Lophophora hybrids

[Inyan]

http://cgi.ebay.de/(-10-)-Samen-Lophophora...228127004r24623

http://www.thegardenforums.org/viewtopic.php?p=142694

Sorry to post a few links to pictures of hybrid seed as well as hybrids. I just thought it might be nice to keep information about hybrids of a specific genus in one place for quick access. Better to go to one place rather than thumb through all the threads in search of bits and pieces. Alright guys, I started this, but I expect contribution from anyone and everyone. Especially you KadaKuda as you've already created some hybrids with this genus. When hybridizing, its important to realize that you may be seeing parthenogenesis with those seeds so you can never be sure from the first cross. Crossing the siblings of your F1 cross should help though if no obvious phenotypical differences arise in the F1 batch of seedlings. This is called your F2 cross. Remember, store your pollen in a dry frozen state for utilization at any time of the year. You don't want to be handicapped by the pollen you happen to have available at the time. Pollen cocktails, mentor pollen, etc. are also good idea's if your simply wanting to add some diversity to your seedlings. You can work out the particulars later if you keep good notes as to what species was in each batch of pollen. Of particular note, use a permanent marker to label your pollens and keep a log book of when a cross was made with a particular batch of pollen, how many attempts were made, what approach was used, etc. If you cut back the style on a few and not on others, you need to know which ones were which as you may find you have parthenogenesis occurring one way and a good cross the other. Please refrain from idle chatter in this thread. I'm hoping this will develop into a serious working thread for those interested in creating hybrids. Please note, attempts at hybridizing with the cross attempted in proper format and resulting seeds or seedlings can be posted here as well without the fear of ostracism or other such ridicule. It is well known that many Lophophora may indeed self pollinate and no cross can be guaranteed even with the best of techniques such as emasculation of the flower before the pollen matures, etc. as we can not rule out parthenogenesis. With that said, I am hopeful that those who are so brave as to list some of their crosses attempted will give dates of pollination as well as dates the fruit matured as fruit can be delayed in many species when the pollen is not one that it is used to. Pods can abort. These things can and should be noted with the understanding that a failure does not mean the cross is not possible. It merely means that more attempts, different stock plants etc. may need to be tried or different techniques.

An example might be:

L. jourdaniana (seed bearer) x M. luethyi (pollen donor) pollinated on 30 Dec. 08. Method employed cut-style method... cross labeled JL1

L. jourdaniana (seed bearer) x M. luethyi pollinated on 30 Dec. 08. Method employed pollen cocktail... cross labeled JL2

L. jourdaniana (seed bearer) x M. luethyi pollinated on 30 Dec. 08. Method employed grafted style. Note: parafilm used and or capillary tube ... cross labeled JL3

L. jourdaniana (seed bearer) x M. luethyi pollinated on 30 Dec. 08. Method employed cut-style method... cross labeled JL4

L. jourdaniana (seed bearer) x M. luethyi pollinated on 30 Dec. 08. Method manual pollination of emasculated flower... cross labeled JL5

M. luethyi (seed bearer) x L. jourdaniana (pollen donor) pollinated on 30 Dec. 08. Method employed cut-style method... cross labeled JL4

The idea being that you can come back and edit, add pictures, etc. to keep everything smooth and in one place.

Protoplast fusion Links

http://www-saps.plantsci.cam.ac.uk/docs/protofusion.pdf

I've put this link in here as it makes protoplast fusion sound easy and goes step by step. You can source the chemicals and more pertinent articles and extrapolate from there how to do this with cacti. Of particular interest of course is the very distant types of crosses made possible with this method. Another useful idea is to stain each cell from a species or genus with a different stain for each experiment. This will allow you to isolate, watch, and select the exact fusion products you are after without the worry of, " Is my fusion product simply Lophophora 22 + Lophophora 22= 44 chromosomes. Another thing to keep in mind is that protoplast fusion utilizing gametes will yield your normal 22 chromosome cacti which may stand a better chance at natural hybridization with another species or even a new species. I'll come back to this and keep adding so keep watching as pertinent information will be edited in.

