| A part of the hairy root-inducing Ri plasmid of Agrobacterium rhizogenes known as T-DNAtransfers the infected plant cell and becomes intergrated with its genomic DNA, and bringshairy root. As compared with normal cell culture, the hairy root culture system is characterizedby high growth rate, free-hormone, genetic stability and production of the higher levels ofsecondary metabolites, which is a perfect cell culture system for production of secondarymetabolites.Medical and pharmological studies povide strong evidence that tea catechins (especiallyEGCG) and momordicosides possess health-protection and pharmacological effects. In recentyears, it has become a hotspot that using hairy-root culture system biosynthesizes bioactivecomponents in plants. It is very difficult to Agrobacterium rhizogenes-mediated transform teaplant due to being multi-year growth wood and plenty of polyphenol. Tea hairy root forminginduced by Ri plasmid is infrequent to succeed, so that there is no a perfect system whichtransform tea plant induced by Ri plasmid and produce secondary metabolites using tea hairyroot culture to be established in domestic and foreign as yet. However, Studies ontransforming bitter gourd to form hairy root induced by Ri plasmid and biosynthesizingfunctional components such as momordicosides and so on using hairy root of bitter gourd wasstill the blank in domestic and foreign.This article investigated the method of identifying Agrobacterium rhizogenes strains activity,inhibition of Agrobacterium rhizogenes by tea catechins and selection of antipolyphenoi strain,the method of a rapid determination of total momordicoside by ultrasonic extraction frombitter gourd, factors affecting high-frequency induction of tea and bitter gourd hairy root, anddetermination of catechins in tea hairy root and mormordicosides in bitter gourd. This papermain findings are as follows:1. Agrobacterium rhizogenes strains with complete plasmid and the vigor incubated Kmresistance culture medium, which colony characteristic was to cover evenly all plate forspread plate, But to have the colony growth in the streaking place for streak plate.2. The catechin (TC80) had the obvious inhibitory action to Agrobacterium rhizogenes,MIC90 was 25μg/ml in liquid media; MIC90 is 100-200μg/ml in solid media.Regardless of the liquid or the solid media, Its MBC (minimum bactericide concentration)This had expounded that the tea tree contains the plenty of catechins and otherpolyphenols in tea was a key factor to restrict the Agrobacterium rhizogenes-mediatedtransformation of tea plant.3. An anti-polyphenol strain R1000AP obtained by anti-polyphenol screening, itsanti-polyphenol capability had the distinct enhancement compared to the primitive strainR1000. Its MIC90 enhances near 40 times. The hairy root frequency induced byanti-polyphenol strain (R1000AP) incresed by 46.5% compared to the original non-anti-polyphenol strain and reached the remarkable difference (P＜0.05).4. The generalized analysis of factors affecting high-frequency induction of tea hairy rootthought that using the anti-phenol strain, the explant selected from the low polyphenolsraw material (such as green tea variety) and the organ (root or stem), utilizatong ofmoderate withering or supersonic pretreatment to create micro injury of cell wall, Thebacterial concentration controlled at OD600=0.8, infection time is 30min as well asaddition of the suitable hormone (for example IBA5.0mg/L)in co-cultivated medium andthe co-cultivated timing control in 2d, Such factor level namely constituted the highfrequency induction system transforming tea plant by Ri plasmid.5. Tea hairy root induced by the R1000AP strain contained rich catechins, which totalquantity reached as high as 15.061mg/g, and also higher nearly 25% compared to two anda bud of Fuding Dabai fresh leaf raw materials as total quantity 12.086mg/g outdoes, inwhich contained nearly 11% ester-type catechin.6. A rapid determination of total momordicosides by ultrasonic extraction from bitter gourd(Momordica charantia) was established in this paper. In the operation of ultrasonicextraction, only 0.1g of sample and 2ml of 80% ethanol were used. Only 30min wasnecessary for the extraction. As compared with the conventional method, the methodrequired shorter extraction time, less solvent reagent and no heating. the determinationefficiency of the ultrasonic method was 10 times higher than the Soxhlet's method. Therewas no significant difference between the analytical results obtained by those twomethods.7. This paper had systemically studied on the factors affecting high-frequency induction ofgenetic transformation of bitter gourd and bitter. The results was shown that the R1000APin 5 strains on trial had the most comprehensive effect to induce bitter gourd hairy root,not only earlier producing the hairy root, also hairy root frequency and hairy root densitybeing both the highest; Rate of the transformation of bitter gourd between different varietydid not have the remarkable difference (P＜0.05); Rate of the transformation of stemexplant was higher 26.3% compared to the cotyledon, and the difference reached theextremely remarkable level (P＜0.01); The tender bitter gourd seedling (seedling age 10-20d)was more sensitive to Agrobacterium rhizogenes, and advantageous in the genetic transformation,but when the seedling age over 40d, the rate of bitter gourd genetic transformation was nearly a zero;The suitable co-culture time is 1-2d, co-culture time over 2d the rate of gennetic transformation wasdrop due to overgrowth of bacteria; Addition of AS to co-culture medium could not remarkablyimprove the effect of genetic transformation of bitter gourd (P＞0.05); Addition of plant hormone toco-culture medium Plant hormone may cause hairy-rooting time ahead of 5d, finally andincreased in the frequence of hairy root, inducing effect of IBM5.0 on the gennetictransformation of bitter gourd was best. in a variety of hormones processing.8. The morphological character of different explants induction to form hairy root had certaindifference, hairy root from stem section with many floss, most of length in 10-25mm, hairyroot density 9.0, however, hairy root from cotyledon section to be thick long, fewfloss, most of hairy root length in 40-50mm, hairy root density 9.0. The growth speed ofhairy root from stem section was approximately 2-5mm/d, and the growth speed of hairyroot from cotyledon section approximately 8-10mm/d.9. With determination of total momordicosides in the root, the stem, the cotyledon of test tube seedling and hairy root by the ultrasonic and alcohol extraction-the vanilla methodcarried on the comparative analysis and the result to indicate that total momordicosidescontent in hairy root of bitter gourd was highest, separately compared to the root, stem andcotyledon of test tube seedling content higher 68.9%, 53.6% and 54.7%, and this kind ofdifference reached the extremely remarkable level.Above results had provided the important scientific basis and the method for establishinghigh-frequency induction system of hairy root and gennetic transformation of tea and bittergourd induced Ri plasmid of Agrobacterium rhizogenes as well as using this hairy root culturesystem to biosynthesize functional components such as tea catechins, momordicosides and soon. |
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