生物類(lèi)英語(yǔ)論文

1、Effect of different carbon source and nitrogen source on the cold resistance of citrus callus inductionHu Siyuan (College of Agronomy, Northwest A&F University, Yangling, Shaanxi 712100,China)Abstract: Citrus callus is ideal material for physical and chemical mutagenesis and genetic transformati

2、on, the carbon source and nitrogen source is a necessary component in the induction and subculture of citrus callus, so the exploration of carbon, nitrogen sources is of great significance to the effect degree of citrus callus induction and effect way. This study choose cold resistance of citrus as

3、test material, through culture vitro tissues, analysis of the effects of carbon source and concentration, nitrogen source and level on the influence of citrus callus induction, To provide the basis for the study of the occurrence and the mechanism of cold resistance of Citrus genetic improvement, so

4、matic embryos.Key words: carbon source; nitrogen source; citrus; callusIntroductionCitrus (Citrus reticulata Blanco) Rutaceae Citrus, small evergreen tree or shrub, about three meters high, weak branches, usually with thorns. Leaves are alternate, leathery, lanceolate to ovate-lanceolate, 5.5-8 cm l

5、ong, 2.9-4 cm wide, apex acuminate, base cuneate, entire or with fine blunt teeth, petiole slender, winged obvious . Flowers small, yellow-white, solitary or clustered in leaf axils, sepals 5; petals 5; stamens 18-24, filaments 3-5 pieces often connate, ovary 9-15 rooms. Citrus fruit flat spherical,

6、 5-7 cm in diameter, orange-yellow or reddish yellow, peel loose flesh meat easily separated.Chenggu citrus cultivation has a long history dating back more than 2,000 years of history. Chenggu County is located in Southern Shaanxi, Central Hanzhong Basin, north subtropical to temperate transition re

7、gion, north of Qinling mountain blocking the invasion of cold northwest, south winds Bashan so warm air can not drive straight. Inversion effect Qinling belt and foothills sloping wall, forming a unique micro-climate regions, fewer winter cold and summer heat less. First, here's citrus high temp

8、erature hot wind does not harm flowering, fruit setting rate; Second cool early autumn, temperature, and is conducive to fruit color, ripe, citrus fruit surface here is gorgeous, high soluble solids content, moderately sweet and sour.Studies1-4 have shown that by changing the carbon source to induce

9、 somatic embryogenesis. As most of the citrus callus has not regenerate embryoid body phenomena 5-7, while the high-frequency induction of somatic embryogenesis of citrus cells for genetic transformation, in vitro mutagenesis, in vitro screening mutants, rapid propagation, etc. work has special sign

10、ificance 8-9. In this paper, sucrose, glucose, soluble starch, three kinds of carbon and nitrogen sources ammonium nitrate one kind of growth and the ability of somatic embryogenesis of citrus callus were studied at different levels, in order to select the optimal culture conditions hardy citrus stu

11、dies provide evidence.1. Purpose and significance 1.1 Purpose 1. Analysis the influence of carbon types and carbon levels on hardy of citrus in vitro cultured explants dedifferentiation;2. Explore the nitrogen source for citrus callus induction effect manner and extent of effect;3. Screening suitabl

12、e carbon, nitrogen conditions of citrus dedifferentiation in vitro callus induction.1.2 Significance Citrus Calli is an ideal material for the physical and chemical mutagenesis and genetic transformation, and carbon and nitrogen is a necessary ingredient in citrus callus induction and subculture, th

13、erefore it is important to explore the extent and the effect of the way on citrus callus induction of carbon source, nitrogen source.1. Explore the carbon and nitrogen sources for citrus callus induction effect, especially on the induction rate and differentiation speed for in-depth study of carbon

14、and nitrogen in the mechanism of tissue culture citrus from the body, and to provide some data to support ;2. Hanzhong citrus varieties for the study material, try to filter out the best of its callus induction of carbon required, the type and content of nitrogen for the production of virus-free cit

15、rus seedlings through callus ideal way to provide relatively basic culture conditions.2. Materials and methods2.1 MaterialsSeeds of the test material explants of the same size, healthy full Chenggu hardy citrus varieties.2.2 Preparation InstrumentHS series horizontal flow bench (Suzhou Antai Air Tec