Bridge crossing or bridge plants is another area one may want to look into.

Suppose one wants to cross Trichocereus x Lophophora and you've tried everything you can and nothing seems to work.

A Bridge cross or two or three may be needed... one finds that a particular Lophophora out of 100 from the same genetic location will cross with a particular M. luethyi your F1 seedlings (L.M.l=F1) from this cross would be Lophophora (seed bearer) x M. luethyi.

Now, you have also discovered that a particular Echinopsis will cross will cross with M. luethyi

These F1 seedlings you label M. luethyi x Echinopsis (pollen cocktail m20%.p60%.t.20%) or F1= M.lE 262)

You continue to cross these F1 seedlings from both of your crosses while simultaneously trying to cross these seedlings to Trichocereus.

Your crosses might look like this: F2= M.lE 262 x L.M.l

These seedlings may then be crossed to a pure Echinopsis and perhaps later to your Trichocereus. Yes, this takes many crosses and back crosses in an attempt to filter out the genes that you are after into the cacti you are attempting to create and perhaps I should have started out with an easier cross or method....

Easier method to visualize the process.

Trichocereus x M. luethyi = f1 TxM.l

Lophophora x M. luethyi = f1 LxM.l

f1 LxM.l x f1 TxM.l = F2 (LxM.l) x (TxM.l) and you have your resulting cross with genetics from both Trichocereus and Lophophora. Yes, unwanted genetics may be in this cross, but you may find that this cross will cross to either species itself in its pure form. Back crossing to Trichocereus and Lophophora and crossing those resulting seedlings to each other or back to their respective sets of parents... Yes, its time consuming work so you really have to love hybridizing and seeing what if anything arises. Pollen cocktails and mentor pollen are both valuable in this type of cross as you can test out many types of pollen or varieties from different species at the same time.

For informational purposes there have been some very wide crosses of this type done. Follow the link for just one such example.

Astrophytum. x Mammillaria. x Gymnocalycium

http://www.lapshin.org/cultivar/N18/exp-e.htm

The above cross is a perfect example of a cross that could be utilized in this manner. Especially so as both Mammillaria and Astrophytum have been reportedly crossed with Lophophora.

[(Astrophytum. x Mammillaria.) x Gymnocalycium] x (Lophophora x Mammillaria) would be another good cross if one assumed that the particular Gymnocalycium or Astrophytum utilized was not able to cross with Lophophora and you desired those particular genetics for your particular project. Unusual form, flower, color, etc. are thus introduced slowly into your Lophophora line which you can then breed back to Lophophora varieties repeatedly while selecting for the trait or traits you were after. In this way, you could eventually create a hybrid that could be considered almost genetically pure Lophophora that had a purple or even a bicolored red/purple flower if that was your goal.

Edited December 31, 2008 by Inyan

[M S Smith]

Just thought I would add this as a contribution to the thread. I can't help much on hybrids otherwise.

~Michael~

[Garbage]

Lol,no genetic testing in those days.

[kadakuda]

good thread, hopefully we will get some of the loph breeders out of their closets here seems to be a fair few here that play a lot with peyote.

it could be nothing at all, but an early observation is that diffusa seems to accept more pollen species than do other lophophora species, at least in my attempts. perhaps its just coincidence, not sure.

Loph hybrids are a little tricky to prove in any kind of timely matter. to see their true phenotype, they should probably be seed grown, 4+ years to flower. if you graft, then you are never really sure if that is the true look or if the grafting end of things muffed it up. at least with cross genera hybrids it is usually more apparent. but with really closely matched hybrids it can be super difficult to tell, maybe even impossible without a lab. either way, we can always speculate and give best guesses .

here are a couple crosses i have done that yielded seeds that sprouted.

L. fricii: Father

L. koehresii: Mother

Resulting fruits

cross worked every time, very good germ rates! seedlings hardy.

there are a few people stateside and in europe with these now as well.

______________________

Lophophora williamsii:Father

Lophophora fricii: mother

5 seeds from 1 fruit, 1 grew and is grafted. still small, photos another day when its sunny and not raining (its outside)

more later.

