Hello Everybody! Sorry for disappearing for a long long time! Today, I'm back! I'm going to blog about plant subculturing which I did for my SIP. It's quite boring though.
In plant subculturing, small pieces of plant tissues are placed on or in a media rich in nutrients and sugar. The major media components are made up of some or all of the followings :
- Macronutrients
- Micronutrients
- Vitamins
- Amino acids or other nitrogen supplements
- Sugar(s)
- Other undefined organic elements
- Solidifying agents
- Growth regulators
Macronutrients :
Macronutrients provide the 6 major elements : nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulfur (S) required for plant cell or tissue growth. Culture media should contain at least 25-60 mM inorganic nitrogen for adequate plant cell growth. Potassium is required in nitrite, sulfate or chloride form at concentration of 20-30 mM. Optimal concentration of phosphorus, magnesium, sulfur and calcium range from 1-3 mM.
Micronutrients :
Essential micronutrients for plant cell growth include iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu) and molybdenum (Mo). Chelated form of iron and zinc are commonly used at minute concentration of uM. Iron maybe the most critical of all the micronutrients.
Vitamins :
Normal plants synthesize the vitamins required for their growth and development. Vitamins are required by plants as catalyze in various metabolic processes. As the plants are cultured in vitro, vitamins may become limiting factors for cell growth. The vitamins most frequently used are thiamine (B1), nicotinic acid, pyridoxine (B6) and myo-inositol. Myo-inositol although it is a carbohydrate not a vitamin, has been shown to stimulate growth. Therefore it is commonly included in many vitamin stock solution at concentration of 50-5000 mg/L.
Amino acids or other nitrogen supplements :
Although culture cells are capable of synthesizing all of the required amino acids, the addition of amino acids may be used to further stimulate cell growth. Amino acids provide plant cells with an immediately available source of nitrogen which generally can be taken up by cells more rapidly than inorganic nitrogen. The most common source of inorganic nitrogen are casein hydrolysate, L-glutamine, L-asparagine and adenine.
Sugar(s) :
Carbohydrates must be supplemented to the culture medium as few cell lines are fully autotropic i.e., capable of supplying their own carbohydrate needs by CO2 assimilation during photosynthesis. Carbon and energy source of plant cell culture media is sucrose. Glucose and fructose maybe substitute in some cases, glucose being as effective as sucrose and fructose being somewhat less effective. The concentration of sucrose normally ranges between 2-3%.
Other undefined organic elements :
Addition of a wide variety of organic extracts to culture media often results in favourable tissue response. Supplements include protein hydrolysate, coconut water, yeast extracts, malt extracts, ground banana, orange and tomato juice. However, undefined organic supplements should be only be used as a last resort. The addition of activated charcoal (AC) to culture media may have beneficial effect. The effect of AC is generally attributed to one of the three factor : absorption of inhibitory compounds, absorption of growth regulators and darkening of the medium.
Solidifying agents :
Agar is the most commonly used gelling agent for preparing semi-solid and solid plant tissue culture medium. First, when agar is mixed with water, it forms a gel that elts at approximately 60-100 deg C and solidify at 45 deg C; thus, agar gel are stable at all incubation temperatures. Another geling agent commonly used is Gelrite. This is a product of bacterial fermentation and should be used at 1.25 - 2.5 g/L, resulting in a clear gel which aids in the detection of contamination.
Growth regulators :
Four broad classes of growth regulators are important in plant tissue culture; the auxins, cytokinins, gibberellins and abscisic acid. Both auxin and cytokinin are usually added to culture media in order to obtain morphogenesis, although the ratio of hormones required is not universally the same. The type of morphogenesis that occurs in a planbt tissue culture largely depends upon the ratio and concentration of auxin and cytokinin present. Root initiation of plantlets, embryogenesis and callus initiation all generally occur when the ratio of auxin to cytokinin is high, whereas shoot proliferation occurs when the ratio is low.
