[8] 微繁殖中的環(huán)境控制

1、Chapter 5 Micropropagation 2. Environmental control in micropropagationl Photoautotrophic micropropagation l Natural ventilation and forced ventilation l Effects of environmental factors on plantlet growthContents CHOCO2CO2CHOPAR sourcePAR sourceHeterotrophyPhotomixotrophyPhotoautotrophyCHO absorpti

2、on from the mediumCO2 fixation (photosynthesis)Nutritional status of in vitro plantletsProduction of transplants-plantlets using micropropagation techniquesMicropropagation, an in vitro vegetative propagation method using pathogen free propagules, plays a vital rule in producing genetically identica

3、l and superior plantlets. Micropropagation has many advantages as a propagation method.1.High production costs due to intensive input of labor.2.Slow plant growth.3.High percent loss of plantlets due to contamination and low percent survival ex vitro.4.Difficulty of mechanization and automation.Prob

4、lems in photomixotrophic micropropagation (PM) using sugar-containing mediumReasons of low survival outside the vessel (ex vitro)nRelative humidity in the vessel is very high (almost 100%).nPlantlets in vitro do not have normal functional stomata and they have little cuticle development.nThey can no

5、t control the water status when they are transplanted in greenhouse conditions.nAcclimatization is necessary to increase the survival percentage in greenhouse.Physiology of in vitro plantletsnLow transpiration rate ( low VPD)nLow net photosynthetic rate ( low CO2 and low PPF)nHigh respiration rate (

6、 high sugar concentration and high temperature)nAbnormal morphological development ( high ethylene concentration)Anatomical characteristics of in vitro plantletsnLittle development of cuticle wax layer nMalfunction of stomatanLess stomatal densitynHyperhydrisity (vitrification)nSmall leaf areanLess

7、development of palisade tissuePhotoautotrophic micropropagationnMicropropagation technique where plants are grown photoautotrophically (without sugar in the medium) under controlled environment favorable to the photosynthesis of the plantlets.nKeys to successnEnhanced ventilation of the vessels (wit

8、h/without CO2 enrichment to growth chamber)nUse of leafy explantsnPorous substrate with liquid medium in place of gelled medium, especially for woody plant speciesConventional (photomixotrophic)Percent water loss from leaves of sweetpotato plantlets immediately after transplanting to ex vitro. Photo

9、autotrophicGrowth and photosynthesis of Momordica grosvenori plantlets grown photoautotrophically in response to light intensityTreatmentcodeLeaf area(mm2)Stem Length (cm)Shoot weight (mg)FreshDryFW/DWControl141276 c4.00.2ab281.015.1c26.22.5d10.7L252802228 b4.00.2ab499.427.0b48.32.8c10.3L503650232 a

10、4.50.2a751.053.5a80.16.9b9.4L1003740270 a4.70.2a846.849.0a97.15.6 a8.7L2002198121 b3.60.2b612.934.7b100.74.1 a6.1Table 2 Effects of four levels of light intensity: 25, 50, 100, 200 mol m-2 s-1 on shoot growth per plantlet cultured on sugar-free medium without NAA for 26 days. The control is a photom

11、ixotrophic culture using sugar- containing medium with NAA (0.5 mg l-1) Effect of four levels of light intensity: 25, 50, 100, 200 mol m-2 s-1 on the growth of Momordica grosvenori plantlets cultured on sugar and NAA-free medium on day 26. The control is a photomixotrophic culture using sugar- and N

12、AA (0.5mg l-1)-containing medium Fig. 3 Effect of four levels of light intensity: 25, 50, 100, 200 mol m-2 s-1 on the chlorophyll content (A) and ratio of chlorophyll a/b (B) of plantlets grown photoautotrophically on day 20. The control is photomixotrophic culture. Vertical bars represent SEControl

13、 L25L50L100L200012345AdcbbaChlorophyll content （mg/FW g）TreatmentsControlL25L50L100L2003.43.5BChlorophyll a/b ratioTreatmentsFig. 4 The Rapid-light curves of the photoautotrophic treatments at four levels of light intensity: 25, 50, 100, 200 mol m-2 s-1 on day 24. The cont

14、rol is photomixotrophic culture. (A) for the second leaf and (B) for the third from the apex of the shoot. Vertical bars represent SE (n=6) 05010015020025030035040004812162024AETR（ mol electron m-2 s-1）PPFD （ mol m-2 s-1） Control L25 L50 L100 L20001002003004005006000481216202428BETR（ mol electron m-

