Inactivation strategy for pseudorabies virus in milk for production of biopharmaceuticals

By selecting pseudorabies virus (PrV) as a model virus, this study assessed the feasibility of applying viral inactivation strategies to manufacturing medicinal products from the milk of transgenic sows. The efficacy of heat, acidic/alkaline and detergent treatments was also evaluated with respect to their ability to inactivate PrV in milk samples. Experimental results indicate that PrV was inactivated obviously at least 7.125 log10 for 30 min at 60 degrees C. At alkaline values of pH 10 and acidic value of pH 4, PrV infectivity was reduced to 3.625 log10 and exceeded 5 log10, respectively. Moreover, PrV virus was inactivated efficiently (> 3.875 log10) by using 0.25-1% of Triton X-100 treatment and without a loss of biological activity of the recombinant human coagulation factor IX (rhFIX). RESULTS: of this study demonstrate the effectiveness of the proposed detergent inactivation method for PrV inactivation of rhFIX production from transgenic products, especially in milk materials.     

Bactericidal effects of ozone at nonspermicidal concentrations.

A study was conducted to assess the use of ozone (O3) to control pathogens or contaminants of concern to animal breeders and regulatory officials. In separate experiments, samples of fresh bovine semen and Pseudomonas aeruginosa, Escherichia coli, or Campylobacter fetus subsp. venerealis were diluted with antibiotic-free milk (10(6) sperm and 10(6) organisms/mL of diluted semen), exposed in the previous day to a constantly monitored level of 5, 10, 15, or 20 micrograms/mL of O3 for 3-5 min. After 10 min at 30 degrees C, sperm motility was assessed and the samples cooled to 5 degrees C. Two and 18 h after the beginning of cooling, aliquots of each semen sample were evaluated for motility and cultured for organisms. Reductions were observed in P. aeruginosa and E. coli colony counts of 2 logs, and in C. fetus of 5 logs, after exposure for 2 h to O3 at a concentration of 5 micrograms/mL that had a moderate effect on sperm motility (reduction of 20%). Fewer than 100 colonies, i.e., a 4 logs reduction of all bacteria, were counted after dilution with ozonized-treated milk at 20 micrograms/mL of O3. However, this concentration of O3 reduced sperm motility by 50% 10 min after dilution. The results of these experiments indicate that a concentration and exposure time to O3 can be selected to reduce P. aeruginosa, E. coli, and C. fetus in contaminated bull semen diluted with milk while having only minimal effects on sperm motility.     

Inhibition of aflatoxin M1 production by bovine hepatocytes after intervention with oltipraz

It is well known that cattle ingesting aflatoxin B1 contaminated feed commodities excrete aflatoxin M1 into their milk. As aflatoxin M1 originates from hepatic metabolism, measures to prevent aflatoxin M1 formation need to be directed to either the immobilization of aflatoxin B1 in the gastrointestinal tract or the modification of hepatic metabolism of aflatoxin B1. Here we studied the influence of oltipraz and a second dithiolthione, (1,2) dithiolo (4,3-c)-1,2-dithiole-3,6 dithione (DDD) on bovine hepatic aflatoxin B1 biotransformation. Oltipraz inhibited aflatoxin B1 metabolism as no aflatoxin M1 and no aflatoxin B1-dihydrodiol, the second metabolite found in bovine hepatocytes, was formed. DDD did not significantly inhibit aflatoxin B1 metabolism. It could be demonstrated that the inhibition of aflatoxin B1 metabolism was due to the inhibition of several cytochrome P450 enzyme activities by oltipraz. In contrast, DDD inhibited only ethoxyresorufin O-deethylation activity. These findings suggest a high efficacy of oltipraz in inhibiting aflatoxin M1 contamination of milk from dairy cows exposed to aflatoxin B1 contaminated feeds.     

