Cloning Plants By Tissue Culture

Bonleesha !!!you're efforts are appreciated. especially the last page ! many minds are about to open up to the inevitable future that tissue culture has in storethanks again !

nitro, that shoot is ready to be cut and transferred, and leave you with a healthy chunk of callus material, to shoot over and over, so you cna keep rooting the shoots, if plants are the intention of course. or you can do all kinds of crazy things with it too.

bonkleesha,
I dont need your lab contact, but could you tell me how much a rose culture costs from the lab. It may help people to put things into perspective in this endeavor.

carolina.com is where the college gets its supplies for tissue culture. the class kit for microprop is about $150, BUT THEY WILL PREMAKE ANY MEDIA TO YOUR SPECIFICATIONS! just email them the formula and they will quote a price.

now... what in the formula needs to change to make it ideal for cannabis? if we can igure it out, i might actually ask for some custom culture tubes.

thats the million dollar question that every body knows, but know body will say,,which in turn=who knows what? im sure you two could put it together,,...its defiantly not on the web..or not in the 1000 studies ive seen..not in a form that i can grasp anyway...didnt i hear carrot callus is pretty close to that med. for mj....for some reason mj and tomatoes come up as one in my brain,,,would a tomato medium have this.....quality?

I GREW A SHOOT, DID U HEAR, IT WORKS!!! I cultured that batch 1/30/12 ,,i can do this math,,,,,in 11 days i am able to split a shoot into 2 plantlets,,do the math 11DAYS,,and i doubled a culture,,,,OH and for all that skipped this whole thread,,, WITH NO KIT!! WITH PICS,, GO BACK AND SEE!


Pharmacoping,,so thats CALLUS stage huh..i thought it may take longer "or" have to be more of a "plant" ,,,but seein how this works,,rooting a BROOMSTICK doesnt sound to hard..HAHA.. theres are about 10 like that, but i cant get pics..theres growth right out of the CUT ends,,little leaves...The P.P.M. is on the way,,hopefully the nadcc will hold out till it arrives...fingers crossed


i have been doing my homework and i have all the micro and macro nuts. plus i have iba/naa and seaweed extract for aux. and cytk. now i need to get the average ppms of micro & macro nuts. to the basic medium for mj or a plant like it..I knows there is a medium to buy that will solve all my guessing,,,but this will be way cooler when 1 X turns into 10+Xs in 30 days!!! IM almost there..STAY TUNED FOR THE NEXT RESULTS,,SOON

Bonkleesha....
carrot medium will get all your callus ready, and then its personal tweaking with cyo-aux to your liking. add both and watch what happens. it's fine, but if more branching is needed.....
just like the china report in this thread.. all of those results are repeatable, there is no one formula, it depends on what you want to do with your explant really. limeon and a bacteria could change things in a big way for you, or maybe you want small bonzai planur ts with 500 branches...all depends. but start with carrot, and basal salts no higher than 150ppm.

Nitro--

150ppm of pure basal salts, available here : http://www.ecogrow.com/16-oz-ecogrow-standard-10814-p-116.html use this exact choice. some will suggest using a bloom formula for all tissue culture, I have, and went bact to this. the nitrogen is sweet. for good.

check this out

and they said it couldnt be done just days ago !!!

To achieve genetic transformation in plants, we need the construction of a vector (genetic vehicle) which transports the genes of interest, flanked by the necessary controlling sequences i.e. promoter and terminator, and deliver the genes into the host plant. The two kinds of gene transfer methods in plants are:

Vector-mediated or indirect gene transfer

Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used. This bacteria is known as “natural genetic engineer” of plants because these bacteria have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The foreign gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted sequences.
To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected and infected with Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The infected tissue is then cultured (co-cultivation) on shoot regeneration medium for 2-3 days during which time the transfer of T-DNA along with foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are transferred onto selection cum plant regeneration medium supplemented with usually lethal concentration of an antibiotic to selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred to root-inducing medium, and after another 3-4 weeks, complete plants are transferred to soil following the hardening (acclimatization) of regenerated plants. The molecular techniques like PCR and southern hybridization are used to detect the presence of foreign genes in the transgenic plants.
Vectorless or direct gene transfer
In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector. The methods used for direct gene transfer in plants are:

Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also used to transfer DNA into cultured cells.

Microinjection where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometerdiameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.
Electroporation involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient (temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA.
The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.
Particle gun/Particle bombardment - In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).The microprojectile bombardment method was initially named as biolistics by its inventor Sanford (198. Two types of plant tissue are commonly used for particle bombardment- Primary explants and the proliferating embryonic tissues.
Transformation - This method is used for introducing foreign DNA into bacterial cells e.g. E. Coli. The transformation frequency (the fraction of cell population that can be transferred) is very good in this method. E.g. the uptake of plasmid DNA by E. coli is carried out in ice cold CaCl2 (0-50C) followed by heat shock treatment at 37-450C for about 90 sec. The transformation efficiency refers to the number of transformants per microgram of added DNA. The CaCl2 breaks the cell wall at certain regions and binds the DNA to the cell surface.
Conjuction - It is a natural microbial recombination process and is used as a method for gene transfer. In conjuction, two live bacteria come together and the single stranded DNA is transferred via cytoplasmic bridges from the donor bacteria to the recipient bacteria.
Liposome mediated gene transfer or Lipofection - Liposomes are circular lipid molecules with an aqueous interior that can carry nucleic acids. Liposomes encapsulate the DNA fragments and then adher to the cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant cells.

Selection of transformed cells from untransformed cells
The selection of transformed plant cells from untransformed cells is an important step in the plant genetic engineering. For this, a marker gene (e.g. for antibiotic resistance) is introduced into the plant along with the transgene followed by the selection of an appropriate selection medium (containing the antibiotic). The segregation and stability of the transgene integration and expression in the subsequent generations can be studied by genetic and molecular analyses (Northern, Southern, Western blot, PCR).