16、hnology Ltd.); the dish; vertical circular chamber pressure steam sterilizer (Shanghai Medical Nuclear Instrument Factory); portable electric pressure steam sterilizer (Shandong Xinhua Medical Instrument) electronic balance (Shanghai balance Instrument Factory); electric distilled water (Shanghai me

17、dical nuclear Instrument Factory). Auxiliary materials(1) In a planned way of cleaning the glassware (Erlenmeyer flasks, petri dishes, etc.), and in natural conditions or oven drying;(2) Take a certain amount of Erlenmeyer flask under seal membrane, with cotton rope is secured; (3) Take a certain am

18、ount of the Erlenmeyer flask was added to the flask to approximately 2/3 of pure water, sealing; (4) Put the filter paper into a petri dish, wrap;(5) Put the sealed Erlenmeyer flasks and Petri dishes into high-pressure steam sterilizer, autoclaved at 1.1 kg/cm2 30min; (6) The sterilization dish, fla

19、sk covered with newspaper spare, finishing laboratory. The basic culture mediumTable 2.1 the basic culture mediumMother liquor categoryCompositionWorking liquid concentration（mg/L）A variety of practical quantity（mg）Constant volume standard（ml）A large number of element classKNO31900190001000NH4N

20、O3165016500MgSO4·7H2O3703700KH2PO41701700CaCl23323319Trace element classMnSO4·H2O22.31115500ZnSO4·7H2O8.6430H3BO36.2310KI0.8341.5Na2MoO4·2H2O0.2512.5CuSO4·5H2O0.0251.25CoCl2·6H2O0.0251.25Lron classNa2EDTA·2H2O37.251862.5500FeSO4·7H2O27.851392.5Organic material

21、 classglycine2.0100500Thiamine hydrochloride0.420Hydrochloric acid pyrazole compound element10500niacin5250inositol10050002.3 Experimental design Variable selection1. Argument:(1) Types of carbon source: sucrose; glucose; soluble starch;(2) The content of carbon source: four levels of sucrose 1

22、5g / L; 30g / L; 45g / L; 60g / L;(3) The content of nitrogen source: KNO3 / NH4NO3 = 1:1 and KNO3 / NH4NO3 = 1:2.2. The dependent variable:(1) Induction rate: the number of callus formation slices/the number of inoculation slices * 100%;(2) Expansion rate: the number of in vitro tissue expansion sl

23、ices /the number of inoculation slices * 100%;(3) Expansion speed: expansion speed = (NK - N0)/K * number of slices. N0 represents the number of in vitro tissue expansion slices at first time, NK represents the number of in vitro tissue expansion slices at Kth day after the first time;(4) Induction

24、speed: induction speed = (NK - N0)/K * number of slices. N0 represents the number of in vitro tissue induction slices at first time, NK represents the number of in vitro tissue induction slices at Kth day after the first time. Design schemeIn this experiment, three kinds of carbon sources, namely su

25、crose, glucose and soluble starch, sucrose density gradient set 4, glucose and soluble starch concentration gradient is not set; nitrogen concentration gradient set 2 were normal 1.65g MT medium / L (KNO3/NH4NO3 = 1:1) and doubling the value of 3.30g / L (KNO3/NH4NO3 = 1:2), the specific content is

26、shown in Table 2.2 table 2.3:Table 2.2 three kinds of carbon source materials in cultureCompositionSerial numberSucrose（g/L）Glucose（g/L）Starch（g/L）NH4NO3（g/L）130001.65230003.30303001.65403003.30500121.65600123.30Table 2.3 four different carbon source level med

27、ium material contentCompositionSerial numberSucrose（g/L）Glucose（g/L）Starch（g/L）NH4NO3（g/L）115001.65230001.65345001.65460001.652.4 Research on Technology Roadmap Access to study material, aseptic inoculation, in vitro culture observation, analysis of carbon and nitrogen source on the effect