Effects of aflatoxin M1 intake at physiologic levels on newborn dairy calves

When aflatoxin-contaminated grain is consumed by dairy cows, aflatoxin M1 is excreted in the milk. Sixteen neonatal male Holstein calves were given milk which had been collected from cows given 5 to 6 mg of aflatoxin B1 each day. The calves were examined for possible detrimental effects of the mycotoxin at pseudophysiologic concentrations. Calves were allotted to 1 of 4 groups given different milk dietary aflatoxin M1 concentrations: group 1--given 0 microgram of aflatoxin M1/L (undetectable); group 2--given 0.5 microgram/L; group 3--given 1 microgram/L; and group 4--given 2 micrograms/L. Whole milk equal to 8% of body weight was fed daily and adjusted each week to maintain this ratio. Water and a 15% crude protein complete calf starter ration were offered ad libitum for the 6-week feeding study. Weekly blood samples were collected via jugular venipuncture and analyzed for serum alkaline phosphatase and aspartate aminotransferase activities. Daily means for milk dry matter intake (in kg) and complete ration intake (in kg) for the calf groups were as follows: 0.46 and 0.36 for group 1; 0.46 and 0.25 for group 2; 0.42 and 0.18 for group 3; and 0.49 and 0.40 for group 4. Significant differences in complete ration and total dry matter intake were noted. The average daily gains (in kg) and gains in height at withers (in cm) were 0.39 and 4.1 for group 1; 0.36 and 4.0 for group 2; 0.29 and 5.7 for group 3; and 0.42 and 5.1 for group 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the inactivation of Salmonella during thermal disinfection of liquid manure]

Technical processes for thermal liquid manure disinfection usually reach temperatures between 50 degrees C and 70 degrees C. The destruction of important infectious micro-organisms can be expected in this temperature range. The purpose of the investigations reported here was to study the thermal resistance of Salmonellae during heat treatment of liquid manure. Salmonella senftenberg 775W survived much longer than each of 12 other strains from 8 different Salmonella serovars. Resulting from a regression analysis kinetics of thermal death were determined for this strain and decimal reduction times were calculated in cases of an exponential die-off. D60-values ranged from 47 to 138 sec depending on the type of slurry used. Further investigations on the inactivation of Salmonella senftenberg 775W at 50 degrees C, 55 degrees C, 60 degrees C, and 65 degrees C were carried out and following D-values were obtained: D50 = 56.7 min, D55 = 11.5 min, D60 = 2.3 min, D65 = 0.47 min. The resulting ZD-value was 7.2 degrees C. Minimum requirements concerning temperature and heating time can be derived from the results of this study. The given recommendations may only be applied, if technical processes work without any functional deficiencies and thermal energy is evenly distributed in the heated slurry. Combinations of temperature and heating time should not fall below the following values: 50 degrees C/15 h, 55 degrees C/3 h, 60 degrees C/30 min, 65 degrees C/5 min.     

Use of the Synpor membrane filter for the separation and determination of the number of somatic cells in the milk of dairy cows using the indole DNA filtration method

The somatic cells of cow's milk were separated by filtering through nitrocellulose membrane filters (Synpor), pore size 2 to 5 micron. An indole reagent was added to the membrane filters with trapped cells, the mixture was warmed in boiling water bath for 20 min., then cooled, centrifuged (3500 X g, 15 min.), and optical density was measured at 490 nm. The relation 8 micrograms = 1 million cells was used for the conversion of DNA content in sample to the counts of cells. The average variation coefficient of the determination was 4.7% and the coefficient of correlation between the indole DNA filter method and direct microscopy on membrane filters was r = 0.997. Using the indole DNA filter method, the counts of somatic cells can be estimated from milk samples kept frozen or from filters with retained cells kept at 4 degrees C, or (for a short time--3 days) at 25 degrees C. The estimation is not distorted when the milk samples are stabilized with K2Cr2O7 or a mixture of K2Cr2O7 and HgCl2, if they are kept at 4 degrees C for eight days.     

