Tissue Culture Cloning: eliminate mother plants & resources they require

greenghost420

greenghost420

Well-Known Member
im about to try and use my pollen thats bout 18 months old. well see how viable it still is, shit was great before being stored. from a few dif males. whats the expiration date on males kept in the fridge via TC?
 
DonTesla

DonTesla

Well-Known Member
greenghost420 said:
im about to try and use my pollen thats bout 18 months old. well see how viable it still is, shit was great before being stored. from a few dif males. whats the expiration date on males kept in the fridge via TC?
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It can be indefinite i believe, the question becomes how long does each petri dish have before it's maxed out and wants a swap up into a new one..

I think jar size, fridge temp, tha plant, and hormone recipe will determine..
 
DonTesla

DonTesla

Well-Known Member
hydroMD said:
Introduction
The starting point for all tissue cultures is plant tissue, called an explant. It can be initiated from any part of a plant - root, stem, petiole, leaf or flower - although the success of any one of these varies between species. It is essential that the surface of the explant is sterilised to remove all microbial contamination. Plant cell division is slow compared to the growth of bacteria and fungi, and even minor contaminants will easily over-grow the plant tissue culture. The explant is then incubated on a sterile nutrient medium to initiate the tissue culture. The composition of the growth medium is designed to both sustain the plant cells, encourage cell division, and control development of either an undifferentiated cell mass, or particular plant organs.

The concentration of the growth regulators in the medium, namely auxin and cytokinin, seems to be the critical factor for determining whether a tissue culture is initiated, and how it subsequently develops. The explant should initially form a callus, from which it is possible to generate multiple embryos and then shoots, forming the basis for plant regeneration and thus the technology of micropropagation. The first stage of tissue culture initiation is vital for information on what combination of media components will give a friable, fast-growing callus, or a green chlorophyllous callus, or embryo, root or shoot formation.

There is at present no way to predict the exact growth medium, and growth protocol, to generate a particular type of callus. These characteristics have to be determined through a carefully designed and observed experiment for each new plant species, and frequently also for each new variety of the species which is taken into tissue culture. The basis of the experiment will be media and protocols that give the desired effect in other plant species, and experience.

The demonstration
The strategy for designing a medium to initiate tissue culture, showing how growth regulators and other factors modulate development, can be demonstrated using the African Violet, a popular house plant. Leaf sections are the source of explants. This demonstration is regularly carried out by a student class, and gives reliable results. Sterile supplies are provided from central facilities, and provision of sterile working areas (for example, in laminar flow hoods) is an advantage, although cultures can be initiated in an open laboratory with careful aseptic technique. The standard precautions used during any laboratory work involving chemicals or microbes should be adopted. If you are in any doubt about safety hazards associated with this demonstration, you should consult your local safety adviser.

Step 1 - selection of the leaves
Leaves are cut from healthy plants, leaving a short length of petiole attached. They should be selected to each yield several explants of leaf squares with approximately 1 cm sides. The youngest and oldest leaves should be avoided.

Wash the dust off the leaves in a beaker of distilled water, holding the leaf stalk with forceps.

Step 2 - surface sterilisation and preparation of the explants
This part of the procedure should be carried out in a sterile working area, or with meticulous aseptic technique.

The leaf, with the petiole still attached, should be immersed in 70% ethanol for 30 seconds, then transferred to a sterile petri dish. Sterile scissors and forceps are then used to cut squares from the leaf as explants, each with approximately 1 cm sides.

The explants are transferred into a 10% hypochlorite bleach solution for 5 minutes, gently agitating once or twice during this time. They are then washed free of bleach by immersing in four successive beakers of sterile distilled water, leaving them for 2-3 minutes in each.

Three explants are placed on each petri dish of growth medium (see table and below), with the upper epidermis pressed gently against the surface of the agar to make good contact.

The petri dishes are sealed with plastic film to prevent moisture loss, and incubated at 25oC in 16h light/8h dark.

