ever heard of clone seeds?

[avant gardener]

In one season I can make more genetics than me and everyone I know could explore in our lifetimes. And that's just one person in his backyard.

So to people that worry about the implications of this technology: cannabis seeds and genetic diversity are here to stay!

Shhhh!
Someone's got to rock a tinfoil hat and play the fearmonger.
Just figured maybe I could get it out of the way before the real whack-jobs found their way to this thread.

 

[Bunrab]

...it isn't 'just' a tissue culture...it is something that people without a sterile lab will be able to work with relatively simply...I love growing out seeds to see what is what...but!...I would also love to be able to order a "selected by the breeder to best represent thier variety" seed clone/whatever it will be called...options for the paying customer can't help but be a good thing...

 

[Phillthy]

sign me up. while i wouldnt want to see them replace regular seeds they would be a great way to back up genetics or share them without troubles. if and when they find a way to "dry" them i would buy into the franchise

 

[MOneYMiKe]

good read I believe tom hill & Chimera are working with this idea right now........

hope so...

 

[ShroomDr]

you can all purchase Tissue culture kits for < $300.

You can make a 'glove box' out of a clear bin and some long rubber gloves.

LC's can sit idle for long periods. A mycelium LC will live indefinitely. A tissue culture would only expire by outgrowing its enclosure (and they can grow REAL slow)

The tissue cultures do have roots. They are rooted in agar, they are just tiny.

This isnt a new idea, and anyone with problems with it certainly does not understand where most common cultivars come from. Even your tequila is from tissue cultured blue agave.

The feds would have ZERO reason to NOT consider each little 'plant-let' a plant. Why wouldnt they?

Its not that i wouldnt want a TC of GreenCrack, KKSC, RKS, etc, but imnsvho....
the balls to do it + the ability to pull it off would eliminate anyone who wasnt smart enough to make 'easier' money.

 

[TLoft13]

I don't think you can take a regular tissue culture, like in a dish, stick it in a jar on the shelf, and come back in a couple of months and revive it.

If these things can sit in suspended animation for while like an actual seed, then I think that is something not being done yet.

Yes they can, IIRC it depends on the solution in the dish. You can store a tissueculture for a long time (months atleast) in an inactive solution(don't remember exactly, think it's a special nutrient/hormone mix), transfer it when you need it in another "active" solution and it starts growing right away, atleast that's what i read. Don't know if seed -like storage times would be feasible for the consumer, probably not.

 

[mal schaun]

http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

 

[Homer J Pimpin]

It wasn't Steve's idea, he found out about it from Chimera but was unable to bring it to market.

finally someone get's it.............

 

[Homer J Pimpin]

hope so...

It's gotta be what they have going on....They are both hush hush,
but putting 2 and 2 together.you get it.

would love to see chimera pop into this thread

 

[C21H30O2]

You can store tissue culture for years at the right temperature and artificial seeds are viable for months to over a year. Something to consider with artificial seeds is that they take a very long time to get going. On the order of a few months. That doesn't negate their value but I still will be buying and making seeds. Sexual propagation is needed for the long term health of desirable genetics. Time, disease, and accidents will take your clones from you eventually, its always good to save genetics sexually and asexually IMO.

 

[TLoft13]

Completly forget, afaik there are two companys with tissue culture clones coming online. One american, one canadian, iirc. Please google yourself if interested.

 

[JKD]

Article regarding synthetic cannabis seeds being made at University of Mississippi.

http://www.medicinalgenomics.com/wp-content/uploads/2011/12/Synthetic-Seeds.pdf

It's already being done. Just not for us... (yet).

JKD

 

[ShroomDr]

Article regarding synthetic cannabis seeds being made at University of Mississippi.

http://www.medicinalgenomics.com/wp-content/uploads/2011/12/Synthetic-Seeds.pdf

It's already being done. Just not for us... (yet).

JKD

...
Introduction
Plant tissue culture techniques have been successfully
applied for rapid clonal multiplication and conservation
of several plant species including Cannabis
plants (Loh et al. 1983; Richez-Dumanois et al. 1986;
Mandolino and Ranalli 1999; Slusarkiewicz-Jarzina
et al. 2005; Bing et al. 2007, Lata et al. 2009a, b,
2010a). Recently, alginate encapsulation technology
for the production of synthetic seeds in conjunction
with micropropagation has become a viable approach
for in vitro germplasm conservation (Lata et al.
2009b). However, the occurrence of somaclonal
variation is a potential drawback when the propagation
of an elite germplasm is intended, where clonal
stability is required to maintain the advantages of
desired elite genotypes. Thus, it is important to assess
the genetic stability of the conserved propagules.
Although many reports are available on the utilization
of synthetic seeds for micro propagation and
conservation of various medicinal plant species
(Mandal et al. 2000; Anand and Bansal 2002; Singh
et al. 2006; Narula et al. 2007; Faisal and Anis 2007;
Ray and Bhattacharyaa 2008; Lata et al. 2009b), there are very few studies on genetic stability of synthetic
seed-derived plantlets exist.