[Teotzlcoatl]

Great works guys!

[Inyan]

Protplast Fusion....This thread will expound on exact chemicals, protocol, etc. for those interested in gamete protoplast fusion or other cell types for 2n=22 or 2n=44 type protoplast fusion products. The idea here is to make this simple enough that anyone could do it. Give me some time to work on this and I'll keep coming back as time permits to edit and add information.

Chemicals sourced:

http://www.ncbe.reading.ac.uk/ncbe/materials/PDF/Price08.pdf

viscozyme...Carbohydrase mix ..Viscozyme® .... £10.00, PectinasePectinase .......Pectinex ... £14.00, Cellulase ....Celluclast® . £10.00

http://www.worthington-biochem.com/PASE/pl.html

Pectinase

http://www.worthington-biochem.com/CEL/pl.html

Cellulase

http://www.sigmaaldrich.com/catalog/Lookup...;ST=RS&F=PR

Viscozyme

http://www.sigmaaldrich.com/catalog/Produc..._KEY&F=SPEC

Plant Protoplast Digest/Wash Solution

http://www.aniara.com/mm5/merchant.mvc?Scr...TOXICITYTESTING

Stain MTT Diphenyltetrazolium Bromide .... turns viable protoplasts red while not needed, will help to differentiate between different protoplasts and ensure you are getting the sought after combination between your different genus/species crosses as otherwise you may end up growing fused protoplasts from the same species/culture.

http://www.sigmaaldrich.com/catalog/Produc..._KEY&F=SPEC

Another dye, while not needed... can help to differentiate between different types of protoplasts.

Evans Blue

Edited December 31, 2008 by Inyan

[Inyan]

Pollination Techniques to overcome fertilization barriers

Mentor pollen is utilized to allow a pollen that is highly unlikely to make it to the ovule to have a teacher that knows the way. Seriously, follow the link or simply type in mentor pollen yourself and google. Macerations of the stigma of the compatible pollen are also sometimes used to inoculate the stigma surface or cut style surface of the plant to be crossed. Whether you want to argue that the stigma recognizes the protein signature from the mentor pollen and produces certain chemicals or from the pollen tube itself is not of importance to me, what is important is that mentor pollen has been successful with many different crosses to allow foreign pollen to introduce its genetic potential to a new genus or species with relative ease when compared to some other methods to overcome these barriers.

"The use of mentor pollen has enabled successful hybridization between cassava, Manihot esculenta Crantz, and the wild species M. pohlii Warwa. Killed pollen of a cross compatible type produced by freeze-thawing was mixed with incompatible pollen and the mixes were dusted on stigmas. This treatment resulted in production of seed in 4.9 % of the total pollinations, compared to 0% in the case of untreated pollinations. The use of a bridge species, M. neusana Nassar, through the hybrid M. pohlii and M. neusana also proved successful in overcoming interspecific barriers between cassava and M. pohlii." ( http://www.geneconserve.pro.br/reprints10.htm )

There are many techniques to create mentor pollen and while freezing dry pollen preserves the integrity of the pollen, repeatedly freezing wet pollen, i.e. freshly collected pollen, irradiating, etc. will render you mentor pollen. Mentor pollen is often mixed into a pollen cocktail that has anywhere from one incompatible pollen or several different types of incompatible pollen.

Pollen cocktails are another valuable technique which may also include mentor pollen. My suggestion is use mentor pollen, but utilize it in a controlled manner such as adding mentor pollen to pollen samples that have been divided into three and only mixing in mentor pollen with 1/3 of your properly labeled pollen containers. Pollen cocktails allow you to quickly find a bridge species or plant. I divide this into bridge species/cacti as you may find a single cacti or an entire species that is capable of cross breeding to either group in question. It is important to keep as accurate records as possible of the particular plants utilized in your pollen cocktail as well as the percentages from each species utilized. After all, if you find a bridge species via a cocktail and are unable to reproduce your effects it does you no good. Noting the individual cacti utilized in each cocktail is just as important as listing the species and %'s of each.

http://mobot.mobot.org/cgi-bin/search_vast

Nice link to chromosome counts for various cacti. While it is a myth that chromosome counts must match to create viable offspring, it does tend to help if chromosome counts match.