Proteolytic inactivation of simian-11 rotavirus: a pilot study

In an effort to develop alternate disinfectants for rotaviruses, pilot studies were conducted to determine if bacterial proteases could render simian-11 (SA-11) rotavirus non-infectious. SA-11 was found to be fairly temperature resistant, retaining a low-level of infectivity following 65 degrees C treatment for 2h at pH 8.5. It also resisted pH 8.5-5 at 45 degrees C for 2h. Alcalase, an alkaline protease, was the most effective of the various proteases (alcalase, durazym, neutrase, and savinase) tested. To analyze specific parameters for alcalase, SA-11 virus (10(5.5) median tissue culture infective dose/ml original titer) was treated at pH 6, 25 and 15 degrees C (simulated field conditions), with 0.1 and 1.0% alcalase. At pH 6.0 and 15 degrees C, 0.1% alcalase reduced SA-11 titer by 0. 75 log in 24h, and by 1.25 log in 120h. At 25 degrees C and pH 6.0, 0.1% alcalase reduced the titer by 2.25 log after 24h, and by 2.75 log in 120h. At pH 6.0 and 15 degrees C, 1% alcalase reduced SA-11 titer by 1.50 log in 24h and by 1.75 log in 120h. At the same enzyme concentration and pH, but at 25 degrees C the titer was reduced by 2. 75 log in the first 24h and by 3.25 log at 120h. These results show that the alkaline protease alcalase is capable of inactivating SA-11 virus to a certain degree depending on conditions. Further definition of operating parameters, demonstration of inactivation under field conditions and analysis whether the demonstrated degree of inactivation would decrease calf morbidity and mortality remain to be explored at this time.     

The effect of heating against Cryptosporidium oocysts

The effect of heat treatment was examined against oocysts of Cryptosporidium parvum, Cryptosporidium muris and chicken Cryptosporidium sp. isolated in Japan. The oocysts of these species were exposed at 50, 55, 60 and 70 degrees C for 5, 15, 30 and 60 sec in water bath, respectively. To determine the infectivity of heated oocysts, the nice and chickens were inoculated with the treated oocysts and the oocyst output in the feces after inoculation was examined. In C. parvum and chicken Cryptosporidium sp., the oocysts were not detected from mice or chickens which were received oocysts heated at 55 degrees C for 30 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec. In C. muris, the oocysts were not detected from mice which were received oocysts heated at 55 degrees C for 15 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec. Consequently, it was clarified that the infectivity of Cryptosporidium oocysts to mice and chickens was lost by heating at 55 degrees C for 30 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec.     

Testing Milkofix, a new preservative preparation for milk samples used for infrared analysis of milk components. II. Verification of its preservative effects in relation to infrared analysis]

For the purposes of the infrared analysis of the basic milk composition, Milkofix, an ecologically friendly preparation used for milk sample preservation (Trzicky, 1990), was compared with untreated samples (N) and with samples preserved with sodium azide (A), bronopol (B) and potassium dichromate (C) at a storage temperature of 20 degrees C (I) and 4 degrees C (II) in samples kept for 14 and 18 days. Pursuant to the recommendations cited in literature, the preservatives had these concentrations: A = 0.0085 g NaN3 and 0.0630 g NaCl; B = 0.0050 g bronopol and 0.0500 g NaCl; C = 0.0330 g K2Cr2O7 and 0.0670 g KCl; M = 0.1250 g, all amounts are per 25 ml milk. Three bulk milk samples were used which were analyzed on an automatic Milko-Scan 133 B infraanalyzer (Foss Electric, Denmark) every day. On the basis of a graphical evaluation of the results by IDF recommendations (1985) the times within which the applicable results could be obtained were determined for the various methods of milk sample treatment (Figs. 1 to 6): N I--0 days; A I--9; B I--10; C I--13; M I--4; N II--10; A II--5; B II--11; C II--15; M II--10 (Tab. I). The results recorded for Milkofix are in agreement with the conclusions drawn in the previous study, where the intervals of four and nine days were determined. The days to milk sample coagulation were as follows: N I--1 day, M I--10 days. The coagulation in A I, B I, C I and N II samples was not observed even after 13-day storage and in A II, B II, C II and M II samples not even after 17 days of storage. The results for particular components (fat, proteins, lactose) of milk samples differently treated in time are presented in Tabs. II, III and IV. A system of evaluating criteria (Tab. V) was used to determine the order beginning from the most convenient method of milk sample treatment for the given purpose: 1. C II, 2. C I, 3. B II and A I, 4. B I, 5. M II, 6. N II, 7. A II, 8. M I and 9. N I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aflatoxin Decontamination of Artificially Contaminated Feeds by Sunlight, γ-Radiation,and Microwave Heating

