Establishment Of Brassica Oleracea L.var. Tissue Culture System And Regulation Of Biosynthesis Metabolism Of Penicillium Elicitor To Sulforaphane In In Vitro Cells

In recent years, broccoli (Brassica oleracea L.var. botrytis L) is popular with peoplebecause it is rich in sulforaphane which can resist cancer so that market’s demand of broccoliis increasing. But it was unable to satisfy the needs of agricultural production becausetraditional breeding methods were complicated, long period and low-yield. This testresearched the system of regeneration, rapid multiplication and cell culture of broccoli usingthe methods of tissue culture in order to provide technical support for the research onobtaining of transgenic broccoli plants with high sulforaphane yield, cloning and cultivation,rapid breeding and metabolic regulation of sulforaphane of broccoli. Test also used thebroccoli callus with rapid multiplication capacity as material, treated with penicillium elicitorand researched the effects of penicillium elicitor on growth of callus, release of endogenousNO and synthetic regulation law of sulforaphane. The main results of this study as follows:Ⅰ、Research on tissue culture system of broccoli(1). Broccoli cotyledons for two weeks had the highest rate of callus-inducing on MSmedium containing2.0mg·L-16-BA+1.0mg·L-12,4-D, which was93.10%.The callus had thehighest rate of redifferentiation on MS medium containing2.5mg·L-16-BA and0.05mg·L-1NAA, which was84.43%, and on the average there were3.41regenerate sprouts on eachcallus.(2). Both of the seeded and regenerated seedlings had the maximum proliferation timeson MS medium containing2.0mg·L-16-BA and0.05mg·L-1NAA, which were13.2and10.5respectively, and the main ways of propagation was clumped buds. Rooting ratios of seededand regenerated seedlings reached the highest value on MS medium containing0.1mg·L-1IBA, were94.52%and89.75%, respectively, and rooting startup time stabled at around5dwith increasing of the subculture times.(3). The determination discovered that content of sulforaphane had pronounceddifferences (P＜0.05) among seeded seedlings, regenerated seedlings, transplanted seedlingsand different parts of seedlings. Sulforaphane content reached the highest value in leaves ofseeded seedlings and roots of regenerated seedlings respectively, which was238.23μg g-1DWand153.24μg g-1DW. The content of sulforaphane also had pronounced differences (P＜0.05) in different parts of transplanted seedlings. Compared with seedlings in vitro, sulforaphanecontent increased in stems and leaves of transplanted seedlings while reduced in rootsobviously (P＜0.05).Ⅱ、Effects of penicillium elicitor on metabolic regulation of sulforaphane in broccolicells(1). Proliferation times of callus induced by broccoli cotyledons enhanced from7.5to18.3after siftings on MS medium with Cu2+whose concentration was1000times and othermicroelements were5or10times higher than normal medium before repeated subculture onnormal proliferation. Besides, callus transformed from clustered and blocky into loose,uniform and granular, and, the speed of callus proliferation also speeded up. Callus was usedas test material after stable subculture for six months.(2). The study found that all of penicillium elicitor with different concentrations inducedby penicillium could lead to synthesis of NO, and the NO synthesis occurred early underpenicillium elicitor stress with high concentration while gently under fungal elicitor stresswith low concentration. The speed of NO synthesis achieved maximum value was26.868μmol·100g-1·h-1under10μg·mL-1penicillium elicitor stress for30h, and this concentrationpenicillium elicitor was active and useful to accumulation of sulforaphane, and the volumewas4.7times more than CK treated with distilled water.(3). AS the quencher of NO, c-PITO not only inhibited the auxo-action of penicilliumelicitor on accumulation of sulforaphane and activity of PAL, but also speeded the decline incell viability induced by penicillium elicitor.(4). Different concentrations of L-NAME and Tungstate-inhibitors of enzymes inpathways of NO synthesis-cooperating with10μg·mL-1penicillium elicitor were used to treatbroccoli cells in vitro, then measured the amount of release and activity of NOS and NR. Theresults found that the release of endogenous NO in broccoli vitro cells induced by penicilliumelicitor mainly through the pathways of NOS and NR, and when one of the pathway wasinhibited, also activity of enzymes in another pathway was inhibited. However, NOS pathwayof NO synthesis was activated when vitro cells treated with both penicillium elicitor and highconcentration of Tungstate-inhibitor of NR, which was due to the stress of high concentrationsof K+in Tungstate. But the NR pathway could not be stimulated by high concentrations of K+ when the NOS pathway of NO synthesis was inhibited. Sulforaphane was affected bysynthesis regulation law of NO, and accumulation of sulforaphane increased when thepathways of NO synthesis were stimulated while the accumulation was inhibited with therestraint of NO synthesis pathways, and there were significant positive correlation betweenNO cumulant and accumulation of sulforaphane (R=0.837P＜0.05). All of the abovedescribed that it was an effective technical approach to promote the accumulation ofsulforaphane through the release of endogenous NO in broccoli induced by penicilliumelicitor.