| In this paper, we studied the callus induction of Zostera marina L. which was ahigher marine flowering submerged plants on the basis of tissue culture technology.Our purpose was to explore the optimal conditions on callus induction of Z.marina L.by the explants of primary meristem and organs, included the effect of the kind ofmediums, culture methods, explants sites, supplyments of mediums and cultureconditios on callus induction of Z.marina L. This could provide adequate source ofgermplasm for somatic embryos indirectly and plant regeneration through callusinduction, and could provide basic technical gaide for culture Z.marina L. in vitrounder artificial conditions. The results were as follows:1Culturing callus inducted by the explants of primary meristem(1) Optimization of culture conditions for seedlings germinationIn this experiment we studied the effect of salinity of culture mediums (5‰,10‰,15‰,20‰,25‰and30‰), culture temperatures (10℃,14℃,18℃,22℃and26℃) and culture illuminations (0Lx,500Lx,1000Lx,2000Lx and4000Lx) onseed germination of Z.marina L. This could provide plenty source of sterile explantsfor tissue culture in vitro.The result showed that lower salinity (5‰-10‰) could promote seeds to geminatein a short time; and the germination in lower temperature (14℃) was higher than thatin other temperatures; higher temperatures and illuminations inhibited the seedgermination.When the medium salinity was5‰-10‰cultured under14℃and500-1000Lxcould provide adequate aseptic explants for culture supplies.(2) Callus inducted by seedlings①The effect of culture mediums, culture methods and explants sites on callus-likeinduction of Z.marina L.In this experiment we studied the effect of culture mediums (MS, N6, PESI, VSEand Sea mediums), culture methods (solide culture, semi-solid culture and liquid culture) and explants sites (radicle, hypocotyls and cotyledon) on callus-like inductionof Z.marina L. on the purpose of providing basis theory guidance for callus inductionof Z.marina L.Our results showed that: there were nothing formed on the semi-solid, liquid MS,N6, PESI, VSE and Sea mediums by the explants of Hypocotyls and Cotyledon. Onlyradicle could form green filament in liquid N6medium, however, many bacteria andfungi existed in the formations. The five solid mediums could promote the seedlingsto form callus-like, MS, N6and Sea mediums were not conducive to callus formation,the callus induction were low and contained a lot of pollutants. VSE and PESImediums were more suitable for radicle to form callus, the callus induction were high,and however, which contained a fewer of staining cells and pollutants. Hypocotylsand cotyledons were not conducive to callus formation compared with radicle.So, radicle could form high induction rate of callus-like on solide VSE mediumwith a certain of plant growth regulaters, carbons and agar, there were a fewer ofstaining cells and pollutants.②The effect of supplyment of medium for callus induction of Z.marina L.In this experiment we studied the effect of the kinds and concentration of plantgrowth regulators (auxin of2,4-D, NAA and IAA and cytokinins of6-BA and KT),carbons (sucrose, mannitol and glycerol), concentration of sucrose (10g/L,20g/L,30g/L,40g/L and50g/L) and concentration of agar (0%,0.1%,0.4%,0.7%,1.0%and1.3%w/v) on callus-like induction of Z.marina L. in order to improve the callusinduction rate and callus quality.Our experiments results showed: plant growth regulators (2,4-D and6-BA)promoted explants of Z.marina L. to form callus, there were no significant betweendifferent treatments of concentrations. Sucrose, mannitol and glycerol were moreeasily promoted the callus-like formation, and sucrose promoted the green callusformation with many staining cells and a few pollutants. There were no significantbetween different concentrations of sucrose, and high concentration of sucrose(40-50g/L) could form dry callus. Maximum growth rate (93.33%) was observed intreatments with0.7%agar (w/v), there were no obvious differences between different treatments. However, high concentration of agar (1.0-1.3%) could form dry callus.In conclusion, When the medium supplied2mg/L2,4-D,1mg/L6-BA,30g/Lsucrose and0.7%(w/v) could were propitious to form high induction rate of callusand there were many staining cells and pollutants in callus.③The effect of culture conditions on callus induction of Z.marina L.In this experiment we studied the effect of temperatures (10℃,15℃,20℃,25℃and30℃) and illuminations (500Lx,1000Lx,2000Lx,4000Lx and8000Lx) oncallus-like induction of Z.marina L. on the purpose of improving the callus inductionrate and callus quality.Our experiments showed: The radicle could form callus-like when the temperatureswere10℃-30℃and maximum growth rate (80%) could occure in the treatment of20℃. When the temperatures varied in500Lx to8000Lx, there were callus-likewere observed over the explants, maximum growth rate (86.67%) was observedunder cultured in500Lx, and the differences were not significant. The callus becamedry and without luster when culture in higher temperatures (25℃-30℃) andilluminations (4000Lx-8000Lx).2Culturing callus inducted by the explants of organs.