The efficiency of decontamination of aflatoxin residues in poultry feeds through exposure to sunlight (solar radiation), γ-radiation (⁶⁰ Co), and microwave heating were investigated in artificially contaminated feed samples. Photodegradation of aflatoxin by sunlight has been found to cause a significant (P < 0.05) decrease in both B1 and the total aflatoxins. Moreover, the degrees of aflatoxins were dependent on exposure time. Both aflatoxin B1 and total aflatoxins were decreased when feed samples exposed to sunlight by 42.3, 39.9, 75.5, and 65.9% for 3 and 30 h of direct sunlight of the treatment T1, whereas feed samples subjected to γ-irradiation and microwave heating caused a significant (P < 0.05) decrease in aflatoxin B1 contents by 42.7 and 32.3% for γ-irradiation and microwave heating (T3 of 25 kGy and 10 min of microwave heating), respectively. Therefore, the solar radiation was more effective in aflatoxin B1 reduction when compared with γ-irradiation and microwave heating.     

Effect of feeding corn naturally contaminated with aflatoxin on feed efficiency, on physiologic, immunologic, and pathologic changes, and on tissue residues in steers

Two of 3 groups of Holstein-Friesian steers (groups II and III; n = 5 each) were fed a ration containing corn naturally contaminated with 800 ng of aflatoxin/g. The other group of steers (group I; n = 5) was fed a ration containing noncontaminated corn. The respective rations were fed for 17.5 weeks, except the ration given to group III; the latter's first diet (contaminated with aflatoxin) was changed to a noncontaminated diet after 15 weeks, continuing for the remaining 2.5 weeks. All steers were killed and tissues and fluids were obtained for aflatoxin analysis. Although aflatoxin B1 and M1 could be detected in blood and urine at several sampling times during the experimental period in groups II and III steers (given the diets containing aflatoxin), there appeared to be no effects on body weight gains and immune phenomena, such as lymphoblastogenesis and antibody production, but there was a waning of the delayed cutaneous hypersensitivity in steers given aflatoxin-contaminated diets. In group III animals (diet was changed to noncontaminated ration at 15 weeks), aflatoxin B1 and M1 disappeared from urine before they were slaughtered. All tissues and fluids, except the rumen contents from these group III steers, were void of detectable aflatoxins B1 and M1 at necropsy. The concentrations of aflatoxin B1 in the rumen content of the latter steers were low. All tissues collected at necropsy from the group II steers fed the aflatoxin diet throughout the 17.5 weeks had detectable aflatoxins B1 or M1 present.     

Improved detection of Mycobacterium avium subsp. paratuberculosis In milk by immunomagnetic PCR

The potential use of a novel immunomagnetic PCR (IMS-PCR) technique as a rapid method to screen milk samples for the presence of Mycobacterium avium subsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IMS) for M. ptb, developed at Queen's University, Belfast, was applied to milk samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS-PCR sensitivity could be further increased by initial centrifugation (2500 g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatment. Following IMS, template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100 degrees C for 15 min. It was estimated that the IMS-PCR assay could detect approximately 10(3)CFU of M. ptb per 50 ml of milk (equivalent to 20 CFU/ml), whereas the minimum detection limit of direct IS900 PCR was estimated at 10(5)CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a total of 40 spiked (10(6)CFU M. ptb) and unspiked, raw and laboratory-pasteurised milk samples were independently tested by IMS-PCR and conventional IS900 PCR. IMS-PCR correctly identified 97. 5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples before and after laboratory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). IMS-PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commercially pasteurised cows' milk.     

牛奶中黄曲霉毒素M1的来源和控制途径

黄曲霉毒素(AFs)是存在于食物或饲料中真菌的有毒代谢物,天然产生的AFs主要有AFB1、AFB2、AFG1和AFG2,而AFM1则是AFB1经动物体内转化而来,具有致癌性,可通过牛奶排出,引起牛奶的安全问题。本文综述了牛奶中AFM1的来源与转化,主要国家和组织对奶中AFM1的限量,奶中AFM1的检测方法和控制途径。     

The significance of mycotoxin assimilation for the contamination of milk and milk products