During the last decades, a tremendous progress has been made in the development of transgenic plants using the various techniques of genetic engineering. The plants, in which a functional foreign gene has been incorporated by any biotechnological methods that generally are not present in the plant, are called transgenic plants. As per estimates recorded in 2002, transgenic crops are cultivated world-wide on about 148 million acres (587 million hectares) land by about 5.5 million farmers. Transgenic plants have many beneficial traits like insect resistance, herbicide tolerance, delayed fruit ripening, improved oil quality, weed control etc.
Some of the commercially grown transgenic plants in developed countries are: “Roundup Ready” soybean, ‘Freedom II squash’, ‘High- lauric’ rapeseed (canola), ‘Flavr Savr’ and ‘Endless Summer’ tomatoes. During 1995, full registration was granted to genetically engineered Bt gene containing insect resistant ‘New Leaf’ (potato), ‘Maximizer’ (corn), ‘BollGard’ (cotton) in USA. Some of the traits introduced in these transgenic plants are as follows:

Stress tolerance

Biotechnology strategies are being developed to overcome problems caused due to biotic stresses (viral, bacterial infections, pests and weeds) and abiotic stresses (physical actors such as temperature, humidity, salinity etc).

Abiotic stress tolerance
The plants show their abiotic stress response reactions by the production of stress related osmolytes like sugars (e.g. trehalose and fructans), sugar alcohols (e.g. mannitol), amino acids (e.g. proline, glycine, betaine) and certain proteins (e.g. antifreeze proteins). Transgenic plants have been produced which over express the genes for one or more of the above mentioned compounds. Such plants show increased tolerance to environmental stresses. Resistance to abiotic stresses includes stress induced by herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone and intense light. These environmental stresses result in the destruction, deterioration of crop plants which leads to low crop productivity. Several strategies have been used and developed to build ressitance in the plants against these stresses.

Herbicide tolerance

Weeds are unwanted plants which decrease the crop yields and by competing with crop plants for light, water and nutrients. Several biotechnological strategies for weed control are being used e.g. the over-production of herbicide target enzyme (usually in the chloroplast) in the plant which makes the plant insensitive to the herbicide. This is done by the introduction of a modified gene that encodes for a resistant form of the enzyme targeted by the herbicide in weeds and crop plants. Roundup Ready crop plants tolerant to herbicide-Roundup, is already being used commercially.
The biological manipulations using genetic engineering to develop herbicide resistant plants are: (a) over-expression of the target protein by integrating multiple copies of the gene or by using a strong promoter., (b) enhancing the plant detoxification system which helps in reducing the effect of herbicide., (c) detoxifying the herbicide by using a foreign gene., and (d) modification of the target protein by mutation.

Some of the examples are:

Glyphosate resistance - Glyphosate is a glycine derivative and is a herbicide which is found to be effective against the 76 of the world’s worst 78 weeds. It kills the plant by being the competitive inhibitor of the enzyme 5-enoyl-pyruvylshikimate 3- phosphate synthase (EPSPS) in the shikimic acid pathway. Due to it’s structural similarity with the substrate phosphoenol pyruvate, glyphosate binds more tightly with EPSPS and thus blocks the shikimic acid pathway.
Certain strategies were used to provide glyphosate resistance to plants.

(a) It was found that EPSPS gene was overexpressed in Petunia due to gene amplification. EPSPS gene was isolated from Petunia
and introduced in to the other plants. These plants could tolerate glyphosate at a dose of 2- 4 times higher than that required to kill wild type plants.
Another example is of Phosphinothricin resistance
Phosphinothricin is a broad spectrum herbicide and is effective against broad-leafed weeds. It acts as a competitive inhibitor
of the enzyme glutamine synthase which results in the inhibition of the enzyme glutamine synthase and accumulation of ammonia and finally the death of the plant. The disturbace in the glutamine synthesis also inhibits the photosynthetic activity.
The enzyme phosphinothricin acetyl transferase ( which was first observed in Streptomyces sp in natural detoxifying mechanism against phosphinothricin) acetylates phosphinothricin, and thus inactivates the herbicide. The gene encoding for phosphinothricin acetyl transferase (bar gene) was introduced in transgenic maize and oil seed rape to provide resistance against phosphinothricin.
Other abiotic stresses
The abiotic stresses due to temperature, drought, and salinity are collectively also known as water deficit stresses. The plants produce osmolytes or osmoprotectants to overcome the osmotic stress. The attempts are on to use genetic engineering strategies to increase the production of osmoprotectants in the plants. The biosynthetic pathways for the production of many osmoprotectants have been established and genes coding the key enzymes have been isolated. E.g. Glycine betaine is a cellular osmolyte which is produced by the participation of a number of key enzymes like choline dehydrogenase, choline monooxygenase etc. The choline oxidase gene from Arthrobacter sp. was used to produce transgenic rice with high levels of glycine betaine giving tolerance against water deficit stress.
Scientists also developed cold-tolerant genes (around 20) in Arabidopsis when this plant was gradually exposed to slowly declining temperature. By introducing the coordinating gene (it encodes a protein which acts as transcription factor for regulating the expression of cold tolerant genes), expression of cold tolerant genes was triggered giving protection to the plants against the cold temperatures.