Molecular studies are well developed in Cannabis
sativa for genetic characterization and marker-assisted
selection and individualization based on random
amplified polymorphic DNA (RAPD), restriction
fragment length polymorphisms (RFLP) analysis,
amplified fragment length polymorphism (AFLP),
microsatellite markers, inter simple sequence repeat
(ISSR) and short tandem repeat (STR) multiplex
(Alghanim and Almirall 2003; Datwyler and Weiblen
2006; Faeti et al. 1996; Gilmore and Peakall 2003;
Hakki et al. 2003; Kojoma et al. 2002; Mendoza et al.
2009; Lata et al. 2010b).

In continuation of our previous work (Lata et al.
2009), we have developed an efficient conservation
protocol to store high-yielding C. sativa elite clones at
low temperature using synthetic seed technology
(unpublished work). Since our goal is to develop a
secure and stable in vitro clonal repository of elite
C. sativa germplasm that will ensure future availability
of desirable pharmacologically active chemotypes, the
importance ofmaintaining stability of in vitro conserved
plants cannot be ignored. To ensure that synthetic seed
technology will indeed conserve the micropropagated
propagules of C. sativa, following their conversion from
encapsulated nodal segment, the genetic fidelity of the
synthetic seed grown in vitro conserved C. sativa
germplasm is assessed using ISSR markers. Furthermore,
biomass samples taken from mature synthetic
seed raised plants andmother plant were also compared
for their major cannabinoids profile and cannabinoids
content using GC-FID to assess differences, if any,
between the two types of plants.
Materials and methods

In vitro propagated C. sativa plantlets were produced
according to a protocol described by Lata et al. (2009a).
Nodal segments (3–5 mm) excised from in vitro
proliferated shoots were encapsulated (Lata et al.
2009b) and kept for germplasm conservation at either
5, 15 or 25C(Lata et al. 2011). After 6 months the seeds
were re-grown in MS ? 0.5 lM TDZ (for shoot
induction) and  MS ? 2.5 lM IBA (for rooting)
media under in vitro conditions for 8 weeks followed by
their hardening and propagation in soil under growroom
condition (Lata et al. 2009a). About 43, 60 and
47% of the seeds had survived at the respective
temperatures over 6 months. After 3 months of vegetative
growth (18 h photoperiod) followed by 7 weeks
of reproductive growth (12 h photoperiod) under controlled
growing conditions (25 ± 3C, 55 ± 5% RH
and PAR 700 ± 24 lmol m-2 s-1 at plant canopy
level), biomass samples were collected from the mature
mother plant and 11 randomly selected clones, representing
all three different storage conditions (5, 15 and
25 C), and all samples were subjected to ISSRanalysis.
DNA extraction
A fresh leaf sample (20 mg) was frozen in liquid N2
and ground in a 2 ml micro-centrifuge tube using
Mixer Mill MM 2000 (Retsch, Newtown PA). The
total genomic DNA was extracted using a DNeasy
plant mini kit (Qiagen) and resuspended in 50 ll
elution buffer. The purified total DNA was quantified
and its quality verified by using a nano-drop 1000
spectrophotometer (Thermo-Scientific, Wilmington,
DE). In the initial screening 14 primers were used for
ISSR analysis.

...
...