(1) The effect of culture mediums, culture methods and explants sites on callusinduction of Z.marina L.In this experiment we studied the effect of culture mediums (MS, N6, PESI, VSEand Sea mediums), culture methods (solide culture, semi-solid culture and liquidculture) and explants sites (roots, rhizomes, leaves and reproductive branches) oncallus induction of Z.marina L. with the purpose of providing basis theory guidancefor callus induction of intermediary meristem.In our experiments we found: There were no callus formed on the solid, semi-solid,liquid MS, N6, PESI, VSE and Sea mediums by the explants of leaves, root andreproductive branches. There was no obvious changing for all rhizomes in Seamediums. Callus could form on the surface of the rhizomes on the MS and N6mediums, the callus induction were67%and100%respectively. However, the resultof staining experiments showed there were only a few large volume cells and many bacteria (or fungi) in the formation. On the other hand, in PESI and VSE mediums,segments developed white callus and filament which grew into the solid mediumsresult in not to remove to stain. Rhizomes could form callus in MS liquid medium, theinduction was80%rhizomes, however, there were a small amount staining branchedfilaments and small-volume cells in the organizations. In the liquid N6mediums,93%of explants could form callus and a large number of staining cells and/or filamentswere observed in the callus formation. Thimbleful macroscopic filamentous typeoutgrowths and white callus were noticed from the node of explants (the callusinduction was73%) cultured in VSE mediums. White and flocculent callus formed onthe whole surface of rhizomes cultured in liquid PESI medium (the callus inductionwas87%), a few staining large-volume cells could be observed in the formations.Conclusion, rhizomes could form a large number of calluses in liquid N6mediumsupplied plant growth regulators and carbons which contained a large number ofstaining cells and/or filaments.(2) The effect of concentration and duration of sodium hypochlorite on obtainingfeasible and sterile explants.In this experiment we studied the effect of different concentration of sodiumhypochlorite (3%,5%,10%and15%) treated different times (5min,10min and15min) on rhizomes were aimed at gaining a large number of feasible and sterileexplants for tissue culture.We found treatment of plant material with5%sodium hypochlorite for15min and10%sodium hypochlorite for5or15min enabled viable explants to be obtained60%for Z. marina L. in our experiments, and when10%sodium hypochlorite treatedexplants for10min could obtain as high as80%sterile rhizomes which could form aplenty of callus.In conclusion, treatment of rhizomes with10%sodium hypochlorite for10minenabled enough viable explants for culture Z. marina L. in vitro.(3)The effect of explants lengh on callus induction of Z. marina L..In this experiment we studied the effect of rhizomes lengh (one node, two node andtree node) on callus induction aimed at obtaing enough explants on the basis of saving natural resource.In our experiments we found: There was no formations observed over the two andthree nodes, however, the liquid medium presented light pink color, and there weremany oligochromasia small-volume cells could be found in the light pink mediums.Callus formed on the cut surface and node of one node rhizome, the stainingexperiment showed there were a large number staining cells in the callus and thosecells together into filamentous type.So, one node of rhizomes was more conducive to callus formation compared withtwo and three node of rhizomes.(4) The effect of supplyment of medium for callus induction of Z.marina L.In this experiment we studied the effect of the kinds and concentration of plantgrowth regulators (auxin of2,4-D, NAA and IAA and cytokinins of6-BA and KT),carbons (sucrose, mannitol and glycerol) and concentration of sucrose (10g/L,20g/L,30g/L,40g/L and50g/L) on callus induction of Z.marina L. with the purpose ofimproving induction rate and increasing callus quality.The resulted showed: Application of plant growth regulators promoted the growthof callus induction evidently; there were a large number callus formation on thesurface of explants and that callus winded together into a ball type, our stainingexperiments showed there were many filaments in callus ball. We could obtain themaximum induction rate (93.33%) treated with2,4-D (2mg/L) and6-BA (1mg/L) inthe experiments of optimization of plant growth regulators, however, there was noobvious difference between the different combinations of concentrations. Sucrosemannitol and glycerol were more easily promoted the callus formation, no significantdifferences were observed in the studied among the different concentrations ofsucrose. However, there ere many contamination and a few staining deep cells exitedin the formations induced by mannitol and glycerol. High contamination of sucrose(40-50g/L) could form a few staining filaments and many pollutants.In brief, when the medium supplied2mg/L2,4-D,1mg/L6-BA and30g/L sucrosecould was suitable to form high induction rate of callus and there were many stainingfilaments in callus. (5) The effect of culture conditions on callus induction of Z.marina L.In this experiment we studied the effect of temperatures (10℃,15℃,20℃,25℃and30℃), illuminations (500Lx,1000Lx,2000Lx,4000Lx and8000Lx) andculture speeds (0rpm,80rpm,120rpm and160rpm) on callus induction ofZ.marina L. at the aim of improving the callus induction rate and callus quality.Maximum growth rate (93.33%) was observed under cultured in15℃, and therewere no obvious differences between different treatments. When the temperatureswere25℃to30℃, there were a few callus and many bacterial formed. Maximumgrowth rate (66.67%) was observed under cultured in2000Lx, and the differenceswere not significant. When the illumination was4000-8000Lx, there were manybacterial formed in the medium. The culture speed of120rpm was more conducive tocallus formation compared with other speed.In a short, rhizomes could obtain a plenty of callus with a large number of stainingfilaments under15℃-20℃,500Lx-2000Lx and120rpm. |
PDF Full Text Request