Insect resistance

A variety of insects, mites and nematodes significantly reduce the yield and quality of the crop plants. The conventional method is to use synthetic pesticides, which also have severe effects on human health and environment. The transgenic technology uses an innovative and eco-friendly method to improve pest control management.About 40 genes obtained from microorganisms of higher plants and animals have been used to provide insect resistance in crop plants
The first genes available for genetic engineering of crop plants for pest resistance were Cry genes (popularly known as Bt genes) from a bacterium Bacillus thuringiensis. These are specific to particular group of insect pests, and are not harmful to other useful insects like butter flies and silk worms. Transgenic crops with Bt genes (e.g. cotton, rice, maize, potato, tomato, brinjal, cauliflower, cabbage, etc.) have been developed. This has proved to be an effective way of controlling the insect pests and has reduced the pesticide use. The most notable example is Bt cotton (which contains CrylAc gene) that is resistant to a notorious insect pest Bollworm (Helicoperpa armigera).. There are certain other insect resistant genes from other microorganisms which have been used for this purpose. Isopentenyl transferase gene from Agrobacterium tumefaciens has been introduced into tobacco and tomato. The transenic plants with this transgene were found to reduce the leaf consumption by tobacco hornworm and decrease the survival of peach potato aphid.
Certain genes from higher plants were also found to result in the synthesis of products possessing insecticidal activity. One of the examples is the Cowpea trypsin inhibitor gene (CpTi) which was introduced into tobacco, potato, and oilseed rape for develping transgenic plants. Earlier it was observed that the wild species of cowpea plants growing in Africa were resistant to attack by a wide range of insects. It was observed that the insecticidal protein was a trypsin inhibitor that was capable of destroying insects belonging to the orders Lepidoptera, Orthaptera etc. Cowpea trypsin inhibitor (CpTi) has no effect on mammalian trypsin, hence it is non-toxic to mammals.

Virus resistance

There are several strategies for engineering plants for viral resistance, and these utilizes the genes from virus itself (e.g. the viral coat protein gene). The virus-derived resistance has given promising results in a number of crop plants such as tobacco, tomato, potato, alfalfa, and papaya. The induction of virus resistance is done by employing virus-encoded genes-virus coat proteins, movement proteins, transmission proteins, satellite RNa, antisense RNAs, and ribozymes. The virus coat protein-mediated approach is the most successful one to provide virus resistance to plants. It was in 1986, transgenic tobacco plants expressing tobacco mosaic virus (TMV) coat protein gene were first developed. These plants exhibited high levels of resistance to TMV.
The transgenic plant providing coat protein-mediated resistance to virus are rice, potato, peanut, sugar beet, alfalfa etc. The viruses that have been used include alfalfa mosaic virus (AIMV), cucumber mosaic virus (CMV), potato virus X (PVX) , potato virus Y (PVY) etc.

Resistance against Fungal and bacterial infections
As a defense strategy against the invading pathogens (fungi and bacteria) the plants accumulate low molecular weight proteins which are collectively known as pathogenesis-related (PR) proteins.
Several transgenic crop plants with increased resistance to fungal pathogens are being raised with genes coding for the different compounds. One of the examples is the Glucanase enzyme that degrades the cell wall of many fungi. The most widely used glucanase is beta-1,4-glucanase. The gene encoding for beta-1,4 glucanase has been isolated from barley, introduced, and expressed in transgenic tobacco plants. This gene provided good protection against soil-borne fungal pathogen Rhizoctonia solani.
Lysozyme degrades chitin and peptidoglycan of cell wall, and in this way fungal infection can be reduced. Transgenic potato plants with lysozyme gene providing resistance to Eswinia carotovora have been developed.

Delayed fruit ripening

The gas hormone, ethylene regulates the ripening of fruits, therefore, ripening can be slowed down by blocking or reducing ethylene production. This can be achieved by introducing ethylene forming gene(s) in a way that will suppress its own expression in the crop plant. Such fruits ripen very slowly (however, they can be ripen by ethylene application) and this helps in exporting the fruits to longer distances without spoilage due to longer-shelf life.
The most common example is the 'Flavr Savr' transgenic tomatoes, which were commercialized in U.S.A in 1994. The main strategy used was the antisense RNA approach. In the normal tomato plant, the PG gene (for the enzyme polygalacturonase) encodes a normal mRNA that produces the enzyme polygalacturonase which is involved in the fruit ripening. The complimentary DNA of PG encodes for antisense mRNA, which is complimentary to normal (sense) mRNA. The hybridization between the sense and antisnse mRNAs renders the sense mRNA ineffective. Consequently, polygalacturonase is not produced causing delay in the fruit ripening. Similarly strategies have been developed to block the ethylene biosynthesis thereby reducing the fruit ripening. E.g. transgenic plants with antisense gene of ACC oxidase (an enzyme involved in the biosynthetic process of ethylene) have been developed. In these plants, production of ethylene was reduced by about 97% with a significant delay in the fruit ripening.
The bacterial gene encoding ACC deaminase (an enzyme that acts on ACC and removes amino group) has been transferred and expressed in tomato plants which showed 90% inhibition in the ethylene biosynthesis.

Male Sterility

The plants may inherit male sterility either from the nucleus or cytoplasm. It is possible to introduce male sterility through genetic manipulations while the female plants maintain fertility. In tobacco plants, these are created by introducing a gene coding for an enzyme (barnase, which is a RNA hydrolyzing enzyme) that inhibits pollen formation. This gene is expressed specifically in the tapetal cells of anther using tapetal specific promoter TA29 to restrict its activity only to the cells involved in pollen production. The restoration of male fertility is done by introducing another gene barstar that suppresses the activity of barnase at the onset of the breeding season. By using this approach, transgenic plants of tobacco, cauliflower, cotton, tomato, corn, lettuce etc. with male sterility have been developed.