The two possible pathways contaminating milk and milk products with mycotoxins are either the secretory or post-secretory route. The latter is of only little importance due to cooling conditions in production and storage. A secretory contamination can only occur with such mycotoxins, which undergo no complete degradation through their passage into the milk. From the mycotoxins, present in cow's feed; virtually only aflatoxin B1 yields a milkborne metabolite, the aflatoxin M1. The carry over rate is low (2 +/- 1%), but can be enhanced by polyhalogenated biphenyls, also present in the forage. Under normal conditions, however, this enhancement will not be measurable due to low equimolar concentrations of both reactants. The aflatoxin M1 content in herd's bulk milk depends exclusively on the content of the precursor aflatoxin B1 in the ration of the cow and is with less than 10 ng/kg fairly low at present in the Federal Republic of Germany. A careful supervision of the imported feed ingredients for mixed feed, however, will ensure to keep those batches out of dairy cow feeding which exceed a certain level of aflatoxin. The legal threshold is 10 micrograms/kg, being even too high to ensure a milk containing less than 10 ng/kg under high energy feeding conditions. The discussed thresholds for aflatoxin M1 in milk are 50 and 10 ng/kg resp., the latter value is scheduled for milk used in infant nutrition. To keep this low concentration the intake of aflatoxin B1 must be less than 2 micrograms/kg of the daily ration.     

Cold storage of wheat. 1. Ergosterol, ochratoxin A and citrinin after inoculation with Penicillium verrucosum]

Seed wheat was inoculated without having been sterilized with an ochratoxin A and citrinin forming strain of Penicillium verrucosum and stored at moisture contents of 18, 20, 22, 24, and 26% at 10 and 4 degrees C. The production of ergosterol, a chemical indicator of fungal biomass, started within the storage time investigated (240 days). Only at 18% H2O/4 degrees C an increase of the ergosterol content was not observed. Ochratoxin A and Citrinin were not detected at 18% H2O/4 degrees C and 20% H2O/4 degrees C within 240 days (detection limit: 10 and 25 micrograms/kg, respectively). At the other combinations of moisture content and temperature the first detection of the two toxins approximately coincided with the onset of ergosterol production. With increasing moisture content and temperature the time up to the start of ergosterol production decreased, whereas the production rates of ergosterol, ochratoxin A and citrinin increased. Both toxins were produced with about the same rate during a first phase of accumulation. At 20-26% H2O there was no influence of moisture content and temperature on the relation between toxin content and the simultaneously reached ergosterol content. It is recommended that wheat highly contaminated with Penicillium verrucosum should not be stored beyond the start of ergosterol production.     

Comparison of the effectiveness of Milkofix, a preservative preparation, with traditional preservative agents in the determination of somatic cell count in milk samples using a fluoro-optic-electronic method]

Milkofix (M), a health friendly preservative substance, to be used for milk sample preservation (Trzicky, 1990), was compared with other preservatives. Untreated milk samples (N) were tested against samples treated with sodium azide (A; 0.0085 g NaN3 and 0.0630 g NaCl), bronopol (B; 0.0100 g bronopol and 0.090 g NaCl), potassium dichromate (C; 0.0330 g K2Cr2O7 and 0.0670 g KCl) and Milkofix (M; 0.1250 g). The doses of the preservatives A, B, C and M are per 25 ml milk. The somatic cell counts (SB) were determined on a FOSSOMATIC 90 apparatus (FOSS ELECTRIC, DENMARK). In the treated milk samples taken from individual cows the values of SB counts were significantly higher than in N samples if determined within eight hours after sampling (Tab. I): in A higher by 18.6%, B by 26.3%, C by 26.4% and M by 24.3% (P less than 0.05). The significantly higher values of SB counts were recorded in bulk milk samples treated with preservatives in comparison with N samples immediately after sampling: in A by 6.0%, in B, C and M by 12.9% (P less than 0.01; Tab. II). After one-day storage of N samples at a temperature of 4 degrees C these differences are insignificant (P greater than 0.05), thus the results of N, A, B, C and M samples obtained after one-day storage at 4 degrees C can be taken as actual and mutually comparable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tests of Milkofix, a new preservative preparation for milk samples used for infrared analysis of milk components. I. Verification of its bacteriostatic and bacteriocidal effects and its interference effects]