15、2 s-1）PPFD（ mol m-2 s-1） Control L25 L50 L100 L200Fig. 5. Effects of sucrose and NAA on growth of Momordica grosvenori plantlets on day 26(Zhang et al., 2009)Advantages of photoautotrophic micropropagationnBiological aspectnPromotion of growth and photosynthesisnHigh survival percentage and smooth t

16、ransition to ex-vitro environmentnElimination of morphological and physiological disordersnLittle loss of plantlets due to contaminationAdvantages of photoautotrophic micropropagationnEngineering aspectnFlexibility in the design of the vessel (larger vessels)nAutomationnSimplification of the micropr

17、opagation systemnPlant growth can be manipulated by controlled environmentLimitations of photoautotrophic micropropagation Relative complexity of techniques and knowledge required for controlling in-vitro environmentExpense for lighting and CO2 enrichment Using explants with larger leaf area for pho

18、tosynthesis Culture vesselnA tissue culture vessel is a system for growing small plants under aseptic conditions. nIn this sense, tissue culture vessels can be considered as miniature greenhouses. nNevertheless, environmental conditions surrounding plantlets are not directly controlled in tissue cul

19、ture. nGreenhouse environmental control contributes to improved growth and quality of plants. nCan in vitro environmental control be similarly beneficial for the plantlet growth and improve the quality of plantlets? Natural ventilation can be achieved by attaching gas-permeable filter disks either o

20、n the side walls or lid of the vessel (Kitaya et al., 1995)l Forced ventilation can be achieved by supplying CO2 enriched air with an air pump into the vessel through a gas-permeable filter disk (Kozai et al., 2000) A small vessel with enhanced natural ventilation using microporous filter disksA lar

21、ge vessel with forced ventilation using microporous filters and air pumpsMicroporous filter diskAir pumpNatural ventilationForced ventilation In comparison with natural ventilation, forced ventilation shows advantages: Change easily ventilation rate during the culture process. Obtain easily an optim

22、um ventilation rate for photosynthesis. A 120-L forcedly ventilated large vessel containing 1500 calla lily plantlets in cell trays Microporus filterEucalyptus camaldulensis (Zobayed et al., 2000)A 20-L vessel containing 500 plantlets 容器換氣次數(shù)的定義是：容器每小時空氣的交換率除以容器的體積。（h-1）上式中T是時間間隔從0-T(h); K在時間初期的氣體濃度(

23、mol mol-1);Ko在時間0的氣體濃度(mol mol-1); Kou容器外的氣體濃度(mol mol-1)；V是容器的體積(L)。容器的換氣次數(shù)容器的換氣次數(shù)Number of air exchanges of the vessel VKouKoKouKTN)ln1(Pn: net photosynthetic rate per plantlet (mol h-1 /plantlet)K: the conversion factor of CO2，即每升CO2的摩爾數(shù)（ 0.0405 mol l-1 at 28 oC ）N: the number of air exchanges of

24、 the vessel (h-1） V: the air volume of the vessel ( L )Cout and Cin: CO2 concentrations (mol mol-1) inside and outside the vessel under steady-state conditions during the photoperiodE: the number of plantlets per vesselEstimating net photosynthetic rate Pn = KNV (Cout Cin ) / E 在傳統(tǒng)的組織培養(yǎng)技術(shù)中，一直把培養(yǎng)基的配方

25、作為研究的重點。包括培養(yǎng)基的類型、植物激素的種類、各種植物激素在培養(yǎng)基中的含量、細(xì)胞生長素和細(xì)胞分裂素之間的比例。 事實上，光照、溫度、濕度、培養(yǎng)基質(zhì)、CO2濃度、植株的密度、培養(yǎng)容器中空氣的流通速度等環(huán)境因素極大的影響小植株的生長發(fā)育。Reasons for environment controlEffects of environmental factors on photosynthesis and plant growthEnvironmental factors in photoautotrophic micropropagation PhotosynthesisLightCO2Ai

26、r temperatureRelative humidityGrowth promotion in vitro High quality plantletsShorten culture period High survival ex-vitro Supporting materialIn vitro environments (1) Low light intensity, typically less than 50 mol m-2 s-1 PPF. High relative humidity, usually 95-100%. Relatively constant air tempe

27、rature, typically 20 to 28 oC (68 to 82 F). Low CO2 concentration during light period and high CO2 concentration during dark period. High ethylene (C2H4) concentration. Little air movement.In vitro environments (2) Presence of sugar, typically at 10 to 30 g/L sucrose concentration. High mineral ion concentrations, 2 to 3 times higher than those of hydroponic nutrient solution. Low dissolved oxygen concentration, especially in gelled media. Accumulation of toxic compounds (such as phenol)容器的換氣次數(shù)對容器內(nèi)容器的換氣次數(shù)對容