Clonal propagation refers to the process of asexual reproduction by multiplication of genetically identical copies of individual plants. The vegetative propagation of plants is labour-intensive, low in productivity and seasonal. The tissue culture methods of plant propagation, known as 'micropropagation' utilizes the culture of apical shoots, axillary buds and meristems on suitable nutrient medium.The regeneration of plantlets in cultured tissue was described by Murashige in 1974. Fossard (1987) gave a detailed account of stages of micropropagation.
The micropropagation is rapid and has been adopted for commercialization of important plants such as banana, apple, pears, strawberry, cardamom, many ornamentals (e.g. Orchids) and other plants.The micropropagation techniques are preferred over the conventional asexual propagation methods because of the following reasons: (a) In the micropropagation method, only a small amount of tissue is required to regenerate millions of clonal plants in a year., (b) micropropagation is also used as a method to develop resistance in many species., (c) in vitro stock can be quickly proliferated as it is season independent,. (d) long term storage of valuable germplasm possible.

The steps in micropropagation method are: a) Initiation of culture - from an explant like shoot tip on a suitable nutrient medium, b) multiple shoots formation from the cultured explant, c) rooting of in vitro developed shoots and, d) transplantation - transplantation to the field following acclimatization.
The factors that affect micropropagation are: (a) genotype and the physiological status of the plant e.g. plants with vigorous germination are more suitable for micropropagation., (b) the culture medium and the culture environment like light, temperature etc. For example an illumination of 16 hours a day and 8 hours night is satisfactory for shoot proliferation and a temperature of 250C is optimal for the growth.
The benefits of micropropagation this method are:
a) rapid multiplication of superior clones can be carried out through out the year, irrespective of seasonal variations.
b) multiplication of disease free plants e.g. virus free plants of sweet potato (Ipomea batatus), cassava (Manihot esculenta)
c) multiplication of sexually derived sterile hybrids
d) It is a cost effective process as it requires minimum growing space.

Somaclonal variation
The genetic variations found in the in vitro cultured cells are collectively referred to as somaclonal variation and the plants derived from such cells are called as ‘somaclones’. It has been observed that the long-term callus and cell suspension culture and plants regenerated from such cultures are often associated with chromosomal variations. It is this property of cultured cells that finds potential application in the crop improvement and in the production of mutants and variants (e.g. disease resistance in potato).
Larkin and Scowcroft (1981) working at the division of Plant Industry, C.S.I.R.O., Australia gave the term 'somaclones' for plant variants obtained from tissue cultures of somatic tissues. Similarly, if the tissue from which the variants have been obtained is having gametophytic origin such as pollen or egg cell, it is known as 'gametoclonal' variation.They explained that it may be due to: (a) reflection of heterogeneity between the cells and explant tissue, (b) a simple representation of spontaneous mutation rate, and (c) activation by culture environment of transposition of genetic materials.
Shepard et al. (1980) also contributed by screening about 100 somaclones produced from leaf protoplasts of Russet Burbank. They found that there was a significant amount of stable variation in compactness of growth habit, maturity, date, tuber uniformity, tuber skin colour and photoperiodic requirements.
Somaclonal Variations has been used in plant breeding programmes where the genetic variations with desired or improved characters are introduced into the plants and new varieties are created that can exhibit disease resistance, improved quality and yield in plants like cereals, legumes, oil seeds tuber crops etc. Somaclonal variation is applicable for seed
Applications of Somaclonal Variations
a) Methodology of introducing somaclonal variations is simpler and easier as compared to recombinant DNA technology.
b) Development and production of plants with disease resistance e.g. rice, wheat, apple, tomato etc.
c) Develop biochemical mutants with abiotic stress resistance e.g. aluminium tolerance in carrot, salt tolerance in tobacco and maize.
d) Development of somaclonal variants with herbicide resistance e.g. tobacco resistant to sulfonylurea
e) Development of seeds with improved quality e.g. a new variety of Lathyrus sativa seeds (Lathyrus Bio L 212) with low content of neurotoxin.
f) Bio-13 – A somaclonal variant of Citronella java (with 37% more oil and 39% more citronellon), a medicinal plant has been released as Bio-13 for commercial cultivation by Central Institute for Medicinal and Aromatic Plants (CIMAP), Lucknow, India.
g) Supertomatoes- Heinz Co. and DNA plant Technology Laboratories (USA) developed Supertomatoes with high solid component by screening somaclones which helped in reducing the shipping and processing costs.

Production of virus free plants
The viral diseases in plants transfer easily and lower the quality and yield of the plants. It is very difficult to treat and cure the virus infected plants therefore te plant breeders are always interested in developing and growing virus free plants.
In some crops like ornamental plants, it has become possible to produce virus free plants through tissue culture at the commercial level. This is done by regenerating plants from cultured tissues derived from a) virus free plants, b) meristems which are generally free of infection - In the elimination of the virus, the size of the meristem used in cultures play a very critical role because most of the viruses exist by establishing a gradient in plant tissues. The regeneration of virus-free plants through cultures is inversely proportional to the size of the meristem used., c) meristems treated with heat shock (34-360C) to inactivate the virus, d) callus, which is usually virus free like meristems.e) chemical treatment of the media- attempts have been made to eradicate the viruses from infected plants by treating the culture medium with chemicals e.g. addition of cytokinins suppressed the multiplication of certain viruses.
Among the culture techniques, meristem-tip culture is the most reliable method for virus and other pathogen elimination.