Hygienic, ecological and health problems of sample preservation for an analysis of basic milk components make us continually to develop a safer chemical preservative substance which will preserve the original sample composition for the time required and which will not influence the analyses. Trzicky (1990) proposed Milkofix (M), a preservative substance on the basis of silver compound. The author reports on minimum risks of the use of this preparation, in comparison with traditional preservatives. Preservative efficiency of Milkofix was compared with other preservatives: K2Cr2O7 (C), NaN3 (A) and bronopol (B). The following concentrations were used: A--0.0085 g NaN3 and 0.0630 g NaCl, B--0.0050 g bronopol and 0.0500 g NaCl, C--0.0330 g K2Cr2O7 and 0.0670 g KCl in tablet, M--0.1250 g of the mixture, all amounts are per 25 ml milk. The observed antibacterial efficiency of M could be seen in a slower decrease in actual acidity, and/or in an increase in titratable acidity in M-treated samples unlike untreated ones (N). From the starting value pH 6.3 (Fig. 1), the value of N treatment dropped to 3.8 after two days, the values of M and A treatments dropped to 4.9 after nine days and to 5.7 after twelve days, respectively. As for SH, the values increased within the same interval from 6.5 (2.5 mmol/l) to 28.6 in N, and to 22.3 in M and 9.4 in A (Fig. 3). There was a similar trend when the milk samples were stored at a temperature of 4 degrees C, but the differences between the preservation methods were not so clear in comparison with storage at a temperature of 20 degrees C (Figs. 1 and 3). The standardized SH value of 9.0 (2.5 mmol) for infraanalyzer measurements was exceeded after 24 hours in N samples, after four days in M samples and after 12 days in A samples at a temperature of 20 degrees C. The observation of the growth of microorganism counts (CPM) showed that this growth was slower in M than in N, but faster in the samples of C treatment (Fig. 5). The generative time of CPM in N made 1.6 hours, in M 2.4 hours and in C 7.9 hours. The lag phase of these mixed cultures was 24 hours in M, 60 hours in C and in N treatment the lag phase was zero.(ABSTRACT TRUNCATED AT 400 WORDS)     

日粮添加不同水平的奶牛专用霉菌毒素吸附剂对粪中双歧杆菌数量和乳中黄曲霉毒素含量的影响

奶牛饲料原料中霉菌毒素广泛存在,并严重地威胁着奶牛健康,影响着奶牛生产性能的发挥。本试验选择年龄、胎次、产奶量、泌乳天数相近的荷斯坦奶牛80头探讨日粮添加4种剂量（0g、10g、15g、20g）的霉菌毒素吸附剂对奶牛粪便中双歧杆菌数量和原料奶中黄曲霉毒素残留量的影响。与对照组（添加霉菌毒素吸附剂0g）相比,实验组奶牛粪便中双歧杆菌的外排量明显降低,但各实验组之间统计差异并不显著,仅有数值上的变化。同时,随着霉菌毒素吸附剂含量的增加,原料奶中体细胞数呈现下降的趋势。与试验前相比,原料奶中黄曲霉毒素含量显著降低（P〈0.05）,且随着添加剂量的增加其黄曲霉毒素残留量更少,但添加20g与添加15g相比并无明显变化。研究表明,随着日粮中霉菌毒素吸附剂不同量的添加,均可有效降低原料奶中黄曲霉毒素的残留量,进而提高原料奶质量。     

红三叶新品系无菌苗培养体系优化

以红三叶(Trifolium pratense L.)新品系为材料,对无菌苗培养的种子消毒方法和培养基组成进行研究,以期为红三叶新品系组培再生体系、分于育种及抗病性研究提供基础依据。结果表明:0.1%HgCl₂,3%NaClO和15%H₂O₂消毒不超过15 min,对红三叶新品系种子发芽率没有显著影响,但消毒效果存在较大差异。红三叶种子消毒的最佳方法为75%酒精预处理,0.1%HgCl₂消毒10 min,发芽率可达89.44%,并彻底无污染。最适培养基为1/4MS+1.5%蔗糖,不添加激素,pH6.0,发芽率可达85.78%,无菌苗生长良好。培养基中无机盐的含量是影响红三叶种子发芽率和无菌苗生长的关键因素。琼脂培养基培养比滤纸培养的无菌苗子叶和下胚轴出愈率显著提高,但不同组分琼脂培养基的无菌苗出愈率无显著差异。