An example of a general media receipe :
Growth regulator : MS (Murashige and Skoog) 6.45g
Vitamin : Myo-inositol 30mL
Organic elements: No name 30mL
Sugar(s): Sucrose 45g
pH 5.8 -5.81
Activated charcoal 1g
Top up to 3L with distilled water
And thats the end of my boring entry this time! Feel Free to ask any questions and sry if I bore u too much! muahahaha...
Royston Tan
0503289A
TG01
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9 comments:
halo! what a long post!!
wana ask abt the narrow pH? why is there a need for such a small and narrow range???
Charmaine
TG01
Hi Roystan
Just wondering, what type of species of plants require agar and what type of species rquire gelrite? Thanks =)
Eugene Wong
TG02
Hi!
To Charmaine,
For the plants in my lab, they require this narrow pH to grow well. This pH is required for the gelling agent to solidify.
To Eugene,
In my plant lab, the orchids are cultured in agar while other plants like ginger plants and heliconias prefer gelrite.
Hope that helps!
Royston
Tg01
yo roy,
I just wanna know how you regulate the pH of the media you use for your experiments.
thanks.
randall
TG02
Hey royston,
How does the auxins, cytokinins, gibberellins and abscisic acid regulate growth?
Thanks
Andre, TG01
Hello Royston. Plants rocks rite?
1)You said that vitamins gets depleted in vitro. So how do you replenish them together with other nutrients?
2)What is callus?
Thankss!!!
Najib Bin Hamid
0503217B
Hey Royston
I just want to ask you 2 qns. hehe. you mentioned that plant tissues are used here right? does that mean it can come from any part of the plant or do you use only a specfic part of the plant? 2nd qns. I was just wondering where's the source of various nutrients that you had stated earlier? heheh..
Thanks!!
Shahirah Tg01
0503174E
hey
how do you know the what types of nutrients or other reagents to add into growing media? where do you get the recipes from? research or online?
elaine
Hi all!!!
To randall,
pH are regulated with the addition of 0.1 M NaOH and HCL to correct the pH in which the pH value is determined by a pH meter.
To Andre,
Auxins are phytohormones that influence cell enlargement, root initiation, and adventitious bud formation. They also suppress the initiation of lateral buds (which is the bud of choice for ensuring genetic stability).
Cytokinins are growth regulators that are required in tissue culture media for cell division, shoot mutiplication, and axillary bud proliferation. They also help delay senescence (aging), and influence auxin transport.
Gibberellins are a group of naturally occurring substances that influence cell enlargement and stem elongation. They also initiate production of enzyme alpha amylase, which converts starches to sugars and stimulates other enzymes.
Abscisic acid is generally added to either inhibit or stimulate callus growth, to enhance shoot or bud proliferation, and to inhibit latter stages of embryo development.
To najib,
All the nutrients will be used up eventually, replenishment is done by subculturing the whole plant culture into container containing fresh media.
Callus is a proliferating mass of disorganized, mostly undifferentiated or undeveloped cells. The callus mass can contain embryoids (embryo-like somatic structures capable of developing into whole plants), or it can contain shoot or root primordia (the earliest developmental stage of an organ or cell).
To shahirah,
Different plant species employs different culture methods. With reference to external source, each individual culture guidelines are given. For e.g. scutellaria barbata (TCM herb) in my lab uses nod for subculturing. A nod is the intersection between the stem and the leaf stalk. The leaves are removed exposing the inner 'flesh' and this explant is placed onto the fresh media.
The source of various nutrients comes from the Murashige and Skoog (MS) media which provides major salts, minor salts, auxins, cytokinins and iron. The quantity of sucrose(sugars), undefined organic element, myo-inositol(vitamins) varies among different plant species.
To Elaine,
The reciples come from external sources such as books and websites which provides culture guides to different plant species. However, the phases in which the plant culture is at and the types of culture methods used, will affect the quantity of the media components. Hence, the research in my lab covers the optmization of the media components and conditions to maximize tissue proliferation.
Hope these help! xD
Royston
TG01
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