Viruses have been eliminated from a number of economically important plant species which has resulted in a significant increase in the yield and production e.g. potato virus X from potato, mosaic virus from cassava etc. These virus free plants are not disease resistant so there is a need to maintain stock plants to multiply virus free plants whenever required.
Production of synthetic seeds

In synthetic seeds, the somatic embryos are encapsulated in a suitable matrix (e.g. sodium alginate), along with substances like mycorrhizae, insecticides, fungicides and herbicides. These artificial seeds can be utilized for the rapid and mass propagation of desired plant species as well as hybrid varieties. The major benefits of synthetic seeds are:
a) They can be stored up to a year with out loss of viability
b) Easy to handle and useful as units of delivery
c) Can be directly sown in the soil like natural seeds and do not need acclimatization in green house.
Mutant selection

An important use of cell cultures is in mutant selection in relation to crop improvement. The frequency of mutations can be increased several fold through mutagenic treatments and millions of cells can be screened. A large number of reports are available where mutants have been selected at cellular level. The cells are often selected directly by adding the toxic substance against which resistance is sought in the mutant cells. Using this method, cell lines resistant to amino acid analogues, antibiotics, herbicides, fungal toxins etc have actually been isolated.

Production of secondary metabolites

The most important chemicals produced using cell culture are secondary metabolites, which are defined as’ those cell constituents which are not essential for survival’. These secondary metabolites include alkaloids, glycosides (steroids and phenolics), terpenoids, latex, tannins etc. It has been observed that as the cells undergo morphological differentiation and maturation during plant growth, some of the cells specialize to produce secondary metabolites. The in vitro production of secondary metabolites is much higher from differentiated tissues when compared to non-differentiated tissues.
The cell cultures contribute in several ways to the production of natural products. These are: (a) a new route of synthesis to establish products e.g. codeine, quinine, pyrethroids, (b) a route of synthesis to a novel product from plants difficult to grow or establish e.g. thebain from Papaver bracteatum, (c) a source of novel chemicals in their own right e.g. rutacultin from culture of Ruta, (d) as biotransformation systems either on their own or as part of a larger chemical process e.g. digoxin synthesis.
The advantages of in vitro production of secondary metabolites
a) The cell cultures and cell growth are easily controlled in order to facilitate improved product formation.
b) The recovery of the product is easy.
c) As the cell culture systems are independent of environmental factors, seasonal variations, pest and microbial diseases, geographical location constraints, it is easy to increase the production of the required metabolite.
d) Mutant cell lines can be developed for the production of novel and commercially useful compounds.
e) Compounds are produced under controlled conditions as per the market demands.
f) The production time is less and cost effective due to minimal labour involved.

Applications of secondary metabolites
Many of these secondary products especially various alkaloids are of immense use in medicine. The yield of these chemicals in cell culture, is though generally lower than in whole plants, it is substantially increased by manipulating physiological and biochemical conditions.
Shikonine is a dye produced by the cells Lithospermum erythrorhizon on a commercial scale. Besides this there are a number of secondary metabolite products that are being widely used for various purposes. Vincristine is used as anticancer agent, digoxin controls cardiovascular disorders, pyrithrins is an insecticide etc. The production of specialty chemicals by plants has become a multibillion industry.
Please refer to the table for some secondary metabolites and their uses.

Production of Somatic hybrids and cybrids
The Somatic cell hybridization/ parasexual hybridization or Protoplast fusion offers an alternative method for obtaining distant hybrids with desirable traits significantly between species or genera, which can not be made to cross by conventional method of sexual hybridization.

Somatic hybridization
Somatic hybridization broadly involves in vitro fusion of isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plant. The process involves: a) fusion of protoplasts, (b) Selection of hybrid cells, (c) identification of hybrid plants.
During the last two decades, a variety of treatments have been used to bring about the fusion of plant protoplasts. Protoplast fusion can be achieved by spontaneous, mechanical, or induced fusion methods.. These treatments include the use of fusogens like NaNO3, high pH with high Ca2++ ion concentration, use of polyethylene glycol (PEG), and electrofusion. These inducing agents used in protoplast fusion are called ‘fusogen’.
PEG treatment is the most widely used method for protoplast fusion as it has certain advantages over others. These are : (a) it results in a reproducible high-frequency of heterokaryon formation., (b) The PEG fusion is non specific and therefore can be used for a wide range of plants., (c) It has low toxicity to the cell and (d) The formation of binucleate heterokaryons is low.

Mechanism of fusion
The fusion of protoplasts takes place in three phases- agglutination, plasma membrane fusion and formation of heterokaryons. When the two protoplasts come in close contact with each other, they adhere to each other. This agglutination can be induced by PEG, high pH and high Ca2+. The protoplast membranes get fused at localized sites at the point of adhesion. This leads to the formation of cytoplasmic bridges between protoplasts. High pH and high Ca2+ ions neutralize the surface charges on the protoplasts which allows closer contact and membrane fusion between agglutinated protoplasts. The fused protoplasts become round as a result of cytoplasmic bridges which leads to the formation of spherical homokaryon or heterokaryon.
Selection of hybrid cells
The methods used for the selection of hybrid cells are biochemical, visual and cytometric methods using fluorescent dyes. The biochemical methods for selection of hybrid cells are based on the use of biochemical compounds in the medium. The drug sensitivity method is useful for the selection hybrids of two plants species, if one of them is sensitive to a drug. Another method, auxotrophic mutant selection method involves the auxotrophs which are mutants that cannot grow on a minimal medium. Therefore specific compounds are added in the medium. The selection of auxotropic mutants is possible only if the hybrid cells can grow on a minimal medium. The visual method involves the identification of heterokaryons under the light microscope. In some of the somatic hybridizations, the chloroplast deficient protoplast of one plant species is fused with the green protoplast of another plant species. The heterokaryons obtained are bigger and green in colour while the parental protoplasts are either small or colourless. The cytometric method uses flow cytometry and flourescent-activated cell sorting techniques for the analysis of plant protoplasts.

Applications of Somatic hybridization

a) Creation of hybrids with disease resistance - Many disease resistance genes (e.g. tobacco mosaic virus, potato virus X, club rot disease) could be successfully transferred from one species to another. E.g resistance has been introduced in tomato against diseases such as TMV, spotted wilt virus and insect pests.
b) Environmental tolerance - using somatic hybridization the genes conferring tolerance for cold, frost and salt were introduced in e.g. in tomato.
c) Cytoplasmic male sterility - using cybridization method, it was possible to transfer cytoplasmic male sterility.
d) Quality characters - somatic hybrids with selective characteristics have been developed e.g. the production of high nicotine content.