Step 3 - assessment of tissue culture development
The explants are incubated for 4 - 6 weeks, and inspected at weekly or fortnightly intervals. The growth of obvious bacterial or fungal colonies indicates contamination, and data from such cultures is obviously suspect. The development of dark brown tissue cultures can also be a consequence of contamination.

The media used in the demonstration are designed to show the effects of auxin, cytokinin, sucrose and mineral salts on development. The media were based on the well-known Murashige and Skoog inorganic medium, with additions as shown in this table.

Typical results

These pictures show typical results, after about 8 weeks on each medium. To summarise, multiple adventitious buds form on the control medium, leading to many small shoots on the upper surface where the leaf is not in contact with the medium.

Absence of sucrose inhibits this production. Shoot production is also limited on the low sucrose concentration, but comparable with the control at high sucrose.

At zero and low levels of cytokinin, callus forms where the leaf surface is in contact with the medium, while at high levels, shoot formation is stimulated.

At zero and low levels of auxins there is a stimulus to shoot formation, but at high concentrations, large numbers of roots are formed.

At low and zero levels of MS salts, there is no growth at all.

These very obvious variations demonstrate the importance of a carbon and inorganic salt source for plant growth, as well as the effect of the auxin:cytokinin ration on the control of plant development.
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Best post yet, thanks doc Hyde!
 
DonTesla

DonTesla

Well-Known Member
KLITE said:
Ok so this has happened

I met with a pharmacist today whos done this type of thing before and shes about to order the hormones and bacteria recquired to do it, ai already have a flow hod with epa. SHe says theres basically two hormones and the different ratios either make roots grow or weird stemy leaves, certain bacteria is recquired to make the process happen or something. She says temperature needs to be kept at certain range otherwise tottally and completely forget about it.
Me and her are gonna play around with it see what happens... i will try to keep you updated if anything goes forth.
According to a reliable source a few ceedbanks in holland already do this, btw..
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I know most wine coolers can have two separate temps set, both for red and white wine.. As well as humidity on some

Great work going out out of your way to to meet with a pharmacist

This is getting sicker by the day..
Healthier and robuster I should say
f^ck sickness
 
thump easy

thump easy

Well-Known Member
ill tell you what its hard for realz about 97 percent of my tryes got sick and died to some type of mold even with the ppm and new pitrie dish is mando.. i lost the other half to trying to get them to go into or to control the their soft bodies after roots also one agur is for roots the other is for shoots it all depends on the agar you made and the hormone you put in i just used a few drops of nutrients and root and shoot hormone and seen both happend.. someone should have the balls and do it and post it..???? i dont have time but am itching to do the research and try again but i dont have time im also late to church
 
DonTesla

DonTesla

Well-Known Member
thump easy said:
ill tell you what ... someone should have the balls and do it and post it..???? i dont have time but am itching to do the research and try again but i dont have time im also late to church
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I got big.. BALLS I CANNOT LIEEEE
all these mothers Gwan denyyyy...

Lets hammer out the recipes I am down to make it happen..buy a wine cooler fridge, make a sterile mini room. Whatever it takes..
 
thump easy

thump easy

Well-Known Member
a dare you guys!!!!!!!!!!!!!! i tripple dare you!!!!!!!!!!!!! dam that sounds like a good og name Tripple Dare You OG??? lolz Loud As Fuck OG?? LAF OG? Mouth OG? Rad Racer OG? Snach Jammer OG??? dang were am i going with this Ogee is always on my mind!!!!!!!!!!!!!! sorry of topic were
 
DonPetro

DonPetro

Well-Known Member
thump easy said:
a dare you guys!!!!!!!!!!!!!! i tripple dare you!!!!!!!!!!!!! dam that sounds like a good og name Tripple Dare You OG??? lolz Loud As Fuck OG?? LAF OG? Mouth OG? Rad Racer OG? Snach Jammer OG??? dang were am i going with this Ogee is always on my mind!!!!!!!!!!!!!! sorry of topic were
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Snatch Jammer...hahahaha
Thump you a legend boi...good to see you in the organics section. And your spelling has improved greatly...lol jk im just bustin your balls man. I enjoy your "Breading Gernal".
 
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