower than that in the control's, the variants maintained higher K+/Na+ ratio, the salt tolerance of the variants was closely related to the higher selective absorption of K+ and K+/Na+ ratio (0.99454**,0.96252*).Under the stress of high concentration of NaCl, the enzyme activities of SOD and POD in the resistant calluses enhanced, while the permeability of membranes and the levels of MDA decreased. The content of proline had a close relationship with the elevated enzyme activity of SOD and POD (r=0.978889**, 0.9143814*), indicating that free proline accumulation played an important role in resisting to oxidative stresses.Compared to the control, a new 24KD band appeared in SDS-PAGE patterns of the variant of Xr2 and Xr3, and the contents of some proteins were higher under the NaCl stress.6. RAPD analysis showed that 51 of 60 random primers could detect polymorphism, displaying quantitative differences and qualitative differences between variant lines and the control. The similarity index and genetic distance analysis (0.0171-0.0277) suggested that the resistant variant calluses and the control had similar genetic background, however a certain variation on DNA level did exist in the variants.