Chromosome number in somatic hybrids
The chromosome number in the somatic hybrids is generally more than the total number of both of the parental protoplasts. If the chromosome number in the hybrid is the sum of the chromosomes of the two parental protoplasts, the hybrid is said to be symmetric hybrid. Asymmetric hybrids have abnormal or wide variations in the chromosome number than the exact total of two species.

In 1972, Carlson and his associates produced the first inter-specific somatic hybrid between Nicotiana glauca and N. langsdorffii. In 1978, Melchers and his co-workers developed the first inter-genetic somatic hybrids between Solanum tuberosum (potato) and Lycopersicon esculentum (tomato). The hybrids are known as ‘Pomatoes or Topatoes’.
Limitations of Somatic Hybridization
a) Somatic hybridization does not always produce plants that give fertile and visible seeds.
b) There is genetic instability associated with protoplast culture.
c) There are limitations in the selection methods of hybrids, as many of them are not efficient.
d) Somatic hybridization between two diploids results in the formation of an amphidiploid which is not favourable therefore haploid protoplasts are recommended in somatic hybridization.
e) It is not certain that a specific character will get expressed in somatic hybridization.
f) Regenerated plants obtained from somatic hybridization are often variable due to somaclonal variations, chromosomal elimination, organelle segregation etc.
g) Protoplast fusion between different species/genus is easy, but the production of viable somatic hybrids is not always possible.

Cybrids
The cytoplasmic hybrids where the nucleus is derived from only one parent and the cytoplasm is derived from both the parents are referred to as cybrids. The process of formation of cybrids is called cybridization. During the process of cybridization and heterokaryon formation, the nuclei are stimulated to segregate so that one protoplast contributes to the cytoplasm while the other contributes nucleus alone. The irradiation with gamma rays and X-rays and use of metabolic inhibitors makes the protoplasts inactive and non-dividing. Some of the genetic traits in certain plants are cytoplasmically controlled. This includes certain types of male sterility, resistance to certain antibiotics and herbicides. Therefore cybrids are important for the transfer of cytoplasmic male sterility (CMS), antibiotic and herbicide resistance in agriculturally useful plants. Cybrids of Brassica raphanus that contain nucleus of B. napus, chloroplasts of atrazinc resistant B. capestris and male sterility from Raphanus sativas have been developed.


In vitro plant germplasm conservation
Germplasm refers to the sum total of all the genes present in a crop and its related species.
The conservation of germplasm involves the preservation of the genetic diversity of a particular plant or genetic stock for it’s use at any time in future. It is important to conserve the endangered plants or else some of the valuable genetic traits present in the existing and primitive plants will be lost. A global organization- International Board of Plant Genetic Resources (IBPGR) has been established for germplasm conservation and provides necessary support for collection, conservation and utilization of plant geneic resources through out the world. The germplasm is preserved by the following two ways:
(a) In-situ conservation- The germplasm is conserved in natural environment by establishing biosphere reserves such as national parks, sanctuaries. This is used in the preservation of land plants in a near natural habitat along with several wild types.
(b) Ex-situ conservation- This method is used for the preservation of germplasm obtained from cultivated and wild plant materials. The genetic material in the form of seeds or in vitro cultures are preserved and stored as gene banks for long term use.
In vivo gene banks have been made to preserve the genetic resources by conventional methods e.g. seeds, vegetative propagules, etc. In vitro gene banks have been made to preserve the genetic resources by non - conventional methods such as cell and tissue culture methods. This will ensure the availability of valuable germplasm to breeder to develop new and improved varieties.

The methods involved in the in vitro conservation of germplasm are:

(a) Cryopreservation- In cryopreservation (Greek-krayos-frost), the cells are preserved in the frozen state. The germplasm is stored at a very low temperature using solid carbon dioxide (at -790C), using low temperature deep freezers (at -800C), using vapour nitrogen (at- 1500C) and liquid nitrogen (at-1960C). The cells stay in completely inactive state and thus can be conserved for long periods. Any tissue from a plant can be used for cryopreservation e.g. meristems, embryos, endosperms, ovules, seeds, cultured plant cells, protoplasts, calluses. Certain compounds like- DMSO (dimethyl sulfoxide), glycerol, ethylene, propylene, sucrose, mannose, glucose, praline, acetamide etc are added during the cryopreservation. These are called cryoprotectants and prevent the damage caused to cells (by freezing or thawing) by reducing the freezing point and super cooling point of water.

(b) Cold Storage- Cold storage is a slow growth germplasm conservation method and conserves the germplasm at a low and non-freezing temperature (1-90C). The growth of the plant material is slowed down in cold storage in contrast to complete stoppage in cryopreservation and thus prevents cryogenic injuries. Long term cold storage is simple, cost effective and yields germplasm with good survival rate. Virus free strawberry plants could be preserved at 100C for about 6 years. Several grape plants have been stored for over 15 years by using a cold storage at temperature around 90C and transferring them in the fresh medium every year.

(c) Low pressure and low oxygen storage- In low- pressure storage, the atmospheric pressure surrounding the plant material is reduced and in the low oxygen storage, the oxygen concentration is reduced. The lowered partial pressure reduces the in vitro growth of plants. In the low-oxygen storage, the oxygen concentration is reduced and the partial pressure of oxygen below 50 mmHg reduces plant tissue growth. Due to the reduced availability of O2, and reduced production of CO2, the photosynthetic activity is reduced which inhibits the plant tissue growth and dimension. This method has also helped in increasing the shelf life of many fruits, vegetables and flowers.