ISSR markers. Data was scored as 1 for being present
and 0 for the absence of DNA band in each
micropropagated and mother plant.
GC analysis
For comparison of phytocannabinoids among the
mother plant and synthetic seeds, biomass samples
were collected at their peak reproductive stage and
were extracted and analyzed (see Ross et al. 1996).
Six major cannabinoids, i.e. D9-tetrahydrocannabinol
(THC), tetrahydrocannabivarin (THCV), cannabidiol
(CBD), cannabichromene (CBC), cannabigerol (CBG)
and cannabinol (CBN) were identified and quantified
by GC (column DB-1; 15 m 9 0.25 mm, 0.25 lm
film thickness; initially at 170C for 1 min then
programmed to 250C at 10C/min; injection at
240C; detector at 260C; with a flame ionization
detector. The concentration of each cannabinoid was
calculated using an internal standard (IS) of 4-androstene-
3,17-dione.
Statistical analysis was performed to assess the
differences, if any, in the chemical constituents
between mother plant (T12) and synthetic seed raised
clones (T1–T11) using SYSTAT software package
(SYSTAT software Inc. Chicago, IL).
Results and discussion
DNA from C. sativa leaves was extracted using
DNeasy plant mini kit from Qiagen. A total of 14
ISSR primers were initially screened with the DNA of
single donor plant of C. sativa and 11 daughter plants as
templates. Based on a criterion of the generation of
distinct bands that were completely reproducible
between the samples, 9 of the 14 primers were selected
as suitable primers for C. sativa, and thus used for the
present study (Table 1).
Eleven randomly selected plants regenerated from
synthetic seeds stored under different storage conditions
(5C—three plants, 15C—five plants and
25C—three plants) derived from encapsulated nodal
segments along with the mother plant were subjected
to ISSR analysis at maturity. These plants showed no
discernible differences among them and as compared
to the mother plant in the ISSR analysis. The general
morphology of the plants grown under the controlled
growroom conditions was also similar. Each tested
primer produced clear and scorable amplification
products in all the plants. Each primer produced a
unique set of amplification products ranging in size
from about 74 bp in UBC 842 to 2093 bp in UBC
825 (Table 1) with an average of 4.4 bands per
primer. A total of 480 (the number of plantlets
analyzed multiplied by the number of bands with all
primers) were generated by the ISSR method, giving
rise to monomorphic patterns across all 12 plantlets
analyzed. No ISSR polymorphism was observed
among MP and the plantlets regenerated from synthetic
seeds after 24 weeks of storage under different
slow growth conditions (Fig. 1).
The number of the primers (9) used in this study,
as well as total number of bands (480) together with
the observed normality and homogeneity of the plants
generated in this study, strongly suggest that the
.....

Acknowledgments This work was supported in part by the
National Institute on Drug Abuse (NIDA), National Institute of
Health (NIH), Department of Health and Human Services,
USA, Contract No. N01DA-10-7773.
References

alginate encapsulation technology.

http://en.wikipedia.org/wiki/Alginic_acid

Alginic acid, also called algin or alginate, is an anionic polysaccharide distributed widely in the cell walls of brown algae, where it, through binding water, forms a viscous gum. In extracted form it absorbs water quickly; it is capable of absorbing 200-300 times its own weight in water.

So... i bolded where it says the technique works, and where it highlights 'the transfer to "normal" substrate'...

 

[avant gardener]

you can all purchase Tissue culture kits for < $300.

http://www.1hydroponichydrosystemsmichigan.org/images/PROPIGATION_plant_tissue_culture_kits.jpg

that's some stoner ingenuity right there.
i'm still not convinced that box supports aseptic technique though.

 

[zymos]

"About 43, 60 and
47% of the seeds had survived at the respective
temperatures over 6 months."

So, not quite there on storage yet...

 

[Illuminate]

the future is here..

 

[TLoft13]

Completly forget, afaik there are two companys with tissue culture clones coming online. One american, one canadian, iirc. Please google yourself if interested.

WTF? I get neg-repped for this? Because i don't have the time and patience right now to google for you?
Lazy bum...

 

[ShroomDr]

that's some stoner ingenuity right there.
i'm still not convinced that box supports aseptic technique though.

well this is just a 'ghetto glove box'. you put all your supplies inside, including lysol, or equivalent.

That was just a random google pick, ive seen 'diy flow hoods' where they add a positive pressure fan with hepa filter.

You can go nuts!

but its basically overkill. A cardboard box, with kitch gloves taped into the sides, and a saran wrap 'viewing window' works too.

Liquid cultures work great with Mushies (mycelium) because the actual mushroom is just the fruiting body. The problem with tissue cultures is transferring them to a regular substrate.

You just break up a mycelium LC, and inject it into a pasteurized substrate. There are no 'nastyies' to compete with the mycelium. Keeping nasty pathogens away from a tissue culture during the 'hardening period' is where the difficultly will come. The 'nastyies' are everywhere!

You think you hear bitching about regular seed germination rates, how many 'half assers' will come out of the wood work? ghetto cardboard glove boxes can work, laminar flow cabinets are a much better option.