Edited January 1, 2009 by Inyan

[Auxin]

Due to fanatical views of my government I wont be working specifically with Lophophora, but these techniques interest me greatly for other work and I think my questions might help the Loph growers too.

Macerations of the stigma of the compatible pollen are also sometimes used to inoculate the stigma surface or cut style surface of the plant to be crossed.

This is one that has been catching my eye, stigmatic isolates used in conjunction with foreign pollen through CSM, GSM, or direct pollination techniques. I've found several clues but its still a little foggy. In reading about breeding lilies and beans I've heard two terms used for the stigmatic isolate used- 'stigmatic fluid' and 'stigmatic exudate' the latter is clearly the thin film of adhesive goo on the stigma whereas I couldnt be sure if the lily breeders were using 'stigmatic fluid' to mean the exudate or if they were cutting/mashing the stigma of a compatible donor and using the fluid inside the tissue. Your use of macerations seems to bypass that uncertainty (simply mush it up on a microscope slide and use the mush to wipe on a film of fluid) but one thing that is still unclear to me is if these various stigmatic isolates should be killed by a freeze-thaw protocol like with mentor pollen to avoid the possibility of introducing wild pollen grains which had adhered to the fluid donor stigma.

I am also unclear on common procedural intricacies of the cut-style method. Its clear that at least some people are completely removing the stigma and a fraction of the style but what is not clear, but seems feasible, is if it is among common techniques to remove the stigma followed by cutting the style at an inward angle from bottom towards top just 3/4 or so the way through, placing the pollen and perhaps stigmatic fluid in that cut, then letting the flap close to in essence merge a cut-style pollination with a grafted technique in order to prevent drying or callousing of the cut-style end which could interfere with pollen germination and growth. If such a technique were successful a modification could be tried in which the stigma is not removed and is instead pollinated with mentor pollen while the foreign pollen is applied in the cut lower down, thus allowing for the chemical signals produced by use of mentor pollen without diluting the foreign pollen. As far as I have read all of that is hypothetical. In practice where the cut-style method is used and the stigma and some of the style is amputated are any efforts made to prevent excessive drying of the pollinated cut? Obviously the plant would be isolated to avoid wild pollen and that isolation would probably increase humidity some but I was thinking perhaps a little 'balloon' of plastic wrap or something like that is formed around the flower to keep the humidity intentionally high to slow callousing?

Thanks

Edited January 2, 2009 by Auxin

[Inyan]

Your proposing something like this?

Stigmatic fluid or exudate..... Don't worry yourself about which is which. Grind the compatible donor stigma up and use the juice. You don't need to think fancy. The same thing goes with the grafted style... don't think fancy. If you know a particular pollen germinates and grows x number of cm's in an hour... you can think of ways to accomplish this and remember, you don't need a lot of tissue for your grafted style/stigma, etc. The main and most important thing to remember is pollen is very much like a seed. It needs moisture to germinate and higher levels of humidity than those that might be encountered in a plants natural setting can often increase the ability of a foreign pollen to germinate. Humidity is of utmost importance for the pollen as well as for the cut style or stigma surface. Even a coke bottle, plastic bag, etc... can keep things more humid. I prefer parafilm myself as it is exceptional for grafting purposes... it breaths, stretches, and falls off when it is no longer needed. As far as how much style or stigma to remove... the zone of inhibition has to be by-passed. Cut the style in half and you've generally accomplished this. Cutting the style in half can be done horizontally or vertically. If done vertically, you can insert an nice chunk of your compatible pistil (compatible with the pollen) ... not the plant or cacti in question. Essentially, you'd be making a grafted style lengthwise instead of horizontally. You have to allow yourself to think outside of the box.