The germplasm conservation through the conventional methods has several limitations such as short-lived seeds, seed dormancy, seed-borne diseases, and high inputs of cost and labour. The techniques of cryo-preservation (freezing cells and tissues at -1960c) and using cold storages help us to overcome these problems.

I think its going to be a good year for some !

+++++++++++++rep!!!!!!!!!!!!!!!!!!!!!!!!!!!

holy shit dude!!!not another one...This one is for the pros...(u guys) i like the china mans dumb dummy study.....BUT tell me???? Does this mean that taking a x-plant down to its nubbin does not take it back to "a seed" like quality??? or will this JUST ensure the quitting of any local things like bugs and mold..ha ha (pharamacopying ssshhhh.) so maybe one could p.m. me ...... or just put this into GRASSHOPPER terms,,,,obviously,,its to much for one to handle at first, but, all the info helps, the start,of what im tryin to learn about...this is the reason i am interested, to purify a plant (not just mj)

heres my take. during and after these cells are washed and sterilized, with success and failures noticed within day to the eye, and then cut up, under sterility, divided, amongst a hundred more vessels, all sterile, over and over, your tru genetics, or at least the ones already affecting the very first plant you excised, are now able to express themselves for the babes they are. Under no genetic stress, caused by the mutagens continuing an attack on your hunny throughout the grow, unnoticed, altering its dna code every time(note the bumps in your stalk) and on trees, this is where the "Genetic Throw Down" occurs, leaving the remainder of its life cycle affected in different ways....these ladies are able to fully report their entire dna code back to you, given their environment is top notch, (described in detail in above post link). Every generation produced is a supergirl compared to the last generation. every leaf is thicker, stalks are shiny and strong from birth and till harvest. the leaves are super prounounced in detail and terpine profiles scream their potential. the very point of this is to accomplish repeatable results, which is a must imo in the medical mj field. every grower knows that there are subtle if not pronounced differences in every clone taken from one mom, seed to seed, generation to generation. A clone fucks up bad eventually, compared to when it was your "best in the world weed". the best way to keep her is in this suspended animation, or a hundred of her in case something goes wrong. not a mom ever changing with clones of varying acceptance(lab results) clones dont get better after that first cut, and even that cut changes many many clones for many growers, as it introduces viruses instantly. In contrast you may infect a plant that grows three fingers and a nose, and may like that trait caused by a viral hijacking somewhere in an ancestor(and could even report more anomalies later!) so you can keep it, purify it, and stop the viral dna hijacking for good, rather than continuing changing traits with every cut, and moment in growth. The finish date of 100% of my flowers is precise, everytime, for years,their finish weight is within 1/4oz evertime, and each one looks exactly like the last of its kind. all are pest resistant, no mites here ever in the room(3 yrs)never a death, or strange plant reaction, or ph issue, no nutrient lock ups, no additives except nutes,silica,zone. the tissue culture has been my link to success. the missing link. its not for many in the field, but the ones growing for their maximum patients, full time, and providing professional results to professional people, know about this, and are now taking a serious look. I've been screaming tissue culture for 20 yrs, to everybody. with very similar results, as here.
so, I would like to thank you guys for chiming in, getting your feet wet, and seeing some possibilities. there are more than we can imagine.


for instance,

If someone(with a card) in MI will pay me for a bio luminescence gene inserted into the dna of a young plant, maybe of your choice. bid now. no pics
until sold, if permissable then.

what do you think nitro ? how high do you think it'll go?

I know, a tall order, but I have seen one, and now can make one ! It's very difficult, with many failures, and a ton of time, for sure an expensive endeavor. But imagine having a mother plant that floresces, as well as all of its cuttings, seedlings, etc. I would venture to say priceless, especially by the way the security of the plant I saw was handled.

I know there are non believers, and I'm a tease...so here is proof that this is being done, and has been done , in a home lab. this is wicked, with my boys(Scott, in Georgia) videos, and some far out stuff. take notes, no secrets divulged, but you'll see a walnut tree, make light. I'm still in school learning these types of manipulations, and their potential applications. I bring marijuana to the lab table, years ago even, and am here now.

Nitro, this wil get you off BIG time !! and is exactly how I will floresce an amazing purified strain hybrid called Ace Of Spades. It may already be the best mj on the planet, it is for me and every person who has used it thusfar.

enjoy the b'jeezus out of this one, and follow the links, all of them...you might even get a glimpse of something special http://www.esf.edu/chestnut/tissue culture.htm#

the one ingredient you're not seeing is PEG. its the link between all of this home lab magic. it will allow layering , like grafting of several different plant species on top of eachother in vitro. this may prove to be a very quick way of growing a multi strain plant/100plants even , with a few different kinds of weed on it for your dialed in pleasure, customized. carrot roots on pot plants

would sure be easier to remove from these damn gro rocks when done !

I can rot fresh shrimp for a bit, and culture the bacteria from its skin. much of this bacteria is bioluminescent in nature,stuck on the skin, and will group together in the soup, until grown, and if taken care of in a jar or petri dish(needs lots of 02), will turn into a nice little herd of glow in the dark stuff.. if anyone has taken this any further, please let me know your experience. rabbits, mice, fish, and other animals are already born and some for sale with these type of bio markers present and visible.

nitro...the medium i used for the shrimp stuff was a snip of beef boullion ! I knew you'd love that !!

Justus von Liebig's Law of the Minimum states that yield is proportional to the amount of the most limiting nutrient, whichever nutrient it may be. From this, it may be inferred that if the deficient nutrient is supplied, yields may be improved to the point that some other nutrient is needed in greater quantity than the soil can provide, and the Law of the Minimum would apply in turn to that nutrient









SHIT,,, this guy thinks anything is possible!!!