28、 of citrus in vitro tissue culture2.4.1 Aseptic inoculationIn the clean bench cut citrus seed cotyledon about the size of 3mm × 3mm random explants inoculated into each culture medium, the body number of each medium was inoculated explant is not less than Culture observationM

29、aterials placed in a temperature 25 ± 2 , relative humidity of 50-60% under conditions of darkness training, periodic statistical swelling number of explants with out a few more, and so calculate the corresponding secondary variables.2.4.3 Statistical analysisRegular observation of statistics,

30、data obtained entry Excel database and then convert the data in SPSS professional platform for single-factor analysis of variance and two factor factorial analysis.3. results and analysis3.1 The effect of carbon species on citrus callus induction In view of the three types of carbon in the expe

31、rimental group were provided with two nitrogen levels, and therefore can not exclude the impact of nitrogen ratio of different carbon sources, it should take two-factor factorial analysis and the results are shown in Table 3.1 and Figure 3.1:Table 3.1 Effect of different carbon sources on&

32、#160;citrus callusTwo-factor factorial analysisSum of squaresDegrees of freedomThe mean squareThe F valueSignificantExpansion rate0.07020.0359.4220.008Induction rate0.04720.0235.6260.030Expansion speed0.00020.0002.1070.184Induction speed2.04E-00521.02E-0054.7110.044Early expansion speed0.00320.

33、0026.6290.020Late expansion speed0.00320.0014.8090.043Early induction speed2.89E-00521.44E-0051.2840.328Late induction speed0.00120.0003.7720.0703210.07250.07000.06750.06500.06250.06000.05750.05500.0525Expansion speedCarbon source type321Carbon source type0.03400.03200.03000.02800.02600.0240Early in

34、duction speedFigure 3.1 Effect of different carbon sources on citrus callus inductionBy the factorial analysis of the results can be seen between the different types of carbon, there are no significant differences in expansion speed, early and late induction speed, but the Expansion r

35、ate (F = 9.422, p <0.01) has significant difference, in the induction rate (F = 5.626,0.01 <p <0.05), the induction speed (F = 4.711,0.01 <p <0.05) and early and late expansion speed (0.01 <p <0.05) has relatively significant difference.3.2 The effect of carbon content on citrus

36、 callus induction This experiment set four carbon source levels and culture conditions were the same in addition to the sucrose content, therefore can use one-way ANOVA for data processing, the results are shown in Table 3.2 and Figure 3.2:Table 3.2 Effect of carbon content on ci

37、trus callus inductionSingle factor analysis of varianceSum of squaresDegrees of freedomThe mean squareThe F valuesignificantExpansion rate0.07430.02528.9850.004Expansion speed0.00130.00023.7540.005Early expansion speed0.00530.00256.8610.001Late expansion speed0.01130.00446.8710.001As can be see

38、n from the results of analysis of variance, between the different levels of carbon sources, there is a very significant differences (F = 28.985, p <0.01) in expansion rate level, there is a very significant difference (F = 23.754, p <0.01) in expansion speed level, as well as the early expansi

39、on speed level (F = 56.861, p <0.01)and the late expansion speed level (F = 46.871, p <0.01) ; from Figure , the expansion rate is the highest when the content is 45g / L, next is 30g / L, 15g / L and 60g / L, expansion speed level is also the highest at 45g / L, followed is 30g / L, 15g / L a

40、nd 60g / L, and the order of early expansion speed level is 30g / L, 15g / L, 45g / L and 60g / L, the order of late expansion speed level is of 45g / L, 60g / L, 30g / L, 15g / L.4321Carbon content0.85000.80000.75000.70000.65000.6000Induction rate0.90004321Carbon content0.090000.08500.08000.07500.0

41、7000.06500Expansion speed0.09500 4321Carbon content0.12000.11000.10000.09000.08000.07000.06000.0500Early expansion speed4321Carbon content0.12000.10000.08000.06000.04000.02000.0000Late expansion speedFigure 3.2 Effect of carbon content on citrus callus induction3.3 The effect of&

42、#160;nitrogen source on citrus callus inductionIn view of the nitrogen in the experimental group were set at three kinds of carbon medium, and therefore can not exclude the impact of different carbon sources of nitrogen, it should take two-factor factorial analysis and the results are show