The mentor pollen, both viable and killed method serve many purposes... one is fruit set. If their is insufficient compatible pollen then your pod will often abort. If there is not enough viable pollen from your mentor pollen... your pod will often abort. You have to experiment and see if 100% dead mentor pollen is okay to trigger things with each and every cross you attempt. If not, then you need to move to minimal pollination utilizing mentor pollen in your pollen cocktail. The idea is this: If you know you need 1-2 grains of viable pollen from your mentor pollen to get a pod not to abort then you use 1-2 pollen grains to accomplish this. Any other seed that forms will be from your non-compatible pollen and thus you have 1-2 seeds that are not hybrids and the rest which are. The problem is of course that many times the resulting hybrid seeds will still abort and or you will find an large mostly empty seed pod with a heavy load of callous tissue surrounding those aborted seeds. I have a few pictures detailing this in my own crosses. Unfortunately, I don't have them small enough to post in here... at least most of them are too large. You are very right in many of your assumptions.. even the experts don't understand 100% why these methods work. I really don't care too much as long as I have data to back those crosses up. I think of many pollen rejection in much the same way as I think of the human immune system. A female with rh- blood may have an rh+ baby with no problem without a rhogam shot. However, the next baby she will not be able to have if it is rh+ if she did not have the shot. Similarly, the body detects foreign antigens when they reach a crucial level and mounts a reaction against them. With this in mind, many plant stigma/style surfaces can be exposed to the proper antigens to signal seed production from dead or minimal levels of viable mentor pollen thereby allowing the foreign pollen antigens to go undetected as they begin to germinate. However, some foreign pollens need the specific antigens found on and in the stigma of their compatible species to germinate. Excessive humidity may bypass this need, but when it does not... this is why stigma fluid is utilized. I'm thinking for our experimental purposes, its probably best to place a a few pollen grains on the stigma prior to macerating it as this allows for that stigma to send added chemical messengers unique to that particular pollens germination to be utilized in/on the cut style of the non-compatible style. Likewise, you can simply apply the compatible pollen to its compatible stigma and then insert this stigma into the stigma/style of your non-compatible cross taking care not to let the stock or scion to dry out in the process. High humidity is a must! Pollen is much more adaptive than many like to think. You can germinate many pollen grains on a slice of onion and so it appears that not all are dependent on stigmatic juices so to speak. You can look at this germination under a microscope if you use a thin peel from the skin itself. Again, be careful not to let it dry out. With that said, minimal pollination has a great advantage in that you utilize a set percentage of pollen types and a set number estimate of pollen grains. Serial dilutions are made of each pollen type and many crosses are made. The idea being that if you know a particular cactus can set seed pods of 20-30 seeds when out-crossed and needs a minimum of 4 seeds to produce a seed pod... you reduce the number of pollen grains down to 10-15 to pollinate each flower with. You use varying percentages or dilutions in relationship to each other with the understanding that any seed pod over 18 seeds is most likely a waste. Those in the 10-15 range though in this scenario may have varying percentages of both hybrid and non hybrid seed that you should be able to uncover the percentages within reason of both compatible and incompatible crosses. This is the theory at least. If you have 4 grains of compatible pollen on a stigma and they are viable and 10 grains of non-compatible pollen and you get 10 seeds to form and 3 abort... you can figure that the first 4 grains did their job of keeping the pod from aborting while the 10 grains of non-compatible pollen went undetected. Perhaps your dilutions work so that you can only get pods of 10 seeds compatible pollen and 2 seeds incompatible polllen... you see why many attempts have to be made and back-crosses to both sets of parents must be done to ensure that you have actually created hybrids when the obvious phenotype has not changed enough to be certain. Its similar in this manner to a mother with rh- blood. She may have an rh+ baby so long as not enough blood/antigen is detected from the baby as she creates antibodies/callous tissue to the resulting foreign tissue/antigen/protein. Why not work with Lophophora pollen? I don't think there is a regulation against shipping pollen yet? You can see a few different examples of how I have tried impossible crossed below. I've tried many more ways mind you and many of them resulted in seed. The majority of the seed aborted early on many of these crosses however. Some did reach pretty impressive sizes before they aborted with this particular cross, albeit, it was the opposite way... enough though for some nice examples. You can extrapolate from my examples and apply them to your own research and hybridizing attempts whatever the species or genus may be.

Edited January 2, 2009 by Inyan