OK,,,,,,,,,,, DO IT UR WAY "DUDES"!?!?!?! THIS IS WHAT I THINK I KNOW. HA HA ,, JUST LIKE CLONES,, YOU NEED TO GET THEM GOIN,,,BUT WHEN CLONES GET GOIN U GET TO ADD SOME N/P/K/......WHEN YOU "MAKE A HOME-MADE MEDIUM,,,, U NEED TO DO ALOT OF RESEARCH,,,,,,THEN THE IDEA IS TO GET ALL OFF THE NUTRIENTS THAT U DISCOVERED AND PUT AS MUCH AS YOU CAN BUT NOT TO MUCH,,,,YUH YUH,,,I KNOW "JUST LIKE ANYTHING",,,,,,NO NO NO!!!,,,THIS TIME YOU WILL NOT BE ABLE TO "ADD" ANYTHING TO IT UNLESS U RE-JAR UR PLANT LET (I THINK)...A CLONE HAS LEAVES AND MORE STEM THAT WILL HELP THE PRODUCTION OF IT TO A POTABLE PLANT,,,A BASIC CULTURE HAS NONE OF THIS,, AND WILL NEED "ALL" THE HELP IT CAN GET THROUGHOUT THE PROCESS..... AND I GATHER THAT THE PLANTLET WILL LEARN HOW TO,,, AND WHEN TO USE THE FOOD SOURCE....SO YOU NEED TO COME UP WITH THAT PERFECT SOLUTION THAT U THINK WILL "DO IT FOR YOU" OR DONT BE A "GRASSHOPPER" AND BUY A MEDIUM THAT U CAN EXPERIENCE THE "GOLD" WITH!!


PHARMACOPING,,,MAN thats cool that u are into all kinds of TC..almost sounds like u ARE a pro...(scientist)..ok i want a 6 tri tip'ed cow!!!GRASS FED PLEASE! one thing at a time,,plants,,then animals...

I MIGHT BE THE LAST STONER TO FIND THIS BUT I THOUGHT IT WAS FUN.. http://www.firstrays.com/fertcalc.htm OH AND HELPFUL,,TOOK ALOT OF GUESSING RIGHT OUT OF MY PLANS!!!

lebigs law doesnt apply anymore. school isnt telling me this, my brain is. we can give these plants 100% of EVERYTHING just about, and the colas arent infinitely big. im sure science people can see the problem with it in the present. plant tissue culture media is a PERFECT example. look at the formula for the mini rose media, and talk to lebig about minimums. no offense to the dude. most scientists were right.... IN THEIR TIME.

This is a standard law in the grow room, however outdated, and provides a set of parameters that need to be addressed. Most continue trying to add things to the mix when they're results are not whats desired, when in fact its most likely one of the major ones needing help- air-water-light-temp-hum-food.

Bonkleesha is right, sorta, about old news. however, food is not the only parameter that is limited in the lab, I'm guessing. A perfect grow atmosphere is described in a bigbuds mag link earlier. ONce all these are alll met, your cola will grow as tall as genetics will allow. But in a shitty room, the best food in the world provided cant make up for the lack of c02,o2 in the root zone,ph imbalances,weak lighting not photosynthesizing enough to completely uptake and burn carbs, etc. I quote the law of minimum often, to anyone at the store trying to "fix" an issue

look at the guitar colas in my pics, they were given the perfectly balanced conditions, and produced perfectly, as expected, each time the same..
not he is not working in a test tube that does not require photosynthesis to flourish. adding nute ppm 's higher than 200ppm(which willl yield slower results from burn) will end the life of the culture. example. One drop of a quality nute, mixed in one liter of water will support callus material for a month in 30 jars!.

worry about your nutrient demands once the plantlet has been released into the veg room. simply stop mixing your own formulas, and buy a professionaly formulated off the shelf one thats already made the wheel turn, Dutch master, advanced nutes, and several others. make sure you follow the instructions from marijuana tested nutes only. My tomatoes do great with the wasted run off nutrients alone, in dirt, outside, or inside.

lesson=more nutrients are never better than less. quality and completeness of your nute system is key. eyes closed, the easiest grow room issue to solve is the nutrient one.

nice calculator for nutrient formulating professionals nitro !!!!

after done fuggin around with spoons and weights, dial this up, leave the rest to the pros, and use that time to experiment with in vitro body parts?

http://www.dutchmaster.com.au/nutrient_calculator.php?language=english

if more people bought the media, i bet they would make it more readily available and it would be cheaper.

I've thought the same thing Bonkleesha, so I started to sell it specifically for mj, about year or two ago. it really is cheap, even retail costs. but the mixing/formulating is a little tricky, and the reason I'm here. Many ways to skin this cat, and I am biased, so these fresh perspectives, unbiased by experience or teachers, is a refreshing way to see new possibilities. working in the lab does not allow these one off types of activities, which is where this all came from in the beginning ! so I take this opportunity to learn form the inexperienced hopefully, working without the constraints of protocol and technique.

it has been great for me, and my lab, with new trial every day.

agar is sold at the health food stores cheap, in bulk. the rest is linked.

In recent years, knowledge of anthocyanin pigments has undergone unprecedented expansion. Indeed, the molecular genetic control of anthocyanin biosynthesis is now one of the best understood of all secondary metabolic pathways. Advances in analytical technology have led to the discovery of many novel anthocyanin compounds, dramatically enriching the palette used by plant breeders to introduce vibrant new colors into horticultural crops. I found these bio flavanoids and anthocyanin pigments used in tissue culture to permanently alter the colors of the flower of plants for sale. salysylic acid(aspirin, willow branch) is used in vitro to induce these, as well as UV light, or heat, for the home lab.

What we are doing is called Bio Hacking