43、n in Table 3.3: Table 3.3 Effect of nitrogen content on citrus callus inductionTwo-factor factorial analysisSum of squaresDegrees of freedomThe mean squareThe F valuesignificantInduction rate0.03210.0327.6250.025Expansion rate0.00310.0030.5350.486Expansion speed8.17E-00518.1

44、7E-0051.4590.262Induction speed3.72E-00613.72E-0061.7200.226Early expansion speed6.65E-00516.65E-0050.2810.610Late expansion speed0.00010.0000.3630.564Early induction speed0.00010.00019.0650.002Late induction speed0.00010.0002.6320.143By the factorial analysis of the results can be seen in different

45、 nitrogen content, there are very significant differences in the levels of the early induction speed (F = 19.065, p <0.01), relatively significant difference exists in the induction rate levels (F = 7.625, 0.01 <p <0.05), while in the expansion rate, expansion speed and the late induction s

46、peed were not significant.4. DiscussionCarbon source is indispensable for plant tissue culture material, which not only provides energy to give the explants, but also to maintain a certain osmotic pressure, commonly carbon fructose, glucose, sucrose, maltose, sorbitol and the like, which can su

47、crose supports most vigorous growth of plant material in vitro culture, has been used as a standard carbon plant tissue culture and widely used recently, other sugars effect on plant tissue culture, caused widespread concern. Many studies have shown that sucrose is not necessarily the best carbon so

48、urce, the type of plant responses are not identical for different saccharides11 Therefore, in the present experimental design selected sucrose, glucose, and soluble starch as carbon three. Most plant tissue cultures except sugar, glucose, soluble starch as the carbon source can also grow well10, but

49、 found in this experiment, sucrose is still the best source of carbon, and its level of concentration on the more differentiation of tissue injury rate will have a huge impact. When sugars (sucrose or glucose) concentration changed from 4% to 1%, the original dense dry condition cotyledon callus bec

50、omes wet mucous membranes and surrounded by soft fluffy layer11. Therefore, when plant tissue culture, the choice of an appropriate concentration of sugar will not only improve the embryogenic callus induction rate, improve the quality of callus and regenerated ways plants can be controlled, thereby

51、 enhancing tissue regeneration frequency.Guo Xiaohong ect. study adventitious tissue culture Salvia () Effects of carbon, nitrogen and phosphorus sources on adventitious root culture of Salvia mentioned in nitrogen have a significant impact on the growth and adventitious Salvia secondary metabolite

52、biosynthesis11. It can be proved that nitrogen in vitro tissue culture should have a significant role, but the results obtained in this experiment, these conclusions have not been fully validated analysis for two reasons, first, the test procedure does not phase uniform and there is a big error in t

53、he experiment, the experimental results will inevitably be affected; Second, the relationship between morphology and differentiation of citrus and Salvia plants in different position in the classification system, callus exists between consistency needs to be further explored.I found that in the cour

54、se of the experiment, the disinfecting solution of choice for disinfection time citrus callus induction is very important, sterilization time is too long will kill tissue cells, so that it can not be normal differentiation, if disinfection time is too short, you can not play to disinfection, vaccina

55、tion material that may be contaminated in a short time, thus losing valuable experimental materials, increasing the cost.In addition, the browning process in plant tissue culture prevalent in12, and fungi contamination, excessive water content of the three major problems and said plant tissue cultur

56、e13. Has a large number of scholars approach with dark browning control explants were studied11-13. I think that the future course of the experiment, can be appropriate in the medium reducing regulators to prevent yam in tissue culture browning process, which is to improve the level of differentiati

57、on is very important yam.5. ConclusionThis study explored citrus callus induction effects and differentiation rate on carbon species, carbon and nitrogen content, the following conclusions:(1) Carbon source was comparison significantly effect on citrus callus induction, in which the effect on t

58、he expanding rate was extremely significant, the effect on the callus induction rate, callus induction speed, the early expansion speed and the late expansion speed was comparison significant, the effect on the expanding speed, the early callus induction speed and the late callus induction speed was not significant; (2) Carbo