Professional lighting solution for tissue culture Micropropagation
Micropropagation is the asexual propagation of plants using the techniques of plant tissue culture (PTC). Plant tissue culture refers to growing and difgerentiation of cells, tissues, and organs isolated from the mother plant, on artifjcial solid or liquid media under aseptic and controlled conditions. The small organs or pieces of tissue plants used in PTC are called explants. Plant tissue culture medium provides inorganic nutrients and usually a carbohydrate to replace the carbon which the plant normally fjxes from the atmosphere by hotosynthesis. When carbon is supplied with sucrose and kept in low light conditions, micropropagated plantlets are not fully dependent on their own photosynthesis.
The PTC techniques provide a new approach to plant propagation, being the best way to produce uniform plant germplasm and the regeneration of pathogen‐free plants. To date,commercial plant micropropagation has shown great productive potential; it is being used in hundreds of commercial laboratories for the propagation of species of agricultural and forestry importance. Commercial micropropagation of difgerent species of economic importance is shown in Figure 1.
The commercial micropropagation process is carried out in the following stages:
Stage 0: Mother plant selection. Donor plants are selected and conditioned to be used to initiate in vitro cultures.
Stage I: In vitro establishing. The choice of the explant and its disinfection is carried out toinitiate an aseptic culture.
Stage II: Multiplication. It is at this stage that mass propagation is performed, obtaining alarge number of new individuals from minimal amounts of tissue.
Stage III: Elongation and rooting.The shoots must form their root system and at the sametime increase their size to facilitate their manipulation and adaptation to the acclimatizationconditions.
Stage IV: Acclimatization. It consists of a slow reduction of the relative humidity and gradual
increases in the luminous intensity for a betuer adaptation to the external environment.
Figure 1. Commercial micropropagation of difgerent species. (a) Stevia rebaudiana, (b) Ananas comosus, (c) Vanilla planifolia and (d) Anthurium andreanum.
Requirements for the completion of each stage of micropropagation vary according to the method being utilized; it is not always necessary to follow each of the prescribed steps.
However, there are factors that afgect the micropropagation process, including:
Factors that depend on the explant:Size, physiological age of the tissue, and explant position.
Factors that depend on the culture medium:Growth regulators, macro‐ and micronutrients,organic nitrogen, and carbon source.
Factors related to the incubation environment:Photoperiod, temperature, humidity, and light source.
Factors related to the incubation environment refer to incubators or growth rooms where temperature, humidity, and light can be controlled. In commercial micropropagation
laboratories, the light source is one of the most important factors controlling plant development. Light quality (spectral quality), quantity, (photon fmux) and photoperiod have a profound infmuence on the morphogenesis, growth and chlorophyll contents of a plant cell,and tissue and organ cultures.
The illumination systems allow wavelengths to be matched to plant photoreceptors to provide more optimal production and to infmuence plant morphology and metabolic composition .Plants use energy between 400 and 700 nm and light in this region is called photosynthetically active radiation (PAR).
The growth and development of plants is dependent on light for:
Photosynthesis:The process whereby light energy is converted to chemical energy in the biosynthesis of chemicals from carbon dioxide and water.
Photomorphogenesis:The light‐induced development of structure or form.
Phototropism:The growth response of plants which is induced by unilateral light.
In recent years, LEDs have emerged as an alternative for commercial micropropagation.LEDs possess various advantages such as less heat radiation, small mass, a monochromatic
spectrum, greater durability, low power consumption, and specifjc wavelength. The fmexibility of matching LED wavelengths to plant photoreceptors may provide more optimal production,infmuencing plant morphology and metabolism.
Spectral quality of LEDs
The traditional light source used for in vitro propagation is fmuorescent lamps (FLs). However,power consumption in FL use is expensive and produces a wide range of wavelengths
(350–750 nm) unnecessary for plant development, whereas monochromatic light‐emituing diodes (LEDs) emit light at specifjc wavelengths. In this sense, LEDs can be fjne‐tuned to only produce the spectrums that plants need for morphogenic responses. The response to LED light in micropropagation systems depends on light irradiance, photoperiod, and wavelength.The wavelength to which in vitro plants are exposed varies according to the species. Recent studies compare the efgect of FLs (545–610 nm) vs white LEDs (460 and 560 nm), red LEDs(660 nm), blue LEDs (460 nm), and the combination of blue and red LED (460 and 660 nm)treatments. LEDs afgect in vitro rooting, number and length of new shoots, chlorophyll and carotenoid pigments, and other characteristics in plants. The spectral irradiance of LEDs is shown in Figure 2.
Figure 2. Spectral curves distribution in relative response of the LEDs and fmuorescent lamps.
LEDs afgect chlorophyll content
Several studies have shown important efgects of LEDs on photosynthetic pigments during micropropagation of difgerent species. Studies show that blue LEDs are a good light source for chlorophyll induction and that red LEDs decrease chlorophyll content. Dewir et al. found that blue LEDs showed greater growth, vigor, and chlorophyll content in Euphorbia milli. Jao et al. reported that blue LEDs promote growth and increase chlorophyll content in Zantedeschia jucunda. The same efgect was observed by Li et al. during in vitro culture of Gossypium hirsutum and Brassica campestris, respectively. Kim et al. and Moon et al. emphasized the role of blue light on chlorophyll formation and chloroplast development in their work with Chrysanthemum and Tripterospermum japonicum, respectively. Monochromatic red LEDs with narrow peak emissions may cause an imbalance in the distribution of light energy between photosystems I and II, and thus be responsible for a reduction in net photosynthesis . According to Li et al., it has been observed that plantlets with lower chlorophyll content utilize the chlorophyll more effjciently than plantlets with higher chloro‐phyll content under red LEDs.
According to Soebo et al. , the possibility exists that red light may inhibit the translocation of photosynthetic products thereby increasing the accumulation of starch. Goins et al. observed higher photosynthetic rates and an increase in stomatal conductance in wheat leaves under mixed red and blue LEDs. Plant growth and development by increasing net photosynthetic rate was also observed in Chrysanthemum under mixed red/blue LED treatments and has been atuributed to the similarities of the spectral energy distribution of red/blue to chlorophyll absorption .
The importance of blue light in stomatal opening has already been studied. It has been proposed that blue light received by phototropins activates a signaling cascade, resulting in fast stomata opening under a red light background . The efgect of light quality on stomatal characteristics has not yet been clearly determined, and difgerential stomatal behavior could be related to photosynthetic activity and plant growth.
According to Topchiy et al. , light quality also plays an important role in hotosynthesis,infmuencing the way in which light is absorbed by chlorophyll. According to George , the level of chlorophyll so far obtained in tissue cultures is well below that found in mesophyll cells of whole plants of the same species, and the rate of chlorophyll formation on exposure of cultured cells to the light is extremely slow compared to the response of etiolated organized tissues. The greening of cultures also tends to be unpredictable, and even within individual cells, a range in the degree of chloroplast development is often found. In the carbon dioxide oncentrations found in culture vessels, green callus tissue is normally photomixotrophic and growth is still partly dependent on the incorporation of sucrose into the medium . However, green photoautotrophic callus cultures have been obtained from several difgerent kinds of plants.